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Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Treatment noncompliance'.	Late response to rosuvastatin and statin-related myalgia due to SLCO1B1, SLCO1B3, ABCB11, and CYP3A5 variants in a patient with Familial Hypercholesterolemia: a case report. Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B11B and5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A53. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. pmcIntroduction Familial hypercholesterolemia (FH) is a genetic metabolic disease that leads to increased high low-density lipoprotein (LDL) cholesterol, which is a risk factor for early atherosclerosis and cardiovascular diseases (1). FH is usually treated with high-dose statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), a key enzyme in cholesterol biosynthesis pathway. Rosuvastatin is one of the most effective statins, probably due its hydrophilicity, that confers selectivity to hepatic cells, higher affinity to HMGR, and lower rates of statin-related adverse events (SRAE) compared to other statins. It is poorly metabolized by CYP2C9 and CYP2C19, while 72% of the non-metabolized molecules are excreted via biliary system. Therefore, rosuvastatin blood levels rely on the activity of membrane transporters, mainly of solute carrier (SLC) and ATP-binding cassette (ABC) families, highly expressed in intestine, liver, and kidney (2). Pharmacogenetic studies have shown that loss-of-function variants in genes encoding OATPs, such as SLCO1B1, SLCO2B1, and SLCO1B3, and ABCs have been associated with variability in low-density lipoprotein cholesterol (LDL-c) reduction and higher risk of SRAE (3). The importance of considering the combined effect of variants in key genes for pharmacogenetic analyses has been increasingly evident (4). In this case report, we discuss how variants in genes participating in different stages of statin pharmacokinetics pathway possibly affected the time to response to rosuvastatin and the risk of SRAE in a female FH patient. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. This case is reported in accordance with the CARE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-5540). Case presentation A 26-year-old Caucasian female patient with definite diagnosis of FH according to Dutch Lipid Clinic Network MEDPED criteria (5) was invited to participate in an intervention study in June 2019. She was previously included in a FH sequencing study (May 2018), in which a panel of 84 genes involved in lipid homeostasis and drug metabolism was sequenced using exon-targeted gene sequencing (NGS). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for both studies. The patient carries the variant LDLR rs28941776 (c.1646G>A, p.Gly549Asp), which has been associated with FH and is classified as pathogenic according to the American College of Medical Genetics (ACMG) guidelines (6). Her clinical history included high levels of total cholesterol and LDL-c since childhood. In 2008, at the age of 15 years, she had an abnormal lipid profile even under a daily treatment with simvastatin 10 mg and ezetimibe 10 mg. Laboratory analyses showed a total cholesterol of 324 mg/dL, LDL-c 264 mg/dL, high-density lipoprotein cholesterol (HDL-c) 46 mg/dL, and triglycerides 71 mg/dL. In 2014, she was diagnosed with hypothyroidism and treated with levothyroxine 25 µg/day, which was gradually increased to 100 µg/day in 2019. She also had a pregnancy history in January 2017. Her therapy history included simvastatin, which led to severe myopathy in 2008, with marked increase in serum creatine kinase (CK) to 1,080 U/L (4.7-fold the upper reference value). The cholesterol-lowering therapy was changed to pravastatin 20 mg and ezetimibe 10 mg daily until May 2011, when she reported another episode of myalgia. Pravastatin was withdrawn and atorvastatin 20 mg was introduced, also associated with ezetimibe 10 mg. Three months later, in August 2011, she reported interrupting atorvastatin treatment due to myalgia. Rosuvastatin 10 mg was then introduced, also associated with ezetimibe 10 mg, after which she showed an LDL-c level of 125 mg/dL and never reported myalgia again. However, her lipid profile worsened throughout the years even under rosuvastatin treatment, with her LDL-c reaching 194 mg/dL with rosuvastatin 20 mg. The patient had no history of liver or kidney impartment, HIV, coronary artery disease (CAD), diabetes, obesity, cardiovascular events, and did not smoke or drink. Her mother and grandmother had a history of FH, but not CAD or cardiovascular events, while her father had hypertension and type 2 diabetes. In the intervention study, the patient was seen four times (V1 to V4) in 5 months, and clinical history and therapy data were obtained. The protocol consisted of a 6-week rosuvastatin wash-out period, after which rosuvastatin was reintroduced for additional 6 weeks, when treatment response was evaluated. Adherence to treatment was assessed in each timepoint using the translated and validated version of the Brief Medication Questionnaire (BMQ) (7) and blood samples were taken in each visit for laboratory testing. The lipid profile during the follow-up is shown in Figure 1. In April 2019 (V1), the patient was taking rosuvastatin 40 mg, ezetimibe 10 mg, and levothyroxine 88 µg daily. She reported experiencing muscle pain after recently increasing rosuvastatin dose from 20 to 40 mg/day. Her lipid profile was altered (total cholesterol 376 mg/dL, LDL-c 263 mg/dL, HDL-c 67 mg/dL, triglycerides 234 mg/dL) without increase in CK levels. She reported being active, running 2 km 2–3 times a week, and had a healthy diet, eating more than five portions of vegetables daily. Her TSH and T4 levels were normal. Rosuvastatin 40 mg was then discontinued for wash-out, ezetimibe was maintained, and levothyroxine dose was increased to 100 µg/day. Figure 1 Plasma lipid profile and pharmacotherapy of the FH patient throughout the study period. EZT, ezetimibe; LVT, levothyroxine; RSV, rosuvastatin; SRAE, statin-related adverse events. In June 2019 (V2), after undergoing a 6-week rosuvastatin wash-out period between V1 and V2, her lipid profile worsened (total cholesterol 512 mg/dL, LDL-c 405 mg/dL, HDL-c 65 mg/dL, triglycerides 213 mg/dL). Because the patient reported myalgia in V1 (rosuvastatin 40 mg), the physician prescribed rosuvastatin 20 mg/day for six weeks. Surprisingly, in August 2019 (V3), the lipid profile (total cholesterol 531 mg/dL, LDL-c 407 mg/dL, HDL-c 67 mg/dL, triglycerides 286 mg/dL) did not change compared to V2. The patient reported experiencing no myalgia to rosuvastatin 20 mg. In September 2019 (V4), her lipid profile improved (total cholesterol 299 mg/dL, LDL-c 208 mg/dL, HDL-c 59 mg/dL, triglycerides 158 mg/dL) and she continued not experiencing myalgia to rosuvastatin. During the follow-up period, serum TSH and T4 levels remained unchanged, suggesting that her hypothyroidism was controlled and did not influence the lipid profile. Moreover, serum CK did not show any abnormality, which indicates no muscle damage due to statin treatment. The patient also reported being adherent to treatment. In the BMQ adherence questionnaire, she reported forgetting the lipid-lowering medications 2 days in the week before V1 (71.4% adherence) and 1 day in the week before V3 (85.7% adherence). The genetic profile of the patient is shown in Table 1. She carries five missense variants in SLCO1B1, SLCO1B3, and ABCB11. She is also homozygote for the CYP3A53 (rs776746) splicing variant. No other missense variants described as impacting rosuvastatin response were found in CYP3A4, CYP2C9, CYP2C19, or other drug transporters, such as ABCG2 (data not shown). Table 1 Variants in pharmacokinetic-related genes of the FH patient with late response to rosuvastatin Gene Variant code Variant type Nucleotide change (Amino acid change) Patient genotype Allele frequency (1,000 genomes, %) Functional impact Effects on rosuvastatin pharmacokinetics References SLCO1B1 rs2306283 (SLCO1B11B) Missense c.388A>G (p.Asn130Asp) AG 1B: 54.4 Comparable to 1A No effect on plasma rosuvastatin levels Ho et al., 2006; Lee et al., 2013 SLCO1B1 rs4149056 (SLCO1B15) Missense c.521T>C p.(Val174Ala) TC 5: 8.8 Reduced activity Increased rosuvastatin plasma levels; Reduced hepatic uptake Kameyama et al., 2005; Lee et al., 2013 SLCO1B1 rs2306283, rs4149056 (SLCO1B115) Missense c.388A>G, c.521T>C (p.Asn130Asp, p.Val174Ala) AG, TC 15: 7.8 Reduced activity Increased rosuvastatin plasma levels; reduced hepatic uptake Kameyama et al., 2005; Birmingham et al., 2015 SLCO1B3 rs4149117 Missense c.334T>G (p.Ser112Ala) GG G: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 SLCO1B3 rs7311358 Missense c.699G>A (p.Met233Ile) AA A: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 ABCB11 rs2287622 Missense c.1331T>C (p.Val444Ala) TC C: 58.9 Reduced activity Increased rosuvastatin plasma levels Soko et al. 2019 CYP3A5 rs776746 (CYP3A53) Splicing c. 6986A>G GG 3: 62.1 No activity No rosuvastatin metabolism; Reduced LDL-c response Bailey et al. 2010 FH, familial hypercholesterolemia; LDL-c, low-density lipoprotein cholesterol. Discussion In heterozygous FH patients, LDL-c level reductions of 47.1% have been observed after a 6-week treatment with rosuvastatin 20 mg (8). The patient, however, did not experience any changes in LDL-c levels at week 6 (V3) of rosuvastatin 20 mg treatment, with a 48.9% LDL-c reduction only at week 12 (V4) of therapy. The delayed rosuvastatin response could be explained by modifications in the therapy scheme during the follow-up period. However, the only change was in levothyroxine dose, that was increased from 88 to 100 µg in V1. It is unlikely that the late response is due to an adaptation to the new levothyroxine dose. The patient was already on treatment with levothyroxine 88 µg before V1; moreover, changes in cholesterol due to an adaptation period should be reflected in her lipid profile in V3, not only in V4. Another possible explanation is a lack of adherence from V2 to V3; however, the patient showed a similar treatment adherence in V3 and V1, which should lead to a similar lipid profile between visits. Furthermore, drug interactions between rosuvastatin, levothyroxine, and ezetimibe that could affect treatment response were not detected, excluding this possibility. Pharmacokinetics-related genes may have contributed to the late response to rosuvastatin (Figure 2). The patient carries two variants in SLCO1B1, c.388A>G (SLCO1B11B) and c.521T>C (SLCO1B15), that are important determinants of rosuvastatin response. SLCO1B15 is a loss-of-function variant that decreases the hepatic uptake and increases blood levels of statins (9) (Table 1). SLCO1B11B has shown comparable activity to the functional 1A variant in in vitro functional studies (10). SLCO1B11B and 5 variants are in linkage disequilibrium (LD) and form the SLCO1B115 haplotype, that also reduced rosuvastatin uptake in functional studies with HEK293 and HeLa cells (11). The decreased liver uptake caused by these SLCO1B1 variants has been associated with increased plasma levels of rosuvastatin in pharmacokinetics studies (9) (Table 1). Figure 2 Proposed mechanism for patient’s late rosuvastatin response and myalgia. 1. The hepatic uptake of rosuvastatin occurs through SLCO1B1 and SLCO1B3 influx transporters, while atorvastatin and simvastatin are internalized through SLCO1B1. The presence of deleterious variants in these transporters (SLCO1B115 and SLCO1B3 c.334T>G and c.699G>A) decreases statin uptake, therefore decreasing their concentration inside the hepatocyte and increasing statin plasma levels. 2. The lack of expression of CYP3A5 due to CYP3A53 also decreases atorvastatin and simvastatin metabolization, which contributes to increasing their plasma levels. This enzyme does not participate markedly in rosuvastatin metabolism. 3. The resulting higher blood statin levels increased the patient’s muscular exposure to statins, that are internalized through SLCO2B1 transporter into the skeletal muscle cell. The high concentrations in the skeletal muscle cell possibly caused patient’s myalgia. 4. Rosuvastatin’s bile excretion occurs through ABCB11 efflux protein. ABCB11 c.1331T>C variant results in a reduced activity ABCB11, which decreases rosuvastatin efflux; this increases rosuvastatin intrahepatic levels and blood levels. Although the patient had reduced function influx transporters, we suggest that the small portion of rosuvastatin absorbed in the beginning of the treatment accumulated due to the loss of function of the ABCB11 variant. This, together with rosuvastatin active metabolites generated by the normal function CYP2C9, allowed HMGR inhibition and therefore cholesterol lowering in the last visit. SLCO1B3 is also an important gene that encodes an influx transporter for rosuvastatin. The patient was homozygous for both SLCO1B3 c.334T>G and c.699G>A, which are in strong LD (12). In an in vitro study, HeLa cells transfected with SLCO1B3 c.334G and c.699A haplotype showed a 13% decrease in rosuvastatin uptake, while for other substrates, such as cholecystokinin-8, an even more marked decrease of 57% was observed (13) (Table 1). Although the effect of SLCO1B3 c.334G and c.699A haplotype in rosuvastatin uptake is not sufficient to explain the delayed response, it might be significant when combined with the effect of the decreased function haplotype SLCO1B115. While SLCO1B15 and SLCO1B115 are associated with higher plasma levels of rosuvastatin, previous studies failed to find an association between these variants and LDL-c reduction in response to short- and long-term rosuvastatin treatments (9). Therefore, the simultaneous presence of decreased function SLCO1B1 and SLCO1B3 haplotypes possibly caused a marked reduction of rosuvastatin intrahepatic concentration, resulting in the lack of response observed in V3. ABCB11 encodes the efflux protein ABCB11, which plays an important role in rosuvastatin bile excretion. In a recent study, ABCB11 c.1331C allele has been associated to increased plasma rosuvastatin levels in healthy subjects (14) (Table 1). This variant possibly causes lower rosuvastatin excretion via bile, which in turn would increase intrahepatic rosuvastatin concentrations. Therefore, this mechanism could explain why even in the presence of low function SLC variants, the patient showed a late but evident LDL-c reduction after 12 weeks of rosuvastatin treatment. The patient also carries the homozygous form of CYP3A53, an intronic variant that results in undetectable expression of CYP3A5 (15). The GEOSTAT-1 study reported that dyslipidemic patients carrying CYP3A53/3 had lower LDL-c reduction after three-month rosuvastatin 10 mg treatment compared to carriers of 1/1 or 1/3 (Table 1). It was suggested that the metabolite produced by CYP3A5 also plays a role in HMGR inhibition, potentiating the response to rosuvastatin, which is why CYP3A5 non-expressors have reduced LDL-c response to rosuvastatin (16). CYP3A53 possibly impaired the patient’s response time to rosuvastatin, but in lower extent, as CYP3A5 does not participate markedly in rosuvastatin metabolism. In addition to the delayed response to rosuvastatin, the patient experienced myalgia associated with rosuvastatin 40 mg/day and other statins, as previously commented. This SRAE may be due to SLCO1B1 variants. SLCO1B15 and SLCO1B115 have been extensively associated with myopathy to simvastatin. A systematic review and meta-analysis reported that carriers of the C allele of SLCO1B15 (c.521T>C) showed a higher risk of myotoxicity (17). Additionally, SLCO1B15 has been associated to rosuvastatin myotoxicity in previous studies (18,19). It has been suggested that it causes higher efflux of statins, increasing statin exposure and, therefore, the risk of myalgia (20). Also, a recent case report showed that variants in SLCO1B3 (c.334T>G and c.699G>A) and ABCB11 (c.1331T>C) and the interaction between rosuvastatin and ticagrelor led to rhabdomyolysis in a patient with chronic kidney disease and other chronic conditions (21), but no other reports were found. CYP3A53 may also have contributed to statin myotoxicity, since it has been associated with increased risk to atorvastatin and rosuvastatin-related myalgia in South-Indian dyslipidemic patients (22). However, this variant was not associated to statin intolerance in another study (23). Most studies have evaluated the effect of individual variants in SRAE, and not the interaction between a group of variants in key genes in statin pharmacokinetics pathway. Therefore, we suggest that the combined effect of the low-activity variants in SLCO1B1 and SLCO1B3, the high-activity variant in ABCB11, and the lack of activity of CYP3A53 predisposed the patient to low hepatic uptake, metabolization and efflux, respectively. The resulting higher rosuvastatin plasma concentration increased its systemic exposure, which may have caused myalgia (Figure 2). Importantly, the patient carries LDLR rs28941776 (c.1646G>A, p.Gly549Asp), a disruptive-missense variant that showed reduced LDL uptake in an in vitro study (24). LDLR variants have been associated with variability in statin response in FH patients (25), but we did not find studies that investigated the association between LDLR variants and time to statin response or myalgia. Nevertheless, this variant could have played a role in patient’s rosuvastatin time to response and it should be considered for further studies. A limitation of this study is that plasma concentrations of rosuvastatin and its metabolites were not measured. However, the adherence of the patient to the prescribed treatment was ensured using a validated adherence questionnaire and regular follow-up calls. In summary, the combination of four low-activity variants in SLC genes, a high-activity variant in ABCB11, and a non-functional variant in CYP3A5 may explain the observed late response to rosuvastatin and the statin-related myalgia. With this case report, we have shown the importance of considering a combination of variants in a pharmacogenetic analysis to predict individual responses to statin treatment and prevent adverse drug events. We believe this study contributes to precision medicine in future clinical settings. Patient perspective “I have had high cholesterol since I was a child and it has been an issue because of the delayed response to treatments and of many adverse reactions to medications, especially simvastatin. The authors have been very attentive towards me throughout the whole study and discovered possible variants that may delay my response to rosuvastatin and influence the pain that I have felt when using statins. I am very happy for knowing the cause of my problem and I would like to thank the authors for this possible diagnosis. This has improved my perspectives of cholesterol treatment.” Supplementary The article’s supplementary files as 10.21037/atm-20-5540 10.21037/atm-20-5540 10.21037/atm-20-5540 Acknowledgments The authors thank Adriana Garofalo, Dr. Hui Tzu Lin Wang, colleagues from the Laboratory of Molecular Investigation in Cardiology, and the Divisions of Dyslipidemia and Pharmacy of the Institute Dante Pazzanese of Cardiology. Their immeasurable technical and logistic support in patient selection and data collection made this study possible. Funding: This work was supported by Sao Paulo Research Foundation (FAPESP), Brazil [Research grant: #2016/12899-6 to MHH]; and National Council for Scientific and Technological Development [CNPq, grant: #447120/2014-0 to MHH], Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDH is a recipient of a fellowship of the São Paulo Research Foundation (FAPESP), grant #2016/25637-0. RCCF, RHB, GMF and VFO are recipients of fellowships from FAPESP, Brazil. AAM is a recipient of fellowship from CAPES, Brazil. ESRM, MHH and RDCH are recipients of fellowships from CNPq, Brazil. BL was a recipient of fellowship from FAPESP, Brazil. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The intervention study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The DNA sequencing study was approved by the Ethics Committees of the Institute Dante Pazzanese of Cardiology (CAAE #4618713.0.1001.5462) and the School of Pharmaceutical Sciences of the University of Sao Paulo (CAAE #24618713.0.3001.0067), Sao Paulo, Brazil. The intervention study was approved by the Ethics Committee of the Institute Dante Pazzanese of Cardiology (CAAE #05234918.4.0000.5462). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees. The patient signed the written informed consents before her enrollment in the studies. In the written informed consent of the DNA sequencing study, the patient was informed that clinical data and blood samples would be collected for laboratory tests and genetic analyses. As for the intervention study, the patient was informed on the intervention protocol and sample collections throughout the visits, and that this data would be used for genetic and epigenetic analyses. Reporting Checklist: The authors have completed the CARE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-5540 Peer Review File: Available at http://dx.doi.org/10.21037/atm-20-5540 Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5540). The authors have no conflicts of interest to declare.	ATORVASTATIN, EZETIMIBE, LEVOTHYROXINE, PRAVASTATIN SODIUM, ROSUVASTATIN, SIMVASTATIN	DrugsGivenReaction	CC BY-NC-ND	33553369	18,931,409	2021-01
What was the administration route of drug 'ATORVASTATIN'?	Late response to rosuvastatin and statin-related myalgia due to SLCO1B1, SLCO1B3, ABCB11, and CYP3A5 variants in a patient with Familial Hypercholesterolemia: a case report. Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B11B and5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A53. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. pmcIntroduction Familial hypercholesterolemia (FH) is a genetic metabolic disease that leads to increased high low-density lipoprotein (LDL) cholesterol, which is a risk factor for early atherosclerosis and cardiovascular diseases (1). FH is usually treated with high-dose statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), a key enzyme in cholesterol biosynthesis pathway. Rosuvastatin is one of the most effective statins, probably due its hydrophilicity, that confers selectivity to hepatic cells, higher affinity to HMGR, and lower rates of statin-related adverse events (SRAE) compared to other statins. It is poorly metabolized by CYP2C9 and CYP2C19, while 72% of the non-metabolized molecules are excreted via biliary system. Therefore, rosuvastatin blood levels rely on the activity of membrane transporters, mainly of solute carrier (SLC) and ATP-binding cassette (ABC) families, highly expressed in intestine, liver, and kidney (2). Pharmacogenetic studies have shown that loss-of-function variants in genes encoding OATPs, such as SLCO1B1, SLCO2B1, and SLCO1B3, and ABCs have been associated with variability in low-density lipoprotein cholesterol (LDL-c) reduction and higher risk of SRAE (3). The importance of considering the combined effect of variants in key genes for pharmacogenetic analyses has been increasingly evident (4). In this case report, we discuss how variants in genes participating in different stages of statin pharmacokinetics pathway possibly affected the time to response to rosuvastatin and the risk of SRAE in a female FH patient. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. This case is reported in accordance with the CARE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-5540). Case presentation A 26-year-old Caucasian female patient with definite diagnosis of FH according to Dutch Lipid Clinic Network MEDPED criteria (5) was invited to participate in an intervention study in June 2019. She was previously included in a FH sequencing study (May 2018), in which a panel of 84 genes involved in lipid homeostasis and drug metabolism was sequenced using exon-targeted gene sequencing (NGS). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for both studies. The patient carries the variant LDLR rs28941776 (c.1646G>A, p.Gly549Asp), which has been associated with FH and is classified as pathogenic according to the American College of Medical Genetics (ACMG) guidelines (6). Her clinical history included high levels of total cholesterol and LDL-c since childhood. In 2008, at the age of 15 years, she had an abnormal lipid profile even under a daily treatment with simvastatin 10 mg and ezetimibe 10 mg. Laboratory analyses showed a total cholesterol of 324 mg/dL, LDL-c 264 mg/dL, high-density lipoprotein cholesterol (HDL-c) 46 mg/dL, and triglycerides 71 mg/dL. In 2014, she was diagnosed with hypothyroidism and treated with levothyroxine 25 µg/day, which was gradually increased to 100 µg/day in 2019. She also had a pregnancy history in January 2017. Her therapy history included simvastatin, which led to severe myopathy in 2008, with marked increase in serum creatine kinase (CK) to 1,080 U/L (4.7-fold the upper reference value). The cholesterol-lowering therapy was changed to pravastatin 20 mg and ezetimibe 10 mg daily until May 2011, when she reported another episode of myalgia. Pravastatin was withdrawn and atorvastatin 20 mg was introduced, also associated with ezetimibe 10 mg. Three months later, in August 2011, she reported interrupting atorvastatin treatment due to myalgia. Rosuvastatin 10 mg was then introduced, also associated with ezetimibe 10 mg, after which she showed an LDL-c level of 125 mg/dL and never reported myalgia again. However, her lipid profile worsened throughout the years even under rosuvastatin treatment, with her LDL-c reaching 194 mg/dL with rosuvastatin 20 mg. The patient had no history of liver or kidney impartment, HIV, coronary artery disease (CAD), diabetes, obesity, cardiovascular events, and did not smoke or drink. Her mother and grandmother had a history of FH, but not CAD or cardiovascular events, while her father had hypertension and type 2 diabetes. In the intervention study, the patient was seen four times (V1 to V4) in 5 months, and clinical history and therapy data were obtained. The protocol consisted of a 6-week rosuvastatin wash-out period, after which rosuvastatin was reintroduced for additional 6 weeks, when treatment response was evaluated. Adherence to treatment was assessed in each timepoint using the translated and validated version of the Brief Medication Questionnaire (BMQ) (7) and blood samples were taken in each visit for laboratory testing. The lipid profile during the follow-up is shown in Figure 1. In April 2019 (V1), the patient was taking rosuvastatin 40 mg, ezetimibe 10 mg, and levothyroxine 88 µg daily. She reported experiencing muscle pain after recently increasing rosuvastatin dose from 20 to 40 mg/day. Her lipid profile was altered (total cholesterol 376 mg/dL, LDL-c 263 mg/dL, HDL-c 67 mg/dL, triglycerides 234 mg/dL) without increase in CK levels. She reported being active, running 2 km 2–3 times a week, and had a healthy diet, eating more than five portions of vegetables daily. Her TSH and T4 levels were normal. Rosuvastatin 40 mg was then discontinued for wash-out, ezetimibe was maintained, and levothyroxine dose was increased to 100 µg/day. Figure 1 Plasma lipid profile and pharmacotherapy of the FH patient throughout the study period. EZT, ezetimibe; LVT, levothyroxine; RSV, rosuvastatin; SRAE, statin-related adverse events. In June 2019 (V2), after undergoing a 6-week rosuvastatin wash-out period between V1 and V2, her lipid profile worsened (total cholesterol 512 mg/dL, LDL-c 405 mg/dL, HDL-c 65 mg/dL, triglycerides 213 mg/dL). Because the patient reported myalgia in V1 (rosuvastatin 40 mg), the physician prescribed rosuvastatin 20 mg/day for six weeks. Surprisingly, in August 2019 (V3), the lipid profile (total cholesterol 531 mg/dL, LDL-c 407 mg/dL, HDL-c 67 mg/dL, triglycerides 286 mg/dL) did not change compared to V2. The patient reported experiencing no myalgia to rosuvastatin 20 mg. In September 2019 (V4), her lipid profile improved (total cholesterol 299 mg/dL, LDL-c 208 mg/dL, HDL-c 59 mg/dL, triglycerides 158 mg/dL) and she continued not experiencing myalgia to rosuvastatin. During the follow-up period, serum TSH and T4 levels remained unchanged, suggesting that her hypothyroidism was controlled and did not influence the lipid profile. Moreover, serum CK did not show any abnormality, which indicates no muscle damage due to statin treatment. The patient also reported being adherent to treatment. In the BMQ adherence questionnaire, she reported forgetting the lipid-lowering medications 2 days in the week before V1 (71.4% adherence) and 1 day in the week before V3 (85.7% adherence). The genetic profile of the patient is shown in Table 1. She carries five missense variants in SLCO1B1, SLCO1B3, and ABCB11. She is also homozygote for the CYP3A53 (rs776746) splicing variant. No other missense variants described as impacting rosuvastatin response were found in CYP3A4, CYP2C9, CYP2C19, or other drug transporters, such as ABCG2 (data not shown). Table 1 Variants in pharmacokinetic-related genes of the FH patient with late response to rosuvastatin Gene Variant code Variant type Nucleotide change (Amino acid change) Patient genotype Allele frequency (1,000 genomes, %) Functional impact Effects on rosuvastatin pharmacokinetics References SLCO1B1 rs2306283 (SLCO1B11B) Missense c.388A>G (p.Asn130Asp) AG 1B: 54.4 Comparable to 1A No effect on plasma rosuvastatin levels Ho et al., 2006; Lee et al., 2013 SLCO1B1 rs4149056 (SLCO1B15) Missense c.521T>C p.(Val174Ala) TC 5: 8.8 Reduced activity Increased rosuvastatin plasma levels; Reduced hepatic uptake Kameyama et al., 2005; Lee et al., 2013 SLCO1B1 rs2306283, rs4149056 (SLCO1B115) Missense c.388A>G, c.521T>C (p.Asn130Asp, p.Val174Ala) AG, TC 15: 7.8 Reduced activity Increased rosuvastatin plasma levels; reduced hepatic uptake Kameyama et al., 2005; Birmingham et al., 2015 SLCO1B3 rs4149117 Missense c.334T>G (p.Ser112Ala) GG G: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 SLCO1B3 rs7311358 Missense c.699G>A (p.Met233Ile) AA A: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 ABCB11 rs2287622 Missense c.1331T>C (p.Val444Ala) TC C: 58.9 Reduced activity Increased rosuvastatin plasma levels Soko et al. 2019 CYP3A5 rs776746 (CYP3A53) Splicing c. 6986A>G GG 3: 62.1 No activity No rosuvastatin metabolism; Reduced LDL-c response Bailey et al. 2010 FH, familial hypercholesterolemia; LDL-c, low-density lipoprotein cholesterol. Discussion In heterozygous FH patients, LDL-c level reductions of 47.1% have been observed after a 6-week treatment with rosuvastatin 20 mg (8). The patient, however, did not experience any changes in LDL-c levels at week 6 (V3) of rosuvastatin 20 mg treatment, with a 48.9% LDL-c reduction only at week 12 (V4) of therapy. The delayed rosuvastatin response could be explained by modifications in the therapy scheme during the follow-up period. However, the only change was in levothyroxine dose, that was increased from 88 to 100 µg in V1. It is unlikely that the late response is due to an adaptation to the new levothyroxine dose. The patient was already on treatment with levothyroxine 88 µg before V1; moreover, changes in cholesterol due to an adaptation period should be reflected in her lipid profile in V3, not only in V4. Another possible explanation is a lack of adherence from V2 to V3; however, the patient showed a similar treatment adherence in V3 and V1, which should lead to a similar lipid profile between visits. Furthermore, drug interactions between rosuvastatin, levothyroxine, and ezetimibe that could affect treatment response were not detected, excluding this possibility. Pharmacokinetics-related genes may have contributed to the late response to rosuvastatin (Figure 2). The patient carries two variants in SLCO1B1, c.388A>G (SLCO1B11B) and c.521T>C (SLCO1B15), that are important determinants of rosuvastatin response. SLCO1B15 is a loss-of-function variant that decreases the hepatic uptake and increases blood levels of statins (9) (Table 1). SLCO1B11B has shown comparable activity to the functional 1A variant in in vitro functional studies (10). SLCO1B11B and 5 variants are in linkage disequilibrium (LD) and form the SLCO1B115 haplotype, that also reduced rosuvastatin uptake in functional studies with HEK293 and HeLa cells (11). The decreased liver uptake caused by these SLCO1B1 variants has been associated with increased plasma levels of rosuvastatin in pharmacokinetics studies (9) (Table 1). Figure 2 Proposed mechanism for patient’s late rosuvastatin response and myalgia. 1. The hepatic uptake of rosuvastatin occurs through SLCO1B1 and SLCO1B3 influx transporters, while atorvastatin and simvastatin are internalized through SLCO1B1. The presence of deleterious variants in these transporters (SLCO1B115 and SLCO1B3 c.334T>G and c.699G>A) decreases statin uptake, therefore decreasing their concentration inside the hepatocyte and increasing statin plasma levels. 2. The lack of expression of CYP3A5 due to CYP3A53 also decreases atorvastatin and simvastatin metabolization, which contributes to increasing their plasma levels. This enzyme does not participate markedly in rosuvastatin metabolism. 3. The resulting higher blood statin levels increased the patient’s muscular exposure to statins, that are internalized through SLCO2B1 transporter into the skeletal muscle cell. The high concentrations in the skeletal muscle cell possibly caused patient’s myalgia. 4. Rosuvastatin’s bile excretion occurs through ABCB11 efflux protein. ABCB11 c.1331T>C variant results in a reduced activity ABCB11, which decreases rosuvastatin efflux; this increases rosuvastatin intrahepatic levels and blood levels. Although the patient had reduced function influx transporters, we suggest that the small portion of rosuvastatin absorbed in the beginning of the treatment accumulated due to the loss of function of the ABCB11 variant. This, together with rosuvastatin active metabolites generated by the normal function CYP2C9, allowed HMGR inhibition and therefore cholesterol lowering in the last visit. SLCO1B3 is also an important gene that encodes an influx transporter for rosuvastatin. The patient was homozygous for both SLCO1B3 c.334T>G and c.699G>A, which are in strong LD (12). In an in vitro study, HeLa cells transfected with SLCO1B3 c.334G and c.699A haplotype showed a 13% decrease in rosuvastatin uptake, while for other substrates, such as cholecystokinin-8, an even more marked decrease of 57% was observed (13) (Table 1). Although the effect of SLCO1B3 c.334G and c.699A haplotype in rosuvastatin uptake is not sufficient to explain the delayed response, it might be significant when combined with the effect of the decreased function haplotype SLCO1B115. While SLCO1B15 and SLCO1B115 are associated with higher plasma levels of rosuvastatin, previous studies failed to find an association between these variants and LDL-c reduction in response to short- and long-term rosuvastatin treatments (9). Therefore, the simultaneous presence of decreased function SLCO1B1 and SLCO1B3 haplotypes possibly caused a marked reduction of rosuvastatin intrahepatic concentration, resulting in the lack of response observed in V3. ABCB11 encodes the efflux protein ABCB11, which plays an important role in rosuvastatin bile excretion. In a recent study, ABCB11 c.1331C allele has been associated to increased plasma rosuvastatin levels in healthy subjects (14) (Table 1). This variant possibly causes lower rosuvastatin excretion via bile, which in turn would increase intrahepatic rosuvastatin concentrations. Therefore, this mechanism could explain why even in the presence of low function SLC variants, the patient showed a late but evident LDL-c reduction after 12 weeks of rosuvastatin treatment. The patient also carries the homozygous form of CYP3A53, an intronic variant that results in undetectable expression of CYP3A5 (15). The GEOSTAT-1 study reported that dyslipidemic patients carrying CYP3A53/3 had lower LDL-c reduction after three-month rosuvastatin 10 mg treatment compared to carriers of 1/1 or 1/3 (Table 1). It was suggested that the metabolite produced by CYP3A5 also plays a role in HMGR inhibition, potentiating the response to rosuvastatin, which is why CYP3A5 non-expressors have reduced LDL-c response to rosuvastatin (16). CYP3A53 possibly impaired the patient’s response time to rosuvastatin, but in lower extent, as CYP3A5 does not participate markedly in rosuvastatin metabolism. In addition to the delayed response to rosuvastatin, the patient experienced myalgia associated with rosuvastatin 40 mg/day and other statins, as previously commented. This SRAE may be due to SLCO1B1 variants. SLCO1B15 and SLCO1B115 have been extensively associated with myopathy to simvastatin. A systematic review and meta-analysis reported that carriers of the C allele of SLCO1B15 (c.521T>C) showed a higher risk of myotoxicity (17). Additionally, SLCO1B15 has been associated to rosuvastatin myotoxicity in previous studies (18,19). It has been suggested that it causes higher efflux of statins, increasing statin exposure and, therefore, the risk of myalgia (20). Also, a recent case report showed that variants in SLCO1B3 (c.334T>G and c.699G>A) and ABCB11 (c.1331T>C) and the interaction between rosuvastatin and ticagrelor led to rhabdomyolysis in a patient with chronic kidney disease and other chronic conditions (21), but no other reports were found. CYP3A53 may also have contributed to statin myotoxicity, since it has been associated with increased risk to atorvastatin and rosuvastatin-related myalgia in South-Indian dyslipidemic patients (22). However, this variant was not associated to statin intolerance in another study (23). Most studies have evaluated the effect of individual variants in SRAE, and not the interaction between a group of variants in key genes in statin pharmacokinetics pathway. Therefore, we suggest that the combined effect of the low-activity variants in SLCO1B1 and SLCO1B3, the high-activity variant in ABCB11, and the lack of activity of CYP3A53 predisposed the patient to low hepatic uptake, metabolization and efflux, respectively. The resulting higher rosuvastatin plasma concentration increased its systemic exposure, which may have caused myalgia (Figure 2). Importantly, the patient carries LDLR rs28941776 (c.1646G>A, p.Gly549Asp), a disruptive-missense variant that showed reduced LDL uptake in an in vitro study (24). LDLR variants have been associated with variability in statin response in FH patients (25), but we did not find studies that investigated the association between LDLR variants and time to statin response or myalgia. Nevertheless, this variant could have played a role in patient’s rosuvastatin time to response and it should be considered for further studies. A limitation of this study is that plasma concentrations of rosuvastatin and its metabolites were not measured. However, the adherence of the patient to the prescribed treatment was ensured using a validated adherence questionnaire and regular follow-up calls. In summary, the combination of four low-activity variants in SLC genes, a high-activity variant in ABCB11, and a non-functional variant in CYP3A5 may explain the observed late response to rosuvastatin and the statin-related myalgia. With this case report, we have shown the importance of considering a combination of variants in a pharmacogenetic analysis to predict individual responses to statin treatment and prevent adverse drug events. We believe this study contributes to precision medicine in future clinical settings. Patient perspective “I have had high cholesterol since I was a child and it has been an issue because of the delayed response to treatments and of many adverse reactions to medications, especially simvastatin. The authors have been very attentive towards me throughout the whole study and discovered possible variants that may delay my response to rosuvastatin and influence the pain that I have felt when using statins. I am very happy for knowing the cause of my problem and I would like to thank the authors for this possible diagnosis. This has improved my perspectives of cholesterol treatment.” Supplementary The article’s supplementary files as 10.21037/atm-20-5540 10.21037/atm-20-5540 10.21037/atm-20-5540 Acknowledgments The authors thank Adriana Garofalo, Dr. Hui Tzu Lin Wang, colleagues from the Laboratory of Molecular Investigation in Cardiology, and the Divisions of Dyslipidemia and Pharmacy of the Institute Dante Pazzanese of Cardiology. Their immeasurable technical and logistic support in patient selection and data collection made this study possible. Funding: This work was supported by Sao Paulo Research Foundation (FAPESP), Brazil [Research grant: #2016/12899-6 to MHH]; and National Council for Scientific and Technological Development [CNPq, grant: #447120/2014-0 to MHH], Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDH is a recipient of a fellowship of the São Paulo Research Foundation (FAPESP), grant #2016/25637-0. RCCF, RHB, GMF and VFO are recipients of fellowships from FAPESP, Brazil. AAM is a recipient of fellowship from CAPES, Brazil. ESRM, MHH and RDCH are recipients of fellowships from CNPq, Brazil. BL was a recipient of fellowship from FAPESP, Brazil. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The intervention study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The DNA sequencing study was approved by the Ethics Committees of the Institute Dante Pazzanese of Cardiology (CAAE #4618713.0.1001.5462) and the School of Pharmaceutical Sciences of the University of Sao Paulo (CAAE #24618713.0.3001.0067), Sao Paulo, Brazil. The intervention study was approved by the Ethics Committee of the Institute Dante Pazzanese of Cardiology (CAAE #05234918.4.0000.5462). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees. The patient signed the written informed consents before her enrollment in the studies. In the written informed consent of the DNA sequencing study, the patient was informed that clinical data and blood samples would be collected for laboratory tests and genetic analyses. As for the intervention study, the patient was informed on the intervention protocol and sample collections throughout the visits, and that this data would be used for genetic and epigenetic analyses. Reporting Checklist: The authors have completed the CARE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-5540 Peer Review File: Available at http://dx.doi.org/10.21037/atm-20-5540 Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5540). The authors have no conflicts of interest to declare.	Oral	DrugAdministrationRoute	CC BY-NC-ND	33553369	18,931,409	2021-01
What was the administration route of drug 'SIMVASTATIN'?	Late response to rosuvastatin and statin-related myalgia due to SLCO1B1, SLCO1B3, ABCB11, and CYP3A5 variants in a patient with Familial Hypercholesterolemia: a case report. Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B11B and5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A53. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. pmcIntroduction Familial hypercholesterolemia (FH) is a genetic metabolic disease that leads to increased high low-density lipoprotein (LDL) cholesterol, which is a risk factor for early atherosclerosis and cardiovascular diseases (1). FH is usually treated with high-dose statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), a key enzyme in cholesterol biosynthesis pathway. Rosuvastatin is one of the most effective statins, probably due its hydrophilicity, that confers selectivity to hepatic cells, higher affinity to HMGR, and lower rates of statin-related adverse events (SRAE) compared to other statins. It is poorly metabolized by CYP2C9 and CYP2C19, while 72% of the non-metabolized molecules are excreted via biliary system. Therefore, rosuvastatin blood levels rely on the activity of membrane transporters, mainly of solute carrier (SLC) and ATP-binding cassette (ABC) families, highly expressed in intestine, liver, and kidney (2). Pharmacogenetic studies have shown that loss-of-function variants in genes encoding OATPs, such as SLCO1B1, SLCO2B1, and SLCO1B3, and ABCs have been associated with variability in low-density lipoprotein cholesterol (LDL-c) reduction and higher risk of SRAE (3). The importance of considering the combined effect of variants in key genes for pharmacogenetic analyses has been increasingly evident (4). In this case report, we discuss how variants in genes participating in different stages of statin pharmacokinetics pathway possibly affected the time to response to rosuvastatin and the risk of SRAE in a female FH patient. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. This case is reported in accordance with the CARE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-5540). Case presentation A 26-year-old Caucasian female patient with definite diagnosis of FH according to Dutch Lipid Clinic Network MEDPED criteria (5) was invited to participate in an intervention study in June 2019. She was previously included in a FH sequencing study (May 2018), in which a panel of 84 genes involved in lipid homeostasis and drug metabolism was sequenced using exon-targeted gene sequencing (NGS). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for both studies. The patient carries the variant LDLR rs28941776 (c.1646G>A, p.Gly549Asp), which has been associated with FH and is classified as pathogenic according to the American College of Medical Genetics (ACMG) guidelines (6). Her clinical history included high levels of total cholesterol and LDL-c since childhood. In 2008, at the age of 15 years, she had an abnormal lipid profile even under a daily treatment with simvastatin 10 mg and ezetimibe 10 mg. Laboratory analyses showed a total cholesterol of 324 mg/dL, LDL-c 264 mg/dL, high-density lipoprotein cholesterol (HDL-c) 46 mg/dL, and triglycerides 71 mg/dL. In 2014, she was diagnosed with hypothyroidism and treated with levothyroxine 25 µg/day, which was gradually increased to 100 µg/day in 2019. She also had a pregnancy history in January 2017. Her therapy history included simvastatin, which led to severe myopathy in 2008, with marked increase in serum creatine kinase (CK) to 1,080 U/L (4.7-fold the upper reference value). The cholesterol-lowering therapy was changed to pravastatin 20 mg and ezetimibe 10 mg daily until May 2011, when she reported another episode of myalgia. Pravastatin was withdrawn and atorvastatin 20 mg was introduced, also associated with ezetimibe 10 mg. Three months later, in August 2011, she reported interrupting atorvastatin treatment due to myalgia. Rosuvastatin 10 mg was then introduced, also associated with ezetimibe 10 mg, after which she showed an LDL-c level of 125 mg/dL and never reported myalgia again. However, her lipid profile worsened throughout the years even under rosuvastatin treatment, with her LDL-c reaching 194 mg/dL with rosuvastatin 20 mg. The patient had no history of liver or kidney impartment, HIV, coronary artery disease (CAD), diabetes, obesity, cardiovascular events, and did not smoke or drink. Her mother and grandmother had a history of FH, but not CAD or cardiovascular events, while her father had hypertension and type 2 diabetes. In the intervention study, the patient was seen four times (V1 to V4) in 5 months, and clinical history and therapy data were obtained. The protocol consisted of a 6-week rosuvastatin wash-out period, after which rosuvastatin was reintroduced for additional 6 weeks, when treatment response was evaluated. Adherence to treatment was assessed in each timepoint using the translated and validated version of the Brief Medication Questionnaire (BMQ) (7) and blood samples were taken in each visit for laboratory testing. The lipid profile during the follow-up is shown in Figure 1. In April 2019 (V1), the patient was taking rosuvastatin 40 mg, ezetimibe 10 mg, and levothyroxine 88 µg daily. She reported experiencing muscle pain after recently increasing rosuvastatin dose from 20 to 40 mg/day. Her lipid profile was altered (total cholesterol 376 mg/dL, LDL-c 263 mg/dL, HDL-c 67 mg/dL, triglycerides 234 mg/dL) without increase in CK levels. She reported being active, running 2 km 2–3 times a week, and had a healthy diet, eating more than five portions of vegetables daily. Her TSH and T4 levels were normal. Rosuvastatin 40 mg was then discontinued for wash-out, ezetimibe was maintained, and levothyroxine dose was increased to 100 µg/day. Figure 1 Plasma lipid profile and pharmacotherapy of the FH patient throughout the study period. EZT, ezetimibe; LVT, levothyroxine; RSV, rosuvastatin; SRAE, statin-related adverse events. In June 2019 (V2), after undergoing a 6-week rosuvastatin wash-out period between V1 and V2, her lipid profile worsened (total cholesterol 512 mg/dL, LDL-c 405 mg/dL, HDL-c 65 mg/dL, triglycerides 213 mg/dL). Because the patient reported myalgia in V1 (rosuvastatin 40 mg), the physician prescribed rosuvastatin 20 mg/day for six weeks. Surprisingly, in August 2019 (V3), the lipid profile (total cholesterol 531 mg/dL, LDL-c 407 mg/dL, HDL-c 67 mg/dL, triglycerides 286 mg/dL) did not change compared to V2. The patient reported experiencing no myalgia to rosuvastatin 20 mg. In September 2019 (V4), her lipid profile improved (total cholesterol 299 mg/dL, LDL-c 208 mg/dL, HDL-c 59 mg/dL, triglycerides 158 mg/dL) and she continued not experiencing myalgia to rosuvastatin. During the follow-up period, serum TSH and T4 levels remained unchanged, suggesting that her hypothyroidism was controlled and did not influence the lipid profile. Moreover, serum CK did not show any abnormality, which indicates no muscle damage due to statin treatment. The patient also reported being adherent to treatment. In the BMQ adherence questionnaire, she reported forgetting the lipid-lowering medications 2 days in the week before V1 (71.4% adherence) and 1 day in the week before V3 (85.7% adherence). The genetic profile of the patient is shown in Table 1. She carries five missense variants in SLCO1B1, SLCO1B3, and ABCB11. She is also homozygote for the CYP3A53 (rs776746) splicing variant. No other missense variants described as impacting rosuvastatin response were found in CYP3A4, CYP2C9, CYP2C19, or other drug transporters, such as ABCG2 (data not shown). Table 1 Variants in pharmacokinetic-related genes of the FH patient with late response to rosuvastatin Gene Variant code Variant type Nucleotide change (Amino acid change) Patient genotype Allele frequency (1,000 genomes, %) Functional impact Effects on rosuvastatin pharmacokinetics References SLCO1B1 rs2306283 (SLCO1B11B) Missense c.388A>G (p.Asn130Asp) AG 1B: 54.4 Comparable to 1A No effect on plasma rosuvastatin levels Ho et al., 2006; Lee et al., 2013 SLCO1B1 rs4149056 (SLCO1B15) Missense c.521T>C p.(Val174Ala) TC 5: 8.8 Reduced activity Increased rosuvastatin plasma levels; Reduced hepatic uptake Kameyama et al., 2005; Lee et al., 2013 SLCO1B1 rs2306283, rs4149056 (SLCO1B115) Missense c.388A>G, c.521T>C (p.Asn130Asp, p.Val174Ala) AG, TC 15: 7.8 Reduced activity Increased rosuvastatin plasma levels; reduced hepatic uptake Kameyama et al., 2005; Birmingham et al., 2015 SLCO1B3 rs4149117 Missense c.334T>G (p.Ser112Ala) GG G: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 SLCO1B3 rs7311358 Missense c.699G>A (p.Met233Ile) AA A: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 ABCB11 rs2287622 Missense c.1331T>C (p.Val444Ala) TC C: 58.9 Reduced activity Increased rosuvastatin plasma levels Soko et al. 2019 CYP3A5 rs776746 (CYP3A53) Splicing c. 6986A>G GG 3: 62.1 No activity No rosuvastatin metabolism; Reduced LDL-c response Bailey et al. 2010 FH, familial hypercholesterolemia; LDL-c, low-density lipoprotein cholesterol. Discussion In heterozygous FH patients, LDL-c level reductions of 47.1% have been observed after a 6-week treatment with rosuvastatin 20 mg (8). The patient, however, did not experience any changes in LDL-c levels at week 6 (V3) of rosuvastatin 20 mg treatment, with a 48.9% LDL-c reduction only at week 12 (V4) of therapy. The delayed rosuvastatin response could be explained by modifications in the therapy scheme during the follow-up period. However, the only change was in levothyroxine dose, that was increased from 88 to 100 µg in V1. It is unlikely that the late response is due to an adaptation to the new levothyroxine dose. The patient was already on treatment with levothyroxine 88 µg before V1; moreover, changes in cholesterol due to an adaptation period should be reflected in her lipid profile in V3, not only in V4. Another possible explanation is a lack of adherence from V2 to V3; however, the patient showed a similar treatment adherence in V3 and V1, which should lead to a similar lipid profile between visits. Furthermore, drug interactions between rosuvastatin, levothyroxine, and ezetimibe that could affect treatment response were not detected, excluding this possibility. Pharmacokinetics-related genes may have contributed to the late response to rosuvastatin (Figure 2). The patient carries two variants in SLCO1B1, c.388A>G (SLCO1B11B) and c.521T>C (SLCO1B15), that are important determinants of rosuvastatin response. SLCO1B15 is a loss-of-function variant that decreases the hepatic uptake and increases blood levels of statins (9) (Table 1). SLCO1B11B has shown comparable activity to the functional 1A variant in in vitro functional studies (10). SLCO1B11B and 5 variants are in linkage disequilibrium (LD) and form the SLCO1B115 haplotype, that also reduced rosuvastatin uptake in functional studies with HEK293 and HeLa cells (11). The decreased liver uptake caused by these SLCO1B1 variants has been associated with increased plasma levels of rosuvastatin in pharmacokinetics studies (9) (Table 1). Figure 2 Proposed mechanism for patient’s late rosuvastatin response and myalgia. 1. The hepatic uptake of rosuvastatin occurs through SLCO1B1 and SLCO1B3 influx transporters, while atorvastatin and simvastatin are internalized through SLCO1B1. The presence of deleterious variants in these transporters (SLCO1B115 and SLCO1B3 c.334T>G and c.699G>A) decreases statin uptake, therefore decreasing their concentration inside the hepatocyte and increasing statin plasma levels. 2. The lack of expression of CYP3A5 due to CYP3A53 also decreases atorvastatin and simvastatin metabolization, which contributes to increasing their plasma levels. This enzyme does not participate markedly in rosuvastatin metabolism. 3. The resulting higher blood statin levels increased the patient’s muscular exposure to statins, that are internalized through SLCO2B1 transporter into the skeletal muscle cell. The high concentrations in the skeletal muscle cell possibly caused patient’s myalgia. 4. Rosuvastatin’s bile excretion occurs through ABCB11 efflux protein. ABCB11 c.1331T>C variant results in a reduced activity ABCB11, which decreases rosuvastatin efflux; this increases rosuvastatin intrahepatic levels and blood levels. Although the patient had reduced function influx transporters, we suggest that the small portion of rosuvastatin absorbed in the beginning of the treatment accumulated due to the loss of function of the ABCB11 variant. This, together with rosuvastatin active metabolites generated by the normal function CYP2C9, allowed HMGR inhibition and therefore cholesterol lowering in the last visit. SLCO1B3 is also an important gene that encodes an influx transporter for rosuvastatin. The patient was homozygous for both SLCO1B3 c.334T>G and c.699G>A, which are in strong LD (12). In an in vitro study, HeLa cells transfected with SLCO1B3 c.334G and c.699A haplotype showed a 13% decrease in rosuvastatin uptake, while for other substrates, such as cholecystokinin-8, an even more marked decrease of 57% was observed (13) (Table 1). Although the effect of SLCO1B3 c.334G and c.699A haplotype in rosuvastatin uptake is not sufficient to explain the delayed response, it might be significant when combined with the effect of the decreased function haplotype SLCO1B115. While SLCO1B15 and SLCO1B115 are associated with higher plasma levels of rosuvastatin, previous studies failed to find an association between these variants and LDL-c reduction in response to short- and long-term rosuvastatin treatments (9). Therefore, the simultaneous presence of decreased function SLCO1B1 and SLCO1B3 haplotypes possibly caused a marked reduction of rosuvastatin intrahepatic concentration, resulting in the lack of response observed in V3. ABCB11 encodes the efflux protein ABCB11, which plays an important role in rosuvastatin bile excretion. In a recent study, ABCB11 c.1331C allele has been associated to increased plasma rosuvastatin levels in healthy subjects (14) (Table 1). This variant possibly causes lower rosuvastatin excretion via bile, which in turn would increase intrahepatic rosuvastatin concentrations. Therefore, this mechanism could explain why even in the presence of low function SLC variants, the patient showed a late but evident LDL-c reduction after 12 weeks of rosuvastatin treatment. The patient also carries the homozygous form of CYP3A53, an intronic variant that results in undetectable expression of CYP3A5 (15). The GEOSTAT-1 study reported that dyslipidemic patients carrying CYP3A53/3 had lower LDL-c reduction after three-month rosuvastatin 10 mg treatment compared to carriers of 1/1 or 1/3 (Table 1). It was suggested that the metabolite produced by CYP3A5 also plays a role in HMGR inhibition, potentiating the response to rosuvastatin, which is why CYP3A5 non-expressors have reduced LDL-c response to rosuvastatin (16). CYP3A53 possibly impaired the patient’s response time to rosuvastatin, but in lower extent, as CYP3A5 does not participate markedly in rosuvastatin metabolism. In addition to the delayed response to rosuvastatin, the patient experienced myalgia associated with rosuvastatin 40 mg/day and other statins, as previously commented. This SRAE may be due to SLCO1B1 variants. SLCO1B15 and SLCO1B115 have been extensively associated with myopathy to simvastatin. A systematic review and meta-analysis reported that carriers of the C allele of SLCO1B15 (c.521T>C) showed a higher risk of myotoxicity (17). Additionally, SLCO1B15 has been associated to rosuvastatin myotoxicity in previous studies (18,19). It has been suggested that it causes higher efflux of statins, increasing statin exposure and, therefore, the risk of myalgia (20). Also, a recent case report showed that variants in SLCO1B3 (c.334T>G and c.699G>A) and ABCB11 (c.1331T>C) and the interaction between rosuvastatin and ticagrelor led to rhabdomyolysis in a patient with chronic kidney disease and other chronic conditions (21), but no other reports were found. CYP3A53 may also have contributed to statin myotoxicity, since it has been associated with increased risk to atorvastatin and rosuvastatin-related myalgia in South-Indian dyslipidemic patients (22). However, this variant was not associated to statin intolerance in another study (23). Most studies have evaluated the effect of individual variants in SRAE, and not the interaction between a group of variants in key genes in statin pharmacokinetics pathway. Therefore, we suggest that the combined effect of the low-activity variants in SLCO1B1 and SLCO1B3, the high-activity variant in ABCB11, and the lack of activity of CYP3A53 predisposed the patient to low hepatic uptake, metabolization and efflux, respectively. The resulting higher rosuvastatin plasma concentration increased its systemic exposure, which may have caused myalgia (Figure 2). Importantly, the patient carries LDLR rs28941776 (c.1646G>A, p.Gly549Asp), a disruptive-missense variant that showed reduced LDL uptake in an in vitro study (24). LDLR variants have been associated with variability in statin response in FH patients (25), but we did not find studies that investigated the association between LDLR variants and time to statin response or myalgia. Nevertheless, this variant could have played a role in patient’s rosuvastatin time to response and it should be considered for further studies. A limitation of this study is that plasma concentrations of rosuvastatin and its metabolites were not measured. However, the adherence of the patient to the prescribed treatment was ensured using a validated adherence questionnaire and regular follow-up calls. In summary, the combination of four low-activity variants in SLC genes, a high-activity variant in ABCB11, and a non-functional variant in CYP3A5 may explain the observed late response to rosuvastatin and the statin-related myalgia. With this case report, we have shown the importance of considering a combination of variants in a pharmacogenetic analysis to predict individual responses to statin treatment and prevent adverse drug events. We believe this study contributes to precision medicine in future clinical settings. Patient perspective “I have had high cholesterol since I was a child and it has been an issue because of the delayed response to treatments and of many adverse reactions to medications, especially simvastatin. The authors have been very attentive towards me throughout the whole study and discovered possible variants that may delay my response to rosuvastatin and influence the pain that I have felt when using statins. I am very happy for knowing the cause of my problem and I would like to thank the authors for this possible diagnosis. This has improved my perspectives of cholesterol treatment.” Supplementary The article’s supplementary files as 10.21037/atm-20-5540 10.21037/atm-20-5540 10.21037/atm-20-5540 Acknowledgments The authors thank Adriana Garofalo, Dr. Hui Tzu Lin Wang, colleagues from the Laboratory of Molecular Investigation in Cardiology, and the Divisions of Dyslipidemia and Pharmacy of the Institute Dante Pazzanese of Cardiology. Their immeasurable technical and logistic support in patient selection and data collection made this study possible. Funding: This work was supported by Sao Paulo Research Foundation (FAPESP), Brazil [Research grant: #2016/12899-6 to MHH]; and National Council for Scientific and Technological Development [CNPq, grant: #447120/2014-0 to MHH], Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDH is a recipient of a fellowship of the São Paulo Research Foundation (FAPESP), grant #2016/25637-0. RCCF, RHB, GMF and VFO are recipients of fellowships from FAPESP, Brazil. AAM is a recipient of fellowship from CAPES, Brazil. ESRM, MHH and RDCH are recipients of fellowships from CNPq, Brazil. BL was a recipient of fellowship from FAPESP, Brazil. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The intervention study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The DNA sequencing study was approved by the Ethics Committees of the Institute Dante Pazzanese of Cardiology (CAAE #4618713.0.1001.5462) and the School of Pharmaceutical Sciences of the University of Sao Paulo (CAAE #24618713.0.3001.0067), Sao Paulo, Brazil. The intervention study was approved by the Ethics Committee of the Institute Dante Pazzanese of Cardiology (CAAE #05234918.4.0000.5462). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees. The patient signed the written informed consents before her enrollment in the studies. In the written informed consent of the DNA sequencing study, the patient was informed that clinical data and blood samples would be collected for laboratory tests and genetic analyses. As for the intervention study, the patient was informed on the intervention protocol and sample collections throughout the visits, and that this data would be used for genetic and epigenetic analyses. Reporting Checklist: The authors have completed the CARE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-5540 Peer Review File: Available at http://dx.doi.org/10.21037/atm-20-5540 Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5540). The authors have no conflicts of interest to declare.	Oral	DrugAdministrationRoute	CC BY-NC-ND	33553369	18,931,409	2021-01
What was the outcome of reaction 'Blood creatine phosphokinase increased'?	Late response to rosuvastatin and statin-related myalgia due to SLCO1B1, SLCO1B3, ABCB11, and CYP3A5 variants in a patient with Familial Hypercholesterolemia: a case report. Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B11B and5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A53. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. pmcIntroduction Familial hypercholesterolemia (FH) is a genetic metabolic disease that leads to increased high low-density lipoprotein (LDL) cholesterol, which is a risk factor for early atherosclerosis and cardiovascular diseases (1). FH is usually treated with high-dose statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), a key enzyme in cholesterol biosynthesis pathway. Rosuvastatin is one of the most effective statins, probably due its hydrophilicity, that confers selectivity to hepatic cells, higher affinity to HMGR, and lower rates of statin-related adverse events (SRAE) compared to other statins. It is poorly metabolized by CYP2C9 and CYP2C19, while 72% of the non-metabolized molecules are excreted via biliary system. Therefore, rosuvastatin blood levels rely on the activity of membrane transporters, mainly of solute carrier (SLC) and ATP-binding cassette (ABC) families, highly expressed in intestine, liver, and kidney (2). Pharmacogenetic studies have shown that loss-of-function variants in genes encoding OATPs, such as SLCO1B1, SLCO2B1, and SLCO1B3, and ABCs have been associated with variability in low-density lipoprotein cholesterol (LDL-c) reduction and higher risk of SRAE (3). The importance of considering the combined effect of variants in key genes for pharmacogenetic analyses has been increasingly evident (4). In this case report, we discuss how variants in genes participating in different stages of statin pharmacokinetics pathway possibly affected the time to response to rosuvastatin and the risk of SRAE in a female FH patient. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. This case is reported in accordance with the CARE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-5540). Case presentation A 26-year-old Caucasian female patient with definite diagnosis of FH according to Dutch Lipid Clinic Network MEDPED criteria (5) was invited to participate in an intervention study in June 2019. She was previously included in a FH sequencing study (May 2018), in which a panel of 84 genes involved in lipid homeostasis and drug metabolism was sequenced using exon-targeted gene sequencing (NGS). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for both studies. The patient carries the variant LDLR rs28941776 (c.1646G>A, p.Gly549Asp), which has been associated with FH and is classified as pathogenic according to the American College of Medical Genetics (ACMG) guidelines (6). Her clinical history included high levels of total cholesterol and LDL-c since childhood. In 2008, at the age of 15 years, she had an abnormal lipid profile even under a daily treatment with simvastatin 10 mg and ezetimibe 10 mg. Laboratory analyses showed a total cholesterol of 324 mg/dL, LDL-c 264 mg/dL, high-density lipoprotein cholesterol (HDL-c) 46 mg/dL, and triglycerides 71 mg/dL. In 2014, she was diagnosed with hypothyroidism and treated with levothyroxine 25 µg/day, which was gradually increased to 100 µg/day in 2019. She also had a pregnancy history in January 2017. Her therapy history included simvastatin, which led to severe myopathy in 2008, with marked increase in serum creatine kinase (CK) to 1,080 U/L (4.7-fold the upper reference value). The cholesterol-lowering therapy was changed to pravastatin 20 mg and ezetimibe 10 mg daily until May 2011, when she reported another episode of myalgia. Pravastatin was withdrawn and atorvastatin 20 mg was introduced, also associated with ezetimibe 10 mg. Three months later, in August 2011, she reported interrupting atorvastatin treatment due to myalgia. Rosuvastatin 10 mg was then introduced, also associated with ezetimibe 10 mg, after which she showed an LDL-c level of 125 mg/dL and never reported myalgia again. However, her lipid profile worsened throughout the years even under rosuvastatin treatment, with her LDL-c reaching 194 mg/dL with rosuvastatin 20 mg. The patient had no history of liver or kidney impartment, HIV, coronary artery disease (CAD), diabetes, obesity, cardiovascular events, and did not smoke or drink. Her mother and grandmother had a history of FH, but not CAD or cardiovascular events, while her father had hypertension and type 2 diabetes. In the intervention study, the patient was seen four times (V1 to V4) in 5 months, and clinical history and therapy data were obtained. The protocol consisted of a 6-week rosuvastatin wash-out period, after which rosuvastatin was reintroduced for additional 6 weeks, when treatment response was evaluated. Adherence to treatment was assessed in each timepoint using the translated and validated version of the Brief Medication Questionnaire (BMQ) (7) and blood samples were taken in each visit for laboratory testing. The lipid profile during the follow-up is shown in Figure 1. In April 2019 (V1), the patient was taking rosuvastatin 40 mg, ezetimibe 10 mg, and levothyroxine 88 µg daily. She reported experiencing muscle pain after recently increasing rosuvastatin dose from 20 to 40 mg/day. Her lipid profile was altered (total cholesterol 376 mg/dL, LDL-c 263 mg/dL, HDL-c 67 mg/dL, triglycerides 234 mg/dL) without increase in CK levels. She reported being active, running 2 km 2–3 times a week, and had a healthy diet, eating more than five portions of vegetables daily. Her TSH and T4 levels were normal. Rosuvastatin 40 mg was then discontinued for wash-out, ezetimibe was maintained, and levothyroxine dose was increased to 100 µg/day. Figure 1 Plasma lipid profile and pharmacotherapy of the FH patient throughout the study period. EZT, ezetimibe; LVT, levothyroxine; RSV, rosuvastatin; SRAE, statin-related adverse events. In June 2019 (V2), after undergoing a 6-week rosuvastatin wash-out period between V1 and V2, her lipid profile worsened (total cholesterol 512 mg/dL, LDL-c 405 mg/dL, HDL-c 65 mg/dL, triglycerides 213 mg/dL). Because the patient reported myalgia in V1 (rosuvastatin 40 mg), the physician prescribed rosuvastatin 20 mg/day for six weeks. Surprisingly, in August 2019 (V3), the lipid profile (total cholesterol 531 mg/dL, LDL-c 407 mg/dL, HDL-c 67 mg/dL, triglycerides 286 mg/dL) did not change compared to V2. The patient reported experiencing no myalgia to rosuvastatin 20 mg. In September 2019 (V4), her lipid profile improved (total cholesterol 299 mg/dL, LDL-c 208 mg/dL, HDL-c 59 mg/dL, triglycerides 158 mg/dL) and she continued not experiencing myalgia to rosuvastatin. During the follow-up period, serum TSH and T4 levels remained unchanged, suggesting that her hypothyroidism was controlled and did not influence the lipid profile. Moreover, serum CK did not show any abnormality, which indicates no muscle damage due to statin treatment. The patient also reported being adherent to treatment. In the BMQ adherence questionnaire, she reported forgetting the lipid-lowering medications 2 days in the week before V1 (71.4% adherence) and 1 day in the week before V3 (85.7% adherence). The genetic profile of the patient is shown in Table 1. She carries five missense variants in SLCO1B1, SLCO1B3, and ABCB11. She is also homozygote for the CYP3A53 (rs776746) splicing variant. No other missense variants described as impacting rosuvastatin response were found in CYP3A4, CYP2C9, CYP2C19, or other drug transporters, such as ABCG2 (data not shown). Table 1 Variants in pharmacokinetic-related genes of the FH patient with late response to rosuvastatin Gene Variant code Variant type Nucleotide change (Amino acid change) Patient genotype Allele frequency (1,000 genomes, %) Functional impact Effects on rosuvastatin pharmacokinetics References SLCO1B1 rs2306283 (SLCO1B11B) Missense c.388A>G (p.Asn130Asp) AG 1B: 54.4 Comparable to 1A No effect on plasma rosuvastatin levels Ho et al., 2006; Lee et al., 2013 SLCO1B1 rs4149056 (SLCO1B15) Missense c.521T>C p.(Val174Ala) TC 5: 8.8 Reduced activity Increased rosuvastatin plasma levels; Reduced hepatic uptake Kameyama et al., 2005; Lee et al., 2013 SLCO1B1 rs2306283, rs4149056 (SLCO1B115) Missense c.388A>G, c.521T>C (p.Asn130Asp, p.Val174Ala) AG, TC 15: 7.8 Reduced activity Increased rosuvastatin plasma levels; reduced hepatic uptake Kameyama et al., 2005; Birmingham et al., 2015 SLCO1B3 rs4149117 Missense c.334T>G (p.Ser112Ala) GG G: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 SLCO1B3 rs7311358 Missense c.699G>A (p.Met233Ile) AA A: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 ABCB11 rs2287622 Missense c.1331T>C (p.Val444Ala) TC C: 58.9 Reduced activity Increased rosuvastatin plasma levels Soko et al. 2019 CYP3A5 rs776746 (CYP3A53) Splicing c. 6986A>G GG 3: 62.1 No activity No rosuvastatin metabolism; Reduced LDL-c response Bailey et al. 2010 FH, familial hypercholesterolemia; LDL-c, low-density lipoprotein cholesterol. Discussion In heterozygous FH patients, LDL-c level reductions of 47.1% have been observed after a 6-week treatment with rosuvastatin 20 mg (8). The patient, however, did not experience any changes in LDL-c levels at week 6 (V3) of rosuvastatin 20 mg treatment, with a 48.9% LDL-c reduction only at week 12 (V4) of therapy. The delayed rosuvastatin response could be explained by modifications in the therapy scheme during the follow-up period. However, the only change was in levothyroxine dose, that was increased from 88 to 100 µg in V1. It is unlikely that the late response is due to an adaptation to the new levothyroxine dose. The patient was already on treatment with levothyroxine 88 µg before V1; moreover, changes in cholesterol due to an adaptation period should be reflected in her lipid profile in V3, not only in V4. Another possible explanation is a lack of adherence from V2 to V3; however, the patient showed a similar treatment adherence in V3 and V1, which should lead to a similar lipid profile between visits. Furthermore, drug interactions between rosuvastatin, levothyroxine, and ezetimibe that could affect treatment response were not detected, excluding this possibility. Pharmacokinetics-related genes may have contributed to the late response to rosuvastatin (Figure 2). The patient carries two variants in SLCO1B1, c.388A>G (SLCO1B11B) and c.521T>C (SLCO1B15), that are important determinants of rosuvastatin response. SLCO1B15 is a loss-of-function variant that decreases the hepatic uptake and increases blood levels of statins (9) (Table 1). SLCO1B11B has shown comparable activity to the functional 1A variant in in vitro functional studies (10). SLCO1B11B and 5 variants are in linkage disequilibrium (LD) and form the SLCO1B115 haplotype, that also reduced rosuvastatin uptake in functional studies with HEK293 and HeLa cells (11). The decreased liver uptake caused by these SLCO1B1 variants has been associated with increased plasma levels of rosuvastatin in pharmacokinetics studies (9) (Table 1). Figure 2 Proposed mechanism for patient’s late rosuvastatin response and myalgia. 1. The hepatic uptake of rosuvastatin occurs through SLCO1B1 and SLCO1B3 influx transporters, while atorvastatin and simvastatin are internalized through SLCO1B1. The presence of deleterious variants in these transporters (SLCO1B115 and SLCO1B3 c.334T>G and c.699G>A) decreases statin uptake, therefore decreasing their concentration inside the hepatocyte and increasing statin plasma levels. 2. The lack of expression of CYP3A5 due to CYP3A53 also decreases atorvastatin and simvastatin metabolization, which contributes to increasing their plasma levels. This enzyme does not participate markedly in rosuvastatin metabolism. 3. The resulting higher blood statin levels increased the patient’s muscular exposure to statins, that are internalized through SLCO2B1 transporter into the skeletal muscle cell. The high concentrations in the skeletal muscle cell possibly caused patient’s myalgia. 4. Rosuvastatin’s bile excretion occurs through ABCB11 efflux protein. ABCB11 c.1331T>C variant results in a reduced activity ABCB11, which decreases rosuvastatin efflux; this increases rosuvastatin intrahepatic levels and blood levels. Although the patient had reduced function influx transporters, we suggest that the small portion of rosuvastatin absorbed in the beginning of the treatment accumulated due to the loss of function of the ABCB11 variant. This, together with rosuvastatin active metabolites generated by the normal function CYP2C9, allowed HMGR inhibition and therefore cholesterol lowering in the last visit. SLCO1B3 is also an important gene that encodes an influx transporter for rosuvastatin. The patient was homozygous for both SLCO1B3 c.334T>G and c.699G>A, which are in strong LD (12). In an in vitro study, HeLa cells transfected with SLCO1B3 c.334G and c.699A haplotype showed a 13% decrease in rosuvastatin uptake, while for other substrates, such as cholecystokinin-8, an even more marked decrease of 57% was observed (13) (Table 1). Although the effect of SLCO1B3 c.334G and c.699A haplotype in rosuvastatin uptake is not sufficient to explain the delayed response, it might be significant when combined with the effect of the decreased function haplotype SLCO1B115. While SLCO1B15 and SLCO1B115 are associated with higher plasma levels of rosuvastatin, previous studies failed to find an association between these variants and LDL-c reduction in response to short- and long-term rosuvastatin treatments (9). Therefore, the simultaneous presence of decreased function SLCO1B1 and SLCO1B3 haplotypes possibly caused a marked reduction of rosuvastatin intrahepatic concentration, resulting in the lack of response observed in V3. ABCB11 encodes the efflux protein ABCB11, which plays an important role in rosuvastatin bile excretion. In a recent study, ABCB11 c.1331C allele has been associated to increased plasma rosuvastatin levels in healthy subjects (14) (Table 1). This variant possibly causes lower rosuvastatin excretion via bile, which in turn would increase intrahepatic rosuvastatin concentrations. Therefore, this mechanism could explain why even in the presence of low function SLC variants, the patient showed a late but evident LDL-c reduction after 12 weeks of rosuvastatin treatment. The patient also carries the homozygous form of CYP3A53, an intronic variant that results in undetectable expression of CYP3A5 (15). The GEOSTAT-1 study reported that dyslipidemic patients carrying CYP3A53/3 had lower LDL-c reduction after three-month rosuvastatin 10 mg treatment compared to carriers of 1/1 or 1/3 (Table 1). It was suggested that the metabolite produced by CYP3A5 also plays a role in HMGR inhibition, potentiating the response to rosuvastatin, which is why CYP3A5 non-expressors have reduced LDL-c response to rosuvastatin (16). CYP3A53 possibly impaired the patient’s response time to rosuvastatin, but in lower extent, as CYP3A5 does not participate markedly in rosuvastatin metabolism. In addition to the delayed response to rosuvastatin, the patient experienced myalgia associated with rosuvastatin 40 mg/day and other statins, as previously commented. This SRAE may be due to SLCO1B1 variants. SLCO1B15 and SLCO1B115 have been extensively associated with myopathy to simvastatin. A systematic review and meta-analysis reported that carriers of the C allele of SLCO1B15 (c.521T>C) showed a higher risk of myotoxicity (17). Additionally, SLCO1B15 has been associated to rosuvastatin myotoxicity in previous studies (18,19). It has been suggested that it causes higher efflux of statins, increasing statin exposure and, therefore, the risk of myalgia (20). Also, a recent case report showed that variants in SLCO1B3 (c.334T>G and c.699G>A) and ABCB11 (c.1331T>C) and the interaction between rosuvastatin and ticagrelor led to rhabdomyolysis in a patient with chronic kidney disease and other chronic conditions (21), but no other reports were found. CYP3A53 may also have contributed to statin myotoxicity, since it has been associated with increased risk to atorvastatin and rosuvastatin-related myalgia in South-Indian dyslipidemic patients (22). However, this variant was not associated to statin intolerance in another study (23). Most studies have evaluated the effect of individual variants in SRAE, and not the interaction between a group of variants in key genes in statin pharmacokinetics pathway. Therefore, we suggest that the combined effect of the low-activity variants in SLCO1B1 and SLCO1B3, the high-activity variant in ABCB11, and the lack of activity of CYP3A53 predisposed the patient to low hepatic uptake, metabolization and efflux, respectively. The resulting higher rosuvastatin plasma concentration increased its systemic exposure, which may have caused myalgia (Figure 2). Importantly, the patient carries LDLR rs28941776 (c.1646G>A, p.Gly549Asp), a disruptive-missense variant that showed reduced LDL uptake in an in vitro study (24). LDLR variants have been associated with variability in statin response in FH patients (25), but we did not find studies that investigated the association between LDLR variants and time to statin response or myalgia. Nevertheless, this variant could have played a role in patient’s rosuvastatin time to response and it should be considered for further studies. A limitation of this study is that plasma concentrations of rosuvastatin and its metabolites were not measured. However, the adherence of the patient to the prescribed treatment was ensured using a validated adherence questionnaire and regular follow-up calls. In summary, the combination of four low-activity variants in SLC genes, a high-activity variant in ABCB11, and a non-functional variant in CYP3A5 may explain the observed late response to rosuvastatin and the statin-related myalgia. With this case report, we have shown the importance of considering a combination of variants in a pharmacogenetic analysis to predict individual responses to statin treatment and prevent adverse drug events. We believe this study contributes to precision medicine in future clinical settings. Patient perspective “I have had high cholesterol since I was a child and it has been an issue because of the delayed response to treatments and of many adverse reactions to medications, especially simvastatin. The authors have been very attentive towards me throughout the whole study and discovered possible variants that may delay my response to rosuvastatin and influence the pain that I have felt when using statins. I am very happy for knowing the cause of my problem and I would like to thank the authors for this possible diagnosis. This has improved my perspectives of cholesterol treatment.” Supplementary The article’s supplementary files as 10.21037/atm-20-5540 10.21037/atm-20-5540 10.21037/atm-20-5540 Acknowledgments The authors thank Adriana Garofalo, Dr. Hui Tzu Lin Wang, colleagues from the Laboratory of Molecular Investigation in Cardiology, and the Divisions of Dyslipidemia and Pharmacy of the Institute Dante Pazzanese of Cardiology. Their immeasurable technical and logistic support in patient selection and data collection made this study possible. Funding: This work was supported by Sao Paulo Research Foundation (FAPESP), Brazil [Research grant: #2016/12899-6 to MHH]; and National Council for Scientific and Technological Development [CNPq, grant: #447120/2014-0 to MHH], Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDH is a recipient of a fellowship of the São Paulo Research Foundation (FAPESP), grant #2016/25637-0. RCCF, RHB, GMF and VFO are recipients of fellowships from FAPESP, Brazil. AAM is a recipient of fellowship from CAPES, Brazil. ESRM, MHH and RDCH are recipients of fellowships from CNPq, Brazil. BL was a recipient of fellowship from FAPESP, Brazil. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The intervention study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The DNA sequencing study was approved by the Ethics Committees of the Institute Dante Pazzanese of Cardiology (CAAE #4618713.0.1001.5462) and the School of Pharmaceutical Sciences of the University of Sao Paulo (CAAE #24618713.0.3001.0067), Sao Paulo, Brazil. The intervention study was approved by the Ethics Committee of the Institute Dante Pazzanese of Cardiology (CAAE #05234918.4.0000.5462). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees. The patient signed the written informed consents before her enrollment in the studies. In the written informed consent of the DNA sequencing study, the patient was informed that clinical data and blood samples would be collected for laboratory tests and genetic analyses. As for the intervention study, the patient was informed on the intervention protocol and sample collections throughout the visits, and that this data would be used for genetic and epigenetic analyses. Reporting Checklist: The authors have completed the CARE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-5540 Peer Review File: Available at http://dx.doi.org/10.21037/atm-20-5540 Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5540). The authors have no conflicts of interest to declare.	Recovered	ReactionOutcome	CC BY-NC-ND	33553369	18,931,409	2021-01
What was the outcome of reaction 'Lipids abnormal'?	Late response to rosuvastatin and statin-related myalgia due to SLCO1B1, SLCO1B3, ABCB11, and CYP3A5 variants in a patient with Familial Hypercholesterolemia: a case report. Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B11B and5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A53. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. pmcIntroduction Familial hypercholesterolemia (FH) is a genetic metabolic disease that leads to increased high low-density lipoprotein (LDL) cholesterol, which is a risk factor for early atherosclerosis and cardiovascular diseases (1). FH is usually treated with high-dose statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), a key enzyme in cholesterol biosynthesis pathway. Rosuvastatin is one of the most effective statins, probably due its hydrophilicity, that confers selectivity to hepatic cells, higher affinity to HMGR, and lower rates of statin-related adverse events (SRAE) compared to other statins. It is poorly metabolized by CYP2C9 and CYP2C19, while 72% of the non-metabolized molecules are excreted via biliary system. Therefore, rosuvastatin blood levels rely on the activity of membrane transporters, mainly of solute carrier (SLC) and ATP-binding cassette (ABC) families, highly expressed in intestine, liver, and kidney (2). Pharmacogenetic studies have shown that loss-of-function variants in genes encoding OATPs, such as SLCO1B1, SLCO2B1, and SLCO1B3, and ABCs have been associated with variability in low-density lipoprotein cholesterol (LDL-c) reduction and higher risk of SRAE (3). The importance of considering the combined effect of variants in key genes for pharmacogenetic analyses has been increasingly evident (4). In this case report, we discuss how variants in genes participating in different stages of statin pharmacokinetics pathway possibly affected the time to response to rosuvastatin and the risk of SRAE in a female FH patient. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. This case is reported in accordance with the CARE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-5540). Case presentation A 26-year-old Caucasian female patient with definite diagnosis of FH according to Dutch Lipid Clinic Network MEDPED criteria (5) was invited to participate in an intervention study in June 2019. She was previously included in a FH sequencing study (May 2018), in which a panel of 84 genes involved in lipid homeostasis and drug metabolism was sequenced using exon-targeted gene sequencing (NGS). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for both studies. The patient carries the variant LDLR rs28941776 (c.1646G>A, p.Gly549Asp), which has been associated with FH and is classified as pathogenic according to the American College of Medical Genetics (ACMG) guidelines (6). Her clinical history included high levels of total cholesterol and LDL-c since childhood. In 2008, at the age of 15 years, she had an abnormal lipid profile even under a daily treatment with simvastatin 10 mg and ezetimibe 10 mg. Laboratory analyses showed a total cholesterol of 324 mg/dL, LDL-c 264 mg/dL, high-density lipoprotein cholesterol (HDL-c) 46 mg/dL, and triglycerides 71 mg/dL. In 2014, she was diagnosed with hypothyroidism and treated with levothyroxine 25 µg/day, which was gradually increased to 100 µg/day in 2019. She also had a pregnancy history in January 2017. Her therapy history included simvastatin, which led to severe myopathy in 2008, with marked increase in serum creatine kinase (CK) to 1,080 U/L (4.7-fold the upper reference value). The cholesterol-lowering therapy was changed to pravastatin 20 mg and ezetimibe 10 mg daily until May 2011, when she reported another episode of myalgia. Pravastatin was withdrawn and atorvastatin 20 mg was introduced, also associated with ezetimibe 10 mg. Three months later, in August 2011, she reported interrupting atorvastatin treatment due to myalgia. Rosuvastatin 10 mg was then introduced, also associated with ezetimibe 10 mg, after which she showed an LDL-c level of 125 mg/dL and never reported myalgia again. However, her lipid profile worsened throughout the years even under rosuvastatin treatment, with her LDL-c reaching 194 mg/dL with rosuvastatin 20 mg. The patient had no history of liver or kidney impartment, HIV, coronary artery disease (CAD), diabetes, obesity, cardiovascular events, and did not smoke or drink. Her mother and grandmother had a history of FH, but not CAD or cardiovascular events, while her father had hypertension and type 2 diabetes. In the intervention study, the patient was seen four times (V1 to V4) in 5 months, and clinical history and therapy data were obtained. The protocol consisted of a 6-week rosuvastatin wash-out period, after which rosuvastatin was reintroduced for additional 6 weeks, when treatment response was evaluated. Adherence to treatment was assessed in each timepoint using the translated and validated version of the Brief Medication Questionnaire (BMQ) (7) and blood samples were taken in each visit for laboratory testing. The lipid profile during the follow-up is shown in Figure 1. In April 2019 (V1), the patient was taking rosuvastatin 40 mg, ezetimibe 10 mg, and levothyroxine 88 µg daily. She reported experiencing muscle pain after recently increasing rosuvastatin dose from 20 to 40 mg/day. Her lipid profile was altered (total cholesterol 376 mg/dL, LDL-c 263 mg/dL, HDL-c 67 mg/dL, triglycerides 234 mg/dL) without increase in CK levels. She reported being active, running 2 km 2–3 times a week, and had a healthy diet, eating more than five portions of vegetables daily. Her TSH and T4 levels were normal. Rosuvastatin 40 mg was then discontinued for wash-out, ezetimibe was maintained, and levothyroxine dose was increased to 100 µg/day. Figure 1 Plasma lipid profile and pharmacotherapy of the FH patient throughout the study period. EZT, ezetimibe; LVT, levothyroxine; RSV, rosuvastatin; SRAE, statin-related adverse events. In June 2019 (V2), after undergoing a 6-week rosuvastatin wash-out period between V1 and V2, her lipid profile worsened (total cholesterol 512 mg/dL, LDL-c 405 mg/dL, HDL-c 65 mg/dL, triglycerides 213 mg/dL). Because the patient reported myalgia in V1 (rosuvastatin 40 mg), the physician prescribed rosuvastatin 20 mg/day for six weeks. Surprisingly, in August 2019 (V3), the lipid profile (total cholesterol 531 mg/dL, LDL-c 407 mg/dL, HDL-c 67 mg/dL, triglycerides 286 mg/dL) did not change compared to V2. The patient reported experiencing no myalgia to rosuvastatin 20 mg. In September 2019 (V4), her lipid profile improved (total cholesterol 299 mg/dL, LDL-c 208 mg/dL, HDL-c 59 mg/dL, triglycerides 158 mg/dL) and she continued not experiencing myalgia to rosuvastatin. During the follow-up period, serum TSH and T4 levels remained unchanged, suggesting that her hypothyroidism was controlled and did not influence the lipid profile. Moreover, serum CK did not show any abnormality, which indicates no muscle damage due to statin treatment. The patient also reported being adherent to treatment. In the BMQ adherence questionnaire, she reported forgetting the lipid-lowering medications 2 days in the week before V1 (71.4% adherence) and 1 day in the week before V3 (85.7% adherence). The genetic profile of the patient is shown in Table 1. She carries five missense variants in SLCO1B1, SLCO1B3, and ABCB11. She is also homozygote for the CYP3A53 (rs776746) splicing variant. No other missense variants described as impacting rosuvastatin response were found in CYP3A4, CYP2C9, CYP2C19, or other drug transporters, such as ABCG2 (data not shown). Table 1 Variants in pharmacokinetic-related genes of the FH patient with late response to rosuvastatin Gene Variant code Variant type Nucleotide change (Amino acid change) Patient genotype Allele frequency (1,000 genomes, %) Functional impact Effects on rosuvastatin pharmacokinetics References SLCO1B1 rs2306283 (SLCO1B11B) Missense c.388A>G (p.Asn130Asp) AG 1B: 54.4 Comparable to 1A No effect on plasma rosuvastatin levels Ho et al., 2006; Lee et al., 2013 SLCO1B1 rs4149056 (SLCO1B15) Missense c.521T>C p.(Val174Ala) TC 5: 8.8 Reduced activity Increased rosuvastatin plasma levels; Reduced hepatic uptake Kameyama et al., 2005; Lee et al., 2013 SLCO1B1 rs2306283, rs4149056 (SLCO1B115) Missense c.388A>G, c.521T>C (p.Asn130Asp, p.Val174Ala) AG, TC 15: 7.8 Reduced activity Increased rosuvastatin plasma levels; reduced hepatic uptake Kameyama et al., 2005; Birmingham et al., 2015 SLCO1B3 rs4149117 Missense c.334T>G (p.Ser112Ala) GG G: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 SLCO1B3 rs7311358 Missense c.699G>A (p.Met233Ile) AA A: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 ABCB11 rs2287622 Missense c.1331T>C (p.Val444Ala) TC C: 58.9 Reduced activity Increased rosuvastatin plasma levels Soko et al. 2019 CYP3A5 rs776746 (CYP3A53) Splicing c. 6986A>G GG 3: 62.1 No activity No rosuvastatin metabolism; Reduced LDL-c response Bailey et al. 2010 FH, familial hypercholesterolemia; LDL-c, low-density lipoprotein cholesterol. Discussion In heterozygous FH patients, LDL-c level reductions of 47.1% have been observed after a 6-week treatment with rosuvastatin 20 mg (8). The patient, however, did not experience any changes in LDL-c levels at week 6 (V3) of rosuvastatin 20 mg treatment, with a 48.9% LDL-c reduction only at week 12 (V4) of therapy. The delayed rosuvastatin response could be explained by modifications in the therapy scheme during the follow-up period. However, the only change was in levothyroxine dose, that was increased from 88 to 100 µg in V1. It is unlikely that the late response is due to an adaptation to the new levothyroxine dose. The patient was already on treatment with levothyroxine 88 µg before V1; moreover, changes in cholesterol due to an adaptation period should be reflected in her lipid profile in V3, not only in V4. Another possible explanation is a lack of adherence from V2 to V3; however, the patient showed a similar treatment adherence in V3 and V1, which should lead to a similar lipid profile between visits. Furthermore, drug interactions between rosuvastatin, levothyroxine, and ezetimibe that could affect treatment response were not detected, excluding this possibility. Pharmacokinetics-related genes may have contributed to the late response to rosuvastatin (Figure 2). The patient carries two variants in SLCO1B1, c.388A>G (SLCO1B11B) and c.521T>C (SLCO1B15), that are important determinants of rosuvastatin response. SLCO1B15 is a loss-of-function variant that decreases the hepatic uptake and increases blood levels of statins (9) (Table 1). SLCO1B11B has shown comparable activity to the functional 1A variant in in vitro functional studies (10). SLCO1B11B and 5 variants are in linkage disequilibrium (LD) and form the SLCO1B115 haplotype, that also reduced rosuvastatin uptake in functional studies with HEK293 and HeLa cells (11). The decreased liver uptake caused by these SLCO1B1 variants has been associated with increased plasma levels of rosuvastatin in pharmacokinetics studies (9) (Table 1). Figure 2 Proposed mechanism for patient’s late rosuvastatin response and myalgia. 1. The hepatic uptake of rosuvastatin occurs through SLCO1B1 and SLCO1B3 influx transporters, while atorvastatin and simvastatin are internalized through SLCO1B1. The presence of deleterious variants in these transporters (SLCO1B115 and SLCO1B3 c.334T>G and c.699G>A) decreases statin uptake, therefore decreasing their concentration inside the hepatocyte and increasing statin plasma levels. 2. The lack of expression of CYP3A5 due to CYP3A53 also decreases atorvastatin and simvastatin metabolization, which contributes to increasing their plasma levels. This enzyme does not participate markedly in rosuvastatin metabolism. 3. The resulting higher blood statin levels increased the patient’s muscular exposure to statins, that are internalized through SLCO2B1 transporter into the skeletal muscle cell. The high concentrations in the skeletal muscle cell possibly caused patient’s myalgia. 4. Rosuvastatin’s bile excretion occurs through ABCB11 efflux protein. ABCB11 c.1331T>C variant results in a reduced activity ABCB11, which decreases rosuvastatin efflux; this increases rosuvastatin intrahepatic levels and blood levels. Although the patient had reduced function influx transporters, we suggest that the small portion of rosuvastatin absorbed in the beginning of the treatment accumulated due to the loss of function of the ABCB11 variant. This, together with rosuvastatin active metabolites generated by the normal function CYP2C9, allowed HMGR inhibition and therefore cholesterol lowering in the last visit. SLCO1B3 is also an important gene that encodes an influx transporter for rosuvastatin. The patient was homozygous for both SLCO1B3 c.334T>G and c.699G>A, which are in strong LD (12). In an in vitro study, HeLa cells transfected with SLCO1B3 c.334G and c.699A haplotype showed a 13% decrease in rosuvastatin uptake, while for other substrates, such as cholecystokinin-8, an even more marked decrease of 57% was observed (13) (Table 1). Although the effect of SLCO1B3 c.334G and c.699A haplotype in rosuvastatin uptake is not sufficient to explain the delayed response, it might be significant when combined with the effect of the decreased function haplotype SLCO1B115. While SLCO1B15 and SLCO1B115 are associated with higher plasma levels of rosuvastatin, previous studies failed to find an association between these variants and LDL-c reduction in response to short- and long-term rosuvastatin treatments (9). Therefore, the simultaneous presence of decreased function SLCO1B1 and SLCO1B3 haplotypes possibly caused a marked reduction of rosuvastatin intrahepatic concentration, resulting in the lack of response observed in V3. ABCB11 encodes the efflux protein ABCB11, which plays an important role in rosuvastatin bile excretion. In a recent study, ABCB11 c.1331C allele has been associated to increased plasma rosuvastatin levels in healthy subjects (14) (Table 1). This variant possibly causes lower rosuvastatin excretion via bile, which in turn would increase intrahepatic rosuvastatin concentrations. Therefore, this mechanism could explain why even in the presence of low function SLC variants, the patient showed a late but evident LDL-c reduction after 12 weeks of rosuvastatin treatment. The patient also carries the homozygous form of CYP3A53, an intronic variant that results in undetectable expression of CYP3A5 (15). The GEOSTAT-1 study reported that dyslipidemic patients carrying CYP3A53/3 had lower LDL-c reduction after three-month rosuvastatin 10 mg treatment compared to carriers of 1/1 or 1/3 (Table 1). It was suggested that the metabolite produced by CYP3A5 also plays a role in HMGR inhibition, potentiating the response to rosuvastatin, which is why CYP3A5 non-expressors have reduced LDL-c response to rosuvastatin (16). CYP3A53 possibly impaired the patient’s response time to rosuvastatin, but in lower extent, as CYP3A5 does not participate markedly in rosuvastatin metabolism. In addition to the delayed response to rosuvastatin, the patient experienced myalgia associated with rosuvastatin 40 mg/day and other statins, as previously commented. This SRAE may be due to SLCO1B1 variants. SLCO1B15 and SLCO1B115 have been extensively associated with myopathy to simvastatin. A systematic review and meta-analysis reported that carriers of the C allele of SLCO1B15 (c.521T>C) showed a higher risk of myotoxicity (17). Additionally, SLCO1B15 has been associated to rosuvastatin myotoxicity in previous studies (18,19). It has been suggested that it causes higher efflux of statins, increasing statin exposure and, therefore, the risk of myalgia (20). Also, a recent case report showed that variants in SLCO1B3 (c.334T>G and c.699G>A) and ABCB11 (c.1331T>C) and the interaction between rosuvastatin and ticagrelor led to rhabdomyolysis in a patient with chronic kidney disease and other chronic conditions (21), but no other reports were found. CYP3A53 may also have contributed to statin myotoxicity, since it has been associated with increased risk to atorvastatin and rosuvastatin-related myalgia in South-Indian dyslipidemic patients (22). However, this variant was not associated to statin intolerance in another study (23). Most studies have evaluated the effect of individual variants in SRAE, and not the interaction between a group of variants in key genes in statin pharmacokinetics pathway. Therefore, we suggest that the combined effect of the low-activity variants in SLCO1B1 and SLCO1B3, the high-activity variant in ABCB11, and the lack of activity of CYP3A53 predisposed the patient to low hepatic uptake, metabolization and efflux, respectively. The resulting higher rosuvastatin plasma concentration increased its systemic exposure, which may have caused myalgia (Figure 2). Importantly, the patient carries LDLR rs28941776 (c.1646G>A, p.Gly549Asp), a disruptive-missense variant that showed reduced LDL uptake in an in vitro study (24). LDLR variants have been associated with variability in statin response in FH patients (25), but we did not find studies that investigated the association between LDLR variants and time to statin response or myalgia. Nevertheless, this variant could have played a role in patient’s rosuvastatin time to response and it should be considered for further studies. A limitation of this study is that plasma concentrations of rosuvastatin and its metabolites were not measured. However, the adherence of the patient to the prescribed treatment was ensured using a validated adherence questionnaire and regular follow-up calls. In summary, the combination of four low-activity variants in SLC genes, a high-activity variant in ABCB11, and a non-functional variant in CYP3A5 may explain the observed late response to rosuvastatin and the statin-related myalgia. With this case report, we have shown the importance of considering a combination of variants in a pharmacogenetic analysis to predict individual responses to statin treatment and prevent adverse drug events. We believe this study contributes to precision medicine in future clinical settings. Patient perspective “I have had high cholesterol since I was a child and it has been an issue because of the delayed response to treatments and of many adverse reactions to medications, especially simvastatin. The authors have been very attentive towards me throughout the whole study and discovered possible variants that may delay my response to rosuvastatin and influence the pain that I have felt when using statins. I am very happy for knowing the cause of my problem and I would like to thank the authors for this possible diagnosis. This has improved my perspectives of cholesterol treatment.” Supplementary The article’s supplementary files as 10.21037/atm-20-5540 10.21037/atm-20-5540 10.21037/atm-20-5540 Acknowledgments The authors thank Adriana Garofalo, Dr. Hui Tzu Lin Wang, colleagues from the Laboratory of Molecular Investigation in Cardiology, and the Divisions of Dyslipidemia and Pharmacy of the Institute Dante Pazzanese of Cardiology. Their immeasurable technical and logistic support in patient selection and data collection made this study possible. Funding: This work was supported by Sao Paulo Research Foundation (FAPESP), Brazil [Research grant: #2016/12899-6 to MHH]; and National Council for Scientific and Technological Development [CNPq, grant: #447120/2014-0 to MHH], Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDH is a recipient of a fellowship of the São Paulo Research Foundation (FAPESP), grant #2016/25637-0. RCCF, RHB, GMF and VFO are recipients of fellowships from FAPESP, Brazil. AAM is a recipient of fellowship from CAPES, Brazil. ESRM, MHH and RDCH are recipients of fellowships from CNPq, Brazil. BL was a recipient of fellowship from FAPESP, Brazil. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The intervention study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The DNA sequencing study was approved by the Ethics Committees of the Institute Dante Pazzanese of Cardiology (CAAE #4618713.0.1001.5462) and the School of Pharmaceutical Sciences of the University of Sao Paulo (CAAE #24618713.0.3001.0067), Sao Paulo, Brazil. The intervention study was approved by the Ethics Committee of the Institute Dante Pazzanese of Cardiology (CAAE #05234918.4.0000.5462). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees. The patient signed the written informed consents before her enrollment in the studies. In the written informed consent of the DNA sequencing study, the patient was informed that clinical data and blood samples would be collected for laboratory tests and genetic analyses. As for the intervention study, the patient was informed on the intervention protocol and sample collections throughout the visits, and that this data would be used for genetic and epigenetic analyses. Reporting Checklist: The authors have completed the CARE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-5540 Peer Review File: Available at http://dx.doi.org/10.21037/atm-20-5540 Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5540). The authors have no conflicts of interest to declare.	Recovered	ReactionOutcome	CC BY-NC-ND	33553369	18,931,409	2021-01
What was the outcome of reaction 'Myalgia'?	Late response to rosuvastatin and statin-related myalgia due to SLCO1B1, SLCO1B3, ABCB11, and CYP3A5 variants in a patient with Familial Hypercholesterolemia: a case report. Statins are the most widely used cholesterol-lowering drugs for cardiovascular diseases prevention. However, some patients are refractory to treatment, whereas others experience statin-related adverse events (SRAE). It has been increasingly important to identify pharmacogenetic biomarkers for predicting statin response and adverse events. This case report describes a female patient with familial hypercholesterolemia (FH) who showed late response to rosuvastatin and experienced myalgia on statin treatment. In the first visit (V1), the patient reported myalgia to rosuvastatin 40 mg, which was interrupted for a 6-week wash-out period. In V2, rosuvastatin 20 mg was reintroduced, but her lipid profile did not show any changes after 6 weeks (V3) (LDL-c: 402 vs. 407 mg/dL). Her lipid profile markedly improved after 12 weeks of treatment (V4) (LDL-c: 208 mg/dL), suggesting a late rosuvastatin response. Her adherence to treatment was similar in V1 and V3 and no drug interactions were detected. Pharmacogenetic analysis revealed that the patient carries low-activity variants in SLCO1B11B and5, SLCO1B3 (rs4149117 and rs7311358), and ABCB11 rs2287622, and the non-functional variant in CYP3A53. The combined effect of variants in pharmacokinetics-related genes may have contributed to the late response to rosuvastatin and statin-related myalgia. Therefore, they should be considered when assessing a patient's response to statin treatment. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. pmcIntroduction Familial hypercholesterolemia (FH) is a genetic metabolic disease that leads to increased high low-density lipoprotein (LDL) cholesterol, which is a risk factor for early atherosclerosis and cardiovascular diseases (1). FH is usually treated with high-dose statins, which are inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), a key enzyme in cholesterol biosynthesis pathway. Rosuvastatin is one of the most effective statins, probably due its hydrophilicity, that confers selectivity to hepatic cells, higher affinity to HMGR, and lower rates of statin-related adverse events (SRAE) compared to other statins. It is poorly metabolized by CYP2C9 and CYP2C19, while 72% of the non-metabolized molecules are excreted via biliary system. Therefore, rosuvastatin blood levels rely on the activity of membrane transporters, mainly of solute carrier (SLC) and ATP-binding cassette (ABC) families, highly expressed in intestine, liver, and kidney (2). Pharmacogenetic studies have shown that loss-of-function variants in genes encoding OATPs, such as SLCO1B1, SLCO2B1, and SLCO1B3, and ABCs have been associated with variability in low-density lipoprotein cholesterol (LDL-c) reduction and higher risk of SRAE (3). The importance of considering the combined effect of variants in key genes for pharmacogenetic analyses has been increasingly evident (4). In this case report, we discuss how variants in genes participating in different stages of statin pharmacokinetics pathway possibly affected the time to response to rosuvastatin and the risk of SRAE in a female FH patient. To the best of our knowledge, this is the first report of a pharmacogenetic analysis on a case of late rosuvastatin response. This case is reported in accordance with the CARE reporting checklist (available at http://dx.doi.org/10.21037/atm-20-5540). Case presentation A 26-year-old Caucasian female patient with definite diagnosis of FH according to Dutch Lipid Clinic Network MEDPED criteria (5) was invited to participate in an intervention study in June 2019. She was previously included in a FH sequencing study (May 2018), in which a panel of 84 genes involved in lipid homeostasis and drug metabolism was sequenced using exon-targeted gene sequencing (NGS). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees and with the Helsinki Declaration (as revised in 2013). Written informed consent was obtained from the patient for both studies. The patient carries the variant LDLR rs28941776 (c.1646G>A, p.Gly549Asp), which has been associated with FH and is classified as pathogenic according to the American College of Medical Genetics (ACMG) guidelines (6). Her clinical history included high levels of total cholesterol and LDL-c since childhood. In 2008, at the age of 15 years, she had an abnormal lipid profile even under a daily treatment with simvastatin 10 mg and ezetimibe 10 mg. Laboratory analyses showed a total cholesterol of 324 mg/dL, LDL-c 264 mg/dL, high-density lipoprotein cholesterol (HDL-c) 46 mg/dL, and triglycerides 71 mg/dL. In 2014, she was diagnosed with hypothyroidism and treated with levothyroxine 25 µg/day, which was gradually increased to 100 µg/day in 2019. She also had a pregnancy history in January 2017. Her therapy history included simvastatin, which led to severe myopathy in 2008, with marked increase in serum creatine kinase (CK) to 1,080 U/L (4.7-fold the upper reference value). The cholesterol-lowering therapy was changed to pravastatin 20 mg and ezetimibe 10 mg daily until May 2011, when she reported another episode of myalgia. Pravastatin was withdrawn and atorvastatin 20 mg was introduced, also associated with ezetimibe 10 mg. Three months later, in August 2011, she reported interrupting atorvastatin treatment due to myalgia. Rosuvastatin 10 mg was then introduced, also associated with ezetimibe 10 mg, after which she showed an LDL-c level of 125 mg/dL and never reported myalgia again. However, her lipid profile worsened throughout the years even under rosuvastatin treatment, with her LDL-c reaching 194 mg/dL with rosuvastatin 20 mg. The patient had no history of liver or kidney impartment, HIV, coronary artery disease (CAD), diabetes, obesity, cardiovascular events, and did not smoke or drink. Her mother and grandmother had a history of FH, but not CAD or cardiovascular events, while her father had hypertension and type 2 diabetes. In the intervention study, the patient was seen four times (V1 to V4) in 5 months, and clinical history and therapy data were obtained. The protocol consisted of a 6-week rosuvastatin wash-out period, after which rosuvastatin was reintroduced for additional 6 weeks, when treatment response was evaluated. Adherence to treatment was assessed in each timepoint using the translated and validated version of the Brief Medication Questionnaire (BMQ) (7) and blood samples were taken in each visit for laboratory testing. The lipid profile during the follow-up is shown in Figure 1. In April 2019 (V1), the patient was taking rosuvastatin 40 mg, ezetimibe 10 mg, and levothyroxine 88 µg daily. She reported experiencing muscle pain after recently increasing rosuvastatin dose from 20 to 40 mg/day. Her lipid profile was altered (total cholesterol 376 mg/dL, LDL-c 263 mg/dL, HDL-c 67 mg/dL, triglycerides 234 mg/dL) without increase in CK levels. She reported being active, running 2 km 2–3 times a week, and had a healthy diet, eating more than five portions of vegetables daily. Her TSH and T4 levels were normal. Rosuvastatin 40 mg was then discontinued for wash-out, ezetimibe was maintained, and levothyroxine dose was increased to 100 µg/day. Figure 1 Plasma lipid profile and pharmacotherapy of the FH patient throughout the study period. EZT, ezetimibe; LVT, levothyroxine; RSV, rosuvastatin; SRAE, statin-related adverse events. In June 2019 (V2), after undergoing a 6-week rosuvastatin wash-out period between V1 and V2, her lipid profile worsened (total cholesterol 512 mg/dL, LDL-c 405 mg/dL, HDL-c 65 mg/dL, triglycerides 213 mg/dL). Because the patient reported myalgia in V1 (rosuvastatin 40 mg), the physician prescribed rosuvastatin 20 mg/day for six weeks. Surprisingly, in August 2019 (V3), the lipid profile (total cholesterol 531 mg/dL, LDL-c 407 mg/dL, HDL-c 67 mg/dL, triglycerides 286 mg/dL) did not change compared to V2. The patient reported experiencing no myalgia to rosuvastatin 20 mg. In September 2019 (V4), her lipid profile improved (total cholesterol 299 mg/dL, LDL-c 208 mg/dL, HDL-c 59 mg/dL, triglycerides 158 mg/dL) and she continued not experiencing myalgia to rosuvastatin. During the follow-up period, serum TSH and T4 levels remained unchanged, suggesting that her hypothyroidism was controlled and did not influence the lipid profile. Moreover, serum CK did not show any abnormality, which indicates no muscle damage due to statin treatment. The patient also reported being adherent to treatment. In the BMQ adherence questionnaire, she reported forgetting the lipid-lowering medications 2 days in the week before V1 (71.4% adherence) and 1 day in the week before V3 (85.7% adherence). The genetic profile of the patient is shown in Table 1. She carries five missense variants in SLCO1B1, SLCO1B3, and ABCB11. She is also homozygote for the CYP3A53 (rs776746) splicing variant. No other missense variants described as impacting rosuvastatin response were found in CYP3A4, CYP2C9, CYP2C19, or other drug transporters, such as ABCG2 (data not shown). Table 1 Variants in pharmacokinetic-related genes of the FH patient with late response to rosuvastatin Gene Variant code Variant type Nucleotide change (Amino acid change) Patient genotype Allele frequency (1,000 genomes, %) Functional impact Effects on rosuvastatin pharmacokinetics References SLCO1B1 rs2306283 (SLCO1B11B) Missense c.388A>G (p.Asn130Asp) AG 1B: 54.4 Comparable to 1A No effect on plasma rosuvastatin levels Ho et al., 2006; Lee et al., 2013 SLCO1B1 rs4149056 (SLCO1B15) Missense c.521T>C p.(Val174Ala) TC 5: 8.8 Reduced activity Increased rosuvastatin plasma levels; Reduced hepatic uptake Kameyama et al., 2005; Lee et al., 2013 SLCO1B1 rs2306283, rs4149056 (SLCO1B115) Missense c.388A>G, c.521T>C (p.Asn130Asp, p.Val174Ala) AG, TC 15: 7.8 Reduced activity Increased rosuvastatin plasma levels; reduced hepatic uptake Kameyama et al., 2005; Birmingham et al., 2015 SLCO1B3 rs4149117 Missense c.334T>G (p.Ser112Ala) GG G: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 SLCO1B3 rs7311358 Missense c.699G>A (p.Met233Ile) AA A: 70.2 Reduced activity Reduced hepatic uptake Schwarz et al. 2011 ABCB11 rs2287622 Missense c.1331T>C (p.Val444Ala) TC C: 58.9 Reduced activity Increased rosuvastatin plasma levels Soko et al. 2019 CYP3A5 rs776746 (CYP3A53) Splicing c. 6986A>G GG 3: 62.1 No activity No rosuvastatin metabolism; Reduced LDL-c response Bailey et al. 2010 FH, familial hypercholesterolemia; LDL-c, low-density lipoprotein cholesterol. Discussion In heterozygous FH patients, LDL-c level reductions of 47.1% have been observed after a 6-week treatment with rosuvastatin 20 mg (8). The patient, however, did not experience any changes in LDL-c levels at week 6 (V3) of rosuvastatin 20 mg treatment, with a 48.9% LDL-c reduction only at week 12 (V4) of therapy. The delayed rosuvastatin response could be explained by modifications in the therapy scheme during the follow-up period. However, the only change was in levothyroxine dose, that was increased from 88 to 100 µg in V1. It is unlikely that the late response is due to an adaptation to the new levothyroxine dose. The patient was already on treatment with levothyroxine 88 µg before V1; moreover, changes in cholesterol due to an adaptation period should be reflected in her lipid profile in V3, not only in V4. Another possible explanation is a lack of adherence from V2 to V3; however, the patient showed a similar treatment adherence in V3 and V1, which should lead to a similar lipid profile between visits. Furthermore, drug interactions between rosuvastatin, levothyroxine, and ezetimibe that could affect treatment response were not detected, excluding this possibility. Pharmacokinetics-related genes may have contributed to the late response to rosuvastatin (Figure 2). The patient carries two variants in SLCO1B1, c.388A>G (SLCO1B11B) and c.521T>C (SLCO1B15), that are important determinants of rosuvastatin response. SLCO1B15 is a loss-of-function variant that decreases the hepatic uptake and increases blood levels of statins (9) (Table 1). SLCO1B11B has shown comparable activity to the functional 1A variant in in vitro functional studies (10). SLCO1B11B and 5 variants are in linkage disequilibrium (LD) and form the SLCO1B115 haplotype, that also reduced rosuvastatin uptake in functional studies with HEK293 and HeLa cells (11). The decreased liver uptake caused by these SLCO1B1 variants has been associated with increased plasma levels of rosuvastatin in pharmacokinetics studies (9) (Table 1). Figure 2 Proposed mechanism for patient’s late rosuvastatin response and myalgia. 1. The hepatic uptake of rosuvastatin occurs through SLCO1B1 and SLCO1B3 influx transporters, while atorvastatin and simvastatin are internalized through SLCO1B1. The presence of deleterious variants in these transporters (SLCO1B115 and SLCO1B3 c.334T>G and c.699G>A) decreases statin uptake, therefore decreasing their concentration inside the hepatocyte and increasing statin plasma levels. 2. The lack of expression of CYP3A5 due to CYP3A53 also decreases atorvastatin and simvastatin metabolization, which contributes to increasing their plasma levels. This enzyme does not participate markedly in rosuvastatin metabolism. 3. The resulting higher blood statin levels increased the patient’s muscular exposure to statins, that are internalized through SLCO2B1 transporter into the skeletal muscle cell. The high concentrations in the skeletal muscle cell possibly caused patient’s myalgia. 4. Rosuvastatin’s bile excretion occurs through ABCB11 efflux protein. ABCB11 c.1331T>C variant results in a reduced activity ABCB11, which decreases rosuvastatin efflux; this increases rosuvastatin intrahepatic levels and blood levels. Although the patient had reduced function influx transporters, we suggest that the small portion of rosuvastatin absorbed in the beginning of the treatment accumulated due to the loss of function of the ABCB11 variant. This, together with rosuvastatin active metabolites generated by the normal function CYP2C9, allowed HMGR inhibition and therefore cholesterol lowering in the last visit. SLCO1B3 is also an important gene that encodes an influx transporter for rosuvastatin. The patient was homozygous for both SLCO1B3 c.334T>G and c.699G>A, which are in strong LD (12). In an in vitro study, HeLa cells transfected with SLCO1B3 c.334G and c.699A haplotype showed a 13% decrease in rosuvastatin uptake, while for other substrates, such as cholecystokinin-8, an even more marked decrease of 57% was observed (13) (Table 1). Although the effect of SLCO1B3 c.334G and c.699A haplotype in rosuvastatin uptake is not sufficient to explain the delayed response, it might be significant when combined with the effect of the decreased function haplotype SLCO1B115. While SLCO1B15 and SLCO1B115 are associated with higher plasma levels of rosuvastatin, previous studies failed to find an association between these variants and LDL-c reduction in response to short- and long-term rosuvastatin treatments (9). Therefore, the simultaneous presence of decreased function SLCO1B1 and SLCO1B3 haplotypes possibly caused a marked reduction of rosuvastatin intrahepatic concentration, resulting in the lack of response observed in V3. ABCB11 encodes the efflux protein ABCB11, which plays an important role in rosuvastatin bile excretion. In a recent study, ABCB11 c.1331C allele has been associated to increased plasma rosuvastatin levels in healthy subjects (14) (Table 1). This variant possibly causes lower rosuvastatin excretion via bile, which in turn would increase intrahepatic rosuvastatin concentrations. Therefore, this mechanism could explain why even in the presence of low function SLC variants, the patient showed a late but evident LDL-c reduction after 12 weeks of rosuvastatin treatment. The patient also carries the homozygous form of CYP3A53, an intronic variant that results in undetectable expression of CYP3A5 (15). The GEOSTAT-1 study reported that dyslipidemic patients carrying CYP3A53/3 had lower LDL-c reduction after three-month rosuvastatin 10 mg treatment compared to carriers of 1/1 or 1/3 (Table 1). It was suggested that the metabolite produced by CYP3A5 also plays a role in HMGR inhibition, potentiating the response to rosuvastatin, which is why CYP3A5 non-expressors have reduced LDL-c response to rosuvastatin (16). CYP3A53 possibly impaired the patient’s response time to rosuvastatin, but in lower extent, as CYP3A5 does not participate markedly in rosuvastatin metabolism. In addition to the delayed response to rosuvastatin, the patient experienced myalgia associated with rosuvastatin 40 mg/day and other statins, as previously commented. This SRAE may be due to SLCO1B1 variants. SLCO1B15 and SLCO1B115 have been extensively associated with myopathy to simvastatin. A systematic review and meta-analysis reported that carriers of the C allele of SLCO1B15 (c.521T>C) showed a higher risk of myotoxicity (17). Additionally, SLCO1B15 has been associated to rosuvastatin myotoxicity in previous studies (18,19). It has been suggested that it causes higher efflux of statins, increasing statin exposure and, therefore, the risk of myalgia (20). Also, a recent case report showed that variants in SLCO1B3 (c.334T>G and c.699G>A) and ABCB11 (c.1331T>C) and the interaction between rosuvastatin and ticagrelor led to rhabdomyolysis in a patient with chronic kidney disease and other chronic conditions (21), but no other reports were found. CYP3A53 may also have contributed to statin myotoxicity, since it has been associated with increased risk to atorvastatin and rosuvastatin-related myalgia in South-Indian dyslipidemic patients (22). However, this variant was not associated to statin intolerance in another study (23). Most studies have evaluated the effect of individual variants in SRAE, and not the interaction between a group of variants in key genes in statin pharmacokinetics pathway. Therefore, we suggest that the combined effect of the low-activity variants in SLCO1B1 and SLCO1B3, the high-activity variant in ABCB11, and the lack of activity of CYP3A53 predisposed the patient to low hepatic uptake, metabolization and efflux, respectively. The resulting higher rosuvastatin plasma concentration increased its systemic exposure, which may have caused myalgia (Figure 2). Importantly, the patient carries LDLR rs28941776 (c.1646G>A, p.Gly549Asp), a disruptive-missense variant that showed reduced LDL uptake in an in vitro study (24). LDLR variants have been associated with variability in statin response in FH patients (25), but we did not find studies that investigated the association between LDLR variants and time to statin response or myalgia. Nevertheless, this variant could have played a role in patient’s rosuvastatin time to response and it should be considered for further studies. A limitation of this study is that plasma concentrations of rosuvastatin and its metabolites were not measured. However, the adherence of the patient to the prescribed treatment was ensured using a validated adherence questionnaire and regular follow-up calls. In summary, the combination of four low-activity variants in SLC genes, a high-activity variant in ABCB11, and a non-functional variant in CYP3A5 may explain the observed late response to rosuvastatin and the statin-related myalgia. With this case report, we have shown the importance of considering a combination of variants in a pharmacogenetic analysis to predict individual responses to statin treatment and prevent adverse drug events. We believe this study contributes to precision medicine in future clinical settings. Patient perspective “I have had high cholesterol since I was a child and it has been an issue because of the delayed response to treatments and of many adverse reactions to medications, especially simvastatin. The authors have been very attentive towards me throughout the whole study and discovered possible variants that may delay my response to rosuvastatin and influence the pain that I have felt when using statins. I am very happy for knowing the cause of my problem and I would like to thank the authors for this possible diagnosis. This has improved my perspectives of cholesterol treatment.” Supplementary The article’s supplementary files as 10.21037/atm-20-5540 10.21037/atm-20-5540 10.21037/atm-20-5540 Acknowledgments The authors thank Adriana Garofalo, Dr. Hui Tzu Lin Wang, colleagues from the Laboratory of Molecular Investigation in Cardiology, and the Divisions of Dyslipidemia and Pharmacy of the Institute Dante Pazzanese of Cardiology. Their immeasurable technical and logistic support in patient selection and data collection made this study possible. Funding: This work was supported by Sao Paulo Research Foundation (FAPESP), Brazil [Research grant: #2016/12899-6 to MHH]; and National Council for Scientific and Technological Development [CNPq, grant: #447120/2014-0 to MHH], Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CDH is a recipient of a fellowship of the São Paulo Research Foundation (FAPESP), grant #2016/25637-0. RCCF, RHB, GMF and VFO are recipients of fellowships from FAPESP, Brazil. AAM is a recipient of fellowship from CAPES, Brazil. ESRM, MHH and RDCH are recipients of fellowships from CNPq, Brazil. BL was a recipient of fellowship from FAPESP, Brazil. Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The intervention study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The DNA sequencing study was approved by the Ethics Committees of the Institute Dante Pazzanese of Cardiology (CAAE #4618713.0.1001.5462) and the School of Pharmaceutical Sciences of the University of Sao Paulo (CAAE #24618713.0.3001.0067), Sao Paulo, Brazil. The intervention study was approved by the Ethics Committee of the Institute Dante Pazzanese of Cardiology (CAAE #05234918.4.0000.5462). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committees. The patient signed the written informed consents before her enrollment in the studies. In the written informed consent of the DNA sequencing study, the patient was informed that clinical data and blood samples would be collected for laboratory tests and genetic analyses. As for the intervention study, the patient was informed on the intervention protocol and sample collections throughout the visits, and that this data would be used for genetic and epigenetic analyses. Reporting Checklist: The authors have completed the CARE reporting checklist. Available at http://dx.doi.org/10.21037/atm-20-5540 Peer Review File: Available at http://dx.doi.org/10.21037/atm-20-5540 Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5540). The authors have no conflicts of interest to declare.	Recovered	ReactionOutcome	CC BY-NC-ND	33553369	18,931,409	2021-01
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Acute hepatic failure'.	Clinical features and potential mechanism of coronavirus disease 2019-associated liver injury. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has posed a serious threat to global public health security. With the increase in the number of confirmed cases globally, the World Health Organization has declared the outbreak of COVID-19 an international public health emergency. Despite atypical pneumonia as the primary symptom, liver dysfunction has also been observed in many clinical cases and is associated with the mortality risk in patients with COVID-19, like severe acute respiratory syndrome and Middle East respiratory syndrome. Here we will provide a schematic overview of the clinical characteristics and the possible mechanisms of liver injury caused by severe acute respiratory syndrome coronavirus 2 infection, which may provide help for optimizing the management of liver injury and reducing mortality in COVID-19 patients. Core Tip: With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with coronavirus disease 2019 (COVID-19). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases. Underlying mechanisms may be viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia. A close monitor of liver function is recommended in COVID-19 patients, especially in critical individuals. INTRODUCTION Since the 21st century, the outbreak of coronaviruses has brought great harm to human society; the most serious of which are the severe acute respiratory syndrome (SARS) in 2003, the Middle East respiratory syndrome (MERS) in 2012 and the novel coronavirus disease in 2019 (COVID-19). The ongoing outbreak of COVID-19 has become a pandemic. As of December 9, 2020, the total number of diagnosed cases globally exceeded 67530912 with a total of more than 1545140 infection-related deaths, carrying a mortality of approximately 2%[1]. Up to now, no specific antiviral therapies have been identified. Thus, an early monitor of critical complications is vital in preventing disease progression and improving survival. With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with COVID-19, making this organ one of the most frequently damaged outside of the respiratory system (summarized in Table 1). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases[2]. However, due to the different design and sample size, the incidence and clinical manifestations of liver injury in these studies are not the same. The mechanism of hepatic damage caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still unclear. Table 1 Main characteristics related to liver injury in patients with coronavirus disease 2019 based on a series of case reports Ref. Sample size Liver injury Elevated ALT Elevated AST Elevated TBIL Elevated ALP Elevated GGT Factors related to liver injury [25] 1099 NA 21.3%: Severe 28.1%, Non-severe 19.8% 22.2%: Severe 39.4%, Non-severe 18.2% 10.5%: Severe 13.3%, Non-severe 9.9% NA NA NA [26] 548 NA 23.1%: Severe 24.1%, Non-severe 22.3% 33.1%: Severe 43.4%, Non-severe 23.3% 4.4%: Severe 6.4%, Non-severe 2.3% NA NA NA [27] 417 21.5% 41.2%: Severe 82.4%, Non-severe 50.2% 47.2%: Severe 75.3%, Non-severe 36.9% 64.2%: Severe 75.3%, Non-severe 60.1% 10.9%: Severe 12.2%, Non-severe 10.5% 48.5%: Severe 75.3%, Non-severe 39.1% Older, male, higher BMI, Underlying liver diseases (NAFLD, alcoholic liver disease and chronic hepatitis B), drugs (lopinavir/ritonavir) [28] 324 NA 15.7% 10.5% 6.5% 1.2% 0.9% NA [29] 298 14.8% NA NA NA NA NA NA [30] 274 NA 22.0%: Deceased 27.0%, Recovered 19.0% 31.0%: Deceased 52.0%, Recovered 16.0% NA NA NA NA [31] 148 37.2% 18.2% 21.6% 6.1% 4.1% 17.6% Male, higher levels of procalcitonin and CRP. PCT, LDH, received lopinavir / ritonavir [32] 85 38.8% 61.2% NA NA NA NA Older, lactic acid, myoglobin, neutrophils, critical illness, aCRP, alymphocyte count [33] 79 36.7% 31.6% 35.4% 5.1% NA NA Male, white blood cell counts, neutrophils, CRP, athe extent of pulmonary alesions on CT [34] 40 55% 52.5% 40% 25% NA NA Many types of drugs, large amounts of hormones, underlying diseases, lymphocyte count, acritical illness [35] 82 78% 30.6% 61.1% 30.6% NA NA NA a Represents independent risk factors for liver injury in coronavirus disease 2019. ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body mass index; COVID-19: Coronavirus disease 2019; CRP: C-reactive protein; CT: Computed tomography; GGT: Gamma-glutamyl transpeptidase; LDH: Lactate dehydrogenase; NA: Not available; NAFLD: Non-alcoholic fatty liver diseases; PCT: Procalcitonin; TBIL: Total bilirubin. SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are the causative agents of SARS, MERS and COVID-19, respectively, and they all belong to the highly pathogenic human beta coronaviruses[3]. Genomics analyses have found that SARS-CoV-2 shares 79.5% genome sequence similarity to SARS-CoV and 50% genome sequence homology to MERS-CoV[4]. SARS-CoV-2 uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV[5]. These common points hint that SARS-CoV-2 may partly mimic SARS-CoV and MERS-CoV infection. In this review, we summarized the characteristics and mechanism of liver injury caused by SARS-CoV-2 infection to provide a reference for further study. Liver injury in SARS and MERS Liver injury is not uncommon in patients infected with SARS-CoV and MERS-CoV according to previous studies[6]. In patients with SARS, liver injury mainly manifests as elevated alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in the early phase of the disease and is associated with the severity of the illness[7-13]. The pathogenesis of hepatic damage caused by SARS-CoV appears to be multifactorial, including a direct injury to the target cells by the virus and an indirect injury mediated by subsequent immune system dysfunction. As the functional receptor for SARS-CoV, ACE2 is abundantly expressed on endothelial cells of the liver, implying that SARS-CoV may directly bind to ACE2 positive cells to dysregulate liver function[5,13]. Liver biopsies in SARS patients demonstrated localization of virus in liver and hepatocyte apoptosis, which further confirmed the direct injury by SARS-CoV[14]. In an analysis of 145 cases of SARS, serum interleukin (IL)-1β, IL-6 and IL-10 were higher in patients with elevated serum levels of ALT than those in ALT normal group, indicating that liver damage was part of the manifestation of system inflammation reactive syndrome induced by SARS-CoV infection[10]. In addition, some studies have also found that hypoxemia and medication are closely related to abnormal liver function[15,16]. Elevated liver enzymes and bilirubin levels, as well as decreased albumin levels were highlighted during the hospital course of MERS-CoV infection in a series of case reports[17-20]. Different from SARS-CoV, MERS-CoV uses dipeptide base peptidase 4 as a cellular receptor to infect cells[21]. In humans, dipeptide base peptidase 4 is expressed constitutively on epithelial cells of liver[22], suggesting a direct hepatic damage caused by MERS-CoV. MERS also involves a mechanism of the upregulation of proinflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, IL-15 and IL-17[23]. However, studies on the relationship between cytokine storm and liver injury are scarce so far. Clinical characteristics of liver injury in patients with COVID-19 Since Chen et al[24] reported that 43 cases of 99 patients (43.4%) in Wuhan Jinyintan Hospital had different degrees of abnormal liver function, the abnormality of liver function test in patients with COVID-19 has aroused widespread concern among clinicians. As shown in Table 1, the incidence of liver injury ranged from 14.8% to 55.0% in recent case studies reporting clinical features of patients with COVID-19[25-34]. In death cases of COVID-19, the rate reached as high as 78.0%[35]. Liver injury presented in 30 out of 113 deceased patients from our previous report[30]. Abnormal liver function mainly manifests as slightly elevated ALT/AST and bilirubin levels, which usually occurs around the second week of the disease course[33-35]. Rarely, severe acute hepatitis associated with COVID-19 has been reported[36]. Contradictory to other hepatitis-induced liver injury, AST-dominant aminotrans-ferase elevation is common in COVID-19, which may provide a clue about the underlying pathophysiology of the impact of COVID-19 on liver. A retrospective study including 60 patients revealed that median AST was higher than ALT at admission (46 U/L vs 30 U/L) and during the hospital course[37]. In a multicenter retrospective cohort-derived data set of 5771 individuals, the elevation of serum AST level was earlier, more frequent and significant than the increase of ALT in severe patients, and AST levels had the highest correlation with mortality when compared with other indicators reflecting liver injury including elevated ALT, alkaline phosphatase (ALP) and total bilirubin (TBIL) levels in patients with COVID-19[38]. Likewise, an elevated baseline AST level has been shown to correlate with intensive care unit (ICU) admission, intubation and death in another study[39]. To our knowledge, three possible reasons may account for this phenomenon. First, given that AST is also distributed in myocardium and skeletal muscle, the American Association for the Study of Liver Diseases has recommended consideration of myositis or cardiac injury as contributors to the AST elevation[40]. Second, recent data identified ribosomal proteins as important host-dependency factors for SARS-CoV-2[41]. Therefore, the virus may directly cause hepatic mitochondrial injury and subsequent AST elevation. Third, AST-predominant aminotransferase elevations have been reported in alcohol-related liver disease, ischemia and cirrhosis. It is possible that hypoxia as well as metabolic changes such as hepatic steatosis may account for AST elevation in COVID-19 patients[42,43]. It is worth noting that liver dysfunction is closely related to the severity of the disease. On the one hand, severe patients have a higher proportion of liver injury: Guan et al[25] extracted a cohort regarding 1099 patients with laboratory-confirmed COVID-19 from 552 hospitals in mainland China. The results showed more patients with severe disease had elevated AST and ALT than those with non-severe disease. Like the result, Wang et al[44] showed that more patients admitted to the ICU had elevated AST levels. Huang et al[45] showed that patients admitted to the ICU had significantly higher ALT levels. On the other hand, patients with abnormal liver tests had higher risks of progressing to a severe disease course: Bloom et al[37] showed that admission AST, peak AST and peak ALT were higher in intubated patients. Of 417 patients with COVID-19, patients with abnormal liver tests of hepatocellular, cholestatic or mixed type at admission had higher odds of progressing to severe pneumonia[27]. Among 148 confirmed SARS-CoV-2-infected patients, the emerging abnormal liver functions after admission caused a prolonged length of stay[31]. In a large United States COVID-19 cohort of 3381 patients, 2273 patients who tested positive for SARS-CoV-2 had higher initial and peak ALT than those who tested negative[46]. Compared with mild [upper limit of normal (ULN) < ALT < two times ULN] and moderate (two times ULN < ALT < five times ULN) liver injury, patients with severe liver injury (ALT > five times ULN) had a more severe clinical course, including higher rates of ICU admission (69%), intubation (65%), renal replacement therapy (33%) and mortality (42%). Other hepatic manifestations in COVID-19 patients were hypoproteinemia and changes in coagulation[47,48]. A large cohort study including 2623 patients reported marked hypoalbuminemia in the critically ill and death groups than non-critically ill patients (38.2%, 71.2% and 82.4% on admission and 45.9%, 77.7% and 95.6% during hospitalization, respectively)[49]. Meanwhile, the patients in this study displayed dramatically prolonged activated partial thromboplastin time in critically ill patients reflected coagulopathy. Further analysis shows that risk factors associated with hepatic damage include older males, a longer time from illness onset to admission, a history of drinking, higher serum levels of C-reactive protein (CRP), white blood cell counts, neutrophils and medication (lopinavir/ritonavir, hormones)[27,31-34,50]. Disease severity (severe/critical), CRP, lymphocyte count and the extent of pulmonary lesions on computed tomography are independent risk factors for liver injury[32-34]. Accumulating data reveals that patients with pre-existing liver diseases are more susceptible to SARS-CoV-2 infection and have poorer prognosis. In our study, viral hepatitis (hepatitis B and hepatitis C) was much more frequent among patients with liver injury than those without[51]. Another study revealed that COVID-19 patients with non-alcoholic fatty liver disease had a significantly higher likelihood of abnormal liver function from admission to discharge when compared to those with non-alcoholic fatty liver disease subjects[52]. Qiu et al[53] first reported a case of acute-on-chronic liver failure due to SARS-COV-2 infection in a patient with decompensated alcoholic cirrhosis. In a multicenter retrospective study, fifty cirrhotic patients with SARS-CoV-2 infection were studied to evaluate the impact of COVID-19 on the clinical outcome[54]. The results showed 30-d mortality rate was higher in cirrhotic patients with COVID-19 than in cirrhotic patients with bacterial infection and in COVID-19 patients without cirrhosis, indicating that COVID-19 was associated with liver function deterioration and elevated mortality in cirrhotic patients. In addition, a study of 2780 COVID-19-positive patients found that those with cirrhosis were at a particularly increased risk for mortality by analyzing a large United States database (risk ratio, 4.6; 95% confidence interval, 2.6-8.3)[55]. Whether the COVID-19 patients with more severe liver injury were positive for hepatotrophis viruses is still controversial. In a retrospective study, the authors analyzed liver function parameters including ALT, AST and TBIL in COVID-19 patients with or without HBV infection and found no significant differences between the two groups[56]. Another study reached a similar conclusion and further proved the longitudinal changes of median values for liver biochemistries were not significantly different between the two groups either[57]. These findings indicated that SARS-CoV-2 will not exacerbate liver injury in patients with HBV co-infection. However, Lin et al[58] drew a completely opposite conclusion. In their cohort, COVID-19 cases with HBV coinfection had higher levels of ALT, AST, TBIL and ALP than the COVID-19 cases without HBV coinfection, showing that inactive HBV carriers with SARS-CoV-2 coinfection are at risk of greater liver injury. Moreover, SARS-CoV-2 was reported to induce HBV reactivation, which may cause severe liver injury in patients with coinfection[57,59]. In addition, a case of COVID-19 with Epstein-Barr virus coinfection was reported recently. On admission, he showed acute liver injury with liver enzymes that were much higher than typically seen solely with COVID-19 infection[60]. Although the evidence is limited, more attention should be paid to COVID-19 patients with other viral coinfections during clinical treatment. Mechanisms of liver injury in patients with COVID-19 Underlying mechanisms involved in liver injury in patients with COVID-19 are complex and interactive, which might include viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia[61] (Figure 1). Figure 1 Potential mechanisms of liver injury in patients with coronavirus disease 2019. 1: Severe acute respiratory syndrome coronavirus-2 may directly bind to angiotensin converting enzyme II positive cholangiocytes to dysregulate liver function; 2: Inflammatory cytokine storm leads to persistent activation of lymphocytes and macrophages that secrete huge amount of inflammatory cytokine, thus contributing to lung as well as liver damage; 3: Drugs including antipyretics, antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids may have potential hepatotoxicity and lead to abnormal liver function; 4: Hepatic ischemia and hypoxia-reperfusion dysfunction induced by complications such as respiratory failure may cause liver damage, especially in critically ill patients. Virus-induced cytopathic effects It is well known that SARS-CoV-2 predominantly enters alveolar epithelial cells through the human ACE2 receptor, thus making the lung the main target organ of SARS-CoV-2 infection[62]. However, previous studies have found that ACE2 receptor is also specifically expressed in bile duct epithelial cells but is rarely expressed in hepatocytes[63,64] and absent of Kupffer cells and hepatic stellate cells[65]. A further study employing single-cell ribonucleic acid-seq suggested that TROP2+ cholangiocytes could be a main target for SARS-CoV-2 infection, leading to impaired liver regeneration and liver function[66]. In a mouse model of acute liver injury, ACE2 was upregulated in liver tissue due to compensatory proliferation of hepatocytes derived from bile duct epithelial cells[64]. During this compensatory process, some newborn hepatocytes still expressed ACE2 receptor and were susceptible to SARS-CoV-2. Recently, Wang et al[67] investigated the patterns of liver impairment by electron microscopy and pathological studies in two COVID-19 cases. In this study, typical coronavirus particles were identified in the cytoplasm of hepatocytes. Histologically, massive hepatic apoptosis and binuclear hepatocytes were observed. Our previous clinical report showed that the bile duct injury related ALP and gamma-glutamyl transpeptidase was elevated in deceased patients[30]. These findings suggest that the liver injury in COVID-19 patients may be due to hepatocyte damage as well as cholangiocyte dysfunction. Other studies reported conflicting results. For example, Qian et al[28] showed that ALP, gamma-glutamyl transpeptidase and TBIL elevations were rare among 324 cases with SARS-CoV-2 pneumonia. Zhang et al[47] reported that after SARS-CoV-2 infection, the overall ALP level is even lower than that with community-acquired pneumonia patients, implying that the duct epithelium injury by SARS-CoV-2 itself is very slight. Thus, SARS-COV-2 infection may not be the major reason related to liver injury. Given the conflicting results above, the role of virus-induced cytopathic effects in COVID-19-related liver injury warrants further investigation. Inflammatory cytokine storm Cell entry of SARS-CoV-2 depends on binding of the viral spike (S) proteins to cellular ACE2 receptor and on spike protein priming by host cell proteases[68]. While the virus enters the cells via fusion with the host membrane, its antigen will be recognized by the antigen presentation cells and then presented to cytotoxic and regulatory T lymphocytes, which initiate an antiviral immune response that includes inflammatory cytokine production and a weak interferon response[69]. In young individuals with an intact immune system, the virus is cleared away during the initial phase, so they show only mild symptoms[45]. However, in the elderly and individuals with underlying chronic diseases, the insufficient viral clearance due to altered immune response will lead to a cytokine storm, which may trigger a violent attack to the body and cause multiple organ failure including the liver[45,70]. Inflammatory cytokine storm is an overactive inflammatory response caused by virus infection, which leads to persistent activation of lymphocytes and macrophages that secrete huge amounts of inflammatory cytokines[71]. For example, SARS-CoV-2 can rapidly activate pathogenic Th1 cells to secrete proinflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor and IL-6[72]. Granulocyte-macrophage colony-stimulating factor further activates CD14+CD16+ inflammatory monocytes to produce large quantities of IL-6, tumor necrosis factor-α and other cytokines. Among these cytokines, IL-6 can bind to sIL-6R to activate STAT3 in nonimmune cells and can bind to membrane-bound IL-6 receptor to lead to pleiotropic effects on acquired and innate immune cells[73]. Meanwhile, sIL-2R may regulate cytotoxic T cells negatively and contribute to lymphopenia through IL-2 signaling inhibition[74]. Accumulating evidence revealed a broad spectrum of proinflammatory cytokines and chemokines dramatically increased in patients with liver dysfunction compared to those with normal liver function[10,32]. Consistent with these results, our data showed the levels of inflammatory markers including high sensitivity CRP, neutrophil-to-lymphocyte ratio, white blood cells, neutrophils, serum ferritin, lactate dehydrogenase, procalcitonin, erythrocyte sedimentation rate and proinflammatory cytokines including IL-2R, IL-6, tumor necrosis factor-α in the liver injury group were significantly higher compared with the group without liver injury[51]. In an analysis of 85 patients with COVID-19, lymphopenia and CRP may even serve as the risk factors related to hepatic injury[32]. Moreover, the postmortem liver biopsy in one patient confirmed that liver injury in COVID-19 is likely immune mediated[43]. These findings indicated that immune-mediated inflammatory response following SARS-CoV-2 infection may cause or contribute to liver damage. In the future, more research is needed to understand the concrete mechanisms involved in cytokine accumulation in COVID-19 and subsequent liver injury. Drug-induced liver injury Fever was one of the most common symptoms on admission and during hospitalization in patients with COVID-19[25]. Therefore, antipyretic therapy is very ordinary in infected patients. Acetaminophen, a common ingredient in antipyretic drugs, is proven to cause significant liver damage or induce liver failure according to a dose-dependent mechanism[75]. Recently, a 27-year-old healthy African American female with a positive SARS-CoV-2 test and acute liver failure secondary to acetaminophen overdose was reported[76]. She had a remote history of focal segmental glomerular sclerosis. To manage her pain, she ingested > 50 tablets of acetaminophen over the 3-4 d preceding presentation. Initial blood work revealed the acetaminophen level was 42 µg/mL (upper limit of normal 30 µg/mL) and elevated aminotransferases with alanine transaminase of 2791 U/L and aspartate transaminase of 3202 U/L. Two days after admission, her hepatic synthetic function worsened significantly, and aminotransferases peaked to an AST 9741 U/L and ALT 11322 U/L. Thus, although paracetamol is a safe and effective first line agent in almost all patients regardless of liver disease etiology[77], the clinicians cannot be too careful in the dose. Although there is currently no specific therapy for COVID-19, many patients especially severe and critical patients, were often treated with multiple drugs, including antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids in clinical practice[78]. These drugs may have potential hepatotoxicity and lead to abnormal liver function. Recent data on liver tests in patients with COVID-19 showed that the use of lopinavir/ritonavir led to increased odds of liver injury[27]. Fan et al[31] reported that among 148 COVID-19 patients, patients receiving treatment with lopinavir/ritonavir were more likely to develop abnormal liver function tests. In another clinical report, liver function injury was more likely to occur in patients who used many types of drugs and large amounts of hormones[34]. Our study also revealed a higher proportion of patients with liver injury had received systemic glucocorticoids (unpublished). The liver biopsy specimens of the patient with COVID-19 showed moderate microvascular steatosis and mild lobular and portal activity, further conforming the possibility of drug-induced hepatic damage[43]. But on the contrary, in a randomized, controlled, open-label trial, Cao et al[79] reported that lopinavir/ritonavir treatment did not significantly increase liver enzymes in patients with serious COVID-19. Due to limited data, we cannot draw a definitive conclusion about whether lopinavir/ritonavir or glucocorticoids increase the risk of developing liver damage. Pneumonia-associated hypoxia Hypoxic hepatitis, also known as ischemic hepatitis or shock liver, is commonly seen in patients with hypotension shock or severe hypoxemia caused by severe heart failure, respiratory failure, surgery, trauma and other causes[80]. Its clinical feature is a massive, rapid rise in serum transaminase (which can exceed 20 × ULN) and is often accompanied by an increase in lactate dehydrogenase. In patients with COVID-19, hypoxia and shock caused by respiratory distress syndrome, system inflammation reactive syndrome, multiple organ dysfunction and other complications can lead to hepatic ischemia and hypoxia-reperfusion dysfunction. Experimental data revealed that hepatocyte death and inflammatory cytokines production caused by hypoxia can be seen in both in vivo and in vitro models of hepatic ischemia and hypoxia[81]. Furthermore, liver histological findings on autopsy of patients with COVID-19 revealed the watery degeneration of some hepatocytes, proving the possibility of hepatic ischemia and hypoxia[27]. However, according to the available evidence, the distribution of aminotransferase levels among patients with COVID-19 do not support pneumonia-associated hypoxia being a common cause of liver injury[82]. Whether hypoxia is related to abnormal liver function in COVID-19 patients remains to be further investigated. Management of COVID-19 patients with liver disease It was reported that about 2%-11% of patients with COVID-19 had underlying chronic liver disease[83]. Chinese Society of Hepatology, Chinese Medical Association, American Association for the Study of Liver Diseases, Asian Pacific Association for the Study of the Liver and European Association for the Study of the Liver have all issued relevant guidelines to help clinicians manage chronic liver disease and liver transplant patients during the epidemic of COVID-19. Because there is no complete and systematic data at present, most of the recommendations for management of COVID-19 patients with liver disease are based on expert consensus. Among these guidelines, the basic principles are infection control, delay of medical treatment, risk classification and supportive management[84-87]. More details are summarized in Table 2. Table 2 Management of coronavirus disease 2019 patients with liver disease Management of COVID-19 patients with liver disease Out-patient care Use telemedicine or visits by phone wherever possible. Consider seeing in person only patients with urgent issues and clinically significant liver disease (e.g., jaundice, elevated ALT or AST > 500 U/L, or recent onset of hepatic decompensation)[40,84,86]. Seeing at the fever clinic[40] Hospital treatment Separate management from non-COVID-19 patients[40,85]. Monitor liver biochemistries regularly, particularly in patients treated with remdesivir or tocilizumab[40]. Avoid ultrasound or other advanced imaging unless it is likely to change management, for example, clinical suspicion for biliary obstruction or venous thrombosis[40]. Hospitalize COVID-19 patients with advanced liver disease as soon as possible[85] Patients with hepatitis B, hepatitis C Document discussion with patient regarding CLD diagnosis and management[84]. Delay starting DAA therapy until after their recovery from COVID-19 disease if there is no suspicion of advanced liver disease[87]. Continue treatment and provide 90-d supplies for HBV oral antiviral drugs or a full course of DAA medications to complete HCV treatment[87] Patients with autoimmune liver disease Continue immunosuppressive therapy in stable patients with AIH[87]. Lower the doses of azathioprine or mycophenolate mofetil when patients develop lymphopenia[87]. Avoid liver biopsy and start empiric therapy in new patients presenting with features of AIH[87]. Avoid high doses of prednisone in AIH patients on corticosteroids[87] Patients with HCC Continue HCC surveillance schedule for high-risk subjects[40]. Document discussion of risks and benefits of delaying surveillance with patient[40]. Proceed with HCC treatments as appropriate[40]. Postpone elective transplant and resection surgery, withhold immunotherapy[84] Pretransplant and post-transplant patients Have low threshold for admitting patients on transplant waiting list diagnosed with COVID-19[40,84]. Consider reduction of immunosuppression therapy as appropriate for posttransplant patients with moderate COVID-19[40,84]. Avoid reductions in immunosuppressive therapy in patients with mild COVID-19 disease[40,84] AIH: Autoimmune hepatitis; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CLD: Chronic liver disease; COVID-19: Coronavirus disease 2019; DAA: Direct acting antiviral; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus. CONCLUSION Liver injury is a common complication in COVID-19 patients and may result from virus-induced cytopathic effects, immune mediated inflammation, drug toxicity and pneumonia-associated hypoxia. Like SARS and MERS, abnormal liver function mainly manifested as transient elevation of serum aminotransferases. Moreover, patients with abnormal liver tests had higher risks of progressing to severe disease. From a clinical perspective, in addition to actively dealing with the primary disease caused by SARS-CoV-2 infection, a close monitor of liver function is recommended in COVID-19 patients, especially in severe/critical individuals. Conflict-of-interest statement: There is no conflict of interest. Manuscript source: Unsolicited manuscript Peer-review started: September 19, 2020 First decision: November 26, 2020 Article in press: December 23, 2020 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Iorio R, Kharbanda KK S-Editor: Zhang L L-Editor: Filipodia P-Editor: Li X	ACETAMINOPHEN	DrugsGivenReaction	CC BY-NC	33553391	19,036,556	2021-01-26
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Overdose'.	Clinical features and potential mechanism of coronavirus disease 2019-associated liver injury. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has posed a serious threat to global public health security. With the increase in the number of confirmed cases globally, the World Health Organization has declared the outbreak of COVID-19 an international public health emergency. Despite atypical pneumonia as the primary symptom, liver dysfunction has also been observed in many clinical cases and is associated with the mortality risk in patients with COVID-19, like severe acute respiratory syndrome and Middle East respiratory syndrome. Here we will provide a schematic overview of the clinical characteristics and the possible mechanisms of liver injury caused by severe acute respiratory syndrome coronavirus 2 infection, which may provide help for optimizing the management of liver injury and reducing mortality in COVID-19 patients. Core Tip: With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with coronavirus disease 2019 (COVID-19). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases. Underlying mechanisms may be viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia. A close monitor of liver function is recommended in COVID-19 patients, especially in critical individuals. INTRODUCTION Since the 21st century, the outbreak of coronaviruses has brought great harm to human society; the most serious of which are the severe acute respiratory syndrome (SARS) in 2003, the Middle East respiratory syndrome (MERS) in 2012 and the novel coronavirus disease in 2019 (COVID-19). The ongoing outbreak of COVID-19 has become a pandemic. As of December 9, 2020, the total number of diagnosed cases globally exceeded 67530912 with a total of more than 1545140 infection-related deaths, carrying a mortality of approximately 2%[1]. Up to now, no specific antiviral therapies have been identified. Thus, an early monitor of critical complications is vital in preventing disease progression and improving survival. With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with COVID-19, making this organ one of the most frequently damaged outside of the respiratory system (summarized in Table 1). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases[2]. However, due to the different design and sample size, the incidence and clinical manifestations of liver injury in these studies are not the same. The mechanism of hepatic damage caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still unclear. Table 1 Main characteristics related to liver injury in patients with coronavirus disease 2019 based on a series of case reports Ref. Sample size Liver injury Elevated ALT Elevated AST Elevated TBIL Elevated ALP Elevated GGT Factors related to liver injury [25] 1099 NA 21.3%: Severe 28.1%, Non-severe 19.8% 22.2%: Severe 39.4%, Non-severe 18.2% 10.5%: Severe 13.3%, Non-severe 9.9% NA NA NA [26] 548 NA 23.1%: Severe 24.1%, Non-severe 22.3% 33.1%: Severe 43.4%, Non-severe 23.3% 4.4%: Severe 6.4%, Non-severe 2.3% NA NA NA [27] 417 21.5% 41.2%: Severe 82.4%, Non-severe 50.2% 47.2%: Severe 75.3%, Non-severe 36.9% 64.2%: Severe 75.3%, Non-severe 60.1% 10.9%: Severe 12.2%, Non-severe 10.5% 48.5%: Severe 75.3%, Non-severe 39.1% Older, male, higher BMI, Underlying liver diseases (NAFLD, alcoholic liver disease and chronic hepatitis B), drugs (lopinavir/ritonavir) [28] 324 NA 15.7% 10.5% 6.5% 1.2% 0.9% NA [29] 298 14.8% NA NA NA NA NA NA [30] 274 NA 22.0%: Deceased 27.0%, Recovered 19.0% 31.0%: Deceased 52.0%, Recovered 16.0% NA NA NA NA [31] 148 37.2% 18.2% 21.6% 6.1% 4.1% 17.6% Male, higher levels of procalcitonin and CRP. PCT, LDH, received lopinavir / ritonavir [32] 85 38.8% 61.2% NA NA NA NA Older, lactic acid, myoglobin, neutrophils, critical illness, aCRP, alymphocyte count [33] 79 36.7% 31.6% 35.4% 5.1% NA NA Male, white blood cell counts, neutrophils, CRP, athe extent of pulmonary alesions on CT [34] 40 55% 52.5% 40% 25% NA NA Many types of drugs, large amounts of hormones, underlying diseases, lymphocyte count, acritical illness [35] 82 78% 30.6% 61.1% 30.6% NA NA NA a Represents independent risk factors for liver injury in coronavirus disease 2019. ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body mass index; COVID-19: Coronavirus disease 2019; CRP: C-reactive protein; CT: Computed tomography; GGT: Gamma-glutamyl transpeptidase; LDH: Lactate dehydrogenase; NA: Not available; NAFLD: Non-alcoholic fatty liver diseases; PCT: Procalcitonin; TBIL: Total bilirubin. SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are the causative agents of SARS, MERS and COVID-19, respectively, and they all belong to the highly pathogenic human beta coronaviruses[3]. Genomics analyses have found that SARS-CoV-2 shares 79.5% genome sequence similarity to SARS-CoV and 50% genome sequence homology to MERS-CoV[4]. SARS-CoV-2 uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV[5]. These common points hint that SARS-CoV-2 may partly mimic SARS-CoV and MERS-CoV infection. In this review, we summarized the characteristics and mechanism of liver injury caused by SARS-CoV-2 infection to provide a reference for further study. Liver injury in SARS and MERS Liver injury is not uncommon in patients infected with SARS-CoV and MERS-CoV according to previous studies[6]. In patients with SARS, liver injury mainly manifests as elevated alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in the early phase of the disease and is associated with the severity of the illness[7-13]. The pathogenesis of hepatic damage caused by SARS-CoV appears to be multifactorial, including a direct injury to the target cells by the virus and an indirect injury mediated by subsequent immune system dysfunction. As the functional receptor for SARS-CoV, ACE2 is abundantly expressed on endothelial cells of the liver, implying that SARS-CoV may directly bind to ACE2 positive cells to dysregulate liver function[5,13]. Liver biopsies in SARS patients demonstrated localization of virus in liver and hepatocyte apoptosis, which further confirmed the direct injury by SARS-CoV[14]. In an analysis of 145 cases of SARS, serum interleukin (IL)-1β, IL-6 and IL-10 were higher in patients with elevated serum levels of ALT than those in ALT normal group, indicating that liver damage was part of the manifestation of system inflammation reactive syndrome induced by SARS-CoV infection[10]. In addition, some studies have also found that hypoxemia and medication are closely related to abnormal liver function[15,16]. Elevated liver enzymes and bilirubin levels, as well as decreased albumin levels were highlighted during the hospital course of MERS-CoV infection in a series of case reports[17-20]. Different from SARS-CoV, MERS-CoV uses dipeptide base peptidase 4 as a cellular receptor to infect cells[21]. In humans, dipeptide base peptidase 4 is expressed constitutively on epithelial cells of liver[22], suggesting a direct hepatic damage caused by MERS-CoV. MERS also involves a mechanism of the upregulation of proinflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, IL-15 and IL-17[23]. However, studies on the relationship between cytokine storm and liver injury are scarce so far. Clinical characteristics of liver injury in patients with COVID-19 Since Chen et al[24] reported that 43 cases of 99 patients (43.4%) in Wuhan Jinyintan Hospital had different degrees of abnormal liver function, the abnormality of liver function test in patients with COVID-19 has aroused widespread concern among clinicians. As shown in Table 1, the incidence of liver injury ranged from 14.8% to 55.0% in recent case studies reporting clinical features of patients with COVID-19[25-34]. In death cases of COVID-19, the rate reached as high as 78.0%[35]. Liver injury presented in 30 out of 113 deceased patients from our previous report[30]. Abnormal liver function mainly manifests as slightly elevated ALT/AST and bilirubin levels, which usually occurs around the second week of the disease course[33-35]. Rarely, severe acute hepatitis associated with COVID-19 has been reported[36]. Contradictory to other hepatitis-induced liver injury, AST-dominant aminotrans-ferase elevation is common in COVID-19, which may provide a clue about the underlying pathophysiology of the impact of COVID-19 on liver. A retrospective study including 60 patients revealed that median AST was higher than ALT at admission (46 U/L vs 30 U/L) and during the hospital course[37]. In a multicenter retrospective cohort-derived data set of 5771 individuals, the elevation of serum AST level was earlier, more frequent and significant than the increase of ALT in severe patients, and AST levels had the highest correlation with mortality when compared with other indicators reflecting liver injury including elevated ALT, alkaline phosphatase (ALP) and total bilirubin (TBIL) levels in patients with COVID-19[38]. Likewise, an elevated baseline AST level has been shown to correlate with intensive care unit (ICU) admission, intubation and death in another study[39]. To our knowledge, three possible reasons may account for this phenomenon. First, given that AST is also distributed in myocardium and skeletal muscle, the American Association for the Study of Liver Diseases has recommended consideration of myositis or cardiac injury as contributors to the AST elevation[40]. Second, recent data identified ribosomal proteins as important host-dependency factors for SARS-CoV-2[41]. Therefore, the virus may directly cause hepatic mitochondrial injury and subsequent AST elevation. Third, AST-predominant aminotransferase elevations have been reported in alcohol-related liver disease, ischemia and cirrhosis. It is possible that hypoxia as well as metabolic changes such as hepatic steatosis may account for AST elevation in COVID-19 patients[42,43]. It is worth noting that liver dysfunction is closely related to the severity of the disease. On the one hand, severe patients have a higher proportion of liver injury: Guan et al[25] extracted a cohort regarding 1099 patients with laboratory-confirmed COVID-19 from 552 hospitals in mainland China. The results showed more patients with severe disease had elevated AST and ALT than those with non-severe disease. Like the result, Wang et al[44] showed that more patients admitted to the ICU had elevated AST levels. Huang et al[45] showed that patients admitted to the ICU had significantly higher ALT levels. On the other hand, patients with abnormal liver tests had higher risks of progressing to a severe disease course: Bloom et al[37] showed that admission AST, peak AST and peak ALT were higher in intubated patients. Of 417 patients with COVID-19, patients with abnormal liver tests of hepatocellular, cholestatic or mixed type at admission had higher odds of progressing to severe pneumonia[27]. Among 148 confirmed SARS-CoV-2-infected patients, the emerging abnormal liver functions after admission caused a prolonged length of stay[31]. In a large United States COVID-19 cohort of 3381 patients, 2273 patients who tested positive for SARS-CoV-2 had higher initial and peak ALT than those who tested negative[46]. Compared with mild [upper limit of normal (ULN) < ALT < two times ULN] and moderate (two times ULN < ALT < five times ULN) liver injury, patients with severe liver injury (ALT > five times ULN) had a more severe clinical course, including higher rates of ICU admission (69%), intubation (65%), renal replacement therapy (33%) and mortality (42%). Other hepatic manifestations in COVID-19 patients were hypoproteinemia and changes in coagulation[47,48]. A large cohort study including 2623 patients reported marked hypoalbuminemia in the critically ill and death groups than non-critically ill patients (38.2%, 71.2% and 82.4% on admission and 45.9%, 77.7% and 95.6% during hospitalization, respectively)[49]. Meanwhile, the patients in this study displayed dramatically prolonged activated partial thromboplastin time in critically ill patients reflected coagulopathy. Further analysis shows that risk factors associated with hepatic damage include older males, a longer time from illness onset to admission, a history of drinking, higher serum levels of C-reactive protein (CRP), white blood cell counts, neutrophils and medication (lopinavir/ritonavir, hormones)[27,31-34,50]. Disease severity (severe/critical), CRP, lymphocyte count and the extent of pulmonary lesions on computed tomography are independent risk factors for liver injury[32-34]. Accumulating data reveals that patients with pre-existing liver diseases are more susceptible to SARS-CoV-2 infection and have poorer prognosis. In our study, viral hepatitis (hepatitis B and hepatitis C) was much more frequent among patients with liver injury than those without[51]. Another study revealed that COVID-19 patients with non-alcoholic fatty liver disease had a significantly higher likelihood of abnormal liver function from admission to discharge when compared to those with non-alcoholic fatty liver disease subjects[52]. Qiu et al[53] first reported a case of acute-on-chronic liver failure due to SARS-COV-2 infection in a patient with decompensated alcoholic cirrhosis. In a multicenter retrospective study, fifty cirrhotic patients with SARS-CoV-2 infection were studied to evaluate the impact of COVID-19 on the clinical outcome[54]. The results showed 30-d mortality rate was higher in cirrhotic patients with COVID-19 than in cirrhotic patients with bacterial infection and in COVID-19 patients without cirrhosis, indicating that COVID-19 was associated with liver function deterioration and elevated mortality in cirrhotic patients. In addition, a study of 2780 COVID-19-positive patients found that those with cirrhosis were at a particularly increased risk for mortality by analyzing a large United States database (risk ratio, 4.6; 95% confidence interval, 2.6-8.3)[55]. Whether the COVID-19 patients with more severe liver injury were positive for hepatotrophis viruses is still controversial. In a retrospective study, the authors analyzed liver function parameters including ALT, AST and TBIL in COVID-19 patients with or without HBV infection and found no significant differences between the two groups[56]. Another study reached a similar conclusion and further proved the longitudinal changes of median values for liver biochemistries were not significantly different between the two groups either[57]. These findings indicated that SARS-CoV-2 will not exacerbate liver injury in patients with HBV co-infection. However, Lin et al[58] drew a completely opposite conclusion. In their cohort, COVID-19 cases with HBV coinfection had higher levels of ALT, AST, TBIL and ALP than the COVID-19 cases without HBV coinfection, showing that inactive HBV carriers with SARS-CoV-2 coinfection are at risk of greater liver injury. Moreover, SARS-CoV-2 was reported to induce HBV reactivation, which may cause severe liver injury in patients with coinfection[57,59]. In addition, a case of COVID-19 with Epstein-Barr virus coinfection was reported recently. On admission, he showed acute liver injury with liver enzymes that were much higher than typically seen solely with COVID-19 infection[60]. Although the evidence is limited, more attention should be paid to COVID-19 patients with other viral coinfections during clinical treatment. Mechanisms of liver injury in patients with COVID-19 Underlying mechanisms involved in liver injury in patients with COVID-19 are complex and interactive, which might include viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia[61] (Figure 1). Figure 1 Potential mechanisms of liver injury in patients with coronavirus disease 2019. 1: Severe acute respiratory syndrome coronavirus-2 may directly bind to angiotensin converting enzyme II positive cholangiocytes to dysregulate liver function; 2: Inflammatory cytokine storm leads to persistent activation of lymphocytes and macrophages that secrete huge amount of inflammatory cytokine, thus contributing to lung as well as liver damage; 3: Drugs including antipyretics, antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids may have potential hepatotoxicity and lead to abnormal liver function; 4: Hepatic ischemia and hypoxia-reperfusion dysfunction induced by complications such as respiratory failure may cause liver damage, especially in critically ill patients. Virus-induced cytopathic effects It is well known that SARS-CoV-2 predominantly enters alveolar epithelial cells through the human ACE2 receptor, thus making the lung the main target organ of SARS-CoV-2 infection[62]. However, previous studies have found that ACE2 receptor is also specifically expressed in bile duct epithelial cells but is rarely expressed in hepatocytes[63,64] and absent of Kupffer cells and hepatic stellate cells[65]. A further study employing single-cell ribonucleic acid-seq suggested that TROP2+ cholangiocytes could be a main target for SARS-CoV-2 infection, leading to impaired liver regeneration and liver function[66]. In a mouse model of acute liver injury, ACE2 was upregulated in liver tissue due to compensatory proliferation of hepatocytes derived from bile duct epithelial cells[64]. During this compensatory process, some newborn hepatocytes still expressed ACE2 receptor and were susceptible to SARS-CoV-2. Recently, Wang et al[67] investigated the patterns of liver impairment by electron microscopy and pathological studies in two COVID-19 cases. In this study, typical coronavirus particles were identified in the cytoplasm of hepatocytes. Histologically, massive hepatic apoptosis and binuclear hepatocytes were observed. Our previous clinical report showed that the bile duct injury related ALP and gamma-glutamyl transpeptidase was elevated in deceased patients[30]. These findings suggest that the liver injury in COVID-19 patients may be due to hepatocyte damage as well as cholangiocyte dysfunction. Other studies reported conflicting results. For example, Qian et al[28] showed that ALP, gamma-glutamyl transpeptidase and TBIL elevations were rare among 324 cases with SARS-CoV-2 pneumonia. Zhang et al[47] reported that after SARS-CoV-2 infection, the overall ALP level is even lower than that with community-acquired pneumonia patients, implying that the duct epithelium injury by SARS-CoV-2 itself is very slight. Thus, SARS-COV-2 infection may not be the major reason related to liver injury. Given the conflicting results above, the role of virus-induced cytopathic effects in COVID-19-related liver injury warrants further investigation. Inflammatory cytokine storm Cell entry of SARS-CoV-2 depends on binding of the viral spike (S) proteins to cellular ACE2 receptor and on spike protein priming by host cell proteases[68]. While the virus enters the cells via fusion with the host membrane, its antigen will be recognized by the antigen presentation cells and then presented to cytotoxic and regulatory T lymphocytes, which initiate an antiviral immune response that includes inflammatory cytokine production and a weak interferon response[69]. In young individuals with an intact immune system, the virus is cleared away during the initial phase, so they show only mild symptoms[45]. However, in the elderly and individuals with underlying chronic diseases, the insufficient viral clearance due to altered immune response will lead to a cytokine storm, which may trigger a violent attack to the body and cause multiple organ failure including the liver[45,70]. Inflammatory cytokine storm is an overactive inflammatory response caused by virus infection, which leads to persistent activation of lymphocytes and macrophages that secrete huge amounts of inflammatory cytokines[71]. For example, SARS-CoV-2 can rapidly activate pathogenic Th1 cells to secrete proinflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor and IL-6[72]. Granulocyte-macrophage colony-stimulating factor further activates CD14+CD16+ inflammatory monocytes to produce large quantities of IL-6, tumor necrosis factor-α and other cytokines. Among these cytokines, IL-6 can bind to sIL-6R to activate STAT3 in nonimmune cells and can bind to membrane-bound IL-6 receptor to lead to pleiotropic effects on acquired and innate immune cells[73]. Meanwhile, sIL-2R may regulate cytotoxic T cells negatively and contribute to lymphopenia through IL-2 signaling inhibition[74]. Accumulating evidence revealed a broad spectrum of proinflammatory cytokines and chemokines dramatically increased in patients with liver dysfunction compared to those with normal liver function[10,32]. Consistent with these results, our data showed the levels of inflammatory markers including high sensitivity CRP, neutrophil-to-lymphocyte ratio, white blood cells, neutrophils, serum ferritin, lactate dehydrogenase, procalcitonin, erythrocyte sedimentation rate and proinflammatory cytokines including IL-2R, IL-6, tumor necrosis factor-α in the liver injury group were significantly higher compared with the group without liver injury[51]. In an analysis of 85 patients with COVID-19, lymphopenia and CRP may even serve as the risk factors related to hepatic injury[32]. Moreover, the postmortem liver biopsy in one patient confirmed that liver injury in COVID-19 is likely immune mediated[43]. These findings indicated that immune-mediated inflammatory response following SARS-CoV-2 infection may cause or contribute to liver damage. In the future, more research is needed to understand the concrete mechanisms involved in cytokine accumulation in COVID-19 and subsequent liver injury. Drug-induced liver injury Fever was one of the most common symptoms on admission and during hospitalization in patients with COVID-19[25]. Therefore, antipyretic therapy is very ordinary in infected patients. Acetaminophen, a common ingredient in antipyretic drugs, is proven to cause significant liver damage or induce liver failure according to a dose-dependent mechanism[75]. Recently, a 27-year-old healthy African American female with a positive SARS-CoV-2 test and acute liver failure secondary to acetaminophen overdose was reported[76]. She had a remote history of focal segmental glomerular sclerosis. To manage her pain, she ingested > 50 tablets of acetaminophen over the 3-4 d preceding presentation. Initial blood work revealed the acetaminophen level was 42 µg/mL (upper limit of normal 30 µg/mL) and elevated aminotransferases with alanine transaminase of 2791 U/L and aspartate transaminase of 3202 U/L. Two days after admission, her hepatic synthetic function worsened significantly, and aminotransferases peaked to an AST 9741 U/L and ALT 11322 U/L. Thus, although paracetamol is a safe and effective first line agent in almost all patients regardless of liver disease etiology[77], the clinicians cannot be too careful in the dose. Although there is currently no specific therapy for COVID-19, many patients especially severe and critical patients, were often treated with multiple drugs, including antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids in clinical practice[78]. These drugs may have potential hepatotoxicity and lead to abnormal liver function. Recent data on liver tests in patients with COVID-19 showed that the use of lopinavir/ritonavir led to increased odds of liver injury[27]. Fan et al[31] reported that among 148 COVID-19 patients, patients receiving treatment with lopinavir/ritonavir were more likely to develop abnormal liver function tests. In another clinical report, liver function injury was more likely to occur in patients who used many types of drugs and large amounts of hormones[34]. Our study also revealed a higher proportion of patients with liver injury had received systemic glucocorticoids (unpublished). The liver biopsy specimens of the patient with COVID-19 showed moderate microvascular steatosis and mild lobular and portal activity, further conforming the possibility of drug-induced hepatic damage[43]. But on the contrary, in a randomized, controlled, open-label trial, Cao et al[79] reported that lopinavir/ritonavir treatment did not significantly increase liver enzymes in patients with serious COVID-19. Due to limited data, we cannot draw a definitive conclusion about whether lopinavir/ritonavir or glucocorticoids increase the risk of developing liver damage. Pneumonia-associated hypoxia Hypoxic hepatitis, also known as ischemic hepatitis or shock liver, is commonly seen in patients with hypotension shock or severe hypoxemia caused by severe heart failure, respiratory failure, surgery, trauma and other causes[80]. Its clinical feature is a massive, rapid rise in serum transaminase (which can exceed 20 × ULN) and is often accompanied by an increase in lactate dehydrogenase. In patients with COVID-19, hypoxia and shock caused by respiratory distress syndrome, system inflammation reactive syndrome, multiple organ dysfunction and other complications can lead to hepatic ischemia and hypoxia-reperfusion dysfunction. Experimental data revealed that hepatocyte death and inflammatory cytokines production caused by hypoxia can be seen in both in vivo and in vitro models of hepatic ischemia and hypoxia[81]. Furthermore, liver histological findings on autopsy of patients with COVID-19 revealed the watery degeneration of some hepatocytes, proving the possibility of hepatic ischemia and hypoxia[27]. However, according to the available evidence, the distribution of aminotransferase levels among patients with COVID-19 do not support pneumonia-associated hypoxia being a common cause of liver injury[82]. Whether hypoxia is related to abnormal liver function in COVID-19 patients remains to be further investigated. Management of COVID-19 patients with liver disease It was reported that about 2%-11% of patients with COVID-19 had underlying chronic liver disease[83]. Chinese Society of Hepatology, Chinese Medical Association, American Association for the Study of Liver Diseases, Asian Pacific Association for the Study of the Liver and European Association for the Study of the Liver have all issued relevant guidelines to help clinicians manage chronic liver disease and liver transplant patients during the epidemic of COVID-19. Because there is no complete and systematic data at present, most of the recommendations for management of COVID-19 patients with liver disease are based on expert consensus. Among these guidelines, the basic principles are infection control, delay of medical treatment, risk classification and supportive management[84-87]. More details are summarized in Table 2. Table 2 Management of coronavirus disease 2019 patients with liver disease Management of COVID-19 patients with liver disease Out-patient care Use telemedicine or visits by phone wherever possible. Consider seeing in person only patients with urgent issues and clinically significant liver disease (e.g., jaundice, elevated ALT or AST > 500 U/L, or recent onset of hepatic decompensation)[40,84,86]. Seeing at the fever clinic[40] Hospital treatment Separate management from non-COVID-19 patients[40,85]. Monitor liver biochemistries regularly, particularly in patients treated with remdesivir or tocilizumab[40]. Avoid ultrasound or other advanced imaging unless it is likely to change management, for example, clinical suspicion for biliary obstruction or venous thrombosis[40]. Hospitalize COVID-19 patients with advanced liver disease as soon as possible[85] Patients with hepatitis B, hepatitis C Document discussion with patient regarding CLD diagnosis and management[84]. Delay starting DAA therapy until after their recovery from COVID-19 disease if there is no suspicion of advanced liver disease[87]. Continue treatment and provide 90-d supplies for HBV oral antiviral drugs or a full course of DAA medications to complete HCV treatment[87] Patients with autoimmune liver disease Continue immunosuppressive therapy in stable patients with AIH[87]. Lower the doses of azathioprine or mycophenolate mofetil when patients develop lymphopenia[87]. Avoid liver biopsy and start empiric therapy in new patients presenting with features of AIH[87]. Avoid high doses of prednisone in AIH patients on corticosteroids[87] Patients with HCC Continue HCC surveillance schedule for high-risk subjects[40]. Document discussion of risks and benefits of delaying surveillance with patient[40]. Proceed with HCC treatments as appropriate[40]. Postpone elective transplant and resection surgery, withhold immunotherapy[84] Pretransplant and post-transplant patients Have low threshold for admitting patients on transplant waiting list diagnosed with COVID-19[40,84]. Consider reduction of immunosuppression therapy as appropriate for posttransplant patients with moderate COVID-19[40,84]. Avoid reductions in immunosuppressive therapy in patients with mild COVID-19 disease[40,84] AIH: Autoimmune hepatitis; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CLD: Chronic liver disease; COVID-19: Coronavirus disease 2019; DAA: Direct acting antiviral; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus. CONCLUSION Liver injury is a common complication in COVID-19 patients and may result from virus-induced cytopathic effects, immune mediated inflammation, drug toxicity and pneumonia-associated hypoxia. Like SARS and MERS, abnormal liver function mainly manifested as transient elevation of serum aminotransferases. Moreover, patients with abnormal liver tests had higher risks of progressing to severe disease. From a clinical perspective, in addition to actively dealing with the primary disease caused by SARS-CoV-2 infection, a close monitor of liver function is recommended in COVID-19 patients, especially in severe/critical individuals. Conflict-of-interest statement: There is no conflict of interest. Manuscript source: Unsolicited manuscript Peer-review started: September 19, 2020 First decision: November 26, 2020 Article in press: December 23, 2020 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Iorio R, Kharbanda KK S-Editor: Zhang L L-Editor: Filipodia P-Editor: Li X	ACETAMINOPHEN	DrugsGivenReaction	CC BY-NC	33553391	19,036,556	2021-01-26
What was the administration route of drug 'ACETAMINOPHEN'?	Clinical features and potential mechanism of coronavirus disease 2019-associated liver injury. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has posed a serious threat to global public health security. With the increase in the number of confirmed cases globally, the World Health Organization has declared the outbreak of COVID-19 an international public health emergency. Despite atypical pneumonia as the primary symptom, liver dysfunction has also been observed in many clinical cases and is associated with the mortality risk in patients with COVID-19, like severe acute respiratory syndrome and Middle East respiratory syndrome. Here we will provide a schematic overview of the clinical characteristics and the possible mechanisms of liver injury caused by severe acute respiratory syndrome coronavirus 2 infection, which may provide help for optimizing the management of liver injury and reducing mortality in COVID-19 patients. Core Tip: With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with coronavirus disease 2019 (COVID-19). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases. Underlying mechanisms may be viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia. A close monitor of liver function is recommended in COVID-19 patients, especially in critical individuals. INTRODUCTION Since the 21st century, the outbreak of coronaviruses has brought great harm to human society; the most serious of which are the severe acute respiratory syndrome (SARS) in 2003, the Middle East respiratory syndrome (MERS) in 2012 and the novel coronavirus disease in 2019 (COVID-19). The ongoing outbreak of COVID-19 has become a pandemic. As of December 9, 2020, the total number of diagnosed cases globally exceeded 67530912 with a total of more than 1545140 infection-related deaths, carrying a mortality of approximately 2%[1]. Up to now, no specific antiviral therapies have been identified. Thus, an early monitor of critical complications is vital in preventing disease progression and improving survival. With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with COVID-19, making this organ one of the most frequently damaged outside of the respiratory system (summarized in Table 1). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases[2]. However, due to the different design and sample size, the incidence and clinical manifestations of liver injury in these studies are not the same. The mechanism of hepatic damage caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still unclear. Table 1 Main characteristics related to liver injury in patients with coronavirus disease 2019 based on a series of case reports Ref. Sample size Liver injury Elevated ALT Elevated AST Elevated TBIL Elevated ALP Elevated GGT Factors related to liver injury [25] 1099 NA 21.3%: Severe 28.1%, Non-severe 19.8% 22.2%: Severe 39.4%, Non-severe 18.2% 10.5%: Severe 13.3%, Non-severe 9.9% NA NA NA [26] 548 NA 23.1%: Severe 24.1%, Non-severe 22.3% 33.1%: Severe 43.4%, Non-severe 23.3% 4.4%: Severe 6.4%, Non-severe 2.3% NA NA NA [27] 417 21.5% 41.2%: Severe 82.4%, Non-severe 50.2% 47.2%: Severe 75.3%, Non-severe 36.9% 64.2%: Severe 75.3%, Non-severe 60.1% 10.9%: Severe 12.2%, Non-severe 10.5% 48.5%: Severe 75.3%, Non-severe 39.1% Older, male, higher BMI, Underlying liver diseases (NAFLD, alcoholic liver disease and chronic hepatitis B), drugs (lopinavir/ritonavir) [28] 324 NA 15.7% 10.5% 6.5% 1.2% 0.9% NA [29] 298 14.8% NA NA NA NA NA NA [30] 274 NA 22.0%: Deceased 27.0%, Recovered 19.0% 31.0%: Deceased 52.0%, Recovered 16.0% NA NA NA NA [31] 148 37.2% 18.2% 21.6% 6.1% 4.1% 17.6% Male, higher levels of procalcitonin and CRP. PCT, LDH, received lopinavir / ritonavir [32] 85 38.8% 61.2% NA NA NA NA Older, lactic acid, myoglobin, neutrophils, critical illness, aCRP, alymphocyte count [33] 79 36.7% 31.6% 35.4% 5.1% NA NA Male, white blood cell counts, neutrophils, CRP, athe extent of pulmonary alesions on CT [34] 40 55% 52.5% 40% 25% NA NA Many types of drugs, large amounts of hormones, underlying diseases, lymphocyte count, acritical illness [35] 82 78% 30.6% 61.1% 30.6% NA NA NA a Represents independent risk factors for liver injury in coronavirus disease 2019. ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body mass index; COVID-19: Coronavirus disease 2019; CRP: C-reactive protein; CT: Computed tomography; GGT: Gamma-glutamyl transpeptidase; LDH: Lactate dehydrogenase; NA: Not available; NAFLD: Non-alcoholic fatty liver diseases; PCT: Procalcitonin; TBIL: Total bilirubin. SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are the causative agents of SARS, MERS and COVID-19, respectively, and they all belong to the highly pathogenic human beta coronaviruses[3]. Genomics analyses have found that SARS-CoV-2 shares 79.5% genome sequence similarity to SARS-CoV and 50% genome sequence homology to MERS-CoV[4]. SARS-CoV-2 uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV[5]. These common points hint that SARS-CoV-2 may partly mimic SARS-CoV and MERS-CoV infection. In this review, we summarized the characteristics and mechanism of liver injury caused by SARS-CoV-2 infection to provide a reference for further study. Liver injury in SARS and MERS Liver injury is not uncommon in patients infected with SARS-CoV and MERS-CoV according to previous studies[6]. In patients with SARS, liver injury mainly manifests as elevated alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in the early phase of the disease and is associated with the severity of the illness[7-13]. The pathogenesis of hepatic damage caused by SARS-CoV appears to be multifactorial, including a direct injury to the target cells by the virus and an indirect injury mediated by subsequent immune system dysfunction. As the functional receptor for SARS-CoV, ACE2 is abundantly expressed on endothelial cells of the liver, implying that SARS-CoV may directly bind to ACE2 positive cells to dysregulate liver function[5,13]. Liver biopsies in SARS patients demonstrated localization of virus in liver and hepatocyte apoptosis, which further confirmed the direct injury by SARS-CoV[14]. In an analysis of 145 cases of SARS, serum interleukin (IL)-1β, IL-6 and IL-10 were higher in patients with elevated serum levels of ALT than those in ALT normal group, indicating that liver damage was part of the manifestation of system inflammation reactive syndrome induced by SARS-CoV infection[10]. In addition, some studies have also found that hypoxemia and medication are closely related to abnormal liver function[15,16]. Elevated liver enzymes and bilirubin levels, as well as decreased albumin levels were highlighted during the hospital course of MERS-CoV infection in a series of case reports[17-20]. Different from SARS-CoV, MERS-CoV uses dipeptide base peptidase 4 as a cellular receptor to infect cells[21]. In humans, dipeptide base peptidase 4 is expressed constitutively on epithelial cells of liver[22], suggesting a direct hepatic damage caused by MERS-CoV. MERS also involves a mechanism of the upregulation of proinflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, IL-15 and IL-17[23]. However, studies on the relationship between cytokine storm and liver injury are scarce so far. Clinical characteristics of liver injury in patients with COVID-19 Since Chen et al[24] reported that 43 cases of 99 patients (43.4%) in Wuhan Jinyintan Hospital had different degrees of abnormal liver function, the abnormality of liver function test in patients with COVID-19 has aroused widespread concern among clinicians. As shown in Table 1, the incidence of liver injury ranged from 14.8% to 55.0% in recent case studies reporting clinical features of patients with COVID-19[25-34]. In death cases of COVID-19, the rate reached as high as 78.0%[35]. Liver injury presented in 30 out of 113 deceased patients from our previous report[30]. Abnormal liver function mainly manifests as slightly elevated ALT/AST and bilirubin levels, which usually occurs around the second week of the disease course[33-35]. Rarely, severe acute hepatitis associated with COVID-19 has been reported[36]. Contradictory to other hepatitis-induced liver injury, AST-dominant aminotrans-ferase elevation is common in COVID-19, which may provide a clue about the underlying pathophysiology of the impact of COVID-19 on liver. A retrospective study including 60 patients revealed that median AST was higher than ALT at admission (46 U/L vs 30 U/L) and during the hospital course[37]. In a multicenter retrospective cohort-derived data set of 5771 individuals, the elevation of serum AST level was earlier, more frequent and significant than the increase of ALT in severe patients, and AST levels had the highest correlation with mortality when compared with other indicators reflecting liver injury including elevated ALT, alkaline phosphatase (ALP) and total bilirubin (TBIL) levels in patients with COVID-19[38]. Likewise, an elevated baseline AST level has been shown to correlate with intensive care unit (ICU) admission, intubation and death in another study[39]. To our knowledge, three possible reasons may account for this phenomenon. First, given that AST is also distributed in myocardium and skeletal muscle, the American Association for the Study of Liver Diseases has recommended consideration of myositis or cardiac injury as contributors to the AST elevation[40]. Second, recent data identified ribosomal proteins as important host-dependency factors for SARS-CoV-2[41]. Therefore, the virus may directly cause hepatic mitochondrial injury and subsequent AST elevation. Third, AST-predominant aminotransferase elevations have been reported in alcohol-related liver disease, ischemia and cirrhosis. It is possible that hypoxia as well as metabolic changes such as hepatic steatosis may account for AST elevation in COVID-19 patients[42,43]. It is worth noting that liver dysfunction is closely related to the severity of the disease. On the one hand, severe patients have a higher proportion of liver injury: Guan et al[25] extracted a cohort regarding 1099 patients with laboratory-confirmed COVID-19 from 552 hospitals in mainland China. The results showed more patients with severe disease had elevated AST and ALT than those with non-severe disease. Like the result, Wang et al[44] showed that more patients admitted to the ICU had elevated AST levels. Huang et al[45] showed that patients admitted to the ICU had significantly higher ALT levels. On the other hand, patients with abnormal liver tests had higher risks of progressing to a severe disease course: Bloom et al[37] showed that admission AST, peak AST and peak ALT were higher in intubated patients. Of 417 patients with COVID-19, patients with abnormal liver tests of hepatocellular, cholestatic or mixed type at admission had higher odds of progressing to severe pneumonia[27]. Among 148 confirmed SARS-CoV-2-infected patients, the emerging abnormal liver functions after admission caused a prolonged length of stay[31]. In a large United States COVID-19 cohort of 3381 patients, 2273 patients who tested positive for SARS-CoV-2 had higher initial and peak ALT than those who tested negative[46]. Compared with mild [upper limit of normal (ULN) < ALT < two times ULN] and moderate (two times ULN < ALT < five times ULN) liver injury, patients with severe liver injury (ALT > five times ULN) had a more severe clinical course, including higher rates of ICU admission (69%), intubation (65%), renal replacement therapy (33%) and mortality (42%). Other hepatic manifestations in COVID-19 patients were hypoproteinemia and changes in coagulation[47,48]. A large cohort study including 2623 patients reported marked hypoalbuminemia in the critically ill and death groups than non-critically ill patients (38.2%, 71.2% and 82.4% on admission and 45.9%, 77.7% and 95.6% during hospitalization, respectively)[49]. Meanwhile, the patients in this study displayed dramatically prolonged activated partial thromboplastin time in critically ill patients reflected coagulopathy. Further analysis shows that risk factors associated with hepatic damage include older males, a longer time from illness onset to admission, a history of drinking, higher serum levels of C-reactive protein (CRP), white blood cell counts, neutrophils and medication (lopinavir/ritonavir, hormones)[27,31-34,50]. Disease severity (severe/critical), CRP, lymphocyte count and the extent of pulmonary lesions on computed tomography are independent risk factors for liver injury[32-34]. Accumulating data reveals that patients with pre-existing liver diseases are more susceptible to SARS-CoV-2 infection and have poorer prognosis. In our study, viral hepatitis (hepatitis B and hepatitis C) was much more frequent among patients with liver injury than those without[51]. Another study revealed that COVID-19 patients with non-alcoholic fatty liver disease had a significantly higher likelihood of abnormal liver function from admission to discharge when compared to those with non-alcoholic fatty liver disease subjects[52]. Qiu et al[53] first reported a case of acute-on-chronic liver failure due to SARS-COV-2 infection in a patient with decompensated alcoholic cirrhosis. In a multicenter retrospective study, fifty cirrhotic patients with SARS-CoV-2 infection were studied to evaluate the impact of COVID-19 on the clinical outcome[54]. The results showed 30-d mortality rate was higher in cirrhotic patients with COVID-19 than in cirrhotic patients with bacterial infection and in COVID-19 patients without cirrhosis, indicating that COVID-19 was associated with liver function deterioration and elevated mortality in cirrhotic patients. In addition, a study of 2780 COVID-19-positive patients found that those with cirrhosis were at a particularly increased risk for mortality by analyzing a large United States database (risk ratio, 4.6; 95% confidence interval, 2.6-8.3)[55]. Whether the COVID-19 patients with more severe liver injury were positive for hepatotrophis viruses is still controversial. In a retrospective study, the authors analyzed liver function parameters including ALT, AST and TBIL in COVID-19 patients with or without HBV infection and found no significant differences between the two groups[56]. Another study reached a similar conclusion and further proved the longitudinal changes of median values for liver biochemistries were not significantly different between the two groups either[57]. These findings indicated that SARS-CoV-2 will not exacerbate liver injury in patients with HBV co-infection. However, Lin et al[58] drew a completely opposite conclusion. In their cohort, COVID-19 cases with HBV coinfection had higher levels of ALT, AST, TBIL and ALP than the COVID-19 cases without HBV coinfection, showing that inactive HBV carriers with SARS-CoV-2 coinfection are at risk of greater liver injury. Moreover, SARS-CoV-2 was reported to induce HBV reactivation, which may cause severe liver injury in patients with coinfection[57,59]. In addition, a case of COVID-19 with Epstein-Barr virus coinfection was reported recently. On admission, he showed acute liver injury with liver enzymes that were much higher than typically seen solely with COVID-19 infection[60]. Although the evidence is limited, more attention should be paid to COVID-19 patients with other viral coinfections during clinical treatment. Mechanisms of liver injury in patients with COVID-19 Underlying mechanisms involved in liver injury in patients with COVID-19 are complex and interactive, which might include viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia[61] (Figure 1). Figure 1 Potential mechanisms of liver injury in patients with coronavirus disease 2019. 1: Severe acute respiratory syndrome coronavirus-2 may directly bind to angiotensin converting enzyme II positive cholangiocytes to dysregulate liver function; 2: Inflammatory cytokine storm leads to persistent activation of lymphocytes and macrophages that secrete huge amount of inflammatory cytokine, thus contributing to lung as well as liver damage; 3: Drugs including antipyretics, antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids may have potential hepatotoxicity and lead to abnormal liver function; 4: Hepatic ischemia and hypoxia-reperfusion dysfunction induced by complications such as respiratory failure may cause liver damage, especially in critically ill patients. Virus-induced cytopathic effects It is well known that SARS-CoV-2 predominantly enters alveolar epithelial cells through the human ACE2 receptor, thus making the lung the main target organ of SARS-CoV-2 infection[62]. However, previous studies have found that ACE2 receptor is also specifically expressed in bile duct epithelial cells but is rarely expressed in hepatocytes[63,64] and absent of Kupffer cells and hepatic stellate cells[65]. A further study employing single-cell ribonucleic acid-seq suggested that TROP2+ cholangiocytes could be a main target for SARS-CoV-2 infection, leading to impaired liver regeneration and liver function[66]. In a mouse model of acute liver injury, ACE2 was upregulated in liver tissue due to compensatory proliferation of hepatocytes derived from bile duct epithelial cells[64]. During this compensatory process, some newborn hepatocytes still expressed ACE2 receptor and were susceptible to SARS-CoV-2. Recently, Wang et al[67] investigated the patterns of liver impairment by electron microscopy and pathological studies in two COVID-19 cases. In this study, typical coronavirus particles were identified in the cytoplasm of hepatocytes. Histologically, massive hepatic apoptosis and binuclear hepatocytes were observed. Our previous clinical report showed that the bile duct injury related ALP and gamma-glutamyl transpeptidase was elevated in deceased patients[30]. These findings suggest that the liver injury in COVID-19 patients may be due to hepatocyte damage as well as cholangiocyte dysfunction. Other studies reported conflicting results. For example, Qian et al[28] showed that ALP, gamma-glutamyl transpeptidase and TBIL elevations were rare among 324 cases with SARS-CoV-2 pneumonia. Zhang et al[47] reported that after SARS-CoV-2 infection, the overall ALP level is even lower than that with community-acquired pneumonia patients, implying that the duct epithelium injury by SARS-CoV-2 itself is very slight. Thus, SARS-COV-2 infection may not be the major reason related to liver injury. Given the conflicting results above, the role of virus-induced cytopathic effects in COVID-19-related liver injury warrants further investigation. Inflammatory cytokine storm Cell entry of SARS-CoV-2 depends on binding of the viral spike (S) proteins to cellular ACE2 receptor and on spike protein priming by host cell proteases[68]. While the virus enters the cells via fusion with the host membrane, its antigen will be recognized by the antigen presentation cells and then presented to cytotoxic and regulatory T lymphocytes, which initiate an antiviral immune response that includes inflammatory cytokine production and a weak interferon response[69]. In young individuals with an intact immune system, the virus is cleared away during the initial phase, so they show only mild symptoms[45]. However, in the elderly and individuals with underlying chronic diseases, the insufficient viral clearance due to altered immune response will lead to a cytokine storm, which may trigger a violent attack to the body and cause multiple organ failure including the liver[45,70]. Inflammatory cytokine storm is an overactive inflammatory response caused by virus infection, which leads to persistent activation of lymphocytes and macrophages that secrete huge amounts of inflammatory cytokines[71]. For example, SARS-CoV-2 can rapidly activate pathogenic Th1 cells to secrete proinflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor and IL-6[72]. Granulocyte-macrophage colony-stimulating factor further activates CD14+CD16+ inflammatory monocytes to produce large quantities of IL-6, tumor necrosis factor-α and other cytokines. Among these cytokines, IL-6 can bind to sIL-6R to activate STAT3 in nonimmune cells and can bind to membrane-bound IL-6 receptor to lead to pleiotropic effects on acquired and innate immune cells[73]. Meanwhile, sIL-2R may regulate cytotoxic T cells negatively and contribute to lymphopenia through IL-2 signaling inhibition[74]. Accumulating evidence revealed a broad spectrum of proinflammatory cytokines and chemokines dramatically increased in patients with liver dysfunction compared to those with normal liver function[10,32]. Consistent with these results, our data showed the levels of inflammatory markers including high sensitivity CRP, neutrophil-to-lymphocyte ratio, white blood cells, neutrophils, serum ferritin, lactate dehydrogenase, procalcitonin, erythrocyte sedimentation rate and proinflammatory cytokines including IL-2R, IL-6, tumor necrosis factor-α in the liver injury group were significantly higher compared with the group without liver injury[51]. In an analysis of 85 patients with COVID-19, lymphopenia and CRP may even serve as the risk factors related to hepatic injury[32]. Moreover, the postmortem liver biopsy in one patient confirmed that liver injury in COVID-19 is likely immune mediated[43]. These findings indicated that immune-mediated inflammatory response following SARS-CoV-2 infection may cause or contribute to liver damage. In the future, more research is needed to understand the concrete mechanisms involved in cytokine accumulation in COVID-19 and subsequent liver injury. Drug-induced liver injury Fever was one of the most common symptoms on admission and during hospitalization in patients with COVID-19[25]. Therefore, antipyretic therapy is very ordinary in infected patients. Acetaminophen, a common ingredient in antipyretic drugs, is proven to cause significant liver damage or induce liver failure according to a dose-dependent mechanism[75]. Recently, a 27-year-old healthy African American female with a positive SARS-CoV-2 test and acute liver failure secondary to acetaminophen overdose was reported[76]. She had a remote history of focal segmental glomerular sclerosis. To manage her pain, she ingested > 50 tablets of acetaminophen over the 3-4 d preceding presentation. Initial blood work revealed the acetaminophen level was 42 µg/mL (upper limit of normal 30 µg/mL) and elevated aminotransferases with alanine transaminase of 2791 U/L and aspartate transaminase of 3202 U/L. Two days after admission, her hepatic synthetic function worsened significantly, and aminotransferases peaked to an AST 9741 U/L and ALT 11322 U/L. Thus, although paracetamol is a safe and effective first line agent in almost all patients regardless of liver disease etiology[77], the clinicians cannot be too careful in the dose. Although there is currently no specific therapy for COVID-19, many patients especially severe and critical patients, were often treated with multiple drugs, including antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids in clinical practice[78]. These drugs may have potential hepatotoxicity and lead to abnormal liver function. Recent data on liver tests in patients with COVID-19 showed that the use of lopinavir/ritonavir led to increased odds of liver injury[27]. Fan et al[31] reported that among 148 COVID-19 patients, patients receiving treatment with lopinavir/ritonavir were more likely to develop abnormal liver function tests. In another clinical report, liver function injury was more likely to occur in patients who used many types of drugs and large amounts of hormones[34]. Our study also revealed a higher proportion of patients with liver injury had received systemic glucocorticoids (unpublished). The liver biopsy specimens of the patient with COVID-19 showed moderate microvascular steatosis and mild lobular and portal activity, further conforming the possibility of drug-induced hepatic damage[43]. But on the contrary, in a randomized, controlled, open-label trial, Cao et al[79] reported that lopinavir/ritonavir treatment did not significantly increase liver enzymes in patients with serious COVID-19. Due to limited data, we cannot draw a definitive conclusion about whether lopinavir/ritonavir or glucocorticoids increase the risk of developing liver damage. Pneumonia-associated hypoxia Hypoxic hepatitis, also known as ischemic hepatitis or shock liver, is commonly seen in patients with hypotension shock or severe hypoxemia caused by severe heart failure, respiratory failure, surgery, trauma and other causes[80]. Its clinical feature is a massive, rapid rise in serum transaminase (which can exceed 20 × ULN) and is often accompanied by an increase in lactate dehydrogenase. In patients with COVID-19, hypoxia and shock caused by respiratory distress syndrome, system inflammation reactive syndrome, multiple organ dysfunction and other complications can lead to hepatic ischemia and hypoxia-reperfusion dysfunction. Experimental data revealed that hepatocyte death and inflammatory cytokines production caused by hypoxia can be seen in both in vivo and in vitro models of hepatic ischemia and hypoxia[81]. Furthermore, liver histological findings on autopsy of patients with COVID-19 revealed the watery degeneration of some hepatocytes, proving the possibility of hepatic ischemia and hypoxia[27]. However, according to the available evidence, the distribution of aminotransferase levels among patients with COVID-19 do not support pneumonia-associated hypoxia being a common cause of liver injury[82]. Whether hypoxia is related to abnormal liver function in COVID-19 patients remains to be further investigated. Management of COVID-19 patients with liver disease It was reported that about 2%-11% of patients with COVID-19 had underlying chronic liver disease[83]. Chinese Society of Hepatology, Chinese Medical Association, American Association for the Study of Liver Diseases, Asian Pacific Association for the Study of the Liver and European Association for the Study of the Liver have all issued relevant guidelines to help clinicians manage chronic liver disease and liver transplant patients during the epidemic of COVID-19. Because there is no complete and systematic data at present, most of the recommendations for management of COVID-19 patients with liver disease are based on expert consensus. Among these guidelines, the basic principles are infection control, delay of medical treatment, risk classification and supportive management[84-87]. More details are summarized in Table 2. Table 2 Management of coronavirus disease 2019 patients with liver disease Management of COVID-19 patients with liver disease Out-patient care Use telemedicine or visits by phone wherever possible. Consider seeing in person only patients with urgent issues and clinically significant liver disease (e.g., jaundice, elevated ALT or AST > 500 U/L, or recent onset of hepatic decompensation)[40,84,86]. Seeing at the fever clinic[40] Hospital treatment Separate management from non-COVID-19 patients[40,85]. Monitor liver biochemistries regularly, particularly in patients treated with remdesivir or tocilizumab[40]. Avoid ultrasound or other advanced imaging unless it is likely to change management, for example, clinical suspicion for biliary obstruction or venous thrombosis[40]. Hospitalize COVID-19 patients with advanced liver disease as soon as possible[85] Patients with hepatitis B, hepatitis C Document discussion with patient regarding CLD diagnosis and management[84]. Delay starting DAA therapy until after their recovery from COVID-19 disease if there is no suspicion of advanced liver disease[87]. Continue treatment and provide 90-d supplies for HBV oral antiviral drugs or a full course of DAA medications to complete HCV treatment[87] Patients with autoimmune liver disease Continue immunosuppressive therapy in stable patients with AIH[87]. Lower the doses of azathioprine or mycophenolate mofetil when patients develop lymphopenia[87]. Avoid liver biopsy and start empiric therapy in new patients presenting with features of AIH[87]. Avoid high doses of prednisone in AIH patients on corticosteroids[87] Patients with HCC Continue HCC surveillance schedule for high-risk subjects[40]. Document discussion of risks and benefits of delaying surveillance with patient[40]. Proceed with HCC treatments as appropriate[40]. Postpone elective transplant and resection surgery, withhold immunotherapy[84] Pretransplant and post-transplant patients Have low threshold for admitting patients on transplant waiting list diagnosed with COVID-19[40,84]. Consider reduction of immunosuppression therapy as appropriate for posttransplant patients with moderate COVID-19[40,84]. Avoid reductions in immunosuppressive therapy in patients with mild COVID-19 disease[40,84] AIH: Autoimmune hepatitis; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CLD: Chronic liver disease; COVID-19: Coronavirus disease 2019; DAA: Direct acting antiviral; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus. CONCLUSION Liver injury is a common complication in COVID-19 patients and may result from virus-induced cytopathic effects, immune mediated inflammation, drug toxicity and pneumonia-associated hypoxia. Like SARS and MERS, abnormal liver function mainly manifested as transient elevation of serum aminotransferases. Moreover, patients with abnormal liver tests had higher risks of progressing to severe disease. From a clinical perspective, in addition to actively dealing with the primary disease caused by SARS-CoV-2 infection, a close monitor of liver function is recommended in COVID-19 patients, especially in severe/critical individuals. Conflict-of-interest statement: There is no conflict of interest. Manuscript source: Unsolicited manuscript Peer-review started: September 19, 2020 First decision: November 26, 2020 Article in press: December 23, 2020 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Iorio R, Kharbanda KK S-Editor: Zhang L L-Editor: Filipodia P-Editor: Li X	Oral	DrugAdministrationRoute	CC BY-NC	33553391	19,036,556	2021-01-26
What was the dosage of drug 'ACETAMINOPHEN'?	Clinical features and potential mechanism of coronavirus disease 2019-associated liver injury. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has posed a serious threat to global public health security. With the increase in the number of confirmed cases globally, the World Health Organization has declared the outbreak of COVID-19 an international public health emergency. Despite atypical pneumonia as the primary symptom, liver dysfunction has also been observed in many clinical cases and is associated with the mortality risk in patients with COVID-19, like severe acute respiratory syndrome and Middle East respiratory syndrome. Here we will provide a schematic overview of the clinical characteristics and the possible mechanisms of liver injury caused by severe acute respiratory syndrome coronavirus 2 infection, which may provide help for optimizing the management of liver injury and reducing mortality in COVID-19 patients. Core Tip: With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with coronavirus disease 2019 (COVID-19). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases. Underlying mechanisms may be viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia. A close monitor of liver function is recommended in COVID-19 patients, especially in critical individuals. INTRODUCTION Since the 21st century, the outbreak of coronaviruses has brought great harm to human society; the most serious of which are the severe acute respiratory syndrome (SARS) in 2003, the Middle East respiratory syndrome (MERS) in 2012 and the novel coronavirus disease in 2019 (COVID-19). The ongoing outbreak of COVID-19 has become a pandemic. As of December 9, 2020, the total number of diagnosed cases globally exceeded 67530912 with a total of more than 1545140 infection-related deaths, carrying a mortality of approximately 2%[1]. Up to now, no specific antiviral therapies have been identified. Thus, an early monitor of critical complications is vital in preventing disease progression and improving survival. With the number of confirmed cases increasing worldwide, abnormal liver function has been observed in many patients with COVID-19, making this organ one of the most frequently damaged outside of the respiratory system (summarized in Table 1). COVID-19-associated liver injury refers to any hepatic damage that occurs during disease progression and treatment in COVID-19 patients with or without underlying liver diseases[2]. However, due to the different design and sample size, the incidence and clinical manifestations of liver injury in these studies are not the same. The mechanism of hepatic damage caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is still unclear. Table 1 Main characteristics related to liver injury in patients with coronavirus disease 2019 based on a series of case reports Ref. Sample size Liver injury Elevated ALT Elevated AST Elevated TBIL Elevated ALP Elevated GGT Factors related to liver injury [25] 1099 NA 21.3%: Severe 28.1%, Non-severe 19.8% 22.2%: Severe 39.4%, Non-severe 18.2% 10.5%: Severe 13.3%, Non-severe 9.9% NA NA NA [26] 548 NA 23.1%: Severe 24.1%, Non-severe 22.3% 33.1%: Severe 43.4%, Non-severe 23.3% 4.4%: Severe 6.4%, Non-severe 2.3% NA NA NA [27] 417 21.5% 41.2%: Severe 82.4%, Non-severe 50.2% 47.2%: Severe 75.3%, Non-severe 36.9% 64.2%: Severe 75.3%, Non-severe 60.1% 10.9%: Severe 12.2%, Non-severe 10.5% 48.5%: Severe 75.3%, Non-severe 39.1% Older, male, higher BMI, Underlying liver diseases (NAFLD, alcoholic liver disease and chronic hepatitis B), drugs (lopinavir/ritonavir) [28] 324 NA 15.7% 10.5% 6.5% 1.2% 0.9% NA [29] 298 14.8% NA NA NA NA NA NA [30] 274 NA 22.0%: Deceased 27.0%, Recovered 19.0% 31.0%: Deceased 52.0%, Recovered 16.0% NA NA NA NA [31] 148 37.2% 18.2% 21.6% 6.1% 4.1% 17.6% Male, higher levels of procalcitonin and CRP. PCT, LDH, received lopinavir / ritonavir [32] 85 38.8% 61.2% NA NA NA NA Older, lactic acid, myoglobin, neutrophils, critical illness, aCRP, alymphocyte count [33] 79 36.7% 31.6% 35.4% 5.1% NA NA Male, white blood cell counts, neutrophils, CRP, athe extent of pulmonary alesions on CT [34] 40 55% 52.5% 40% 25% NA NA Many types of drugs, large amounts of hormones, underlying diseases, lymphocyte count, acritical illness [35] 82 78% 30.6% 61.1% 30.6% NA NA NA a Represents independent risk factors for liver injury in coronavirus disease 2019. ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: Body mass index; COVID-19: Coronavirus disease 2019; CRP: C-reactive protein; CT: Computed tomography; GGT: Gamma-glutamyl transpeptidase; LDH: Lactate dehydrogenase; NA: Not available; NAFLD: Non-alcoholic fatty liver diseases; PCT: Procalcitonin; TBIL: Total bilirubin. SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are the causative agents of SARS, MERS and COVID-19, respectively, and they all belong to the highly pathogenic human beta coronaviruses[3]. Genomics analyses have found that SARS-CoV-2 shares 79.5% genome sequence similarity to SARS-CoV and 50% genome sequence homology to MERS-CoV[4]. SARS-CoV-2 uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV[5]. These common points hint that SARS-CoV-2 may partly mimic SARS-CoV and MERS-CoV infection. In this review, we summarized the characteristics and mechanism of liver injury caused by SARS-CoV-2 infection to provide a reference for further study. Liver injury in SARS and MERS Liver injury is not uncommon in patients infected with SARS-CoV and MERS-CoV according to previous studies[6]. In patients with SARS, liver injury mainly manifests as elevated alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in the early phase of the disease and is associated with the severity of the illness[7-13]. The pathogenesis of hepatic damage caused by SARS-CoV appears to be multifactorial, including a direct injury to the target cells by the virus and an indirect injury mediated by subsequent immune system dysfunction. As the functional receptor for SARS-CoV, ACE2 is abundantly expressed on endothelial cells of the liver, implying that SARS-CoV may directly bind to ACE2 positive cells to dysregulate liver function[5,13]. Liver biopsies in SARS patients demonstrated localization of virus in liver and hepatocyte apoptosis, which further confirmed the direct injury by SARS-CoV[14]. In an analysis of 145 cases of SARS, serum interleukin (IL)-1β, IL-6 and IL-10 were higher in patients with elevated serum levels of ALT than those in ALT normal group, indicating that liver damage was part of the manifestation of system inflammation reactive syndrome induced by SARS-CoV infection[10]. In addition, some studies have also found that hypoxemia and medication are closely related to abnormal liver function[15,16]. Elevated liver enzymes and bilirubin levels, as well as decreased albumin levels were highlighted during the hospital course of MERS-CoV infection in a series of case reports[17-20]. Different from SARS-CoV, MERS-CoV uses dipeptide base peptidase 4 as a cellular receptor to infect cells[21]. In humans, dipeptide base peptidase 4 is expressed constitutively on epithelial cells of liver[22], suggesting a direct hepatic damage caused by MERS-CoV. MERS also involves a mechanism of the upregulation of proinflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, IL-15 and IL-17[23]. However, studies on the relationship between cytokine storm and liver injury are scarce so far. Clinical characteristics of liver injury in patients with COVID-19 Since Chen et al[24] reported that 43 cases of 99 patients (43.4%) in Wuhan Jinyintan Hospital had different degrees of abnormal liver function, the abnormality of liver function test in patients with COVID-19 has aroused widespread concern among clinicians. As shown in Table 1, the incidence of liver injury ranged from 14.8% to 55.0% in recent case studies reporting clinical features of patients with COVID-19[25-34]. In death cases of COVID-19, the rate reached as high as 78.0%[35]. Liver injury presented in 30 out of 113 deceased patients from our previous report[30]. Abnormal liver function mainly manifests as slightly elevated ALT/AST and bilirubin levels, which usually occurs around the second week of the disease course[33-35]. Rarely, severe acute hepatitis associated with COVID-19 has been reported[36]. Contradictory to other hepatitis-induced liver injury, AST-dominant aminotrans-ferase elevation is common in COVID-19, which may provide a clue about the underlying pathophysiology of the impact of COVID-19 on liver. A retrospective study including 60 patients revealed that median AST was higher than ALT at admission (46 U/L vs 30 U/L) and during the hospital course[37]. In a multicenter retrospective cohort-derived data set of 5771 individuals, the elevation of serum AST level was earlier, more frequent and significant than the increase of ALT in severe patients, and AST levels had the highest correlation with mortality when compared with other indicators reflecting liver injury including elevated ALT, alkaline phosphatase (ALP) and total bilirubin (TBIL) levels in patients with COVID-19[38]. Likewise, an elevated baseline AST level has been shown to correlate with intensive care unit (ICU) admission, intubation and death in another study[39]. To our knowledge, three possible reasons may account for this phenomenon. First, given that AST is also distributed in myocardium and skeletal muscle, the American Association for the Study of Liver Diseases has recommended consideration of myositis or cardiac injury as contributors to the AST elevation[40]. Second, recent data identified ribosomal proteins as important host-dependency factors for SARS-CoV-2[41]. Therefore, the virus may directly cause hepatic mitochondrial injury and subsequent AST elevation. Third, AST-predominant aminotransferase elevations have been reported in alcohol-related liver disease, ischemia and cirrhosis. It is possible that hypoxia as well as metabolic changes such as hepatic steatosis may account for AST elevation in COVID-19 patients[42,43]. It is worth noting that liver dysfunction is closely related to the severity of the disease. On the one hand, severe patients have a higher proportion of liver injury: Guan et al[25] extracted a cohort regarding 1099 patients with laboratory-confirmed COVID-19 from 552 hospitals in mainland China. The results showed more patients with severe disease had elevated AST and ALT than those with non-severe disease. Like the result, Wang et al[44] showed that more patients admitted to the ICU had elevated AST levels. Huang et al[45] showed that patients admitted to the ICU had significantly higher ALT levels. On the other hand, patients with abnormal liver tests had higher risks of progressing to a severe disease course: Bloom et al[37] showed that admission AST, peak AST and peak ALT were higher in intubated patients. Of 417 patients with COVID-19, patients with abnormal liver tests of hepatocellular, cholestatic or mixed type at admission had higher odds of progressing to severe pneumonia[27]. Among 148 confirmed SARS-CoV-2-infected patients, the emerging abnormal liver functions after admission caused a prolonged length of stay[31]. In a large United States COVID-19 cohort of 3381 patients, 2273 patients who tested positive for SARS-CoV-2 had higher initial and peak ALT than those who tested negative[46]. Compared with mild [upper limit of normal (ULN) < ALT < two times ULN] and moderate (two times ULN < ALT < five times ULN) liver injury, patients with severe liver injury (ALT > five times ULN) had a more severe clinical course, including higher rates of ICU admission (69%), intubation (65%), renal replacement therapy (33%) and mortality (42%). Other hepatic manifestations in COVID-19 patients were hypoproteinemia and changes in coagulation[47,48]. A large cohort study including 2623 patients reported marked hypoalbuminemia in the critically ill and death groups than non-critically ill patients (38.2%, 71.2% and 82.4% on admission and 45.9%, 77.7% and 95.6% during hospitalization, respectively)[49]. Meanwhile, the patients in this study displayed dramatically prolonged activated partial thromboplastin time in critically ill patients reflected coagulopathy. Further analysis shows that risk factors associated with hepatic damage include older males, a longer time from illness onset to admission, a history of drinking, higher serum levels of C-reactive protein (CRP), white blood cell counts, neutrophils and medication (lopinavir/ritonavir, hormones)[27,31-34,50]. Disease severity (severe/critical), CRP, lymphocyte count and the extent of pulmonary lesions on computed tomography are independent risk factors for liver injury[32-34]. Accumulating data reveals that patients with pre-existing liver diseases are more susceptible to SARS-CoV-2 infection and have poorer prognosis. In our study, viral hepatitis (hepatitis B and hepatitis C) was much more frequent among patients with liver injury than those without[51]. Another study revealed that COVID-19 patients with non-alcoholic fatty liver disease had a significantly higher likelihood of abnormal liver function from admission to discharge when compared to those with non-alcoholic fatty liver disease subjects[52]. Qiu et al[53] first reported a case of acute-on-chronic liver failure due to SARS-COV-2 infection in a patient with decompensated alcoholic cirrhosis. In a multicenter retrospective study, fifty cirrhotic patients with SARS-CoV-2 infection were studied to evaluate the impact of COVID-19 on the clinical outcome[54]. The results showed 30-d mortality rate was higher in cirrhotic patients with COVID-19 than in cirrhotic patients with bacterial infection and in COVID-19 patients without cirrhosis, indicating that COVID-19 was associated with liver function deterioration and elevated mortality in cirrhotic patients. In addition, a study of 2780 COVID-19-positive patients found that those with cirrhosis were at a particularly increased risk for mortality by analyzing a large United States database (risk ratio, 4.6; 95% confidence interval, 2.6-8.3)[55]. Whether the COVID-19 patients with more severe liver injury were positive for hepatotrophis viruses is still controversial. In a retrospective study, the authors analyzed liver function parameters including ALT, AST and TBIL in COVID-19 patients with or without HBV infection and found no significant differences between the two groups[56]. Another study reached a similar conclusion and further proved the longitudinal changes of median values for liver biochemistries were not significantly different between the two groups either[57]. These findings indicated that SARS-CoV-2 will not exacerbate liver injury in patients with HBV co-infection. However, Lin et al[58] drew a completely opposite conclusion. In their cohort, COVID-19 cases with HBV coinfection had higher levels of ALT, AST, TBIL and ALP than the COVID-19 cases without HBV coinfection, showing that inactive HBV carriers with SARS-CoV-2 coinfection are at risk of greater liver injury. Moreover, SARS-CoV-2 was reported to induce HBV reactivation, which may cause severe liver injury in patients with coinfection[57,59]. In addition, a case of COVID-19 with Epstein-Barr virus coinfection was reported recently. On admission, he showed acute liver injury with liver enzymes that were much higher than typically seen solely with COVID-19 infection[60]. Although the evidence is limited, more attention should be paid to COVID-19 patients with other viral coinfections during clinical treatment. Mechanisms of liver injury in patients with COVID-19 Underlying mechanisms involved in liver injury in patients with COVID-19 are complex and interactive, which might include viral infection in liver cells, systemic inflammation induced by cytokine storm, drug induced liver injury or pneumonia-associated hypoxia[61] (Figure 1). Figure 1 Potential mechanisms of liver injury in patients with coronavirus disease 2019. 1: Severe acute respiratory syndrome coronavirus-2 may directly bind to angiotensin converting enzyme II positive cholangiocytes to dysregulate liver function; 2: Inflammatory cytokine storm leads to persistent activation of lymphocytes and macrophages that secrete huge amount of inflammatory cytokine, thus contributing to lung as well as liver damage; 3: Drugs including antipyretics, antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids may have potential hepatotoxicity and lead to abnormal liver function; 4: Hepatic ischemia and hypoxia-reperfusion dysfunction induced by complications such as respiratory failure may cause liver damage, especially in critically ill patients. Virus-induced cytopathic effects It is well known that SARS-CoV-2 predominantly enters alveolar epithelial cells through the human ACE2 receptor, thus making the lung the main target organ of SARS-CoV-2 infection[62]. However, previous studies have found that ACE2 receptor is also specifically expressed in bile duct epithelial cells but is rarely expressed in hepatocytes[63,64] and absent of Kupffer cells and hepatic stellate cells[65]. A further study employing single-cell ribonucleic acid-seq suggested that TROP2+ cholangiocytes could be a main target for SARS-CoV-2 infection, leading to impaired liver regeneration and liver function[66]. In a mouse model of acute liver injury, ACE2 was upregulated in liver tissue due to compensatory proliferation of hepatocytes derived from bile duct epithelial cells[64]. During this compensatory process, some newborn hepatocytes still expressed ACE2 receptor and were susceptible to SARS-CoV-2. Recently, Wang et al[67] investigated the patterns of liver impairment by electron microscopy and pathological studies in two COVID-19 cases. In this study, typical coronavirus particles were identified in the cytoplasm of hepatocytes. Histologically, massive hepatic apoptosis and binuclear hepatocytes were observed. Our previous clinical report showed that the bile duct injury related ALP and gamma-glutamyl transpeptidase was elevated in deceased patients[30]. These findings suggest that the liver injury in COVID-19 patients may be due to hepatocyte damage as well as cholangiocyte dysfunction. Other studies reported conflicting results. For example, Qian et al[28] showed that ALP, gamma-glutamyl transpeptidase and TBIL elevations were rare among 324 cases with SARS-CoV-2 pneumonia. Zhang et al[47] reported that after SARS-CoV-2 infection, the overall ALP level is even lower than that with community-acquired pneumonia patients, implying that the duct epithelium injury by SARS-CoV-2 itself is very slight. Thus, SARS-COV-2 infection may not be the major reason related to liver injury. Given the conflicting results above, the role of virus-induced cytopathic effects in COVID-19-related liver injury warrants further investigation. Inflammatory cytokine storm Cell entry of SARS-CoV-2 depends on binding of the viral spike (S) proteins to cellular ACE2 receptor and on spike protein priming by host cell proteases[68]. While the virus enters the cells via fusion with the host membrane, its antigen will be recognized by the antigen presentation cells and then presented to cytotoxic and regulatory T lymphocytes, which initiate an antiviral immune response that includes inflammatory cytokine production and a weak interferon response[69]. In young individuals with an intact immune system, the virus is cleared away during the initial phase, so they show only mild symptoms[45]. However, in the elderly and individuals with underlying chronic diseases, the insufficient viral clearance due to altered immune response will lead to a cytokine storm, which may trigger a violent attack to the body and cause multiple organ failure including the liver[45,70]. Inflammatory cytokine storm is an overactive inflammatory response caused by virus infection, which leads to persistent activation of lymphocytes and macrophages that secrete huge amounts of inflammatory cytokines[71]. For example, SARS-CoV-2 can rapidly activate pathogenic Th1 cells to secrete proinflammatory cytokines, such as granulocyte-macrophage colony-stimulating factor and IL-6[72]. Granulocyte-macrophage colony-stimulating factor further activates CD14+CD16+ inflammatory monocytes to produce large quantities of IL-6, tumor necrosis factor-α and other cytokines. Among these cytokines, IL-6 can bind to sIL-6R to activate STAT3 in nonimmune cells and can bind to membrane-bound IL-6 receptor to lead to pleiotropic effects on acquired and innate immune cells[73]. Meanwhile, sIL-2R may regulate cytotoxic T cells negatively and contribute to lymphopenia through IL-2 signaling inhibition[74]. Accumulating evidence revealed a broad spectrum of proinflammatory cytokines and chemokines dramatically increased in patients with liver dysfunction compared to those with normal liver function[10,32]. Consistent with these results, our data showed the levels of inflammatory markers including high sensitivity CRP, neutrophil-to-lymphocyte ratio, white blood cells, neutrophils, serum ferritin, lactate dehydrogenase, procalcitonin, erythrocyte sedimentation rate and proinflammatory cytokines including IL-2R, IL-6, tumor necrosis factor-α in the liver injury group were significantly higher compared with the group without liver injury[51]. In an analysis of 85 patients with COVID-19, lymphopenia and CRP may even serve as the risk factors related to hepatic injury[32]. Moreover, the postmortem liver biopsy in one patient confirmed that liver injury in COVID-19 is likely immune mediated[43]. These findings indicated that immune-mediated inflammatory response following SARS-CoV-2 infection may cause or contribute to liver damage. In the future, more research is needed to understand the concrete mechanisms involved in cytokine accumulation in COVID-19 and subsequent liver injury. Drug-induced liver injury Fever was one of the most common symptoms on admission and during hospitalization in patients with COVID-19[25]. Therefore, antipyretic therapy is very ordinary in infected patients. Acetaminophen, a common ingredient in antipyretic drugs, is proven to cause significant liver damage or induce liver failure according to a dose-dependent mechanism[75]. Recently, a 27-year-old healthy African American female with a positive SARS-CoV-2 test and acute liver failure secondary to acetaminophen overdose was reported[76]. She had a remote history of focal segmental glomerular sclerosis. To manage her pain, she ingested > 50 tablets of acetaminophen over the 3-4 d preceding presentation. Initial blood work revealed the acetaminophen level was 42 µg/mL (upper limit of normal 30 µg/mL) and elevated aminotransferases with alanine transaminase of 2791 U/L and aspartate transaminase of 3202 U/L. Two days after admission, her hepatic synthetic function worsened significantly, and aminotransferases peaked to an AST 9741 U/L and ALT 11322 U/L. Thus, although paracetamol is a safe and effective first line agent in almost all patients regardless of liver disease etiology[77], the clinicians cannot be too careful in the dose. Although there is currently no specific therapy for COVID-19, many patients especially severe and critical patients, were often treated with multiple drugs, including antiviral medications (lopinavir/ritonavir), antibiotics (macrolides, quinolones) and steroids in clinical practice[78]. These drugs may have potential hepatotoxicity and lead to abnormal liver function. Recent data on liver tests in patients with COVID-19 showed that the use of lopinavir/ritonavir led to increased odds of liver injury[27]. Fan et al[31] reported that among 148 COVID-19 patients, patients receiving treatment with lopinavir/ritonavir were more likely to develop abnormal liver function tests. In another clinical report, liver function injury was more likely to occur in patients who used many types of drugs and large amounts of hormones[34]. Our study also revealed a higher proportion of patients with liver injury had received systemic glucocorticoids (unpublished). The liver biopsy specimens of the patient with COVID-19 showed moderate microvascular steatosis and mild lobular and portal activity, further conforming the possibility of drug-induced hepatic damage[43]. But on the contrary, in a randomized, controlled, open-label trial, Cao et al[79] reported that lopinavir/ritonavir treatment did not significantly increase liver enzymes in patients with serious COVID-19. Due to limited data, we cannot draw a definitive conclusion about whether lopinavir/ritonavir or glucocorticoids increase the risk of developing liver damage. Pneumonia-associated hypoxia Hypoxic hepatitis, also known as ischemic hepatitis or shock liver, is commonly seen in patients with hypotension shock or severe hypoxemia caused by severe heart failure, respiratory failure, surgery, trauma and other causes[80]. Its clinical feature is a massive, rapid rise in serum transaminase (which can exceed 20 × ULN) and is often accompanied by an increase in lactate dehydrogenase. In patients with COVID-19, hypoxia and shock caused by respiratory distress syndrome, system inflammation reactive syndrome, multiple organ dysfunction and other complications can lead to hepatic ischemia and hypoxia-reperfusion dysfunction. Experimental data revealed that hepatocyte death and inflammatory cytokines production caused by hypoxia can be seen in both in vivo and in vitro models of hepatic ischemia and hypoxia[81]. Furthermore, liver histological findings on autopsy of patients with COVID-19 revealed the watery degeneration of some hepatocytes, proving the possibility of hepatic ischemia and hypoxia[27]. However, according to the available evidence, the distribution of aminotransferase levels among patients with COVID-19 do not support pneumonia-associated hypoxia being a common cause of liver injury[82]. Whether hypoxia is related to abnormal liver function in COVID-19 patients remains to be further investigated. Management of COVID-19 patients with liver disease It was reported that about 2%-11% of patients with COVID-19 had underlying chronic liver disease[83]. Chinese Society of Hepatology, Chinese Medical Association, American Association for the Study of Liver Diseases, Asian Pacific Association for the Study of the Liver and European Association for the Study of the Liver have all issued relevant guidelines to help clinicians manage chronic liver disease and liver transplant patients during the epidemic of COVID-19. Because there is no complete and systematic data at present, most of the recommendations for management of COVID-19 patients with liver disease are based on expert consensus. Among these guidelines, the basic principles are infection control, delay of medical treatment, risk classification and supportive management[84-87]. More details are summarized in Table 2. Table 2 Management of coronavirus disease 2019 patients with liver disease Management of COVID-19 patients with liver disease Out-patient care Use telemedicine or visits by phone wherever possible. Consider seeing in person only patients with urgent issues and clinically significant liver disease (e.g., jaundice, elevated ALT or AST > 500 U/L, or recent onset of hepatic decompensation)[40,84,86]. Seeing at the fever clinic[40] Hospital treatment Separate management from non-COVID-19 patients[40,85]. Monitor liver biochemistries regularly, particularly in patients treated with remdesivir or tocilizumab[40]. Avoid ultrasound or other advanced imaging unless it is likely to change management, for example, clinical suspicion for biliary obstruction or venous thrombosis[40]. Hospitalize COVID-19 patients with advanced liver disease as soon as possible[85] Patients with hepatitis B, hepatitis C Document discussion with patient regarding CLD diagnosis and management[84]. Delay starting DAA therapy until after their recovery from COVID-19 disease if there is no suspicion of advanced liver disease[87]. Continue treatment and provide 90-d supplies for HBV oral antiviral drugs or a full course of DAA medications to complete HCV treatment[87] Patients with autoimmune liver disease Continue immunosuppressive therapy in stable patients with AIH[87]. Lower the doses of azathioprine or mycophenolate mofetil when patients develop lymphopenia[87]. Avoid liver biopsy and start empiric therapy in new patients presenting with features of AIH[87]. Avoid high doses of prednisone in AIH patients on corticosteroids[87] Patients with HCC Continue HCC surveillance schedule for high-risk subjects[40]. Document discussion of risks and benefits of delaying surveillance with patient[40]. Proceed with HCC treatments as appropriate[40]. Postpone elective transplant and resection surgery, withhold immunotherapy[84] Pretransplant and post-transplant patients Have low threshold for admitting patients on transplant waiting list diagnosed with COVID-19[40,84]. Consider reduction of immunosuppression therapy as appropriate for posttransplant patients with moderate COVID-19[40,84]. Avoid reductions in immunosuppressive therapy in patients with mild COVID-19 disease[40,84] AIH: Autoimmune hepatitis; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CLD: Chronic liver disease; COVID-19: Coronavirus disease 2019; DAA: Direct acting antiviral; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus. CONCLUSION Liver injury is a common complication in COVID-19 patients and may result from virus-induced cytopathic effects, immune mediated inflammation, drug toxicity and pneumonia-associated hypoxia. Like SARS and MERS, abnormal liver function mainly manifested as transient elevation of serum aminotransferases. Moreover, patients with abnormal liver tests had higher risks of progressing to severe disease. From a clinical perspective, in addition to actively dealing with the primary disease caused by SARS-CoV-2 infection, a close monitor of liver function is recommended in COVID-19 patients, especially in severe/critical individuals. Conflict-of-interest statement: There is no conflict of interest. Manuscript source: Unsolicited manuscript Peer-review started: September 19, 2020 First decision: November 26, 2020 Article in press: December 23, 2020 Specialty type: Medicine, research and experimental Country/Territory of origin: China Peer-review report’s scientific quality classification Grade A (Excellent): 0 Grade B (Very good): 0 Grade C (Good): C, C Grade D (Fair): 0 Grade E (Poor): 0 P-Reviewer: Iorio R, Kharbanda KK S-Editor: Zhang L L-Editor: Filipodia P-Editor: Li X	MORE THAN 50TABLETS OVER THE 3?4 D PRECEDING PRESENTATION	DrugDosageText	CC BY-NC	33553391	19,036,556	2021-01-26
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Hepatitis B'.	Hemophagocytic Lymphohistiocytosis Associated With Hepatitis B and HIV Coinfection With Resultant Liver Failure. Hemophagocytic lymphohistiocytosis is a syndrome of excessive immune activation frequently attributed to infection. We report a case of hemophagocytic lymphohistiocytosis secondary to hepatitis B in a patient with human immunodeficiency virus coinfection and subsequent liver failure. INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome of immune dysregulation and macrophage activation resulting in a hyperinflammatory state associated with high mortality. It is characterized by pyrexia, cytopenias, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, elevated interleukin (IL)-2, low natural killer (NK) activity, and evidence of hemophagocytosis. HLH is frequently attributed to viral infections, most commonly Epstein-Barr and cytomegalovirus.1,2 The following is an unusual case of HLH diagnosed in a patient with active hepatitis B virus (HBV) and human immunodeficiency virus (HIV), ultimately resulting in liver failure and death. CASE REPORT A 33-year-old man presented with complaints of fever, nausea, and productive cough and was admitted for severe septic shock. He was recently diagnosed with chronic HBV (anti-HBc and HBsAg positive, IgM anti-HBc negative) and HIV. He was started on bictegravir, emtricitabine, and tenofovir a month before his admission. Of note, the patient had several recent hospitalizations for pancytopenia and sepsis without a clear cause. On arrival, his vital signs were as follows: temperature 103.1°F, heart rate 152 beats per minute, blood pressure 133/63 mm Hg, and SpO2 97% on 2 L nasal cannula with respiratory rate 60 breaths per minute. Shortly after arrival, he became hypoxic (SpO2 85%) and hypotensive (90s/40s mm Hg), requiring intubation and vasopressor support. His admission laboratory test results were notable for hemoglobin 6.5 g/dL, white blood cells 3.1 10 × 9/L, platelet count 30 10 × 9/L, Cr 1.34 mg/dL, albumin 2 g/dL, bilirubin 0.9 mg/dL, aminotransferase aminotransferase (AST)/alanine aminotransferase 72/38 IU/L, and international normalized ratio 1.7. HBV viral load on admission was 511,000 copies, with CD4 count of 15 and HIV viral load 10,500 copies. Thoracic X-ray and computed tomography were concerning for pneumonia. Abdominal and pelvic computed tomography demonstrated hepatosplenomegaly without cirrhotic morphology, with spleen and liver measuring 16 cm and 24 cm, respectively, with small abdominal ascites. He received a blood transfusion and was treated with broad-spectrum antibiotics (meropenem and vancomycin) as well as empiric treatment for Pneumocystis jiroveci pneumonia and corticosteroids for septic shock. He received renally dosed dolutegravir, tenofovir, and emtricitabine for his HIV. Despite these measures, the patient continued fever and require frequent blood transfusions. He underwent an extensive infectious and autoimmune disease evaluation including negative blood, acid-fast Bacilli blood, urine, and bronchial alveolar lavage cultures, respiratory virus and pneumonia panels, fungal and parasitic serologies, herpes virus and cytomegalovirus polymerase chain reactions, with evidence of previous Epstein-Barr virus and parvovirus infections. He also had negative leukemia and lymphoma panel. The patient was noted to have increasing bilirubin (5.6) and AST (120), with scleral icterus on examination. His HBV viral load was redrawn, increasing to 12,500,000. Entecavir was started in addition to tenofovir and emtricitabine, with subsequent decrease in his viral load to 5,600 over 4 weeks. HBV genotype revealed no resistance. HIV viral load became undetectable. Immune reconstitution inflammatory syndrome was considered, although this diagnosis could not explain his increasing HBV viral load. Given his persistent pyrexia and elevated ferritin, HLH was suspected. Further evaluation revealed ferritin of 17,918 ng/mL, triglyceride 434 mm/dL, fibrinogen 151 mg/dL, and soluble IL-2 receptor 27,900 pg/mL (high), with low NK activity. Our pathologists reviewed a bone marrow biopsy previously performed at an outside hospital with scant hemophagocytosis findings (Figure 1). Figure 1. Hematoxylin and eosin stain of bone marrow biopsy with hemophagocytosis. Arrows demonstrate hemophagocytosis of red blood cells. The diagnosis of HLH was made, and the patient was treated with a dexamethasone taper initially resulting in reduced transfusion requirements as well as decreased pyrexia. However, transfusion requirements and pyrexia returned. The decision was made to begin treatment with renally dosed etoposide per HLH-94 protocol resulting in significant clinical improvement.3 His hospital course was complicated by acute renal failure requiring temporary dialysis, a gastrointestinal bleed secondary to a duodenal ulcer requiring endoscopic intervention, encephalopathy with subarachnoid hemorrhages, and 2 episodes of acute hypoxic respiratory failure requiring intubation and ventilatory support. He eventually recovered enough to be discharged home on a steroid taper. Unfortunately, patient was unable to obtain his HBV and HIV medications on discharge and presented 5 days later with acute hypoxic respiratory failure again requiring intubation. He continued to decompensate, with ferritin above assay limits of 40,000, bilirubin peaking at 26, international normalized ratio 2.5, AST 358, and alanine aminotransferase 180, with persistent fevers and a high daily blood product requirement. The family decided to move to comfort measures, and he succumbed to his illness. DISCUSSION HLH is a rare immune-mediated life-threatening syndrome that can involve multiple organ systems. HLH is classified into primary (genetic) and secondary (reactive), which is further subclassified into infectious and autoimmune, although about a third of adult cases are multifactorial.1 HLH overlaps clinically with many more common conditions frequently leading to a delay in diagnosis. Our patient met 8/8 criteria for HLH, with hyperferritenemia, hypofibrinogenemia, hypertriglyceridemia, pancytopenia, hepatosplenomegaly, low NK activity, elevated IL-2, and evidence of hemophagocytosis on bone marrow biopsy. Liver injury is very common in HLH, with up to 85% of patients with secondary HLH having abnormal liver function.4 The liver injury is possibly secondary to cytokine storm and infiltration of the parenchyma by activated hemophagocytic histiocytes.5 Liver transplantation is frequently contraindicated in these patients secondary to severe systemic illness. Our patient's HLH was most likely secondary to his active HBV infection, as his viral load increased 25-fold, likely inciting the aberrant immune response. The reason for his rapid increase in HBV viral load is unclear, since genotype revealed no mutations and he was on 2 active antivirals. Extensive evaluation for additional infectious causes or malignancy was unremarkable. Although HLH has been associated with HIV infection, his HIV was less likely contributory, as his viral load became undetectable on treatment over his hospital course.2 The brief period without antiviral medications after discharge could have resulted in acute HBV hepatitis and subsequent worsening of HLH because laboratory markers for HLH dramatically worsened on his second admission. On literature review, HLH was attributed to HBV in 13 cases.6–10 Two detailed case reports treated the patient with etoposide and dexamethasone per HLH-94 protocol.8,9 To the best of our knowledge, this is the first reported case of HLH attributed to HBV with HIV coinfection. As about 10% of patients with HIV have HBV coinfection, HLH should be considered in coinfected febrile patients with cytopenias.11 Early recognition and treatment of HLH is essential for survival. DISCLOSURES Author contributions: H. Blaney wrote the manuscript. D. Thotakura reviewed the literature. L. Sisco edited the manuscript and reviewed the literature and is the author guarantor. Financial disclosure: None to report. Informed consent was obtained for this case report.	BICTEGRAVIR, DOLUTEGRAVIR, EMTRICITABINE, TENOFOVIR ALAFENAMIDE FUMARATE, TENOFOVIR DISOPROXIL FUMARATE	DrugsGivenReaction	CC BY-NC-ND	33553464	20,925,366	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Therapy interrupted'.	Hemophagocytic Lymphohistiocytosis Associated With Hepatitis B and HIV Coinfection With Resultant Liver Failure. Hemophagocytic lymphohistiocytosis is a syndrome of excessive immune activation frequently attributed to infection. We report a case of hemophagocytic lymphohistiocytosis secondary to hepatitis B in a patient with human immunodeficiency virus coinfection and subsequent liver failure. INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome of immune dysregulation and macrophage activation resulting in a hyperinflammatory state associated with high mortality. It is characterized by pyrexia, cytopenias, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, elevated interleukin (IL)-2, low natural killer (NK) activity, and evidence of hemophagocytosis. HLH is frequently attributed to viral infections, most commonly Epstein-Barr and cytomegalovirus.1,2 The following is an unusual case of HLH diagnosed in a patient with active hepatitis B virus (HBV) and human immunodeficiency virus (HIV), ultimately resulting in liver failure and death. CASE REPORT A 33-year-old man presented with complaints of fever, nausea, and productive cough and was admitted for severe septic shock. He was recently diagnosed with chronic HBV (anti-HBc and HBsAg positive, IgM anti-HBc negative) and HIV. He was started on bictegravir, emtricitabine, and tenofovir a month before his admission. Of note, the patient had several recent hospitalizations for pancytopenia and sepsis without a clear cause. On arrival, his vital signs were as follows: temperature 103.1°F, heart rate 152 beats per minute, blood pressure 133/63 mm Hg, and SpO2 97% on 2 L nasal cannula with respiratory rate 60 breaths per minute. Shortly after arrival, he became hypoxic (SpO2 85%) and hypotensive (90s/40s mm Hg), requiring intubation and vasopressor support. His admission laboratory test results were notable for hemoglobin 6.5 g/dL, white blood cells 3.1 10 × 9/L, platelet count 30 10 × 9/L, Cr 1.34 mg/dL, albumin 2 g/dL, bilirubin 0.9 mg/dL, aminotransferase aminotransferase (AST)/alanine aminotransferase 72/38 IU/L, and international normalized ratio 1.7. HBV viral load on admission was 511,000 copies, with CD4 count of 15 and HIV viral load 10,500 copies. Thoracic X-ray and computed tomography were concerning for pneumonia. Abdominal and pelvic computed tomography demonstrated hepatosplenomegaly without cirrhotic morphology, with spleen and liver measuring 16 cm and 24 cm, respectively, with small abdominal ascites. He received a blood transfusion and was treated with broad-spectrum antibiotics (meropenem and vancomycin) as well as empiric treatment for Pneumocystis jiroveci pneumonia and corticosteroids for septic shock. He received renally dosed dolutegravir, tenofovir, and emtricitabine for his HIV. Despite these measures, the patient continued fever and require frequent blood transfusions. He underwent an extensive infectious and autoimmune disease evaluation including negative blood, acid-fast Bacilli blood, urine, and bronchial alveolar lavage cultures, respiratory virus and pneumonia panels, fungal and parasitic serologies, herpes virus and cytomegalovirus polymerase chain reactions, with evidence of previous Epstein-Barr virus and parvovirus infections. He also had negative leukemia and lymphoma panel. The patient was noted to have increasing bilirubin (5.6) and AST (120), with scleral icterus on examination. His HBV viral load was redrawn, increasing to 12,500,000. Entecavir was started in addition to tenofovir and emtricitabine, with subsequent decrease in his viral load to 5,600 over 4 weeks. HBV genotype revealed no resistance. HIV viral load became undetectable. Immune reconstitution inflammatory syndrome was considered, although this diagnosis could not explain his increasing HBV viral load. Given his persistent pyrexia and elevated ferritin, HLH was suspected. Further evaluation revealed ferritin of 17,918 ng/mL, triglyceride 434 mm/dL, fibrinogen 151 mg/dL, and soluble IL-2 receptor 27,900 pg/mL (high), with low NK activity. Our pathologists reviewed a bone marrow biopsy previously performed at an outside hospital with scant hemophagocytosis findings (Figure 1). Figure 1. Hematoxylin and eosin stain of bone marrow biopsy with hemophagocytosis. Arrows demonstrate hemophagocytosis of red blood cells. The diagnosis of HLH was made, and the patient was treated with a dexamethasone taper initially resulting in reduced transfusion requirements as well as decreased pyrexia. However, transfusion requirements and pyrexia returned. The decision was made to begin treatment with renally dosed etoposide per HLH-94 protocol resulting in significant clinical improvement.3 His hospital course was complicated by acute renal failure requiring temporary dialysis, a gastrointestinal bleed secondary to a duodenal ulcer requiring endoscopic intervention, encephalopathy with subarachnoid hemorrhages, and 2 episodes of acute hypoxic respiratory failure requiring intubation and ventilatory support. He eventually recovered enough to be discharged home on a steroid taper. Unfortunately, patient was unable to obtain his HBV and HIV medications on discharge and presented 5 days later with acute hypoxic respiratory failure again requiring intubation. He continued to decompensate, with ferritin above assay limits of 40,000, bilirubin peaking at 26, international normalized ratio 2.5, AST 358, and alanine aminotransferase 180, with persistent fevers and a high daily blood product requirement. The family decided to move to comfort measures, and he succumbed to his illness. DISCUSSION HLH is a rare immune-mediated life-threatening syndrome that can involve multiple organ systems. HLH is classified into primary (genetic) and secondary (reactive), which is further subclassified into infectious and autoimmune, although about a third of adult cases are multifactorial.1 HLH overlaps clinically with many more common conditions frequently leading to a delay in diagnosis. Our patient met 8/8 criteria for HLH, with hyperferritenemia, hypofibrinogenemia, hypertriglyceridemia, pancytopenia, hepatosplenomegaly, low NK activity, elevated IL-2, and evidence of hemophagocytosis on bone marrow biopsy. Liver injury is very common in HLH, with up to 85% of patients with secondary HLH having abnormal liver function.4 The liver injury is possibly secondary to cytokine storm and infiltration of the parenchyma by activated hemophagocytic histiocytes.5 Liver transplantation is frequently contraindicated in these patients secondary to severe systemic illness. Our patient's HLH was most likely secondary to his active HBV infection, as his viral load increased 25-fold, likely inciting the aberrant immune response. The reason for his rapid increase in HBV viral load is unclear, since genotype revealed no mutations and he was on 2 active antivirals. Extensive evaluation for additional infectious causes or malignancy was unremarkable. Although HLH has been associated with HIV infection, his HIV was less likely contributory, as his viral load became undetectable on treatment over his hospital course.2 The brief period without antiviral medications after discharge could have resulted in acute HBV hepatitis and subsequent worsening of HLH because laboratory markers for HLH dramatically worsened on his second admission. On literature review, HLH was attributed to HBV in 13 cases.6–10 Two detailed case reports treated the patient with etoposide and dexamethasone per HLH-94 protocol.8,9 To the best of our knowledge, this is the first reported case of HLH attributed to HBV with HIV coinfection. As about 10% of patients with HIV have HBV coinfection, HLH should be considered in coinfected febrile patients with cytopenias.11 Early recognition and treatment of HLH is essential for survival. DISCLOSURES Author contributions: H. Blaney wrote the manuscript. D. Thotakura reviewed the literature. L. Sisco edited the manuscript and reviewed the literature and is the author guarantor. Financial disclosure: None to report. Informed consent was obtained for this case report.	BICTEGRAVIR, DOLUTEGRAVIR, EMTRICITABINE, TENOFOVIR ALAFENAMIDE FUMARATE, TENOFOVIR DISOPROXIL FUMARATE	DrugsGivenReaction	CC BY-NC-ND	33553464	20,925,366	2021-02
What was the outcome of reaction 'Hepatitis B'?	Hemophagocytic Lymphohistiocytosis Associated With Hepatitis B and HIV Coinfection With Resultant Liver Failure. Hemophagocytic lymphohistiocytosis is a syndrome of excessive immune activation frequently attributed to infection. We report a case of hemophagocytic lymphohistiocytosis secondary to hepatitis B in a patient with human immunodeficiency virus coinfection and subsequent liver failure. INTRODUCTION Hemophagocytic lymphohistiocytosis (HLH) is a rare clinical syndrome of immune dysregulation and macrophage activation resulting in a hyperinflammatory state associated with high mortality. It is characterized by pyrexia, cytopenias, hepatosplenomegaly, hypertriglyceridemia, hypofibrinogenemia, elevated interleukin (IL)-2, low natural killer (NK) activity, and evidence of hemophagocytosis. HLH is frequently attributed to viral infections, most commonly Epstein-Barr and cytomegalovirus.1,2 The following is an unusual case of HLH diagnosed in a patient with active hepatitis B virus (HBV) and human immunodeficiency virus (HIV), ultimately resulting in liver failure and death. CASE REPORT A 33-year-old man presented with complaints of fever, nausea, and productive cough and was admitted for severe septic shock. He was recently diagnosed with chronic HBV (anti-HBc and HBsAg positive, IgM anti-HBc negative) and HIV. He was started on bictegravir, emtricitabine, and tenofovir a month before his admission. Of note, the patient had several recent hospitalizations for pancytopenia and sepsis without a clear cause. On arrival, his vital signs were as follows: temperature 103.1°F, heart rate 152 beats per minute, blood pressure 133/63 mm Hg, and SpO2 97% on 2 L nasal cannula with respiratory rate 60 breaths per minute. Shortly after arrival, he became hypoxic (SpO2 85%) and hypotensive (90s/40s mm Hg), requiring intubation and vasopressor support. His admission laboratory test results were notable for hemoglobin 6.5 g/dL, white blood cells 3.1 10 × 9/L, platelet count 30 10 × 9/L, Cr 1.34 mg/dL, albumin 2 g/dL, bilirubin 0.9 mg/dL, aminotransferase aminotransferase (AST)/alanine aminotransferase 72/38 IU/L, and international normalized ratio 1.7. HBV viral load on admission was 511,000 copies, with CD4 count of 15 and HIV viral load 10,500 copies. Thoracic X-ray and computed tomography were concerning for pneumonia. Abdominal and pelvic computed tomography demonstrated hepatosplenomegaly without cirrhotic morphology, with spleen and liver measuring 16 cm and 24 cm, respectively, with small abdominal ascites. He received a blood transfusion and was treated with broad-spectrum antibiotics (meropenem and vancomycin) as well as empiric treatment for Pneumocystis jiroveci pneumonia and corticosteroids for septic shock. He received renally dosed dolutegravir, tenofovir, and emtricitabine for his HIV. Despite these measures, the patient continued fever and require frequent blood transfusions. He underwent an extensive infectious and autoimmune disease evaluation including negative blood, acid-fast Bacilli blood, urine, and bronchial alveolar lavage cultures, respiratory virus and pneumonia panels, fungal and parasitic serologies, herpes virus and cytomegalovirus polymerase chain reactions, with evidence of previous Epstein-Barr virus and parvovirus infections. He also had negative leukemia and lymphoma panel. The patient was noted to have increasing bilirubin (5.6) and AST (120), with scleral icterus on examination. His HBV viral load was redrawn, increasing to 12,500,000. Entecavir was started in addition to tenofovir and emtricitabine, with subsequent decrease in his viral load to 5,600 over 4 weeks. HBV genotype revealed no resistance. HIV viral load became undetectable. Immune reconstitution inflammatory syndrome was considered, although this diagnosis could not explain his increasing HBV viral load. Given his persistent pyrexia and elevated ferritin, HLH was suspected. Further evaluation revealed ferritin of 17,918 ng/mL, triglyceride 434 mm/dL, fibrinogen 151 mg/dL, and soluble IL-2 receptor 27,900 pg/mL (high), with low NK activity. Our pathologists reviewed a bone marrow biopsy previously performed at an outside hospital with scant hemophagocytosis findings (Figure 1). Figure 1. Hematoxylin and eosin stain of bone marrow biopsy with hemophagocytosis. Arrows demonstrate hemophagocytosis of red blood cells. The diagnosis of HLH was made, and the patient was treated with a dexamethasone taper initially resulting in reduced transfusion requirements as well as decreased pyrexia. However, transfusion requirements and pyrexia returned. The decision was made to begin treatment with renally dosed etoposide per HLH-94 protocol resulting in significant clinical improvement.3 His hospital course was complicated by acute renal failure requiring temporary dialysis, a gastrointestinal bleed secondary to a duodenal ulcer requiring endoscopic intervention, encephalopathy with subarachnoid hemorrhages, and 2 episodes of acute hypoxic respiratory failure requiring intubation and ventilatory support. He eventually recovered enough to be discharged home on a steroid taper. Unfortunately, patient was unable to obtain his HBV and HIV medications on discharge and presented 5 days later with acute hypoxic respiratory failure again requiring intubation. He continued to decompensate, with ferritin above assay limits of 40,000, bilirubin peaking at 26, international normalized ratio 2.5, AST 358, and alanine aminotransferase 180, with persistent fevers and a high daily blood product requirement. The family decided to move to comfort measures, and he succumbed to his illness. DISCUSSION HLH is a rare immune-mediated life-threatening syndrome that can involve multiple organ systems. HLH is classified into primary (genetic) and secondary (reactive), which is further subclassified into infectious and autoimmune, although about a third of adult cases are multifactorial.1 HLH overlaps clinically with many more common conditions frequently leading to a delay in diagnosis. Our patient met 8/8 criteria for HLH, with hyperferritenemia, hypofibrinogenemia, hypertriglyceridemia, pancytopenia, hepatosplenomegaly, low NK activity, elevated IL-2, and evidence of hemophagocytosis on bone marrow biopsy. Liver injury is very common in HLH, with up to 85% of patients with secondary HLH having abnormal liver function.4 The liver injury is possibly secondary to cytokine storm and infiltration of the parenchyma by activated hemophagocytic histiocytes.5 Liver transplantation is frequently contraindicated in these patients secondary to severe systemic illness. Our patient's HLH was most likely secondary to his active HBV infection, as his viral load increased 25-fold, likely inciting the aberrant immune response. The reason for his rapid increase in HBV viral load is unclear, since genotype revealed no mutations and he was on 2 active antivirals. Extensive evaluation for additional infectious causes or malignancy was unremarkable. Although HLH has been associated with HIV infection, his HIV was less likely contributory, as his viral load became undetectable on treatment over his hospital course.2 The brief period without antiviral medications after discharge could have resulted in acute HBV hepatitis and subsequent worsening of HLH because laboratory markers for HLH dramatically worsened on his second admission. On literature review, HLH was attributed to HBV in 13 cases.6–10 Two detailed case reports treated the patient with etoposide and dexamethasone per HLH-94 protocol.8,9 To the best of our knowledge, this is the first reported case of HLH attributed to HBV with HIV coinfection. As about 10% of patients with HIV have HBV coinfection, HLH should be considered in coinfected febrile patients with cytopenias.11 Early recognition and treatment of HLH is essential for survival. DISCLOSURES Author contributions: H. Blaney wrote the manuscript. D. Thotakura reviewed the literature. L. Sisco edited the manuscript and reviewed the literature and is the author guarantor. Financial disclosure: None to report. Informed consent was obtained for this case report.	Fatal	ReactionOutcome	CC BY-NC-ND	33553464	20,925,366	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Ascites'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	AMIKACIN, AZITHROMYCIN ANHYDROUS, CEFOXITIN SODIUM, CYTARABINE, ETOPOSIDE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN, MELPHALAN, PIPERACILLIN SODIUM\TAZOBACTAM SODIUM, TIGECYCLINE	DrugsGivenReaction	CC BY-NC-ND	33553476	19,985,973	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'C-reactive protein increased'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	CYTARABINE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN	DrugsGivenReaction	CC BY-NC-ND	33553476	19,882,896	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Cellulitis'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	AMIKACIN, AZITHROMYCIN ANHYDROUS, CEFOXITIN SODIUM, CYTARABINE, ETOPOSIDE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN, MELPHALAN, PIPERACILLIN SODIUM\TAZOBACTAM SODIUM, TIGECYCLINE	DrugsGivenReaction	CC BY-NC-ND	33553476	19,985,973	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chronic graft versus host disease in eye'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	CYTARABINE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN	DrugsGivenReaction	CC BY-NC-ND	33553476	19,882,896	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chronic graft versus host disease in skin'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	CYTARABINE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN	DrugsGivenReaction	CC BY-NC-ND	33553476	19,882,896	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chronic graft versus host disease oral'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	CYTARABINE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN	DrugsGivenReaction	CC BY-NC-ND	33553476	19,882,896	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Febrile neutropenia'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	CYTARABINE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN	DrugsGivenReaction	CC BY-NC-ND	33553476	19,882,896	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Gastrointestinal disorder'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	AMIKACIN, AZITHROMYCIN ANHYDROUS, CEFOXITIN SODIUM, CYTARABINE, ETOPOSIDE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN, MELPHALAN, PIPERACILLIN SODIUM\TAZOBACTAM SODIUM, TIGECYCLINE	DrugsGivenReaction	CC BY-NC-ND	33553476	19,985,973	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Graft versus host disease in liver'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	CYTARABINE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN	DrugsGivenReaction	CC BY-NC-ND	33553476	19,882,896	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Wound decomposition'.	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	AMIKACIN, AZITHROMYCIN ANHYDROUS, CEFOXITIN SODIUM, CYTARABINE, ETOPOSIDE, FILGRASTIM, FLUDARABINE PHOSPHATE, IDARUBICIN, MELPHALAN, PIPERACILLIN SODIUM\TAZOBACTAM SODIUM, TIGECYCLINE	DrugsGivenReaction	CC BY-NC-ND	33553476	19,985,973	2021-02
What was the dosage of drug 'FLUDARABINE PHOSPHATE'?	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	INDUCTION CHEMOTHERAPY ; IDA-FLAG REGIMEN	DrugDosageText	CC BY-NC-ND	33553476	19,985,973	2021-02
What was the dosage of drug 'IDARUBICIN'?	Mycobacterium abscessus Gastric Band Infection Complicated by Immune Reconstitution Inflammatory Syndrome and Cured in the Context of Allogeneic Hematopoietic Stem Cell Transplantation. We present a case of abdominal gastric band-associated Mycobacterium abscessus infection, manifesting after the onset of acute myeloid leukemia, complicated by immune reconstitution inflammatory syndrome (IRIS), and cured while receiving an allogeneic hematopoietic stem cell transplant. IRIS should be considered in less classical situations where there is unexplained clinical deterioration. Mycobacterium abscessus complex is a group of rapidly growing mycobacteria (RGM) that cause predominantly cutaneous and pulmonary infection, and postsurgical infection including foreign body involvement is well described [1]. There is a variety of clinical manifestations of RGM in solid organ transplant recipients, with central venous catheter (CVC) infection being the predominant manifestation in hematopoietic stem cell transplant (HSCT) recipients [2]. Immune reconstitution inflammatory syndrome (IRIS) is an uncommon phenomenon in nontuberculous mycobacteria (NTM) infection in non-HIV patients. We present an unusual case of M. abscessus complex infection associated with gastric band infection, complicated by IRIS, and cured in the context of allogeneic HSCT for acute myeloid leukemia (AML). CASE REPORT A 54-year-old woman had undergone gastric band insertion in 2008 as a weight loss procedure without prior surgical complications and no port manipulation since insertion. She developed AML in July 2014 and was treated with idarubicin, cytarabine, etoposide induction, and 2 high-dose cytarabine consolidation chemotherapy cycles. In March 2015, she presented with relapse of AML and 1 month of epigastric pain. Her subsequent progress is detailed in Figure 1. Upper gastrointestinal endoscopy demonstrated erosion of the gastric band into the gastric lumen, which was laparoscopically removed but not sent for culture. Following surgical recovery, she had induction IDA-FLAG (idarubicin, high dose cytarabine, fludarabine, filgrastim) chemotherapy, complicated by wound breakdown and cellulitis at the right and left abdominal laparoscopic port sites. Computed tomography (CT) of the abdomen showed abdominal wall collections deep to the port sites (Figure 2), phlegmon adjacent to the lesser curvature of the stomach suggestive of persistent leak at the site of the previous gastric band, and moderate ascites. Abdominal wall tissue and ascite samples on cytology showed an acute inflammatory infiltrate of predominantly polymorphonuclear leucocytes and lymphocytes, by microscopy 3+ acid fast bacilli were seen, and on blood and chocolate agar grew an RGM, which on matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was Mycobacterium abscessus (score >2.0) and was identified by in-house polymerase chain reaction (PCR) as Mycobacterium abscessus complex (the PCR is validated for identification of M. abscessus complex but not to the species level). Phenotypic susceptibility testing by the Sensititre RAPMYCO microdilution panel (Thermo Fisher, Inc., Cleveland, OH, USA) showed a minimum inhibitory concentration for amikacin of 16 mg/L (susceptible), for cefoxitin of 32 mg/L (intermediate), for imipenem of 32 mg/L (intermediate), for tigecycline of 0.5 mg/L (no breakpoint), for linezolid of 16 mg/L (resistant), for trimethoprim/sulfamethoxazole of ≥8/152 mg/L (resistant), for ciprofloxacin of ≥4 mg/L (resistant), for moxifloxacin of ≥8 mg/L (resistant), and for minocycline of ≥8 mg/L (resistant). Clarithromycin showed inducible resistance (2 mg/L at 5 days and >16 mg/L at 14 days after incubation). Azithromycin phenotypic testing and erm gene determination were not performed. Notably, the patient had a history of rash with meropenem and was treated from April 21, 2015 (Day 0), with cefoxitin 12 g daily, tigecycline 100 mg daily, and azithromycin 500 mg daily. Azithromycin was continued in the regimen, as it is well tolerated and there is uncertainty regarding whether azithromycin induces erm gene expression to the same extent as clarithromycin [3, 4]. Neutrophil count recovered to >1.0 ×109/L by Day +20 (May 11, 2015), following which she developed worsening abdominal wall abscesses with new imaging findings of peritoneal nodularity (Figure 3). C-reactive protein (CRP) was mildly elevated (30 mg/L) and had not significantly increased. Repeat mycobacterial wound cultures were negative, and there was no response several weeks after intravenous amikacin 15 mg/kg/d was added to the regimen on June 4, 2015. We suspected IRIS, and there was prompt clinical, CRP (reduced to <1 mg/L), and radiological improvement to oral prednisolone 60 mg daily commenced Day +55 (June 15, 2015), which was reduced 10 mg per week. Amikacin was ceased. AML was in remission, and she proceeded to sibling allogeneic HSCT with fludarabrine/melphalan conditioning on Day +113 (August 12, 2015). With engraftment, there was a rapid rise of CRP to 200 mg/L with transient fever. The patient was given piperacillin-tazobactam for 5 days, and her CRP fell to 20–50 mg/L, at which point there was deterioration in the abdominal wounds, which were culture negative, and there was a rapid response in wounds and reduction in CRP after increasing prednisolone to 25 mg daily on Day +139 (September 7, 2015) with slow tapering. The dose was increased to 25 mg daily on Day +239 (December 15, 2015) for possible liver graft-vs-host-disease (GVHD) and was slowly weaned. Cefoxitin, tigecycline, and azithromycin were continued through until Day +281 (January 27, 2016; 6 months post-transplant). Following antibiotic cessation, there was no recurrence of infection. She subsequently developed chronic GVHD of skin, eyes, and mouth, but at last review in November 2020, she was alive and free of leukemia. Figure 1. Timeline of patient progress from the diagnosis of relapsed acute myeloid leukemia and laparoscopic band–associated Mycobacterium abscessus complex infection, March 2015, demonstrating periods of deterioration due to immune reconstitution inflammatory syndrome following neutrophil recovery after IDA-FLAG chemotherapy (June 4, 2015) and following engraftment after allogeneic hematopoietic stem cell transplant (August 29, 2015). The left y-axis shows units for neutrophil count (×109/L) and prednisolone dose (prescribed dose is 5 times the represented units in mg). The right y-axis shows units for C-reactive protein (mg/L). Abbreviations: AML, acute myeloid leukemia; CT, computed tomography; HSCT, hematopoietic stem cell transplant; IDA-FLAG, idarubicin, high dose cytarabine, fludarabine, filgrastim; IRIS, immune reconstitution inflammatory syndrome. Figure 2. Computed tomography of the abdomen at diagnosis, April 21, 2015, demonstrating abdominal wall collections deep to laparoscopic port sites. Figure 3. Computed tomography of the abdomen, June 15, 2015, demonstrating peritoneal nodularity (arrow) at ~2 months after the commencement of mycobacterial treatment and following neutrophil recovery. DISCUSSION M. abscessus complex has caused infections of foreign bodies such as tympanostomy tubes, peritoneal dialysis catheters, breast implants, prosthetic joints, and prosthetic vascular grafts [1]. In our case, we infer that M. abscessus complex infection arose primarily from the gastric band because it initially manifested by erosion into the stomach, there was radiological evidence of infection at the lesser curvature of the stomach and peritoneum, and ascite culture was positive. Our conclusion is that abdominal wall and laparoscopic port site infection were secondary manifestations of deeper involvement. In the largest case series of RGM gastric band infections reported on the time period 2005–2011, 11 cases were due to Mycobacterium fortuitum and 7 were due to M. abscessus. Although the time from band placement to infection ranged from 21 days to 8 years, the majority presented within 3 months, manifested by peritonitis, band erosion, or chronic ulceration at the port site. The port was considered the primary location of infection in 10 patients (56%) [5]. We cannot be sure when infection of the gastric band occurred in our patient; however, it is possible that it occurred at insertion in 2008, remaining latent until the immunocompromise incurred 7 years later from AML and chemotherapy. We did not perform hospital environmental sampling for M. abscessus culture, but we believe that hospital-acquired infection is unlikely, as there were no other contemporaneous M. abscessus cases identified, nor any prior or subsequent M. abscessus cases related to the hospital environment or water supply. Nontuberculous mycobacterial infections complicate 0.4% to 4.9% of HSCTs. Central venous catheter (CVC)–related infections with RGM and pulmonary infections with Mycobacterium avium complex (MAC) and other slow-growing mycobacteria are the predominant presentations, with cutaneous and disseminated infection being less common [2]. The largest series of RGM CVC infections (n = 23, M. abscessus = 10) after HSCT occurred at a median of 61 days post-transplant. The catheter was removed in all but 2 cases. Combination antibiotic therapy was given for a median of 6–7 weeks in the case of tunnel infection or bacteremia and 3 weeks for exit site infection. Cure was achieved in 21 patients, while 2 patients died of unrelated causes [6]. In the other major publication of RGM infection after HSCT, 6/7 were CVC-related, presenting 7–90 days post-transplant, and 6/7 infections resolved [7]. In our reported case, there was no suggestion of CVC infection, and blood cultures were negative. M. abscessus complex is notable for its antibiotic resistance, often only susceptible to amikacin and tigecycline, with intermediate susceptibility to cefoxitin and imipenem. Macrolide susceptibility is species dependent. We were highly concerned about proceeding to HSCT in the context of extensive active infection with this very resistant and virulent organism, but we had no other option. We have demonstrated that cure of this difficult infection can occur even through HSCT. Likewise, favorable results were obtained for 3 RGM CVC-related infections diagnosed shortly before HSCT [6]. A highly illustrative aspect of the case was the occurrence of IRIS. There are no agreed-upon diagnostic criteria for IRIS even where it has been more extensively studied in HIV patients. Recognized features are a new or worsening inflammatory condition after reconstitution of immunity that is not explained by another cause such as drug-resistant infection, superinfection, drug allergy, or noncompliance [8]. It is commonly responsive to corticosteroids, although this is not a component of proposed diagnostic criteria [8–10]. In our case, IRIS was diagnosed first following neutrophil recovery after chemotherapy and second after engraftment post-HSCT, when there was increased inflammation and deterioration in the wounds, radiological deterioration, sterile cultures, modestly raised CRP, no response to antibiotic augmentation, and rapid response to systemic corticosteroids. NTM-associated IRIS has been predominantly described with MAC infection in HIV patients [11], with few reports in neutropenic or transplant patients [9, 10, 12, 13]. The largest report by Manion and colleagues described 3 patients with primary immunodeficiency and disseminated MAC infection who underwent allogeneic HSCT. They had features of IRIS related to various stimuli such as neutrophil recovery, donor lymphocyte infusion, and when immunosuppression for GVHD was reduced. At times of IRIS, they demonstrated elevated CRP, interferon-γ, tumor necrosis factor–α, interleukin-6, interleukin-18, and acquisition of a CD4+ T lymphocyte MAC-specific cytokine response. Mycobacterial and IRIS treatment were not described [13]. RGM-associated IRIS has been reported once before in an AIDS patient who was diagnosed with disseminated M. abscessus complex infection 4 weeks after commencing antiretroviral therapy (ART), who improved with azithromycin, standard 4-drug tuberculosis (TB) therapy, and ART continuation, but without steroids. Mycobacterial species and antibiotic susceptibilities were not reported. This appears to have been a case of “unmasking” IRIS [14]. Prednisolone 20–40 daily for 4–8 weeks has been suggested for moderate to severe MAC-associated IRIS in HIV patients when there is no response to nonsteroidal anti-inflammatory drugs. Tumor necrosis factor inhibitors and thalidomide have been utilized in steroid-refractory TB-associated IRIS in HIV infection [15]. In summary, we present a highly unusual and illustrative case of M. abscessus complex infection associated with distant gastric band insertion, manifesting after the onset of AML and chemotherapy, complicated by IRIS, that was controlled and finally cured while receiving HSCT. Outcomes are not always poor in patients with difficult infection requiring HSCT, and we are reminded to consider IRIS in less classical situations where there is unexplained clinical deterioration. Acknowledgments Financial support. There was no funding for this manuscript. Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Patient consent. Written informed consent was obtained from the patient. The design of the work accords to the Australian National Statement on Ethical Conduct in Human Research [16], and was approved by Fiona Stanley Hospital.	INDUCTION CHEMOTHERAPY ; IDA-FLAG REGIMEN	DrugDosageText	CC BY-NC-ND	33553476	19,985,973	2021-02
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Off label use'.	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	CARBOPLATIN, ETOPOSIDE, IFOSFAMIDE, MEMANTINE HYDROCHLORIDE	DrugsGivenReaction	CC BY-NC-ND	33553701	19,043,197	2021-03
What was the dosage of drug 'CARBOPLATIN'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553701	19,053,942	2021-03
What was the dosage of drug 'ETOPOSIDE'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553701	19,053,942	2021-03
What was the dosage of drug 'IFOSFAMIDE'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553701	19,053,942	2021-03
What was the dosage of drug 'MEMANTINE'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553701	19,053,942	2021-03
What was the outcome of reaction 'Demyelination'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	Recovered	ReactionOutcome	CC BY-NC-ND	33553701	19,185,214	2021-03
What was the outcome of reaction 'Disturbance in attention'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	Recovered	ReactionOutcome	CC BY-NC-ND	33553701	19,154,034	2021-03
What was the outcome of reaction 'Memory impairment'?	Chemoradiation induced multiple sclerosis-like demyelination. We report the case of a 28-year-old man, diagnosed with a non-secreting, non-metastatic suprasellar germinoma treated with chemoradiation who developed, four months after completion of radiation therapy, multiple discrete demyelinating lesions mimicking multiple sclerosis (MS). The patient had no previous diagnosis of MS and the neuroimaging studies performed both at the time of diagnosis and after chemotherapy, pre-irradiation, showed no evidence of white matter lesions. He remained asymptomatic, with no focal neurological deficits. Biochemical analysis of the CSF was positive for the intrathecal synthesis of IgG with oligoclonal bands. Follow-up MRI six months later showed a spontaneous decrease in lesion size and resolution of associated inflammatory signs, with lesions remaining stable in number. We discuss the potential origin of these white matter lesions, which may correspond to MS-like late-delayed demyelination secondary to chemoradiation therapy, in a previously predisposed patient. 1 Introduction Chemotherapy and radiation therapy-induced neurotoxicity is a well-known secondary effect in cancer patients with both treatments having a negative impact upon neural precursor cells, mainly of oligodendrocyte lineage affecting axonal myelination [1,2]. Recent research has shown that chemotherapy depletes oligodendrocyte lineage cells in humans and leads to a persistent try-glial dysregulation via microglial activation and induction of a chronic inflammatory state that disrupts the gliogenic microenvironement and glial homeostasis [1]. This mechanism resembles other neurological diseases featuring myelin dysfunction such as MS [3] and Alzheimer's disease [4]. Activated microglia blocks the proliferation and dysregulates the differentiation of oligodendrocyte precursor cells (OPCs) leading to dysmyelination. Moreover, the activation of reactive astrocytes, promotes oligodendrocyte death increasing neurotoxicity [1]. A similar process takes place after radiation exposure with cranial irradiation inducing chronic microglial inflammation and leading to decreased hippocampal neurogenesis [7,8]. Radiotherapy can lead to necrosis of white matter tracts, axonal degeneration and vascular injury [9]. Demyelination, one of the hallmarks of this radiation-induced neurotoxicity, is presumed to result from the enhanced radiosensitivity of OPCs [2,10]. Moreover, radiation-induced damage to the microvasculature, prompting to hemorrhagic and ischemic events, local necrosis and blood-brain-barrier disruption (with resulting vasogenic edema), facilitates CNS influx of inflammatory cells, further contributing to a pro-inflammatory state and persistent demyelination [9]. A diffusion tensor MR imaging study has shown that early demyelination is dose-dependent, affecting regions exposed to high radiation doses, up to three months after radiotherapy. However, this process is continuous and progressive diffuse demyelination, not limited to high-dose volumes, can be seen 4 to 6 months following radiotherapy [11]. This case report concerns a patient with no prior clinical or radiological signs of MS who, 4 months after being treated with chemoradiation for a suprasellar germinoma, developed demyelinating lesions diagnostic of MS, according to MAGNIMS criteria [12]. To the best of our knowledge MS-like demyelinating plaques have not been previously described as a direct consequence of chemotherapy and/or radiotherapy in non-MS patients. 2 Case report The patient is a previously healthy 28 year-old-man who presented with progressive fatigue, polyuria, polydipsia and anejaculation. His neurological and neuroophthalmological exams were unremarkable and his family history was non-contributory. Laboratory investigation disclosed hypopituitarism including diabetes insipida, hypogonadothrophic hypopituitarism and central hypothyroidism. Magnetic resonance imaging (MRI) of the brain, sella turcica and neuroaxis (Fig. 1) revealed a mass lesion in the pituitary infundibulum and pituitary stalk, showing moderate enhancement after gadolinium administration. The brain parenchyma was unremarkable and there were no signs of subependymal or leptomeningeal enhancement to suggest cerebrospinal fluid (CSF) seeding.Fig. 1 MRI of the brain at diagnosis: Sagittal T1W (A), T2W (B) and CE T1W (C) and coronal CE T1W (D) images demonstrate an enhancing mass lesion in the infundibulum and pituitary stalk protruding into the suprasellar cistern (arrows). An incidental peripheral enhancing epiphyseal cyst is also noted (dashed arrows). Axial FLAIR images (E) throughout the brain at this stage were unremarkable with no evidence of demyelinating WM lesions. Fig. 1 Lumbar puncture disclosed normal opening pressure and crystalline CSF. Cytologic analysis was negative for neoplastic cells and biochemical analysis showed the presence of intrathecal synthesis of IgG with oligoclonal bands (IgG 3.78 mg/dl, Freedman pattern 2). Bacteriologic and virologic CSF testing were also negative. Seric and CSF levels of α-fetoprotein and β-HCG were normal. Surgical biopsy of the pituitary stalk mass, performed under neuronavigation revealed a germinoma. With a diagnosis of a non-secreting, non-metastatic supra-sellar germinoma the patient was treated according to the SIOP (International Society of Paediatric Oncology) protocol with a 3 multidrug chemotherapy regimen including carboplatin, etoposide and ifosfamide followed by radiation therapy. MRI performed 10 days after completing the chemotherapy regimen showed a complete macroscopic response and no signs of complication (Supplementary Fig. 1). The patient then received whole-ventricular irradiation (24 Gy given in 15 fractions of 1.6 Gy/cycle/day) using Volumetric Modulated Arc Therapy (VMAT) with concomitant memantine. According to Common Terminology Criteria for Adverse Events (CTCAE), toxicity included grade 1 hepatotoxicity, grade 3 neutropenia and grade 4 thrombocytopenia during CT and grade 2 headache and vomiting during RT. Four months after completing the treatment protocol, MRI of the brain and spine (Fig. 2) showed complete tumor response and was remarkable for the interval appearance of multiple discrete white matter lesions affecting the posterior fossa and supratentorium, distributed throughout the deep and periventricular white matter with a typical orientation perpendicular to the body of the lateral ventricles and involving the calloso-septal interface (“Dawson's fingers”). Some of these lesions showed a subtle halo of restricted diffusion and perilesional edema suggesting inflammatory activity. No lesions were found in the spinal cord or optic nerves.Fig. 2 MRI of the brain 3 months after completion of the CRT protocol: Pre- (A) and post‑gadolinium (B) axial T1W, axial T2W (C), axial FLAIR (D) and DWI images, b1000 (E) and ADC maps (F) demonstrate the interval appearance of multiple discrete deep and periventricular white matter lesions hypointense on T1 and hyperintense on T2W images, with no contrast enhancement on post‑gadolinium T1W images, most with facilitated diffusion and a few with a thin rim of restricted diffusion. Most lesions are located in the deep white matter of the centrum semi-ovale, some affecting the pericallosal region oriented perpendicular to the body of the lateral ventricles (arrows), with the largest lesion in the peri-atrial white matter on the left side (dashed arrows). This lesion shows a peripheral digitiform T2W/FLAIR hyperintense rim consistent with peripheral edema with no significant mass effect upon the ventricular trigone or adjacent sulci. Also noted are 2 lesions in the posterior fossa, one in the left lateral aspect of the pons and the other in the posterior aspect of the middle cerebellar peduncle (short arrows) and a lesion in the left temporal lobe adjacent to the lateral margin of the temporal horn (arrowhead). Fig. 2 A second lumbar puncture continued to show oligoclonal bands and intrathecal synthesis of IgG in the CSF (IgG 2.01 mg/dl, Freedman pattern 2) with no additional biocytochemical changes. Panel of infection, autoimmunity, including autoimmune encephalitis and anti-neuronal antibodies (Ab), were negative. Visual evoked potentials (VEP) showed normal amplitude and median latencies of the main peak (P100) with no asymmetries. Clinical evaluation did not reveal focal neurological deficits. The patient complained of mild memory impairment recalling words, difficulty concentrating which prevented him from resuming his professional life and, although he was a sportsman before, he had no thrive for sports. No active treatment was deemed appropriate and the patient remained under surveillance. Subsequent MRI, performed 10 months after treatment (Supplementary Fig. 2), showed a slight decrease in the size of the largest demyelinating lesion located in the peri-atrial white matter and resolution of the associated vasogenic edema. It also showed interval disappearance of the faint peripheral contrast enhancement and restricted diffusion of the lesions. No new demyelinating lesions and no evidence of tumor recurrence were seen. On the last follow up visit, one year after treatment, the patient remained asymptomatic with no focal neurologic deficits, specifically denying memory and concentration difficulties. He resumed his full-time job and his normal social habits. 3 Discussion This case is remarkable for the appearance of a neuroimaging picture compatible with MS, 4 months after chemoradiotherapy (CRT) for a suprasellar germinoma, in a previously healthy young adult with no family history of MS and no previous white matter lesions, showing intrathecal synthesis of IgG and oligoclonal bands in the CSF. There are 2 potential explanations for this occurrence: a toxic effect from CRT leading to an unusual demyelinating pattern simulating MS or the coincidental development of a clinically silent MS in a previously predisposed patient. The following discussion will address the existing evidence for both these hypotheses.1. CRT-induced toxicity simulating MS Neurotoxicity is a well-known side effect of both chemo and radiation therapy and share a common denominator: depletion of oligodendrocyte precursor cells and disruption of oligodendrocyte lineage dynamics leading to axonal demyelination, triggered by microglial activation and inflammation [1]. Several chemotherapy agents, in particular antimetabolites and alkylating drugs, have been shown to induce an acute and most often reversible leukoencephalopathy via microglial activation and inflammation [3,5,6]. Carboplatin and ifosfamide are both neurotoxic alkylating agents. The former most often associated with neurovascular dysregulation leading to posterior reversible leukoencephalopathy [13,14] and, the latter, responsible for a toxic leukeoencephalopathy syndrome seen in 10–20% of patients which is not usually associated with structural white matter changes on conventional MRI studies [15,16]. Neuroimaging findings of radiation-induced leukoencephalopathy comprise discrete or diffuse and confluent white matter lesions, solid contrast enhancing lesions, and necrotic lesions with thick, irregular, contrast-enhancing borders eliciting vasogenic brain edema which, in a chronic stage, may evolve to cystic porencephaly and brain atrophy [[17], [18], [19]]. Advanced diffusion tensor MR imaging techniques, have been shown to depict early microstructural white matter changes, related to increased vascular permeability and neuroinflammation across all radiation doses, even below 10 Gy [20]. In our case the demyelinating lesions disclosed on the MRI scan 4 months after RT, strongly suggest an acute/subacute inflammatory demyelinating process with multiple discrete lesions showing restricted diffusion and vasogenic edema. Interestingly, the superimposition of the irradiated volumes on the post-RT axial FLAIR MR images (Fig. 3), demonstrates that almost all demyelinating lesions lay within the 20 Gy isodose curve. Therefore, although the distribution and pattern of the white matter lesions resemble that of MS, it is conceivable that they may have resulted from the superimposed neurotoxic effect of radiation therapy upon an already susceptible ground of microglial inflammation induced by the previous chemotherapy.2. Coincidental clinically silent MS in a predisposed patient Fig. 3 Dose distribution after registration of CT planning upon axial FLAIR images of the follow-up MRI scan obtained 3 months after treatment. The 24 Gy isodose curve (red) shows the volume irradiated with the prescribed dose. The 20 Gy isodose curve (blue) represents the volume that received at least 20 Gy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 3 The morphology and distribution of the demyelinating lesions disclosed on the post-radiation MRI scan have a typical pattern for MS according to the MAGNIMS criteria [12]. These neuroimaging findings, in an otherwise asymptomatic patient, strongly favor a radiologically isolated syndrome (RIS), a clinically silent form of MS. Multiple factors (clinical, laboratory and radiologic) have been associated with the likelihood to progress from subclinical to clinical MS including the presence of OCBs, younger age, male gender, positive family history and abnormal visual evoked potentials [21]. On MRI, the presence of gadolinium-enhancing and spinal cord lesions are predictors of conversion to a full-blown MS [12,21]. Interestingly, our patient had oligoclonal bands in the CSF prior to the development of the WM lesions. While not exclusive for MS, OCBs have a reported positive predictive value (PPV) ranging between 61 and 94% depending on the reference population and on the integration with other CSF findings [22]. However, OCBs are present in 6% of cancer patients and have been reported in at least 6 cases of germinoma [[23], [24], [25]]. Although the development of this RIS could have been coincidental, the temporal relationship with the CRT is hard to be neglected. Eventhough the effects of brain irradiation in MS patients remain elusive, it seems intuitive that MS patients carry a higher risk of chemoradiation-induced neurotoxicity as both processes target oligodendrocytes. In addition, it is likely that RT-induced BBB disruption, facilitates the influx of autoreactive T-lymphocytes in MS-predisposed patients. Previous studies have reported an increased susceptibility of MS patients to radiation-induced demyelination, in some cases, precipitating disease reactivation in patients with long-lasting quiescent disease [[26], [27], [28], [29], [30], [31]]. The largest review study found in the literature is a retrospective evaluation of 15 MS patients, treated with external beam radiation therapy between 1976 and 2014 [32].This study supported the impression that MS patients are at higher risk for neurotoxicity compared to non-MS patients. Moreover, 3 patients who had probable MS, evolved to full-blown MS after irradiation. It is conceivable that since the use of more conformal radiotherapy techniques IMRT, VMAT and radiosurgery, sparing healthy brain tissue, these results may not be reproduced. Large retrospective studies will be required to clarify this issue. In our case, while irradiating the whole ventricular system, the periventricular WM included in the low-dose bath encompasses most of the lesions, making it quite likely that radiation therapy was the trigger for the development of the white matter lesions following prior chemo-sensitization in a potentially predisposed patient (with CSF OCBs). The weight of each independent factor is hard to determine. In fact, we favor this hypothesis as the most likely explanation for the appearance of the demyelinating lesions. To our knowledge such a pattern of demyelination has not yet been described in association with radiation nor with the chemotherapy agents used in this multidrug regimen (etoposide, carboplatin and ifosfamide). Since the clinical and imaging features and the temporal evolution of the demyelinating lesions of our patient did not suggest other demyelinating diseases such as acute disseminated encephalomyelitis (ADEM) or neuromyelitis optica (NMO) we did not search for aquaporin 4 (AQP4) or myelin oligodendrocyte glycoprotein (MOG) antibodies. In fact, an MRI of the neuro-axis excluded spinal cord lesions and the visual evoked potentials were normal. However, since MOG antibody-associated inflammatory demyelinating diseases represent an oligodendropathy [33,34], it would be interesting to find whether or not these antibodies were present in our patient. In a short follow-up period of one year, the patient did not develop neurological symptoms and there has been no progression of the neuroimaging findings. He will remain in close surveillance to ascertain whether or not he will evolve to a full-blown MS. The following are the supplementary data related to this article.Supplementary Fig. 1 MRI of the brain after 4 cycles of chemotherapy (cisplatin, etoposide and ifosfamide) and before radiation therapy: Sagittal T1W (A), Axial T2W (B) and FLAIR (C) images throughout the brain show complete macroscopic response of the pituitary stalk lesion and no signs of treatment complication namely, no evidence of white matter lesions. Supplementary Fig. 1Supplementary Fig. 2 Follow-up MRI of the brain 6 months after CRT: Post-gadolinium axial T1W (A) and axial FLAIR (B) images demonstrate a slight decrease in the size of the largest peri-trigonal lesion with resolution of the peripheral edema and restricted diffusion (dashed arrows). The remainder demyelinating lesions remain similar in size and number and no new lesions appeared in the interval. Supplementary Fig. 2 Study funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. CRediT authorship contribution statement Alexandra Borges: Conceptualization, Data curation, Writing - original draft, Writing - review & editing. Daniela Garcez: Conceptualization, Data curation, Writing - review & editing. Cátia Pedro: Data curation, Writing - review & editing. João Passos: Conceptualization, Supervision, Writing - review & editing. Declaration of Competing Interest None.	Recovered	ReactionOutcome	CC BY-NC-ND	33553701	19,154,034	2021-03
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Corneal oedema'.	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	AFLIBERCEPT, MOXIFLOXACIN HYDROCHLORIDE	DrugsGivenReaction	CC BY-NC-ND	33553806	19,401,202	2021-03
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Eye pain'.	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	AFLIBERCEPT	DrugsGivenReaction	CC BY-NC-ND	33553806	19,403,157	2021-03
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Fibrin'.	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	AFLIBERCEPT, MOXIFLOXACIN HYDROCHLORIDE	DrugsGivenReaction	CC BY-NC-ND	33553806	19,401,202	2021-03
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pain'.	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	AFLIBERCEPT, MOXIFLOXACIN HYDROCHLORIDE	DrugsGivenReaction	CC BY-NC-ND	33553806	19,401,202	2021-03
What was the administration route of drug 'AFLIBERCEPT'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	Intraocular	DrugAdministrationRoute	CC BY-NC-ND	33553806	19,403,157	2021-03
What was the administration route of drug 'MOXIFLOXACIN HYDROCHLORIDE'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	Topical	DrugAdministrationRoute	CC BY-NC-ND	33553806	19,401,202	2021-03
What was the administration route of drug 'POVIDONE-IODINE'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	Sunconjunctival	DrugAdministrationRoute	CC BY-NC-ND	33553806	19,475,982	2021-03
What was the administration route of drug 'PROPARACAINE'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	Topical	DrugAdministrationRoute	CC BY-NC-ND	33553806	19,475,982	2021-03
What was the dosage of drug 'LIDOCAINE'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553806	19,475,982	2021-03
What was the dosage of drug 'POVIDONE-IODINE'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553806	19,475,982	2021-03
What was the dosage of drug 'PROPARACAINE'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	UNKNOWN	DrugDosageText	CC BY-NC-ND	33553806	19,475,982	2021-03
What was the outcome of reaction 'Retinal haemorrhage'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	Recovering	ReactionOutcome	CC BY-NC-ND	33553806	19,403,157	2021-03
What was the outcome of reaction 'Visual acuity reduced'?	Nutritionally variant streptococci causing endophthalmitis associated with intravitreal anti-vascular endothelial growth factor injection. To describe the clinical course and microbial properties of the first two reported cases of nutritionally variant Streptococci (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI). A 74 year-old female developed Granulicatella adiacens endophthalmitis following IVI. The patient underwent a pars plana vitrectomy and visual acuity recovered to 20/30 in six weeks. Similarly, an 88 year-old male developed Abiotrophia defectiva endophthalmitis after IVI. After a pars plana vitrectomy, the visual acuity recovered to 20/60 at five weeks. Endophthalmitis due to Streptococcus species has traditionally resulted in uniformly poor visual outcomes. However, nutritionally variant Streptococci, now reclassified as Granulicatella and Abiotrophia species, appear to have a less aggressive clinical course and better visual acuity outcomes. To the authors' knowledge, these are the first reports of nutritionally variant Streptococci following IVI related endophthalmitis. 1 Introduction Infectious endophthalmitis following intravitreal anti-vascular endothelial growth factor injection (IVI) is a rare but potentially catastrophic complication. The reported incidence of endophthalmitis after IVI remains low, about 0.02% in retrospective reviews.1,2 Coagulase negative Staphylococcus and Streptococcus species are well recognized as the most common isolates, with generally poor visual outcomes associated with Streptococcus species.2, 3, 4 We report the first cases of nutritionally variant Streptococci (NVS) (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis after IVI injection. The visual acuity outcomes were better than expected for Streptococcus species. 2 Findings See summarized findings in Table 1.Table 1 Clinical features of patients with nutritionally variant Streptococcus endophthalmitis following intravitreal anti-VEGF injection. Table 1Patient Diagnosis Causative Organism Medication Days to Presentation Pre Injection VA VA at Presentation Final VA 1a AMD Granulicatella adiacens Aflibercept 2 20/25 HM 20/30 2b AMD Abiotrophia defectiva Aflibercept 2 20/20 LP 20/60 Key: AMD = age related macular degeneration, VA = visual acuity, HM = hand motion, LP = light perception. a Patient 1 underwent vitreous tap/injection with intravitreal injection of vancomycin, ceftazidime, and triamcinolone acetonide at initial presentation, and pars plana vitrectomy with intravitreal triamcinolone acetonide 9 days after presentation. b Patient 2 underwent vitreous tap/injection with intravitreal injection of vancomycin and ceftazidime at initial presentation, and pars plana vitrectomy with intravitreal vancomycin and ceftazidime 6 days after presentation. 2.1 Case 1 A healthy 74 year-old female with a history of neovascular age-related macular degeneration (AMD) who had undergone approximately 131 intravitreal anti-VEGF injections to both eyes received an intravitreal injection of aflibercept in the right eye for persistent subretinal fluid. During the injection, both the patient and physician wore a mask. The IVI injection technique consisted of inferotemporal subconjunctival lidocaine, followed by topical 5% povidone-iodine (PVI) eyelid scrubs and topical PVI to the conjunctiva fornix. After a lid speculum was placed, additional conjunctival PVI was applied, followed by a topical PVI-soaked pledget held on the injection site for 15 seconds prior to injection. The patient presented two days later with right eye pain, redness, and a decrease in vision. At presentation, best corrected visual acuity (BCVA) of the right eye was hand motion (decreased from 20/25) and examination revealed conjunctival injection, hypopyon, dense vitritis, and intraretinal hemorrhages (Fig. 1). The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL), ceftazidime (2.25 mg/0.1 mL), and triamcinolone acetonide (0.40 mg/0.1 mL). No oral antibiotics were prescribed. Because of persistent vitreous opacities and inflammation, nine days after initial presentation the patient underwent pars plana vitrectomy with injection of triamcinolone. Six weeks after initial presentation, the BCVA improved to 20/30 with a marked reduction in intraretinal hemorrhages (Fig. 2). Final microbiology report of the original vitreous sample showed moderate growth of Granulicatella adiacens. Sensitivities were not performed.Fig. 1 Vitreous opacities and intraretinal hemorrhages on day of presentation with Granulicatella adiacens endophthalmitis after IVI. Fig. 1Fig. 2 Fundus photo 6 weeks after initial presentation demonstrating resolved vitritis following vitreous tap/inject of antibiotics and later vitrectomy for IVI endophthalmitis. Fig. 2 2.2 Case 2 An 88 year-old male with a history of neovascular AMD received an intravitreal injection of aflibercept in the left eye and presented two days later with pain, redness, and decrease in vision. The injection protocol consisted of the physician wearing a mask, application of topical proparacaine 0.5% drops, followed by instillation of PVI 5% and lidocaine 4% drops. The eyelids were swabbed with PVI 10% swabs. Supplemental lidocaine and PVI drops were applied and a 4% lidocaine-soaked cotton tip applicator was applied using pressure to the injection site on the sclera. A drop of topical moxifloxacin was applied immediately after the injection. A speculum was not used. At presentation, BCVA of the left eye was light perception (decreased from 20/20) and intraocular pressure was 13 mmHg. Examination showed conjunctival injection, corneal edema with keratic precipitates, hypopyon, fibrin, and dense vitritis with vitreous membranes on B-scan ultrasonography. The patient was diagnosed with endophthalmitis and underwent a vitreous tap and injection of intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2.25 mg/0.1 mL). He was started on topical prednisolone every 2 hours and no oral antibiotics were prescribed. Due to non-improving status, 6 days after initial presentation the patient underwent a pars plana vitrectomy with repeat intravitreal vancomycin and ceftazidime that demonstrated vascular attenuation and widespread intraretinal hemorrhages. Five weeks after intravitreal treatment, the BCVA was 20/60. Eight months later at last follow up, the BCVA remained stable and the intraretinal hemorrhages were markedly improved, though not completely absent. Vitreous cultures from the original vitreous tap returned positive for Abiotrophia defectiva, susceptible to vancomycin (MIC < 0.50), ceftriaxone (MIC < 0.75), and cefuroxime (MIC < 0.50); and resistant to benzylpenicillin (MIC > 0.25), and levofloxacin. (MIC > 3). 3 Discussion The first two cases of NVS endophthalmitis after IVI are described in this report. Infectious endophthalmitis following IVI is uncommon, with a rate of 0.038%–0.056% in large meta-analyses and approximately 0.02% in retrospective reviews.1, 2, 3,5 Coagulase negative Staphylococcus and Streptococcus species have been well recognized as the most common causative organisms, with poor outcomes in eyes infected with Streptococcus species.1, 2, 3,6 McCannel also reported higher rates of Streptococcus species endophthalmitis after IVI compared to other intraocular surgeries, hypothesizing that dispersion of aerosolized moisture droplets from the upper respiratory tract could result in contamination of the injection field.5 NVS have long been known to be normal inhabitants of the human oropharynx, urogenital, and gastrointestinal tracts, and are recognized causes of bacterial endocarditis. Based on genetic characteristics, the NVS were re-classified into two genera Granulicatella and Abiotrophia and four different species, two of which are Granulicatella adiacens and Abiotrophia defectiva.7 In the ophthalmic literature, Granulicatella adiacens has been implicated in chronic dacryocystitis and post-traumatic orbital abscess, while Abiotrophia defectiva has been reported in cases of infectious keratitis and in bleb-associated, Ozudex-associated, and post cataract extraction endophthalmitis.8, 9, 10, 11, 12, 13 To the authors’ knowledge, there have been no cases of NVS endophthalmitis reported after IVI. In the current literature, visual outcomes cases of Abiotrophia defectiva associated endophthalmitis have been poor with visual acuities generally worse than 20/100.8, 9 However, the two patients in this series both achieved visual acuities better than or equal to 20/60 at last follow up, suggesting that NVS may be less virulent than other Streptococcus species. It is possible that a pars plana vitrectomy played a role in the improved outcome in these two cases, such that early vitrectomy may be considered in NVS associated endophthalmitis. 4 Conclusions In summary, the first two cases of NVS (Granulicatella adiacens and Abiotrophia defectiva) endophthalmitis following IVI are presented in this report. The authors propose dispersion of aerosolized NVS droplets from the oropharynx may have contaminated the injection field and that a firmly taped mask on both the patient and physician may reduce the risk of NVS associated endophthalmitis after IVI. Following standard diagnostic and clinical management, outcomes of NVS associated endophthalmitis may be better than expected for Streptococcus species. Patient consent Written consent was obtained to publish case details. Funding This study was supported in part by an unrestricted grant from 10.13039/100001818Research to Prevent Blindness (New York, New York) and 10.13039/100000002NIH Center Core Grant P30EY014801 (Bethesda, Maryland). The sponsor or funding organization had no role in the design or conduct of this study. Authorship All authors attest that they meet the current ICMJE criteria for Authorship. Declaration of competing interest The following authors have no financial disclosures: RS, CS, DPR, HLB, JDS, DM, HWF.	Recovering	ReactionOutcome	CC BY-NC-ND	33553806	19,403,157	2021-03
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Coronary artery dissection'.	Postpartum multi-vessel spontaneous coronary artery dissection in the setting of cocaine and amphetamine use: a case report. Spontaneous coronary artery dissection (SCAD) is a recognized cause of acute coronary syndrome (ACS). Pregnancy, the postpartum period, and illicit drug use have all been reported as potential triggers. We describe the case of a 41-year-old patient who presented to the emergency department with chest pain in the setting of recent cocaine and amphetamine use. The patient was 4 months postpartum following an uncomplicated pregnancy. Past medical history was non-contributory, with no known risk factors for ischaemic heart disease. Electrocardiogram was normal but high-sensitivity troponin T was significantly elevated. Coronary angiography revealed multi-vessel SCAD. This was managed conservatively as the patient remained clinically stable and pain free without high-risk anatomy (left main stem or proximal two-vessel coronary artery dissection). Spontaneous coronary artery dissection must be considered in a postpartum patient presenting with ACS, particularly in the context of environmental stressors such as illicit drug use. Coronary angiography is key to determine diagnosis and guide management. Conservative therapy is favoured, except for patients with ongoing ischaemia, haemodynamic instability, and left main stem involvement. In this case, we suspect SCAD occurred due to the haemodynamic effects of cocaine and amphetamines in the context of structural arterial changes of the postpartum state. Learning points Spontaneous coronary artery dissection (SCAD) must be considered in a postpartum patient presenting with acute coronary syndrome, particularly in the context of environmental stressors such as illicit drug use. Coronary angiography is key to determine diagnosis and guide management of SCAD. Conservative management of SCAD is favoured, except for patients with ongoing ischaemia, haemodynamic instability, and left main stem involvement. Introduction Spontaneous coronary artery dissection (SCAD) is a recognized cause of acute coronary syndrome (ACS) and constitutes 2–4% of ACS presentations overall. However, the incidence is higher in women under 50 years presenting with ACS, estimated at 24–35%. Pregnancy, the postpartum period, and illicit drug use have been reported as triggers. We present a case of SCAD in a 41-year-old postpartum female in the setting of both cocaine and 3,4-methylenedioxymethamphetamine (MDMA) use (an amphetamine derivative). To our knowledge, this is the first reported case of SCAD with all three of these potential precipitating factors coinciding in a single patient. Timeline Day Event 0 Ingestion of cocaine and amphetamines Patient is 4 months postpartum 3 Presents to ED with chest pain ECG shows normal sinus rhythm with no ST changes. Initial Troponin T is elevated at 196ng/L Echocardiography shows preserved left ventricular function with normal valves and no regional wall motion abnormalities 4 Coronary angiogram demonstrates type 2a dissection of the mid to distal left anterior descending artery There is also type 2b dissection of the distal posterior descending artery of the right coronary artery 6 Magnetic resonance angiography of the renal and carotid/vertebral arteries is normal 7 Discharged home on medical therapy 40 Patient well at follow-up in clinic Case presentation A 41-year-old female presented to the emergency department (ED) with chest pain. It was described as substernal chest tightness, 5/10 in severity with radiation to the left arm and jaw. Past medical history was non-contributory, with no known risk factors for ischaemic heart disease. The patient was 4 months postpartum following an uncomplicated pregnancy. She admitted to using cocaine and MDMA 3 days prior to presentation. Physical examination was normal with a blood pressure of 130/80 and a heart rate of 76 b.p.m. The patient was pain free on arrival to ED. An electrocardiogram (ECG) on presentation showed normal sinus rhythm with no ST changes. Initial Troponin T was elevated at 196 ng/L, peaking at 1116 ng/L at 12 h (normal range: 0–14 ng/L). Echocardiography showed preserved left ventricular (LV) function with normal valvular morphology and no regional wall motion abnormalities. Coronary angiography demonstrated type 2a dissection of the mid to distal left anterior descending artery without extension to the apex (Figures 1 and 2). There was also type 2b dissection of the distal posterior descending artery of the right coronary artery (Figure 3). Intracoronary glyceryl trinitrate (GTN) did not affect these findings. The left main stem and circumflex artery were angiographically normal. Intracoronary imaging was not performed in order to avoid the risk of propagation of dissection. This was managed conservatively as the patient was clinically stable and pain free without high-risk anatomy (left main stem or proximal two-vessel coronary artery dissection). Figure 1 Coronary angiogram demonstrating type 2a dissection of the mid to distal left anterior descending (LAD) artery without extension to the apex. Figure 2 Coronary angiogram demonstrating type 2a dissection of the mid to distal left anterior descending (LAD) artery without extension to the apex. Figure 3 Coronary angiogram demonstrating type 2b dissection of the distal posterior descending artery (PDA) of the right coronary artery (RCA). The patient was treated with aspirin and beta-blocker therapy. Magnetic resonance angiography of the renal and carotid/vertebral arteries was normal. The patient was discharged after 7 days and was well at 6 weeks follow-up in clinic. There were no further episodes of chest pain and excellent compliance with medications. The patient has been enrolled in cardiac rehab and counselled on the importance of illicit drug use cessation. We plan to see her in clinic in 3 months. Discussion Spontaneous coronary artery dissection was first described by Pretty at autopsy in 1931.1 The predominant mechanism of myocardial injury is coronary artery obstruction by intramural haematoma or intimal disruption, rather than atherosclerotic plaque rupture or intraluminal thrombus.2 Secondary causes (cardiac catheterization, chest trauma, aortic dissection, and cardiac surgery) must be excluded before a coronary artery dissection is deemed ‘spontaneous’. The aetiology of SCAD is multifactorial. Potential contributors include underlying arteriopathy, genetics, hormonal influences, systemic inflammatory diseases, and environmental stressors. The most dominant association reported is fibromuscular dysplasia with a prevalence of up to 86% in SCAD cohorts who were routinely screened.3 Pregnancy-related SCAD (P-SCAD) accounts for <5% of SCAD cases.3 However, P-SCAD is implicated in ∼40% of pregnancy-related myocardial infarction.4 It occurs most commonly in the 3rd trimester of pregnancy and the early postpartum period. High progesterone levels during pregnancy can weaken the tunica media through loss of normal corrugation of elastic fibres, a decrease in acid mucopolysaccharides, and impairment of collagen synthesis.5 Haemodynamic changes during late pregnancy, such as augmented cardiac output and circulating volume, predispose to SCAD. These changes increase arterial wall shear stress, causing micro-structural changes in the aorta, which can extend to the coronary arteries.6 Cocaine is associated with a number of cardiovascular conditions, including SCAD.7 Jaffe et al.8 described the first case of cocaine-related SCAD in 1994. Cocaine acts as a sympathomimetic, resulting in increased inotropic and chronotropic response, and vasoconstriction via alpha-receptor stimulation. Consequent high arterial wall shear stress may lead to dissection. Cocaine also has a prothrombotic effect through increased platelet activity and aggregation.9 Amphetamine-related SCAD has also been described.10 Amphetamines have similar cardiovascular sequelae to cocaine, relating predominantly to coronary vasospasm and platelet activation-mediated thrombus formation. In this case, we suspect SCAD occurred due to the haemodynamic effects of cocaine and MDMA in the context of structural arterial changes of the postpartum state. This ‘second hit’ phenomenon has been suggested previously, with environmental factors precipitating SCAD in vulnerable individuals. This is based on the concept that sequential insults, which are individually innocuous, can lead to overwhelming physiologic reactions. Coronary angiography is the gold standard for the diagnosis of SCAD, regardless of aetiology. Minimal contrast injections must be used when performing angiography to mitigate the risk of propagation of dissection. Spontaneous coronary artery dissection is divided into three types using the Saw angiographic classification. Type 1 is characterized by multiple radiolucent lumens. Type 2 is described as intramural haematoma and diffuse stenosis. This diffuse narrowing may be bordered by normal artery segments proximally and distally (type 2a), or it may extend to the apical tip of the artery (type 2b). Type 3 mimics atherosclerosis.11 Intravascular imaging can be a useful adjunct to coronary angiography. It has been suggested that optical coherence tomography or intravascular ultrasound can be considered for type 2 SCAD and is required for the diagnosis of type 3 SCAD.4 Intracoronary imaging may provide a definitive diagnosis; however, there are potential catastrophic risks, including extension of the dissection and catheter-induced vessel occlusion. It may be reserved for cases where there is diagnostic ambiguity and to aid percutaneous intervention. The optimal management strategy for SCAD remains undetermined and there is a paucity of randomized controlled data in this area. Observational data have indicated angiographic healing of SCAD lesions in the majority of patients (70–97%) who were restudied weeks to months after a conservatively managed index episode.11,12 Medical therapy includes beta-blockers, antiplatelets, and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. Beta-blockers should be considered in patients with SCAD who have LV dysfunction or ventricular arrhythmias. Some advocate for their routine use on the basis of extrapolation from benefits in aortic dissection.4 The exact role of antiplatelet therapy for SCAD is unknown. Most experts recommend aspirin for at least 1 year.2 In light of increased bleeding risks with antiplatelet agents, and uncertain benefits and risks, careful individual selection of suitability for dual-antiplatelet therapy in conservatively managed patients is recommended. Renin-angiotensin system antagonists should be used when MI is complicated by LV systolic dysfunction. Female patients of reproductive age must be warned of the teratogenicity of these medications.2 The value of follow-up angiography for SCAD is undefined. Computed tomography coronary angiography has been proposed as a potential alternative to conventional angiography although current data are limited to single case discussions and one small series.13 In terms of screening for potential predisposing conditions, the European Society of Cardiology (ESC) 2018 position paper advocates imaging of extra-coronary vascular beds in patients with SCAD.14 Genetic screening and blood work to screen for inflammatory or connective tissue disease are of low yield and not routinely recommended. Overall, cardiovascular outcomes for SCAD patients are relatively good with acute in-hospital mortality reported as <5%. Recurrent SCAD is frequent, occurring in ∼15% of patients at 2-year follow-up.3 In patients with LV dysfunction, ejection fraction has been observed to improve after the acute presentation.15 This may represent normalization of stunned myocardium following spontaneous resolution of SCAD. Conclusion This case highlights the importance of considering SCAD in a postpartum patient presenting with ACS, particularly in the context of environmental stressors such as illicit drug use. Coronary angiography is key to determine diagnosis and guide management. Conservative therapy is favoured, except for patients with ongoing ischaemia, haemodynamic instability, and left main stem involvement. Lead author biography Dr Laurna McGovern is a Specialist Registrar in Cardiology in Ireland. Supplementary material Supplementary material is available at European Heart Journal - Case Reports online. Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Supplementary data. Consent: The authors confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patient in line with COPE guidance. Conflict of interest: none declared. Funding: none declared.	AMPHETAMINE SULFATE	DrugsGivenReaction	CC BY-NC	33554015	19,405,701	2021-01
What was the outcome of reaction 'Coronary artery dissection'?	Postpartum multi-vessel spontaneous coronary artery dissection in the setting of cocaine and amphetamine use: a case report. Spontaneous coronary artery dissection (SCAD) is a recognized cause of acute coronary syndrome (ACS). Pregnancy, the postpartum period, and illicit drug use have all been reported as potential triggers. We describe the case of a 41-year-old patient who presented to the emergency department with chest pain in the setting of recent cocaine and amphetamine use. The patient was 4 months postpartum following an uncomplicated pregnancy. Past medical history was non-contributory, with no known risk factors for ischaemic heart disease. Electrocardiogram was normal but high-sensitivity troponin T was significantly elevated. Coronary angiography revealed multi-vessel SCAD. This was managed conservatively as the patient remained clinically stable and pain free without high-risk anatomy (left main stem or proximal two-vessel coronary artery dissection). Spontaneous coronary artery dissection must be considered in a postpartum patient presenting with ACS, particularly in the context of environmental stressors such as illicit drug use. Coronary angiography is key to determine diagnosis and guide management. Conservative therapy is favoured, except for patients with ongoing ischaemia, haemodynamic instability, and left main stem involvement. In this case, we suspect SCAD occurred due to the haemodynamic effects of cocaine and amphetamines in the context of structural arterial changes of the postpartum state. Learning points Spontaneous coronary artery dissection (SCAD) must be considered in a postpartum patient presenting with acute coronary syndrome, particularly in the context of environmental stressors such as illicit drug use. Coronary angiography is key to determine diagnosis and guide management of SCAD. Conservative management of SCAD is favoured, except for patients with ongoing ischaemia, haemodynamic instability, and left main stem involvement. Introduction Spontaneous coronary artery dissection (SCAD) is a recognized cause of acute coronary syndrome (ACS) and constitutes 2–4% of ACS presentations overall. However, the incidence is higher in women under 50 years presenting with ACS, estimated at 24–35%. Pregnancy, the postpartum period, and illicit drug use have been reported as triggers. We present a case of SCAD in a 41-year-old postpartum female in the setting of both cocaine and 3,4-methylenedioxymethamphetamine (MDMA) use (an amphetamine derivative). To our knowledge, this is the first reported case of SCAD with all three of these potential precipitating factors coinciding in a single patient. Timeline Day Event 0 Ingestion of cocaine and amphetamines Patient is 4 months postpartum 3 Presents to ED with chest pain ECG shows normal sinus rhythm with no ST changes. Initial Troponin T is elevated at 196ng/L Echocardiography shows preserved left ventricular function with normal valves and no regional wall motion abnormalities 4 Coronary angiogram demonstrates type 2a dissection of the mid to distal left anterior descending artery There is also type 2b dissection of the distal posterior descending artery of the right coronary artery 6 Magnetic resonance angiography of the renal and carotid/vertebral arteries is normal 7 Discharged home on medical therapy 40 Patient well at follow-up in clinic Case presentation A 41-year-old female presented to the emergency department (ED) with chest pain. It was described as substernal chest tightness, 5/10 in severity with radiation to the left arm and jaw. Past medical history was non-contributory, with no known risk factors for ischaemic heart disease. The patient was 4 months postpartum following an uncomplicated pregnancy. She admitted to using cocaine and MDMA 3 days prior to presentation. Physical examination was normal with a blood pressure of 130/80 and a heart rate of 76 b.p.m. The patient was pain free on arrival to ED. An electrocardiogram (ECG) on presentation showed normal sinus rhythm with no ST changes. Initial Troponin T was elevated at 196 ng/L, peaking at 1116 ng/L at 12 h (normal range: 0–14 ng/L). Echocardiography showed preserved left ventricular (LV) function with normal valvular morphology and no regional wall motion abnormalities. Coronary angiography demonstrated type 2a dissection of the mid to distal left anterior descending artery without extension to the apex (Figures 1 and 2). There was also type 2b dissection of the distal posterior descending artery of the right coronary artery (Figure 3). Intracoronary glyceryl trinitrate (GTN) did not affect these findings. The left main stem and circumflex artery were angiographically normal. Intracoronary imaging was not performed in order to avoid the risk of propagation of dissection. This was managed conservatively as the patient was clinically stable and pain free without high-risk anatomy (left main stem or proximal two-vessel coronary artery dissection). Figure 1 Coronary angiogram demonstrating type 2a dissection of the mid to distal left anterior descending (LAD) artery without extension to the apex. Figure 2 Coronary angiogram demonstrating type 2a dissection of the mid to distal left anterior descending (LAD) artery without extension to the apex. Figure 3 Coronary angiogram demonstrating type 2b dissection of the distal posterior descending artery (PDA) of the right coronary artery (RCA). The patient was treated with aspirin and beta-blocker therapy. Magnetic resonance angiography of the renal and carotid/vertebral arteries was normal. The patient was discharged after 7 days and was well at 6 weeks follow-up in clinic. There were no further episodes of chest pain and excellent compliance with medications. The patient has been enrolled in cardiac rehab and counselled on the importance of illicit drug use cessation. We plan to see her in clinic in 3 months. Discussion Spontaneous coronary artery dissection was first described by Pretty at autopsy in 1931.1 The predominant mechanism of myocardial injury is coronary artery obstruction by intramural haematoma or intimal disruption, rather than atherosclerotic plaque rupture or intraluminal thrombus.2 Secondary causes (cardiac catheterization, chest trauma, aortic dissection, and cardiac surgery) must be excluded before a coronary artery dissection is deemed ‘spontaneous’. The aetiology of SCAD is multifactorial. Potential contributors include underlying arteriopathy, genetics, hormonal influences, systemic inflammatory diseases, and environmental stressors. The most dominant association reported is fibromuscular dysplasia with a prevalence of up to 86% in SCAD cohorts who were routinely screened.3 Pregnancy-related SCAD (P-SCAD) accounts for <5% of SCAD cases.3 However, P-SCAD is implicated in ∼40% of pregnancy-related myocardial infarction.4 It occurs most commonly in the 3rd trimester of pregnancy and the early postpartum period. High progesterone levels during pregnancy can weaken the tunica media through loss of normal corrugation of elastic fibres, a decrease in acid mucopolysaccharides, and impairment of collagen synthesis.5 Haemodynamic changes during late pregnancy, such as augmented cardiac output and circulating volume, predispose to SCAD. These changes increase arterial wall shear stress, causing micro-structural changes in the aorta, which can extend to the coronary arteries.6 Cocaine is associated with a number of cardiovascular conditions, including SCAD.7 Jaffe et al.8 described the first case of cocaine-related SCAD in 1994. Cocaine acts as a sympathomimetic, resulting in increased inotropic and chronotropic response, and vasoconstriction via alpha-receptor stimulation. Consequent high arterial wall shear stress may lead to dissection. Cocaine also has a prothrombotic effect through increased platelet activity and aggregation.9 Amphetamine-related SCAD has also been described.10 Amphetamines have similar cardiovascular sequelae to cocaine, relating predominantly to coronary vasospasm and platelet activation-mediated thrombus formation. In this case, we suspect SCAD occurred due to the haemodynamic effects of cocaine and MDMA in the context of structural arterial changes of the postpartum state. This ‘second hit’ phenomenon has been suggested previously, with environmental factors precipitating SCAD in vulnerable individuals. This is based on the concept that sequential insults, which are individually innocuous, can lead to overwhelming physiologic reactions. Coronary angiography is the gold standard for the diagnosis of SCAD, regardless of aetiology. Minimal contrast injections must be used when performing angiography to mitigate the risk of propagation of dissection. Spontaneous coronary artery dissection is divided into three types using the Saw angiographic classification. Type 1 is characterized by multiple radiolucent lumens. Type 2 is described as intramural haematoma and diffuse stenosis. This diffuse narrowing may be bordered by normal artery segments proximally and distally (type 2a), or it may extend to the apical tip of the artery (type 2b). Type 3 mimics atherosclerosis.11 Intravascular imaging can be a useful adjunct to coronary angiography. It has been suggested that optical coherence tomography or intravascular ultrasound can be considered for type 2 SCAD and is required for the diagnosis of type 3 SCAD.4 Intracoronary imaging may provide a definitive diagnosis; however, there are potential catastrophic risks, including extension of the dissection and catheter-induced vessel occlusion. It may be reserved for cases where there is diagnostic ambiguity and to aid percutaneous intervention. The optimal management strategy for SCAD remains undetermined and there is a paucity of randomized controlled data in this area. Observational data have indicated angiographic healing of SCAD lesions in the majority of patients (70–97%) who were restudied weeks to months after a conservatively managed index episode.11,12 Medical therapy includes beta-blockers, antiplatelets, and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers. Beta-blockers should be considered in patients with SCAD who have LV dysfunction or ventricular arrhythmias. Some advocate for their routine use on the basis of extrapolation from benefits in aortic dissection.4 The exact role of antiplatelet therapy for SCAD is unknown. Most experts recommend aspirin for at least 1 year.2 In light of increased bleeding risks with antiplatelet agents, and uncertain benefits and risks, careful individual selection of suitability for dual-antiplatelet therapy in conservatively managed patients is recommended. Renin-angiotensin system antagonists should be used when MI is complicated by LV systolic dysfunction. Female patients of reproductive age must be warned of the teratogenicity of these medications.2 The value of follow-up angiography for SCAD is undefined. Computed tomography coronary angiography has been proposed as a potential alternative to conventional angiography although current data are limited to single case discussions and one small series.13 In terms of screening for potential predisposing conditions, the European Society of Cardiology (ESC) 2018 position paper advocates imaging of extra-coronary vascular beds in patients with SCAD.14 Genetic screening and blood work to screen for inflammatory or connective tissue disease are of low yield and not routinely recommended. Overall, cardiovascular outcomes for SCAD patients are relatively good with acute in-hospital mortality reported as <5%. Recurrent SCAD is frequent, occurring in ∼15% of patients at 2-year follow-up.3 In patients with LV dysfunction, ejection fraction has been observed to improve after the acute presentation.15 This may represent normalization of stunned myocardium following spontaneous resolution of SCAD. Conclusion This case highlights the importance of considering SCAD in a postpartum patient presenting with ACS, particularly in the context of environmental stressors such as illicit drug use. Coronary angiography is key to determine diagnosis and guide management. Conservative therapy is favoured, except for patients with ongoing ischaemia, haemodynamic instability, and left main stem involvement. Lead author biography Dr Laurna McGovern is a Specialist Registrar in Cardiology in Ireland. Supplementary material Supplementary material is available at European Heart Journal - Case Reports online. Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Supplementary data. Consent: The authors confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patient in line with COPE guidance. Conflict of interest: none declared. Funding: none declared.	Recovered	ReactionOutcome	CC BY-NC	33554015	19,405,701	2021-01
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Anaemia'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Blood bilirubin increased'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Blood creatine increased'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Fatigue'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Haemorrhage'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Leukopenia'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Palmar-plantar erythrodysaesthesia syndrome'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Thrombocytopenia'.	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FLUOROURACIL, LEUCOVORIN, OXALIPLATIN, PANITUMUMAB	DrugsGivenReaction	CC BY	33555359	18,965,694	2021-05
What was the administration route of drug 'BEVACIZUMAB'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	Intravenous (not otherwise specified)	DrugAdministrationRoute	CC BY	33555359	18,979,549	2021-05
What was the administration route of drug 'CAPECITABINE'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	Oral	DrugAdministrationRoute	CC BY	33555359	18,979,549	2021-05
What was the administration route of drug 'GIMERACIL\OTERACIL\TEGAFUR'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	Oral	DrugAdministrationRoute	CC BY	33555359	18,979,549	2021-05
What was the administration route of drug 'PANITUMUMAB'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	Other	DrugAdministrationRoute	CC BY	33555359	18,965,694	2021-05
What was the dosage of drug 'BEVACIZUMAB'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	ON DAY 1	DrugDosageText	CC BY	33555359	18,979,549	2021-05
What was the dosage of drug 'CAPECITABINE'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	EVENING OF DAY 1 TO THE MORNING OF DAY 15	DrugDosageText	CC BY	33555359	18,979,549	2021-05
What was the dosage of drug 'FLUOROURACIL'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	OVER 46 HOURS	DrugDosageText	CC BY	33555359	18,979,549	2021-05
What was the dosage of drug 'GIMERACIL\OTERACIL\TEGAFUR'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	FROM THE EVENING OF DAY 1 TO THE MORNING OF DAY 15	DrugDosageText	CC BY	33555359	18,979,549	2021-05
What was the dosage of drug 'LEUCOVORIN'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	OVER 2 H ON DAY 1	DrugDosageText	CC BY	33555359	18,979,549	2021-05
What was the dosage of drug 'OXALIPLATIN'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	ON DAY 1	DrugDosageText	CC BY	33555359	18,979,549	2021-05
What was the dosage of drug 'PANITUMUMAB'?	Phase II study of an oxaliplatin-based regimen for relapsed colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy (INSPIRE study). The aim of this study was to evaluate the efficacy and safety of first-line chemotherapy with re-introduction of oxaliplatin (OX) more than 6 months after adjuvant chemotherapy including OX. Stage II/III colon cancer patients with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX were considered eligible. Eligible patients were treated with 5-fluorouracil, l-leucovorin and OX plus molecularly targeted agents or capecitabine and OX plus bevacizumab (BV) or S-1 and OX plus BV. The primary endpoint was the progression-free survival (PFS), and the secondary endpoints were the overall survival (OS), response rate (RR) and toxicity. A total of 50 patients were enrolled between September 2013 and May 2019. Twelve patients received 5-fluorouracil, l-leucovorin and OX (FOLFOX) plus BV, 21 patients received capecitabine and OX plus BV, 10 patients received S-1 and OX plus BV and 7 patients received FOLFOX plus cetuximab or panitumumab. The median PFS was 11.5 months (95% confidence interval [CI] 8.3-16.0), the median OS was 45.4 months (95% CI 37.4-NA), and the RR was 56.0% (95% CI 42.3-68.8). Adverse events of grade ≥ 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). First-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX can be used safely with expected efficacy for relapsed colon cancer patients. Introduction Colon cancer is the third-most commonly diagnosed cancer, with an estimated 1,400,000 new cases and 700,000 deaths globally each year [1]. Chemotherapy is an essential method of colon cancer treatment [2–4]. Among the various chemotherapy agents, oxaliplatin (OX) is one of the most substantial key agents for colon cancer treatment in both adjuvant and unresectable-metastatic disease settings. Thus far, three pivotal studies have shown that OX-based adjuvant chemotherapy, such as infusional 5-fluorouracil, l-leucovorin and OX (FOLFOX) or capecitabine and OX (CAPOX), for colon cancer significantly improved both the overall survival (OS) and disease-free survival [5–7]. OX-based adjuvant chemotherapy for colon cancer has been widely accepted and performed in clinical practice, and FOLFOX and CAPOX are also widely used in both the first and the second lines for metastatic colon cancer [8–10]. However, there is little supporting evidence available, and few studies have evaluated the efficacy and safety of OX re-introduction as the first-line treatment for relapsed colon cancer after OX-based adjuvant chemotherapy [11, 12]. To establish the optimal use of OX for colon cancer treatment, it is necessary to investigate the clinical benefit of OX re-introduction as the first-line treatment for relapsed disease after OX-based adjuvant chemotherapy. The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than 6 months after the completion of adjuvant chemotherapy with an OX-containing regimen. Patients and methods Study design This study was a single-arm, multicenter, phase II study to evaluate the efficacy and safety of physician’s choice OX-based regimen for colon cancer patients with neuropathies of grade < 1 who relapsed more than 6 months after OX-based adjuvant chemotherapy. Study data and informed consent were obtained in accordance with the Declaration of Helsinki. The Certified Clinical Research Review Board of Aichi Medical University Hospital approved this study protocol. This trial was registered with the UMIN Clinical Trials Registry as UMIN 000011348 https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000013300. This trial was registered with the Japan Registry of Clinical Trials as jRCTs041180118. https://jrct.niph.go.jp/latest-detail/jRCTs041180118; all patients were given a written explanation and provided their written informed consent before participating. Inclusion and exclusion criteria Tumors were staged according to the UICC version 7 [13]. The inclusion criteria were as follows: (1) stage II/III colon cancer with neuropathies of grade ≤ 1 who relapsed more than 6 months after adjuvant chemotherapy including OX; (2) performance status of 0–1; (3) ≧ 20 years of age; (4) presence of at least one measurable lesion using the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; (5) past history of adjuvant chemotherapy including OX with a cumulative dose of more than 300 mg/m2; (6) adequate hematologic, liver, and coagulation profiles and normal electrocardiogram findings; and (7) consent given to participate in this clinical study. The exclusion criteria were as follows: (1) serious coexisting morbidities; (2) active synchronous or metachronous malignant disease; (3) pregnant or lactating; (4) not considered suitable for participation for any other reason. Treatment methods Eligible patients were treated with infusional FOLFOX plus molecularly targeted agents or CAPOX plus bevacizumab (BV) or S-1 and OX (SOX) plus BV. Selection of OX-based regimen was decided by the attending physician at registration of each patient. FOLFOX was administered as a 2-h OX 85 mg/m2 infusion on day 1 in tandem with a 2-h l-leucovorin 200 mg/m2 infusion on day 1 and 5-FU as a 400-mg/m2 bolus followed by a 46-h 2400 mg/m2 infusion on days 1 to 3, every 2 weeks. In addition, BV (5 mg/kg on day 1) or cetuximab (400 mg/m2 as the initial dose and 250 mg/m2 as the subsequent doses on days 1 and 8) or panitumumab (6 mg/kg on day 1) was combined with FOLFOX. CAPOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral capecitabine 1000 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. SOX plus BV was administered as intravenous OX 130 mg/m2 on day 1, oral S-1 40 mg/m2 twice daily from the evening of day 1 to the morning of day 15 and BV 7.5 mg/kg on day 1, every 3 weeks. Endpoints The primary endpoint was the progression-free survival (PFS). The secondary endpoints were the OS, response rate (RR) and the safety of the combination therapy. Radiographic image studies were performed every eight weeks. The RR was evaluated by the RECIST 1.1 criteria [14]. All adverse events recorded were graded according to the Common Terminology Criteria for Adverse Events of the National Cancer Institute (CTCAE) version 4.0 [15]. The PFS was defined as the period between the day of registration and progression or death, whichever came first. Patients were censored at the last point when no progression was confirmed if the patients did not experience any event associated with the PFS. The OS was defined as the period between the day of registration and death. The data of patients who had not experienced an event were censored at the date of the final observation. Statistical analyses We set the threshold median PFS at 7 months and the expected median PFS at 10.5 months based on the results of a previous study [16–19]. Given a 2-sided alpha of 0.05 and statistical power of 80% with about 10% ineligible or dropout patients, we set 50 patients as the target sample size in this study. The analytical population for efficacy was defined as all eligible patients, and that of safety was defined as all eligible patients who received treatment at least once. In the present study, disease control rate (DCR) was defined as the percentage of complete response, partial response, and stable disease in full set analysis. The PFS and OS curves were calculated using the Kaplan–Meier method, and the 95% confidence interval (CI) was estimated using the Brookmeyer and Crowley method with log–log transformation. All analyses were implemented by SAS 9.4, SAS/STAT 14.2 (SAS Institute, Cary, NC,USA). Results Patients’ background characteristics From September 2013 to May 2019, 50 patients were registered from 21 institutions. The intension-to-treat analysis and safety analysis were carried out on those 50 patients. Table 1 shows the patients’ background characteristics. Twenty-eight patients were male, and 22 were female, with a median age of 69.5 years (range 27–82 years). The time until recurrence from the completion of adjuvant therapy was 6–12 months in 16 patients, 12–24 months in 15 patients and more than 24 months in 19 patients. The median total dose of OX for adjuvant chemotherapy were 1136 (470–1904) mg/body. The most common metastatic site was the lung (22 patients, 44%), lymph node (19 patients, 38%), peritoneal metastasis (13 patients, 26%) and liver (11 patients, 22%). The median follow-up was 34.3 months (range 20.8–63.7 months). Twelve patients received FOLFOX plus BV, 21 patients received CAPOX plus BV, 10 patients received SOX plus BV, and 7 patients received FOLFOX plus cetuximab or panitumumab.Table 1 Patient characteristics Characteristics No. of patients (%) Gender Male 28 56.0 Female 22 44.0 Age (years) Median 69.5 Range 27–82 Performance status (PS) 0 44 88.0 1 6 12.0 Cancer location Colon 29 58.0 Rectum 21 42.0 Previous adjuvant chemotherapy FOLFOX 16 32.0 CAPOX 32 64.0 Other 2 4.0 Time from adjuvant chemotherapy 6 -12 months 16 32.0 12–24 months 15 30.0 More than 24 months 19 38.0 Oxaliplatin free interval 6–12 months 15 30.0 12–24 months 14 28.0 More than 24 months 21 42.0 Baseline peripheral sensory neuropathy 0 31 62.0 1 19 38.0 Number of relapse site 0 0 0 1 32 64.0 > 2 18 36.0 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, Intention to treat population, n = 50 Efficacy All follow-up data were collected by Dec/2019 and the median follow-up period was 34.3 months. The median PFS was 11.5 months (95% CI 8.3–16.0 months) (Fig. 1). The median PFS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [13.0 months (95% CI 7.0–19.2)/11.0 months (95% CI 7.5–19.9)/12.7 months (95% CI 7.8–17.7), respectively]. The median OS was 45.4 months (95% CI 37.4 months–NA) (Fig. 2). The reasons for discontinuing the study treatment included progression of the primary disease in 26 patients (54.2%), adverse events in 5 patients (10.4%) (Platelet count decreased was 2 patients, Urine protein was 1 patient, Neutropenia was 1 patient, Anorexia was 1 patient), discretion of the physician in 4 patients (8.3%), refusal by 6 patients (12.5%) and withdrawal of 3 patients (6.3%) due to confirmation of complete response (CR). Two patients continued the protocol treatment. The median OS among subgroups based on time from the completion of adjuvant chemotherapy (6–12 months/12–24 months/more than 24 months) was comparable [44.6 months (95% CI, 24.6-NA)/45.4 months (95% CI, 27.3-NA)/61.3 months (95% CI, 18.6-NA), respectively]. According to the subgroup analysis for OX-free interval, the median PFS and OS were 13.4 months (95% CI 7.0–19.2) and NA months (95% CI 41.9–NA) respectively for 6–12 months, 10.4 months (95% CI 7.4–19.9) and 37.4 months (95% CI 18.7–NA) respectively for 12–24 months, and 12.1 months (95% CI 7.7–17.5) and 45.4 months (95% CI 29.8–NA) respectively for more than 24 months.Fig. 1 The progression-free survival Fig. 2 The overall survival Table 2 shows the efficacy data. The best overall RR was 56.0% (95% CI 42.3–68.8%). The disease control rate (DCR) was 86.0% (95% CI 73.5–93.4%). In the present study, the best overall RR for OX-free interval was 53.3% (8/15) for 6–12 months, 71.4% (10/14) for 12–24 months and 47.6% (10/21) for more than 24 months. Four patients were converted to be resectable and underwent curative resection.Table 2 Efficacy data Parameter Number of patients (%) Best overall response rate Complete response (CR) 5 10.0 Partial response (PR) 23 46.0 Stable disease (SD) 15 30.0 Progressive disease (PD) 4 8.0 Not assessable 3 6.0 Best overall response rate (CR + PR) 28 56.0 95% CI 42.3–68.8 Disease control rate (CR + PR + SD) 43 86.0 95% CI 73.5–93.4 Treatment compliance and safety Table 3 shows the treatment exposure. The median total dose of OX was 525 mg/m2 (85–1690 mg/m2). The median total dose of OX was 348 mg/m2 (85–1615 mg/m2) for FOLFOX plus BV, 650 mg/m2 (130–1645 mg/m2) for CAPOX plus BV, 525 mg/m2 (260–1690 mg/m2) for SOX plus BV and 770 mg/m2 (170–1235 mg/m2) for FOLFOX plus cetuximab or panitumumab. The median course of the study treatment was 14 cycles in FOLFOX plus BV, 10 cycles in CAPOX plus BV, 6 cycles in SOX plus BV and 15 cycles in FOLFOX plus cetuximab or panitumumab.Table 3 Treatment exposure of oxaliplatin Oxaliplatin total dose (mg/m2) Regimen FOLFOX plus BV CAPOX plus BV SOX plus BV FOLFOX plus Cmab or Pmab n 12 21 10 7 Mean 540 701 718 710 Std 475 392 501 406 Min 85 130 260 170 Median 348 650 525 770 Max 1615 1645 1690 1235 FOLFOX infusional 5-fluorouracil, l-leucovorin and oxaliplatin, CAPOX capecitabine and oxaliplatin, SOX S-1 and oxaliplatin, BV Bevacizumab, Cmab Cetuximab, Pmab Panitumumab Adverse events (AEs) of any grade were observed in 88.0% (44/50 patients) of patients. Table 4 shows the details of the AEs. Adverse events of grade 3 that occurred in ≥ 5% of cases were neutropenia in 6 patients (12%), peripheral sensory neuropathy in 5 patients (10%), diarrhea in 4 patients (8%), hypertension in 4 patients (8%), anorexia in 3 patients (6%) and allergic reactions in 3 patients (6%). There was no case of grade 4 adverse event or treatment-related death.Table 4 Relevant adverse events occurring in ≥ 10% of patients (highest grade per patients) Adverse event Grade 3/4 All Grade Number of patients (%) Number of patients (%) Hematological Leukopenia 0 0 25 50.0 Neutropenia 6 12.0 26 52.0 Anemia 0 0 26 52.0 Thrombocytopenia 0 0 28 56.0 No hematological ALP increased 1 2.0 20 40.0 Blood bilirubin increased 0 0 21 42.0 Creatine increased 0 0 11 22.0 Peripheral sensory neuropathy 5 10.0 45 90.0 Peripheral motor neuropathy 2 4.0 17 34.0 Stomatitis 1 2.0 24 48.0 Nausea 2 4.0 29 58.0 Vomiting 1 2.0 10 20.0 Diarrhea 4 8.0 20 40.0 Rash 1 2.0 20 40.0 Paronychia 1 2.0 8 16.0 Anorexia 3 6.0 36 72.0 Fatigue 0 0 35 70.0 Allergic reaction 3 6.0 11 22.0 Hand foot syndrome 0 0 27 54.0 Hypertension 4 8.0 18 36.0 Hemorrhage 0 0 8 16.0 Discussion The present study evaluated the efficacy and safety of first-line chemotherapy with re-introduction of OX more than six months after the completion of adjuvant chemotherapy including OX. Our findings suggested that first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX could be used safely with the expected efficacy for relapsed colon cancer patients. Therefore, the re-introduction of OX treatment is a viable option for relapsed colon cancer patients who have already been treated with OX-based adjuvant chemotherapy. The present study showed that the median PFS was 11.5 months for first-line chemotherapy with re-introduction of OX after adjuvant chemotherapy including OX for colon cancer. In the adjuvant setting, very recently, Kotaka et al. showed the similar results. They evaluate the efficacy of reintroducing FOLFOX or CAPOX with or without BV in relapsed 31 colorectal cancer patient who treated OX as adjuvant chemotherapy between October 2012 and October 2016 [12]. They found that median PFS was 10.8 months (95% CI 6.9–18.8 months). In a metastatic setting, recently, a few studies have evaluated the clinical effects of the re-introduction of OX for colorectal cancer after chemotherapy including OX. de Gramont et al. performed an additional analysis of the OPTIMisation of OXaliplatin (OPTIMOX) trial to evaluate the efficacy of OX re-introduction for metastatic colorectal cancer patients. They found that OX re-introduction had an independent and significant impact on the OS (hazard ratio: 0.56, P = 0.009) [20]. In addition, Chibaudel et al. evaluated the clinical effects of the re-introduction of OX-based chemotherapy and the OX-free interval (OFI; cut-off value: 6 months) on tumor sensitivity to OX re-introduction in initially unresectable colorectal cancer who received first-line OX-based chemotherapy (OPTIMOX trial) [20–23]. The PFS and OS were 3.0 and 8.8 months in patients with an OFI < 6 months, respectively, and 5.5 and 16.8 months in patients with an OFI ≥ 6 months, respectively. Furthermore, an OFI of ≥ 6 months improved the survival. Given these results, even after chemotherapy including OX, the re-introduction of OX might improve the survival among colon cancer patients, according to the OFI. In the present study, the best overall RR and DCR were 56.0% and 86.0%, respectively. Although the patient background characteristics and treatment lines have differed among studies, there have been some showing OX sensitivity in patients after OX-based chemotherapy in both adjuvant and metastatic setting. Table 5 summarized the efficacy of the present study and previous studies. In adjuvant setting, Kotaka et al. reported that the RR was 62.1% (95% CI 42.3–79.3) and the DCR was 82.8% (95% CI 64.2–94.2). The RR for oxaliplatin-free interval was 100.0% in months 6–12 and 56.0% after 12 months. In metastatic setting, Suenaga et al. evaluated the re-introduction of OX-based chemotherapy in 33 metastatic colorectal cancer refractory to standard treatment [24]. They reported that the RR was 6.1% (95% CI 2.5–14.7%) and the DCR 66.7% (95% CI 49.7–83.6%). Goebel et al. investigated FOLFOX re-introduction after a break in treatment or following disease progression on another regimen in 29 cases of metastatic colorectal cancer. They found that the re-introduction of OX was feasible and achieved a response or stabilization in 73% of patients [21]. In addition, the OPTIMOX-1 and OPTIMOX-2 studies showed an RR of 19% and DCR of 58%. Interestingly, the OPTIMOX-1 and OPTIMOX-2 studies also showed that the tumor sensitivity differed between the patients with an OFI < 6 months and ≥ 6 months. The respective DCR and RR were 14% and 45% in those with an OFI < 6 months and 22% and 63% in those with an OFI ≥ 6 months. In addition, the progression disease rate sharply decreased from 52% in the patients with an OFI < 6 months to 23% in those with an OFI ≥ 6 months. Although it is difficult to directly compare the results due to differences in the patient profiles and treatments, even after OX-based chemotherapy, the patients still have a potentially OX-sensitive tumor. Furthermore, the tumor sensitivity might also change depending on the OFI.Table 5 Summary of the efficacy of the present study and previous studies Present study REACT study [Ref. 12] RE-OPEN study [Ref. 24] Goebel et al. [Ref. 21] Study population setting OX-based adjuvant chemotherapy OX-based adjuvant chemotherapy OX-based chemotherapy for metastatic setting OX-based chemotherapy for metastatic setting Sample size 50 patients 31 patients 33 patients 29 patients Progression free survival 11.5 months 10.8 months 98 days 18 weeks Overall survival 45.4 months 28.7 months 300 days 42 weeks Response rate 56.0% 62.1% 6.1% 21% Disease control rate 86.0% 82.8% 39.4% 73% The present study showed that AEs of any grade were observed in 88% of patients. The incidence of both peripheral sensory and motor neuropathies were not increased. According to previous reports, the incidence of the AEs was acceptable. On other hands, in the previous similar reports, the rate of grade 1/2 and 3 allergic reaction was 12.9% and 3.2%, respectively [12]. The rate of grade 1/2 and 3 allergic reaction of the present study was higher than in the previous study. However, the allergic reaction was not main reason for discontinuation of treatment. Therefore, first-line chemotherapy with re-introduction of OX more than 6 months after adjuvant chemotherapy including OX seems able to be used safely for relapsed colorectal cancer patients. Several limitations associated with the present study warrant mention. First, there might have been some selection bias. This study was a single-arm, multicenter, phase II study and thus might only have included patients considered suited for OX-based chemotherapy. Second, the optimal OFI was unclear. In the present study, we set the OFI as 6 months according to previous studies. It is unclear whether or not a longer OFI affects the survival and OX sensitivity. However, this issue is a difficult problem to solve, because the early relapse after adjuvant chemotherapy is related to more aggressive tumor. Third, we did not collect the proportion against the expected dose of OX in the adjuvant chemotherapy. Although the median dose of the OX in the present study was similar to previous study; the proportion against the expected dose of OX in the adjuvant chemotherapy was important information for sensitivity in OX re-introduction as the first-line treatment after OX-based adjuvant chemotherapy. Considering these, the further study will clarify these issues. In conclusion, first-line chemotherapy with re-introduction of OX more than 6 months after completion of adjuvant chemotherapy that had included OX was able to be used safely with the expected efficacy for relapsed colon cancer patients. The re-introduction of OX treatment appears to be a viable treatment option for relapsed colon cancer patients treated with OX-based adjuvant chemotherapy. Acknowledgements This study was supported, in part, by the non-profit organization Epidemiological & Clinical Research Information Network (ECRIN). We are grateful to Ms. Yumi Miyashita for her excellent contributions as the clinical research coordinator of this study. We presented the study results previously at ESMO World Congress on Gastrointestinal Cancer 2020, in VIRTUAL 1-4 July 2020 and ESMO VIRTUAL CONGRESS 2020 19-21 September 2020. Funding The trial was funded by Yakult Honsha Co., Ltd. under contract. Yakult Honsha played no role in the design, collection, analysis or interpretation of the data, or writing of this manuscript. Compliance with ethical standards Conflict of interest Hironaga Satake has received research funding from Ono Pharmaceutical Co. Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Sanofi Co., Ltd., and honoraria from Bayer Co., Ltd., Bristol-Myers Squibb Co., Ltd., Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Co., Ltd., Eli Lilly Japan Co., Ltd., Merck Bio Pharma Co., Ltd., MSD Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi Co., Ltd., Taiho Pharmaceutical Co., Ltd., Takeda Co., Ltd. and Yakult Honsha Co., Ltd. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Keiichiro Ishibashi and Toru Aoyama contributed equally to this article.	ON DAY 1	DrugDosageText	CC BY	33555359	18,979,549	2021-05
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Disease progression'.	Pseudotumor cerebri syndrome in a child with Alagille syndrome: intracranial pressure dynamics and treatment outcome after ventriculoperitoneal shunting. Alagille syndrome (AS) is a rare multisystem disease of the liver, heart, eyes, face, skeleton, kidneys, and vascular system. The occurrence of pseudotumor cerebri syndrome (PTCS) in patients with AS has been reported only exceptionally. Owning to its rarity and a mostly atypical presentation, the diagnosis and natural history of affected patients remain uncertain. We report an atypical case of PTCS in a 4-year-old boy with a known history of AS who presented with bilateral papilledema (PE) on a routine ophthalmological examination. Visual findings deteriorated after treatment with acetazolamide. Continuous intracranial pressure (ICP) monitoring was then utilized to investigate ICP dynamics. Successful treatment with resolution of PE was achieved after ventriculoperitoneal shunting but relapsed due to growth-related dislocation of the ventricular catheter. This report brings new insights into the ICP dynamics and the resulting treatment in this possibly underdiagnosed subgroup of PTCS patients. It also demonstrates that ventriculoperitoneal shunting can provide long-term improvement of symptoms for more than 10 years. pmcAlagille syndrome (AS) or arteriohepatic dysplasia is a rare (1:30,000) autosomal-dominant inherited multisystem disorder [15]. It primarily affects the liver, heart, eyes, face, skeleton, kidneys, and vascular system, whereas its expressivity (phenotypic severity) is highly variable, ranging from no apparent clinical involvement to severe disease that requires liver transplantation [1, 8, 16, 19]. Pseudotumor cerebri syndrome (PTCS) is a disorder characterized by elevated intracranial pressure (ICP) with normal cerebrospinal fluid (CSF) composition in the absence of hydrocephalus, mass lesions, or structural abnormalities. Common presenting signs and symptoms of elevated ICP include papilledema (PE), visual disturbances, and headache. PTCS can be differentiated in a primary form of unknown etiology, which is also termed idiopathic intracranial hypertension (IIH), and a secondary form in which intracranial hypertension results from a medical condition or an exogenous agent [2, 11, 12, 24]. The occurrence of PTCS in AS is exceedingly rare [9, 10, 20, 21]. Thus far ICP monitoring has not been reported in this subgroup of PTCS. Furthermore, owing to their rarity, the natural history and treatment of PTCS in AS patients remain unclear. Here, we present the clinical findings, ICP dynamics, and treatment outcome in a 4-yead-old boy with AS and associated PTCS. Furthermore, previous reports are being reviewed and the possible pathophysiology is discussed. Case report History and examination This 4-year-old boy had a known history of AS. He was born full term at 40 weeks’ gestation by Cesarean section secondary to cephalopelvic disproportion following an uncomplicated pregnancy. At 28 days of age, he underwent a Kasai procedure for biliary atresia. Due to progression of his liver disease, a liver transplantation was performed at 11 months of age after AS had been diagnosed. At 4 years of age, bilateral PE was detected in a routine ophthalmological examination. He was then referred to the neuropediatric department for further evaluation. On clinical examination, he had a normal blood pressure, his weight was 16.5 kg, and his height 96 cm. Neuropediatric examination was normal. Laboratory studies including renal and liver function parameters were normal. An echocardiogram revealed no cardiovascular abnormalities. Medication consisted of immunosuppressive therapy with ciclosporine (70 mg/day), mycophenolate mofetil (400 mg/day), and prednisolone (1 mg/day). The cyclosporine level was within normal range. An MRI scan was performed in which hydrocephalus, mass effect, or structural lesion was ruled out. Subsequently, a lumbar puncture in propofol sedation was achieved revealing a recumbent opening pressure of 48 cm H2O. Examination of the CSF including bacteriological and viral panels showed no abnormalities. Consequently acetazolamide treatment was initiated at a 2 × 125 mg dosage. After 6 weeks, the PE persisted and also deterioration of the visual acuity was documented. In a repeat LP, the opening pressure was still elevated at 34 cm H2O. Repeat neuroimaging assessment remained unremarkable. Given the progressive visual loss and the PE being refractory to medical treatment, the patient was referred to the Department of Neurosurgery for further evaluation and measurement of ICP dynamics. Intracranial pressure monitoring An epidural sensor (Neurodur®, Raumedic, Münchberg, Germany) was implanted over the right frontal area via a precoronal burrhole, and the ICP was monitored for 2 days. ICP monitoring demonstrated massive dynamic ICP changes over time with markedly increased ICP values most of the time (Fig. 1). The decomposition of pressure traces revealed pressure waves with amplitudes reaching values up to 60 mmHg for several minutes. B-waves dominated ICP dynamics superposing the slower ICP fluctuations in time. Additionally, a relatively high number of A-wave pressure transients were scattered in the traces. The majority of ICP values were measured to be within the range of 15 to 70 mmHg (83%).Fig. 1 Intracranial pressure dynamics in a 4-year-old boy with PTCS in Alagille syndrome. The traces show distinctive pathological pressure transients of high amplitude. Data are outlined as traces indicating mean pressure, and as smoothed histograms in a vertical layout. The upper limit of physiological ICP was set at 15 mmHg and is presented as dashed line. On the right side four samples of ICP transients (1–4) demonstrate the complex dynamics of the elevated ICP. Dotted line: zero pressure; brackets: time intervals of 5 min Shunt surgery and postoperative course Subsequent to ICP monitoring, a ventriculoperitoneal shunt was implanted using an optic-guided neuronavigation system (Medtronic, Minneapolis, MN, USA). A proGAV® valve with an integrated shuntassistant® (Aesculap-Miethke, Tuttlingen, Germany) was implanted at an opening pressure of 10 cm H20 [25]. The CT scan on the first postoperative day demonstrated an accurately placed ventricular catheter, and the patient was discharged on the seventh postoperative day. One month after discharge, PE had completely resolved. Further follow-ups were unremarkable until a growth-related dislocation of the ventricular catheter was noted 3.5 years after the shunting procedure. An ophthalmological examination showed relapse of bilateral PE. The ventricular catheter was revised and the immediate postoperative course was uneventful. One month following discharge, the shunt system was explanted due to an infection with Staphylococcus aureus and an external ventricular drain was placed. Antibiotics were administrated intravenously for 3 weeks. When serial CSF bacterial cultures were negative, a new ventriculoperitoneal shunt was inserted guided by electromagnetic neuronavigation (AxiEM) [14]. The valve was set at an opening pressure of 6 cm H2O. Postoperative CT confirmed an accurately placed ventricular catheter. At long-term follow-up (12 years), there was no recurrence and both ophthalmological and neurological findings were unremarkable. The opening pressure has not been adjusted during the follow-up period. Discussion The natural course of AS is associated with high morbidity and mortality if not being diagnosed timely and treated appropriately [16]. AS is associated with a plethora of ophthalmological abnormalities, predominately posterior embryotoxon, optic disc drusen, angulated retinal vessels, and pigmentary retinopathy [17]. In the majority of patients, normal vision can be secured [18]. PE is the most common sign of elevated ICP in PCTS, which can lead to progressive visual loss, when left untreated [26]. Although PE is not associated commonly with AS, concomitant congenital optic disc anomalies can be misinterpreted as PE and consequently result in misdiagnosis and unnecessary treatment [5, 17]. The typical patient with primary PTCS or IIH is a young obese woman in childbearing age [12, 29]. Establishing the diagnosis of primary PCTS in a typical patient is mostly a straightforward process based on the 2013 revision of the Friedman & Jacobson criteria [12]. These require that in the presence of PE the opening pressure measured at lumbar puncture is elevated, the CSF composition is normal, there is no evidence of neurological deficits except for cranial nerve abnormalities, and there are no pathological findings in MRI with and without gadolinium [5, 11, 12]. In contrast, the clinical profile of pediatric PCTS differs widely. Pediatric patients frequently remain asymptomatic but PE is incidentally diagnosed in up to 30% during a routine eye examination [4, 13, 27, 28]. Correct diagnosis in pediatric patients is challenging. Opposite to adults, in children gender distribution is equal and weight is not a contributing factor for disease development. It has been estimated that up to 30% of children with PTCS and a normal body weight harbor a secondary etiology; therefore, appropriate identification of causative factors and conditions is essential prior further treatment [3, 27]. The optimal treatment of PTCS in children remains debatable. The main stay of PTCS medical treatment in adults is acetazolamide. Surgical treatment (CSF shunting procedures, optic nerve sheath fenestration, and venous stenting) is indicated when visual loss is progressive and/or symptoms are intractable despite maximal medical management [14, 23, 28]. Although continuous ICP-monitoring is not routinely performed for diagnosing PTCS, it has been shown that its application is a helpful adjunct especially in atypical cases and when surgical treatment is considered [23, 30]. Especially in pediatric patients, it can provide an accurate measurement of ICP, which is commonly overestimated through a lumbar puncture [6]. An increased baseline ICP, large ICP fluctuations, and the presence of ICP-oscillations (A- and B-waves) during recordings further can confirm the diagnosis [23, 30]. The occurrence of PCTS in patients with AS is exceedingly rare with only few patients having been reported thus far (Table 1). Identifying true risk factors is important for establishing the diagnosis of PCTS and also for understanding the pathophysiology of the disorder [7].Table 1 Summary of patients with PTCS and Alagille syndrome Authors and year Age at presentation/sex Clinical presentation Opening pressure/cm H20 Time between LT and PTCS development/immunosuppression Vitamin A level/supplementation Therapy Outcome Follow-up duration (years) Emerick et al., 2005 3 yrs./M symptomatic, PE Raised/24 Not transplanted ns/ns Acetazolamide and dexamethasone Full recovery ns Narula et al., 2006 6 yrs./NS symptomatic, PE Raised/ns 4 yrs./tacrolimus and steroids Normal/no Acetazolamide and prednisolone, LPS after 15 mo PE resolved, normal vision ns 25 mo/NS asymptomatic PE Raised/ns 16 mo/tacrolimus (changed from cyclosporine due to early rejection) Elevated before PTCS development, normal after PTCS development/ns Furosemide Normal CSF pressure and vision ns 20 mo/NS Asymptomatic PE Raised/ns Not transplanted Normal/no Repeated LP (3×) Normal development and vision ns Ertekin et al., 2010 5 yrs./M Symptomatic Raised/29 Not transplanted Elevated/yes, less than recommended levels Repeated LPs, acetazolamide Symptom free ns Mouzaki et al., 2010 25 mo/F Asymptomatic PE Raised/37 Not transplanted Elevated/no Acetazolamide Improvement of PE 0.5 13 mo/M Asymptomatic PE Raised/35 Not transplanted Elevated/no Acetazolamide Improvement of PE ns Polemikos et al., 2020 4 yrs./M Asymptomatic PE Raised/48 3 yrs./cyclosporine, steroids, mucophenolat mofetil Normal/no Acetazolamide, VPS after 6 weeks Complete resolution of PE 12 F, female; M, male; LP, lumbar puncture; LPS, lumboperitoneal shunt; LT, liver transplantation; ns, not stated; PE, papilledema; VPS, ventriculoperitoneal shunt; PTCS, pseudotumor cerebri syndrome Both hypervitaminosis and hypovitaminosis A are risk factors for PCTS, and may be relevant in AS since most patients have a fat-soluble vitamin deficiency of variable degree which necessitates supplementation [15]. Previous reports have considered elevated vitamin A levels as causative agent of PCTS in AS, two of them in the absence of vitamin A supplementation [21] and one while on receiving vitamin A and D in less than recommended levels [10]. From a pathophysiological point of view, it has been hypothesized that an increased production of CSF or an increased resistance to CSF outflow is the underlying mechanisms for PTCS, whereas most evidence supports the latter, possibly due to a field effect involving epithelial membranes [20]. Sheldon et al. proposed a neuroendocrine pathomechanism for pediatric PTCS. They hypothesized that in PTCS hormonal and metabolic factors regulate CSF production and CSF absorption ultimately leading to elevated ICP [24]. Furthermore, genetic or epigenetic factors appear to be relevant for developing PCTS, based on previously reported familial cases of PTCS [23]. In 89% of cases, AS is caused by mutations/deletions in the Notch signaling pathway ligand gene, JAGGED1, or the gene for its receptor, NOTCH2 [16]. The Notch signaling pathway has an important role in vascular development. Disruption of this pathway results in abnormal vascular development and signaling, which may also contribute to the development of IIH [15, 22]. Mouzaki et al. speculated that abnormalities in the microvasculature of the choroid plexus in AS patients could lead to abnormal CSF production or absorption, which in turn would cause intracranial hypertension [21]. The occurrence of PTCS in AS patients after liver transplantation has been previously described in 2 cases by Narula et al. [22]. While these patients were on tacrolimus, our patient was receiving cyclosporine, which has been related to the development raised ICP after renal, bone marrow, and heart transplantation. Nevertheless, based on the occurrence of intracranial hypertension in AS patients without liver transplant, it appears unlikely that the immunosuppressive medication solely resulted in PCTS, although it could have been a contributing factor [22]. Due to its rarity, knowledge about treatment of PTCS in AS remains very limited. This is reflected by the few previously reported cases as outlined in Table 1. While medical treatment with acetazolamide, furosemide, and steroids was effective, there is only very little information on long-term efficacy. We here show that ventriculoperitoneal shunting may provide long-term relief and prevent recurrent PE. Relapse of PE due to growth-related dislocation of the ventricular catheter reinforces the need of ophthalmological and radiological follow-up examinations. PTCS in AS is likely to be underdiagnosed because ophthalmological examinations are not performed routinely after initial diagnostic workup and due to the fact that the majority of patients remain asymptomatic [22]. Conclusions Our report emphasizes the need for careful evaluation of PCTS in pediatric AS. Through the utility of continuous ICP monitoring, we offer new insights regarding ICP dynamics of this population. We also demonstrate that CSF shunting may provide reliable relief of symptoms on long-term follow-up. Our findings along with other reported cases suggest that a genetic predisposition makes AS patients susceptible for developing PCTS, whereas associated medical conditions and exogenous factors can trigger or exacerbate intracranial hypertension. The differentiation of PTCS in AS as primary or secondary PCTS is a subject deserving further discussion with findings thus far being supportive for the first. Abbreviations AS Alagille syndrome CSF Cerebrospinal fluid ICP Intracranial pressure IIH Idiopathic intracranial hypertension PE Papilledema PTCS Pseudotumor cerebri syndrome Funding Open Access funding enabled and organized by Projekt DEAL. Data availability Not applicable. Declarations Conflict of interest The authors declare no competing interests. Code availability Not applicable. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	ACETAZOLAMIDE	DrugsGivenReaction	CC BY	33555437	20,805,420	2021-09
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Drug ineffective'.	Pseudotumor cerebri syndrome in a child with Alagille syndrome: intracranial pressure dynamics and treatment outcome after ventriculoperitoneal shunting. Alagille syndrome (AS) is a rare multisystem disease of the liver, heart, eyes, face, skeleton, kidneys, and vascular system. The occurrence of pseudotumor cerebri syndrome (PTCS) in patients with AS has been reported only exceptionally. Owning to its rarity and a mostly atypical presentation, the diagnosis and natural history of affected patients remain uncertain. We report an atypical case of PTCS in a 4-year-old boy with a known history of AS who presented with bilateral papilledema (PE) on a routine ophthalmological examination. Visual findings deteriorated after treatment with acetazolamide. Continuous intracranial pressure (ICP) monitoring was then utilized to investigate ICP dynamics. Successful treatment with resolution of PE was achieved after ventriculoperitoneal shunting but relapsed due to growth-related dislocation of the ventricular catheter. This report brings new insights into the ICP dynamics and the resulting treatment in this possibly underdiagnosed subgroup of PTCS patients. It also demonstrates that ventriculoperitoneal shunting can provide long-term improvement of symptoms for more than 10 years. pmcAlagille syndrome (AS) or arteriohepatic dysplasia is a rare (1:30,000) autosomal-dominant inherited multisystem disorder [15]. It primarily affects the liver, heart, eyes, face, skeleton, kidneys, and vascular system, whereas its expressivity (phenotypic severity) is highly variable, ranging from no apparent clinical involvement to severe disease that requires liver transplantation [1, 8, 16, 19]. Pseudotumor cerebri syndrome (PTCS) is a disorder characterized by elevated intracranial pressure (ICP) with normal cerebrospinal fluid (CSF) composition in the absence of hydrocephalus, mass lesions, or structural abnormalities. Common presenting signs and symptoms of elevated ICP include papilledema (PE), visual disturbances, and headache. PTCS can be differentiated in a primary form of unknown etiology, which is also termed idiopathic intracranial hypertension (IIH), and a secondary form in which intracranial hypertension results from a medical condition or an exogenous agent [2, 11, 12, 24]. The occurrence of PTCS in AS is exceedingly rare [9, 10, 20, 21]. Thus far ICP monitoring has not been reported in this subgroup of PTCS. Furthermore, owing to their rarity, the natural history and treatment of PTCS in AS patients remain unclear. Here, we present the clinical findings, ICP dynamics, and treatment outcome in a 4-yead-old boy with AS and associated PTCS. Furthermore, previous reports are being reviewed and the possible pathophysiology is discussed. Case report History and examination This 4-year-old boy had a known history of AS. He was born full term at 40 weeks’ gestation by Cesarean section secondary to cephalopelvic disproportion following an uncomplicated pregnancy. At 28 days of age, he underwent a Kasai procedure for biliary atresia. Due to progression of his liver disease, a liver transplantation was performed at 11 months of age after AS had been diagnosed. At 4 years of age, bilateral PE was detected in a routine ophthalmological examination. He was then referred to the neuropediatric department for further evaluation. On clinical examination, he had a normal blood pressure, his weight was 16.5 kg, and his height 96 cm. Neuropediatric examination was normal. Laboratory studies including renal and liver function parameters were normal. An echocardiogram revealed no cardiovascular abnormalities. Medication consisted of immunosuppressive therapy with ciclosporine (70 mg/day), mycophenolate mofetil (400 mg/day), and prednisolone (1 mg/day). The cyclosporine level was within normal range. An MRI scan was performed in which hydrocephalus, mass effect, or structural lesion was ruled out. Subsequently, a lumbar puncture in propofol sedation was achieved revealing a recumbent opening pressure of 48 cm H2O. Examination of the CSF including bacteriological and viral panels showed no abnormalities. Consequently acetazolamide treatment was initiated at a 2 × 125 mg dosage. After 6 weeks, the PE persisted and also deterioration of the visual acuity was documented. In a repeat LP, the opening pressure was still elevated at 34 cm H2O. Repeat neuroimaging assessment remained unremarkable. Given the progressive visual loss and the PE being refractory to medical treatment, the patient was referred to the Department of Neurosurgery for further evaluation and measurement of ICP dynamics. Intracranial pressure monitoring An epidural sensor (Neurodur®, Raumedic, Münchberg, Germany) was implanted over the right frontal area via a precoronal burrhole, and the ICP was monitored for 2 days. ICP monitoring demonstrated massive dynamic ICP changes over time with markedly increased ICP values most of the time (Fig. 1). The decomposition of pressure traces revealed pressure waves with amplitudes reaching values up to 60 mmHg for several minutes. B-waves dominated ICP dynamics superposing the slower ICP fluctuations in time. Additionally, a relatively high number of A-wave pressure transients were scattered in the traces. The majority of ICP values were measured to be within the range of 15 to 70 mmHg (83%).Fig. 1 Intracranial pressure dynamics in a 4-year-old boy with PTCS in Alagille syndrome. The traces show distinctive pathological pressure transients of high amplitude. Data are outlined as traces indicating mean pressure, and as smoothed histograms in a vertical layout. The upper limit of physiological ICP was set at 15 mmHg and is presented as dashed line. On the right side four samples of ICP transients (1–4) demonstrate the complex dynamics of the elevated ICP. Dotted line: zero pressure; brackets: time intervals of 5 min Shunt surgery and postoperative course Subsequent to ICP monitoring, a ventriculoperitoneal shunt was implanted using an optic-guided neuronavigation system (Medtronic, Minneapolis, MN, USA). A proGAV® valve with an integrated shuntassistant® (Aesculap-Miethke, Tuttlingen, Germany) was implanted at an opening pressure of 10 cm H20 [25]. The CT scan on the first postoperative day demonstrated an accurately placed ventricular catheter, and the patient was discharged on the seventh postoperative day. One month after discharge, PE had completely resolved. Further follow-ups were unremarkable until a growth-related dislocation of the ventricular catheter was noted 3.5 years after the shunting procedure. An ophthalmological examination showed relapse of bilateral PE. The ventricular catheter was revised and the immediate postoperative course was uneventful. One month following discharge, the shunt system was explanted due to an infection with Staphylococcus aureus and an external ventricular drain was placed. Antibiotics were administrated intravenously for 3 weeks. When serial CSF bacterial cultures were negative, a new ventriculoperitoneal shunt was inserted guided by electromagnetic neuronavigation (AxiEM) [14]. The valve was set at an opening pressure of 6 cm H2O. Postoperative CT confirmed an accurately placed ventricular catheter. At long-term follow-up (12 years), there was no recurrence and both ophthalmological and neurological findings were unremarkable. The opening pressure has not been adjusted during the follow-up period. Discussion The natural course of AS is associated with high morbidity and mortality if not being diagnosed timely and treated appropriately [16]. AS is associated with a plethora of ophthalmological abnormalities, predominately posterior embryotoxon, optic disc drusen, angulated retinal vessels, and pigmentary retinopathy [17]. In the majority of patients, normal vision can be secured [18]. PE is the most common sign of elevated ICP in PCTS, which can lead to progressive visual loss, when left untreated [26]. Although PE is not associated commonly with AS, concomitant congenital optic disc anomalies can be misinterpreted as PE and consequently result in misdiagnosis and unnecessary treatment [5, 17]. The typical patient with primary PTCS or IIH is a young obese woman in childbearing age [12, 29]. Establishing the diagnosis of primary PCTS in a typical patient is mostly a straightforward process based on the 2013 revision of the Friedman & Jacobson criteria [12]. These require that in the presence of PE the opening pressure measured at lumbar puncture is elevated, the CSF composition is normal, there is no evidence of neurological deficits except for cranial nerve abnormalities, and there are no pathological findings in MRI with and without gadolinium [5, 11, 12]. In contrast, the clinical profile of pediatric PCTS differs widely. Pediatric patients frequently remain asymptomatic but PE is incidentally diagnosed in up to 30% during a routine eye examination [4, 13, 27, 28]. Correct diagnosis in pediatric patients is challenging. Opposite to adults, in children gender distribution is equal and weight is not a contributing factor for disease development. It has been estimated that up to 30% of children with PTCS and a normal body weight harbor a secondary etiology; therefore, appropriate identification of causative factors and conditions is essential prior further treatment [3, 27]. The optimal treatment of PTCS in children remains debatable. The main stay of PTCS medical treatment in adults is acetazolamide. Surgical treatment (CSF shunting procedures, optic nerve sheath fenestration, and venous stenting) is indicated when visual loss is progressive and/or symptoms are intractable despite maximal medical management [14, 23, 28]. Although continuous ICP-monitoring is not routinely performed for diagnosing PTCS, it has been shown that its application is a helpful adjunct especially in atypical cases and when surgical treatment is considered [23, 30]. Especially in pediatric patients, it can provide an accurate measurement of ICP, which is commonly overestimated through a lumbar puncture [6]. An increased baseline ICP, large ICP fluctuations, and the presence of ICP-oscillations (A- and B-waves) during recordings further can confirm the diagnosis [23, 30]. The occurrence of PCTS in patients with AS is exceedingly rare with only few patients having been reported thus far (Table 1). Identifying true risk factors is important for establishing the diagnosis of PCTS and also for understanding the pathophysiology of the disorder [7].Table 1 Summary of patients with PTCS and Alagille syndrome Authors and year Age at presentation/sex Clinical presentation Opening pressure/cm H20 Time between LT and PTCS development/immunosuppression Vitamin A level/supplementation Therapy Outcome Follow-up duration (years) Emerick et al., 2005 3 yrs./M symptomatic, PE Raised/24 Not transplanted ns/ns Acetazolamide and dexamethasone Full recovery ns Narula et al., 2006 6 yrs./NS symptomatic, PE Raised/ns 4 yrs./tacrolimus and steroids Normal/no Acetazolamide and prednisolone, LPS after 15 mo PE resolved, normal vision ns 25 mo/NS asymptomatic PE Raised/ns 16 mo/tacrolimus (changed from cyclosporine due to early rejection) Elevated before PTCS development, normal after PTCS development/ns Furosemide Normal CSF pressure and vision ns 20 mo/NS Asymptomatic PE Raised/ns Not transplanted Normal/no Repeated LP (3×) Normal development and vision ns Ertekin et al., 2010 5 yrs./M Symptomatic Raised/29 Not transplanted Elevated/yes, less than recommended levels Repeated LPs, acetazolamide Symptom free ns Mouzaki et al., 2010 25 mo/F Asymptomatic PE Raised/37 Not transplanted Elevated/no Acetazolamide Improvement of PE 0.5 13 mo/M Asymptomatic PE Raised/35 Not transplanted Elevated/no Acetazolamide Improvement of PE ns Polemikos et al., 2020 4 yrs./M Asymptomatic PE Raised/48 3 yrs./cyclosporine, steroids, mucophenolat mofetil Normal/no Acetazolamide, VPS after 6 weeks Complete resolution of PE 12 F, female; M, male; LP, lumbar puncture; LPS, lumboperitoneal shunt; LT, liver transplantation; ns, not stated; PE, papilledema; VPS, ventriculoperitoneal shunt; PTCS, pseudotumor cerebri syndrome Both hypervitaminosis and hypovitaminosis A are risk factors for PCTS, and may be relevant in AS since most patients have a fat-soluble vitamin deficiency of variable degree which necessitates supplementation [15]. Previous reports have considered elevated vitamin A levels as causative agent of PCTS in AS, two of them in the absence of vitamin A supplementation [21] and one while on receiving vitamin A and D in less than recommended levels [10]. From a pathophysiological point of view, it has been hypothesized that an increased production of CSF or an increased resistance to CSF outflow is the underlying mechanisms for PTCS, whereas most evidence supports the latter, possibly due to a field effect involving epithelial membranes [20]. Sheldon et al. proposed a neuroendocrine pathomechanism for pediatric PTCS. They hypothesized that in PTCS hormonal and metabolic factors regulate CSF production and CSF absorption ultimately leading to elevated ICP [24]. Furthermore, genetic or epigenetic factors appear to be relevant for developing PCTS, based on previously reported familial cases of PTCS [23]. In 89% of cases, AS is caused by mutations/deletions in the Notch signaling pathway ligand gene, JAGGED1, or the gene for its receptor, NOTCH2 [16]. The Notch signaling pathway has an important role in vascular development. Disruption of this pathway results in abnormal vascular development and signaling, which may also contribute to the development of IIH [15, 22]. Mouzaki et al. speculated that abnormalities in the microvasculature of the choroid plexus in AS patients could lead to abnormal CSF production or absorption, which in turn would cause intracranial hypertension [21]. The occurrence of PTCS in AS patients after liver transplantation has been previously described in 2 cases by Narula et al. [22]. While these patients were on tacrolimus, our patient was receiving cyclosporine, which has been related to the development raised ICP after renal, bone marrow, and heart transplantation. Nevertheless, based on the occurrence of intracranial hypertension in AS patients without liver transplant, it appears unlikely that the immunosuppressive medication solely resulted in PCTS, although it could have been a contributing factor [22]. Due to its rarity, knowledge about treatment of PTCS in AS remains very limited. This is reflected by the few previously reported cases as outlined in Table 1. While medical treatment with acetazolamide, furosemide, and steroids was effective, there is only very little information on long-term efficacy. We here show that ventriculoperitoneal shunting may provide long-term relief and prevent recurrent PE. Relapse of PE due to growth-related dislocation of the ventricular catheter reinforces the need of ophthalmological and radiological follow-up examinations. PTCS in AS is likely to be underdiagnosed because ophthalmological examinations are not performed routinely after initial diagnostic workup and due to the fact that the majority of patients remain asymptomatic [22]. Conclusions Our report emphasizes the need for careful evaluation of PCTS in pediatric AS. Through the utility of continuous ICP monitoring, we offer new insights regarding ICP dynamics of this population. We also demonstrate that CSF shunting may provide reliable relief of symptoms on long-term follow-up. Our findings along with other reported cases suggest that a genetic predisposition makes AS patients susceptible for developing PCTS, whereas associated medical conditions and exogenous factors can trigger or exacerbate intracranial hypertension. The differentiation of PTCS in AS as primary or secondary PCTS is a subject deserving further discussion with findings thus far being supportive for the first. Abbreviations AS Alagille syndrome CSF Cerebrospinal fluid ICP Intracranial pressure IIH Idiopathic intracranial hypertension PE Papilledema PTCS Pseudotumor cerebri syndrome Funding Open Access funding enabled and organized by Projekt DEAL. Data availability Not applicable. Declarations Conflict of interest The authors declare no competing interests. Code availability Not applicable. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	ACETAZOLAMIDE	DrugsGivenReaction	CC BY	33555437	20,805,420	2021-09
What is the weight of the patient?	Pseudotumor cerebri syndrome in a child with Alagille syndrome: intracranial pressure dynamics and treatment outcome after ventriculoperitoneal shunting. Alagille syndrome (AS) is a rare multisystem disease of the liver, heart, eyes, face, skeleton, kidneys, and vascular system. The occurrence of pseudotumor cerebri syndrome (PTCS) in patients with AS has been reported only exceptionally. Owning to its rarity and a mostly atypical presentation, the diagnosis and natural history of affected patients remain uncertain. We report an atypical case of PTCS in a 4-year-old boy with a known history of AS who presented with bilateral papilledema (PE) on a routine ophthalmological examination. Visual findings deteriorated after treatment with acetazolamide. Continuous intracranial pressure (ICP) monitoring was then utilized to investigate ICP dynamics. Successful treatment with resolution of PE was achieved after ventriculoperitoneal shunting but relapsed due to growth-related dislocation of the ventricular catheter. This report brings new insights into the ICP dynamics and the resulting treatment in this possibly underdiagnosed subgroup of PTCS patients. It also demonstrates that ventriculoperitoneal shunting can provide long-term improvement of symptoms for more than 10 years. pmcAlagille syndrome (AS) or arteriohepatic dysplasia is a rare (1:30,000) autosomal-dominant inherited multisystem disorder [15]. It primarily affects the liver, heart, eyes, face, skeleton, kidneys, and vascular system, whereas its expressivity (phenotypic severity) is highly variable, ranging from no apparent clinical involvement to severe disease that requires liver transplantation [1, 8, 16, 19]. Pseudotumor cerebri syndrome (PTCS) is a disorder characterized by elevated intracranial pressure (ICP) with normal cerebrospinal fluid (CSF) composition in the absence of hydrocephalus, mass lesions, or structural abnormalities. Common presenting signs and symptoms of elevated ICP include papilledema (PE), visual disturbances, and headache. PTCS can be differentiated in a primary form of unknown etiology, which is also termed idiopathic intracranial hypertension (IIH), and a secondary form in which intracranial hypertension results from a medical condition or an exogenous agent [2, 11, 12, 24]. The occurrence of PTCS in AS is exceedingly rare [9, 10, 20, 21]. Thus far ICP monitoring has not been reported in this subgroup of PTCS. Furthermore, owing to their rarity, the natural history and treatment of PTCS in AS patients remain unclear. Here, we present the clinical findings, ICP dynamics, and treatment outcome in a 4-yead-old boy with AS and associated PTCS. Furthermore, previous reports are being reviewed and the possible pathophysiology is discussed. Case report History and examination This 4-year-old boy had a known history of AS. He was born full term at 40 weeks’ gestation by Cesarean section secondary to cephalopelvic disproportion following an uncomplicated pregnancy. At 28 days of age, he underwent a Kasai procedure for biliary atresia. Due to progression of his liver disease, a liver transplantation was performed at 11 months of age after AS had been diagnosed. At 4 years of age, bilateral PE was detected in a routine ophthalmological examination. He was then referred to the neuropediatric department for further evaluation. On clinical examination, he had a normal blood pressure, his weight was 16.5 kg, and his height 96 cm. Neuropediatric examination was normal. Laboratory studies including renal and liver function parameters were normal. An echocardiogram revealed no cardiovascular abnormalities. Medication consisted of immunosuppressive therapy with ciclosporine (70 mg/day), mycophenolate mofetil (400 mg/day), and prednisolone (1 mg/day). The cyclosporine level was within normal range. An MRI scan was performed in which hydrocephalus, mass effect, or structural lesion was ruled out. Subsequently, a lumbar puncture in propofol sedation was achieved revealing a recumbent opening pressure of 48 cm H2O. Examination of the CSF including bacteriological and viral panels showed no abnormalities. Consequently acetazolamide treatment was initiated at a 2 × 125 mg dosage. After 6 weeks, the PE persisted and also deterioration of the visual acuity was documented. In a repeat LP, the opening pressure was still elevated at 34 cm H2O. Repeat neuroimaging assessment remained unremarkable. Given the progressive visual loss and the PE being refractory to medical treatment, the patient was referred to the Department of Neurosurgery for further evaluation and measurement of ICP dynamics. Intracranial pressure monitoring An epidural sensor (Neurodur®, Raumedic, Münchberg, Germany) was implanted over the right frontal area via a precoronal burrhole, and the ICP was monitored for 2 days. ICP monitoring demonstrated massive dynamic ICP changes over time with markedly increased ICP values most of the time (Fig. 1). The decomposition of pressure traces revealed pressure waves with amplitudes reaching values up to 60 mmHg for several minutes. B-waves dominated ICP dynamics superposing the slower ICP fluctuations in time. Additionally, a relatively high number of A-wave pressure transients were scattered in the traces. The majority of ICP values were measured to be within the range of 15 to 70 mmHg (83%).Fig. 1 Intracranial pressure dynamics in a 4-year-old boy with PTCS in Alagille syndrome. The traces show distinctive pathological pressure transients of high amplitude. Data are outlined as traces indicating mean pressure, and as smoothed histograms in a vertical layout. The upper limit of physiological ICP was set at 15 mmHg and is presented as dashed line. On the right side four samples of ICP transients (1–4) demonstrate the complex dynamics of the elevated ICP. Dotted line: zero pressure; brackets: time intervals of 5 min Shunt surgery and postoperative course Subsequent to ICP monitoring, a ventriculoperitoneal shunt was implanted using an optic-guided neuronavigation system (Medtronic, Minneapolis, MN, USA). A proGAV® valve with an integrated shuntassistant® (Aesculap-Miethke, Tuttlingen, Germany) was implanted at an opening pressure of 10 cm H20 [25]. The CT scan on the first postoperative day demonstrated an accurately placed ventricular catheter, and the patient was discharged on the seventh postoperative day. One month after discharge, PE had completely resolved. Further follow-ups were unremarkable until a growth-related dislocation of the ventricular catheter was noted 3.5 years after the shunting procedure. An ophthalmological examination showed relapse of bilateral PE. The ventricular catheter was revised and the immediate postoperative course was uneventful. One month following discharge, the shunt system was explanted due to an infection with Staphylococcus aureus and an external ventricular drain was placed. Antibiotics were administrated intravenously for 3 weeks. When serial CSF bacterial cultures were negative, a new ventriculoperitoneal shunt was inserted guided by electromagnetic neuronavigation (AxiEM) [14]. The valve was set at an opening pressure of 6 cm H2O. Postoperative CT confirmed an accurately placed ventricular catheter. At long-term follow-up (12 years), there was no recurrence and both ophthalmological and neurological findings were unremarkable. The opening pressure has not been adjusted during the follow-up period. Discussion The natural course of AS is associated with high morbidity and mortality if not being diagnosed timely and treated appropriately [16]. AS is associated with a plethora of ophthalmological abnormalities, predominately posterior embryotoxon, optic disc drusen, angulated retinal vessels, and pigmentary retinopathy [17]. In the majority of patients, normal vision can be secured [18]. PE is the most common sign of elevated ICP in PCTS, which can lead to progressive visual loss, when left untreated [26]. Although PE is not associated commonly with AS, concomitant congenital optic disc anomalies can be misinterpreted as PE and consequently result in misdiagnosis and unnecessary treatment [5, 17]. The typical patient with primary PTCS or IIH is a young obese woman in childbearing age [12, 29]. Establishing the diagnosis of primary PCTS in a typical patient is mostly a straightforward process based on the 2013 revision of the Friedman & Jacobson criteria [12]. These require that in the presence of PE the opening pressure measured at lumbar puncture is elevated, the CSF composition is normal, there is no evidence of neurological deficits except for cranial nerve abnormalities, and there are no pathological findings in MRI with and without gadolinium [5, 11, 12]. In contrast, the clinical profile of pediatric PCTS differs widely. Pediatric patients frequently remain asymptomatic but PE is incidentally diagnosed in up to 30% during a routine eye examination [4, 13, 27, 28]. Correct diagnosis in pediatric patients is challenging. Opposite to adults, in children gender distribution is equal and weight is not a contributing factor for disease development. It has been estimated that up to 30% of children with PTCS and a normal body weight harbor a secondary etiology; therefore, appropriate identification of causative factors and conditions is essential prior further treatment [3, 27]. The optimal treatment of PTCS in children remains debatable. The main stay of PTCS medical treatment in adults is acetazolamide. Surgical treatment (CSF shunting procedures, optic nerve sheath fenestration, and venous stenting) is indicated when visual loss is progressive and/or symptoms are intractable despite maximal medical management [14, 23, 28]. Although continuous ICP-monitoring is not routinely performed for diagnosing PTCS, it has been shown that its application is a helpful adjunct especially in atypical cases and when surgical treatment is considered [23, 30]. Especially in pediatric patients, it can provide an accurate measurement of ICP, which is commonly overestimated through a lumbar puncture [6]. An increased baseline ICP, large ICP fluctuations, and the presence of ICP-oscillations (A- and B-waves) during recordings further can confirm the diagnosis [23, 30]. The occurrence of PCTS in patients with AS is exceedingly rare with only few patients having been reported thus far (Table 1). Identifying true risk factors is important for establishing the diagnosis of PCTS and also for understanding the pathophysiology of the disorder [7].Table 1 Summary of patients with PTCS and Alagille syndrome Authors and year Age at presentation/sex Clinical presentation Opening pressure/cm H20 Time between LT and PTCS development/immunosuppression Vitamin A level/supplementation Therapy Outcome Follow-up duration (years) Emerick et al., 2005 3 yrs./M symptomatic, PE Raised/24 Not transplanted ns/ns Acetazolamide and dexamethasone Full recovery ns Narula et al., 2006 6 yrs./NS symptomatic, PE Raised/ns 4 yrs./tacrolimus and steroids Normal/no Acetazolamide and prednisolone, LPS after 15 mo PE resolved, normal vision ns 25 mo/NS asymptomatic PE Raised/ns 16 mo/tacrolimus (changed from cyclosporine due to early rejection) Elevated before PTCS development, normal after PTCS development/ns Furosemide Normal CSF pressure and vision ns 20 mo/NS Asymptomatic PE Raised/ns Not transplanted Normal/no Repeated LP (3×) Normal development and vision ns Ertekin et al., 2010 5 yrs./M Symptomatic Raised/29 Not transplanted Elevated/yes, less than recommended levels Repeated LPs, acetazolamide Symptom free ns Mouzaki et al., 2010 25 mo/F Asymptomatic PE Raised/37 Not transplanted Elevated/no Acetazolamide Improvement of PE 0.5 13 mo/M Asymptomatic PE Raised/35 Not transplanted Elevated/no Acetazolamide Improvement of PE ns Polemikos et al., 2020 4 yrs./M Asymptomatic PE Raised/48 3 yrs./cyclosporine, steroids, mucophenolat mofetil Normal/no Acetazolamide, VPS after 6 weeks Complete resolution of PE 12 F, female; M, male; LP, lumbar puncture; LPS, lumboperitoneal shunt; LT, liver transplantation; ns, not stated; PE, papilledema; VPS, ventriculoperitoneal shunt; PTCS, pseudotumor cerebri syndrome Both hypervitaminosis and hypovitaminosis A are risk factors for PCTS, and may be relevant in AS since most patients have a fat-soluble vitamin deficiency of variable degree which necessitates supplementation [15]. Previous reports have considered elevated vitamin A levels as causative agent of PCTS in AS, two of them in the absence of vitamin A supplementation [21] and one while on receiving vitamin A and D in less than recommended levels [10]. From a pathophysiological point of view, it has been hypothesized that an increased production of CSF or an increased resistance to CSF outflow is the underlying mechanisms for PTCS, whereas most evidence supports the latter, possibly due to a field effect involving epithelial membranes [20]. Sheldon et al. proposed a neuroendocrine pathomechanism for pediatric PTCS. They hypothesized that in PTCS hormonal and metabolic factors regulate CSF production and CSF absorption ultimately leading to elevated ICP [24]. Furthermore, genetic or epigenetic factors appear to be relevant for developing PCTS, based on previously reported familial cases of PTCS [23]. In 89% of cases, AS is caused by mutations/deletions in the Notch signaling pathway ligand gene, JAGGED1, or the gene for its receptor, NOTCH2 [16]. The Notch signaling pathway has an important role in vascular development. Disruption of this pathway results in abnormal vascular development and signaling, which may also contribute to the development of IIH [15, 22]. Mouzaki et al. speculated that abnormalities in the microvasculature of the choroid plexus in AS patients could lead to abnormal CSF production or absorption, which in turn would cause intracranial hypertension [21]. The occurrence of PTCS in AS patients after liver transplantation has been previously described in 2 cases by Narula et al. [22]. While these patients were on tacrolimus, our patient was receiving cyclosporine, which has been related to the development raised ICP after renal, bone marrow, and heart transplantation. Nevertheless, based on the occurrence of intracranial hypertension in AS patients without liver transplant, it appears unlikely that the immunosuppressive medication solely resulted in PCTS, although it could have been a contributing factor [22]. Due to its rarity, knowledge about treatment of PTCS in AS remains very limited. This is reflected by the few previously reported cases as outlined in Table 1. While medical treatment with acetazolamide, furosemide, and steroids was effective, there is only very little information on long-term efficacy. We here show that ventriculoperitoneal shunting may provide long-term relief and prevent recurrent PE. Relapse of PE due to growth-related dislocation of the ventricular catheter reinforces the need of ophthalmological and radiological follow-up examinations. PTCS in AS is likely to be underdiagnosed because ophthalmological examinations are not performed routinely after initial diagnostic workup and due to the fact that the majority of patients remain asymptomatic [22]. Conclusions Our report emphasizes the need for careful evaluation of PCTS in pediatric AS. Through the utility of continuous ICP monitoring, we offer new insights regarding ICP dynamics of this population. We also demonstrate that CSF shunting may provide reliable relief of symptoms on long-term follow-up. Our findings along with other reported cases suggest that a genetic predisposition makes AS patients susceptible for developing PCTS, whereas associated medical conditions and exogenous factors can trigger or exacerbate intracranial hypertension. The differentiation of PTCS in AS as primary or secondary PCTS is a subject deserving further discussion with findings thus far being supportive for the first. Abbreviations AS Alagille syndrome CSF Cerebrospinal fluid ICP Intracranial pressure IIH Idiopathic intracranial hypertension PE Papilledema PTCS Pseudotumor cerebri syndrome Funding Open Access funding enabled and organized by Projekt DEAL. Data availability Not applicable. Declarations Conflict of interest The authors declare no competing interests. Code availability Not applicable. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	16.5 kg.	Weight	CC BY	33555437	20,770,619	2021-09
What was the outcome of reaction 'Idiopathic intracranial hypertension'?	Pseudotumor cerebri syndrome in a child with Alagille syndrome: intracranial pressure dynamics and treatment outcome after ventriculoperitoneal shunting. Alagille syndrome (AS) is a rare multisystem disease of the liver, heart, eyes, face, skeleton, kidneys, and vascular system. The occurrence of pseudotumor cerebri syndrome (PTCS) in patients with AS has been reported only exceptionally. Owning to its rarity and a mostly atypical presentation, the diagnosis and natural history of affected patients remain uncertain. We report an atypical case of PTCS in a 4-year-old boy with a known history of AS who presented with bilateral papilledema (PE) on a routine ophthalmological examination. Visual findings deteriorated after treatment with acetazolamide. Continuous intracranial pressure (ICP) monitoring was then utilized to investigate ICP dynamics. Successful treatment with resolution of PE was achieved after ventriculoperitoneal shunting but relapsed due to growth-related dislocation of the ventricular catheter. This report brings new insights into the ICP dynamics and the resulting treatment in this possibly underdiagnosed subgroup of PTCS patients. It also demonstrates that ventriculoperitoneal shunting can provide long-term improvement of symptoms for more than 10 years. pmcAlagille syndrome (AS) or arteriohepatic dysplasia is a rare (1:30,000) autosomal-dominant inherited multisystem disorder [15]. It primarily affects the liver, heart, eyes, face, skeleton, kidneys, and vascular system, whereas its expressivity (phenotypic severity) is highly variable, ranging from no apparent clinical involvement to severe disease that requires liver transplantation [1, 8, 16, 19]. Pseudotumor cerebri syndrome (PTCS) is a disorder characterized by elevated intracranial pressure (ICP) with normal cerebrospinal fluid (CSF) composition in the absence of hydrocephalus, mass lesions, or structural abnormalities. Common presenting signs and symptoms of elevated ICP include papilledema (PE), visual disturbances, and headache. PTCS can be differentiated in a primary form of unknown etiology, which is also termed idiopathic intracranial hypertension (IIH), and a secondary form in which intracranial hypertension results from a medical condition or an exogenous agent [2, 11, 12, 24]. The occurrence of PTCS in AS is exceedingly rare [9, 10, 20, 21]. Thus far ICP monitoring has not been reported in this subgroup of PTCS. Furthermore, owing to their rarity, the natural history and treatment of PTCS in AS patients remain unclear. Here, we present the clinical findings, ICP dynamics, and treatment outcome in a 4-yead-old boy with AS and associated PTCS. Furthermore, previous reports are being reviewed and the possible pathophysiology is discussed. Case report History and examination This 4-year-old boy had a known history of AS. He was born full term at 40 weeks’ gestation by Cesarean section secondary to cephalopelvic disproportion following an uncomplicated pregnancy. At 28 days of age, he underwent a Kasai procedure for biliary atresia. Due to progression of his liver disease, a liver transplantation was performed at 11 months of age after AS had been diagnosed. At 4 years of age, bilateral PE was detected in a routine ophthalmological examination. He was then referred to the neuropediatric department for further evaluation. On clinical examination, he had a normal blood pressure, his weight was 16.5 kg, and his height 96 cm. Neuropediatric examination was normal. Laboratory studies including renal and liver function parameters were normal. An echocardiogram revealed no cardiovascular abnormalities. Medication consisted of immunosuppressive therapy with ciclosporine (70 mg/day), mycophenolate mofetil (400 mg/day), and prednisolone (1 mg/day). The cyclosporine level was within normal range. An MRI scan was performed in which hydrocephalus, mass effect, or structural lesion was ruled out. Subsequently, a lumbar puncture in propofol sedation was achieved revealing a recumbent opening pressure of 48 cm H2O. Examination of the CSF including bacteriological and viral panels showed no abnormalities. Consequently acetazolamide treatment was initiated at a 2 × 125 mg dosage. After 6 weeks, the PE persisted and also deterioration of the visual acuity was documented. In a repeat LP, the opening pressure was still elevated at 34 cm H2O. Repeat neuroimaging assessment remained unremarkable. Given the progressive visual loss and the PE being refractory to medical treatment, the patient was referred to the Department of Neurosurgery for further evaluation and measurement of ICP dynamics. Intracranial pressure monitoring An epidural sensor (Neurodur®, Raumedic, Münchberg, Germany) was implanted over the right frontal area via a precoronal burrhole, and the ICP was monitored for 2 days. ICP monitoring demonstrated massive dynamic ICP changes over time with markedly increased ICP values most of the time (Fig. 1). The decomposition of pressure traces revealed pressure waves with amplitudes reaching values up to 60 mmHg for several minutes. B-waves dominated ICP dynamics superposing the slower ICP fluctuations in time. Additionally, a relatively high number of A-wave pressure transients were scattered in the traces. The majority of ICP values were measured to be within the range of 15 to 70 mmHg (83%).Fig. 1 Intracranial pressure dynamics in a 4-year-old boy with PTCS in Alagille syndrome. The traces show distinctive pathological pressure transients of high amplitude. Data are outlined as traces indicating mean pressure, and as smoothed histograms in a vertical layout. The upper limit of physiological ICP was set at 15 mmHg and is presented as dashed line. On the right side four samples of ICP transients (1–4) demonstrate the complex dynamics of the elevated ICP. Dotted line: zero pressure; brackets: time intervals of 5 min Shunt surgery and postoperative course Subsequent to ICP monitoring, a ventriculoperitoneal shunt was implanted using an optic-guided neuronavigation system (Medtronic, Minneapolis, MN, USA). A proGAV® valve with an integrated shuntassistant® (Aesculap-Miethke, Tuttlingen, Germany) was implanted at an opening pressure of 10 cm H20 [25]. The CT scan on the first postoperative day demonstrated an accurately placed ventricular catheter, and the patient was discharged on the seventh postoperative day. One month after discharge, PE had completely resolved. Further follow-ups were unremarkable until a growth-related dislocation of the ventricular catheter was noted 3.5 years after the shunting procedure. An ophthalmological examination showed relapse of bilateral PE. The ventricular catheter was revised and the immediate postoperative course was uneventful. One month following discharge, the shunt system was explanted due to an infection with Staphylococcus aureus and an external ventricular drain was placed. Antibiotics were administrated intravenously for 3 weeks. When serial CSF bacterial cultures were negative, a new ventriculoperitoneal shunt was inserted guided by electromagnetic neuronavigation (AxiEM) [14]. The valve was set at an opening pressure of 6 cm H2O. Postoperative CT confirmed an accurately placed ventricular catheter. At long-term follow-up (12 years), there was no recurrence and both ophthalmological and neurological findings were unremarkable. The opening pressure has not been adjusted during the follow-up period. Discussion The natural course of AS is associated with high morbidity and mortality if not being diagnosed timely and treated appropriately [16]. AS is associated with a plethora of ophthalmological abnormalities, predominately posterior embryotoxon, optic disc drusen, angulated retinal vessels, and pigmentary retinopathy [17]. In the majority of patients, normal vision can be secured [18]. PE is the most common sign of elevated ICP in PCTS, which can lead to progressive visual loss, when left untreated [26]. Although PE is not associated commonly with AS, concomitant congenital optic disc anomalies can be misinterpreted as PE and consequently result in misdiagnosis and unnecessary treatment [5, 17]. The typical patient with primary PTCS or IIH is a young obese woman in childbearing age [12, 29]. Establishing the diagnosis of primary PCTS in a typical patient is mostly a straightforward process based on the 2013 revision of the Friedman & Jacobson criteria [12]. These require that in the presence of PE the opening pressure measured at lumbar puncture is elevated, the CSF composition is normal, there is no evidence of neurological deficits except for cranial nerve abnormalities, and there are no pathological findings in MRI with and without gadolinium [5, 11, 12]. In contrast, the clinical profile of pediatric PCTS differs widely. Pediatric patients frequently remain asymptomatic but PE is incidentally diagnosed in up to 30% during a routine eye examination [4, 13, 27, 28]. Correct diagnosis in pediatric patients is challenging. Opposite to adults, in children gender distribution is equal and weight is not a contributing factor for disease development. It has been estimated that up to 30% of children with PTCS and a normal body weight harbor a secondary etiology; therefore, appropriate identification of causative factors and conditions is essential prior further treatment [3, 27]. The optimal treatment of PTCS in children remains debatable. The main stay of PTCS medical treatment in adults is acetazolamide. Surgical treatment (CSF shunting procedures, optic nerve sheath fenestration, and venous stenting) is indicated when visual loss is progressive and/or symptoms are intractable despite maximal medical management [14, 23, 28]. Although continuous ICP-monitoring is not routinely performed for diagnosing PTCS, it has been shown that its application is a helpful adjunct especially in atypical cases and when surgical treatment is considered [23, 30]. Especially in pediatric patients, it can provide an accurate measurement of ICP, which is commonly overestimated through a lumbar puncture [6]. An increased baseline ICP, large ICP fluctuations, and the presence of ICP-oscillations (A- and B-waves) during recordings further can confirm the diagnosis [23, 30]. The occurrence of PCTS in patients with AS is exceedingly rare with only few patients having been reported thus far (Table 1). Identifying true risk factors is important for establishing the diagnosis of PCTS and also for understanding the pathophysiology of the disorder [7].Table 1 Summary of patients with PTCS and Alagille syndrome Authors and year Age at presentation/sex Clinical presentation Opening pressure/cm H20 Time between LT and PTCS development/immunosuppression Vitamin A level/supplementation Therapy Outcome Follow-up duration (years) Emerick et al., 2005 3 yrs./M symptomatic, PE Raised/24 Not transplanted ns/ns Acetazolamide and dexamethasone Full recovery ns Narula et al., 2006 6 yrs./NS symptomatic, PE Raised/ns 4 yrs./tacrolimus and steroids Normal/no Acetazolamide and prednisolone, LPS after 15 mo PE resolved, normal vision ns 25 mo/NS asymptomatic PE Raised/ns 16 mo/tacrolimus (changed from cyclosporine due to early rejection) Elevated before PTCS development, normal after PTCS development/ns Furosemide Normal CSF pressure and vision ns 20 mo/NS Asymptomatic PE Raised/ns Not transplanted Normal/no Repeated LP (3×) Normal development and vision ns Ertekin et al., 2010 5 yrs./M Symptomatic Raised/29 Not transplanted Elevated/yes, less than recommended levels Repeated LPs, acetazolamide Symptom free ns Mouzaki et al., 2010 25 mo/F Asymptomatic PE Raised/37 Not transplanted Elevated/no Acetazolamide Improvement of PE 0.5 13 mo/M Asymptomatic PE Raised/35 Not transplanted Elevated/no Acetazolamide Improvement of PE ns Polemikos et al., 2020 4 yrs./M Asymptomatic PE Raised/48 3 yrs./cyclosporine, steroids, mucophenolat mofetil Normal/no Acetazolamide, VPS after 6 weeks Complete resolution of PE 12 F, female; M, male; LP, lumbar puncture; LPS, lumboperitoneal shunt; LT, liver transplantation; ns, not stated; PE, papilledema; VPS, ventriculoperitoneal shunt; PTCS, pseudotumor cerebri syndrome Both hypervitaminosis and hypovitaminosis A are risk factors for PCTS, and may be relevant in AS since most patients have a fat-soluble vitamin deficiency of variable degree which necessitates supplementation [15]. Previous reports have considered elevated vitamin A levels as causative agent of PCTS in AS, two of them in the absence of vitamin A supplementation [21] and one while on receiving vitamin A and D in less than recommended levels [10]. From a pathophysiological point of view, it has been hypothesized that an increased production of CSF or an increased resistance to CSF outflow is the underlying mechanisms for PTCS, whereas most evidence supports the latter, possibly due to a field effect involving epithelial membranes [20]. Sheldon et al. proposed a neuroendocrine pathomechanism for pediatric PTCS. They hypothesized that in PTCS hormonal and metabolic factors regulate CSF production and CSF absorption ultimately leading to elevated ICP [24]. Furthermore, genetic or epigenetic factors appear to be relevant for developing PCTS, based on previously reported familial cases of PTCS [23]. In 89% of cases, AS is caused by mutations/deletions in the Notch signaling pathway ligand gene, JAGGED1, or the gene for its receptor, NOTCH2 [16]. The Notch signaling pathway has an important role in vascular development. Disruption of this pathway results in abnormal vascular development and signaling, which may also contribute to the development of IIH [15, 22]. Mouzaki et al. speculated that abnormalities in the microvasculature of the choroid plexus in AS patients could lead to abnormal CSF production or absorption, which in turn would cause intracranial hypertension [21]. The occurrence of PTCS in AS patients after liver transplantation has been previously described in 2 cases by Narula et al. [22]. While these patients were on tacrolimus, our patient was receiving cyclosporine, which has been related to the development raised ICP after renal, bone marrow, and heart transplantation. Nevertheless, based on the occurrence of intracranial hypertension in AS patients without liver transplant, it appears unlikely that the immunosuppressive medication solely resulted in PCTS, although it could have been a contributing factor [22]. Due to its rarity, knowledge about treatment of PTCS in AS remains very limited. This is reflected by the few previously reported cases as outlined in Table 1. While medical treatment with acetazolamide, furosemide, and steroids was effective, there is only very little information on long-term efficacy. We here show that ventriculoperitoneal shunting may provide long-term relief and prevent recurrent PE. Relapse of PE due to growth-related dislocation of the ventricular catheter reinforces the need of ophthalmological and radiological follow-up examinations. PTCS in AS is likely to be underdiagnosed because ophthalmological examinations are not performed routinely after initial diagnostic workup and due to the fact that the majority of patients remain asymptomatic [22]. Conclusions Our report emphasizes the need for careful evaluation of PCTS in pediatric AS. Through the utility of continuous ICP monitoring, we offer new insights regarding ICP dynamics of this population. We also demonstrate that CSF shunting may provide reliable relief of symptoms on long-term follow-up. Our findings along with other reported cases suggest that a genetic predisposition makes AS patients susceptible for developing PCTS, whereas associated medical conditions and exogenous factors can trigger or exacerbate intracranial hypertension. The differentiation of PTCS in AS as primary or secondary PCTS is a subject deserving further discussion with findings thus far being supportive for the first. Abbreviations AS Alagille syndrome CSF Cerebrospinal fluid ICP Intracranial pressure IIH Idiopathic intracranial hypertension PE Papilledema PTCS Pseudotumor cerebri syndrome Funding Open Access funding enabled and organized by Projekt DEAL. Data availability Not applicable. Declarations Conflict of interest The authors declare no competing interests. Code availability Not applicable. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	Recovered	ReactionOutcome	CC BY	33555437	20,770,619	2021-09
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Abdominal pain'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Abortion spontaneous'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Alveolitis'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Bacterial infection'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Chills'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Exposure during pregnancy'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Eyelid oedema'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Foetal exposure during pregnancy'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Gastritis'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Gastrointestinal inflammation'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Haemorrhage'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Intestinal malrotation'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Intrauterine infection'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Low birth weight baby'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Nuchal rigidity'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Oropharyngeal pain'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Premature baby'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Pyrexia'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Renal tubular disorder'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Renal tubular necrosis'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Stillbirth'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,145,774	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Stomatitis'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Tremor'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
Give an alphabetized list of all active substances of drugs taken by the patient who experienced 'Vaginal haemorrhage'.	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	HYDROXYCHLOROQUINE SULFATE	DrugsGivenReaction	CC BY	33557386	20,101,534	2021-02-04
What was the administration route of drug 'HYDROXYCHLOROQUINE SULFATE'?	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	Transplacental	DrugAdministrationRoute	CC BY	33557386	20,145,774	2021-02-04
What was the outcome of reaction 'Alveolitis'?	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	Fatal	ReactionOutcome	CC BY	33557386	20,145,774	2021-02-04
What was the outcome of reaction 'Eyelid oedema'?	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	Fatal	ReactionOutcome	CC BY	33557386	20,145,774	2021-02-04
What was the outcome of reaction 'Gastritis'?	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	Fatal	ReactionOutcome	CC BY	33557386	20,145,774	2021-02-04
What was the outcome of reaction 'Gastrointestinal inflammation'?	Kingella kingae Intrauterine Infection: An Unusual Cause of Chorioamnionitis and Miscarriage in a Patient with Undifferentiated Connective Tissue Disease. Kingella kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. In children less than 4 years old, K. kingae invasive infection can induce septic arthritis and osteomyelitis, and more rarely endocarditis, meningitis, ocular infections, and pneumonia. In adults, it may be a cause of endocarditis. To date, K. kingae acute chorioamnionitis (AC) leading to preterm rupture of membranes (PPROM) and miscarriage has never been reported. Herein, we describe a case of intrauterine fetal death (IUFD) at 22 weeks' gestation due to K. kingae infection occurred in a patient affected by undifferentiated connective tissue disease (UCTD) in lupus erythematosus systemic (LES) evolution with severe neutropenia. K. kingae was isolated in placental subamnionic swab and tissue cultures as well as fetal ear, nose, and pharyngeal swabs. Placental histological examination showed necrotizing AC and funisitis. In the fetus, neutrophils were observed within the alveoli and in the gastrointestinal lumen. Maternal medical treatment for UCTD was modified according to the K. kingae invasive infection. In the event of IUFD due to AC, microbiological cultures on placenta and fetal tissues should always be carried out in order to isolate the etiologic agent and target the correct medical treatment. 1. Introduction Kingella kingae is a Gram-negative, facultatively anaerobic coccobacillus of the Neisseriaceae family. It is a slowly growing bacterium also a member of the HACEK group (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) [1,2]. K. kingae is currently recognized as the most common etiology of septic arthritis and osteomyelitis in children between the ages of 6 and 48 months. More rarely, it can cause complicated endocarditis, meningitis, ocular infections, pericarditis, peritonitis, and pneumonia [3,4]. To the best of our knowledge, K. kingae infection has never been reported in pregnancy. Only a case of early onset sepsis (EOS) in a premature infant has been described [5]. Herein, we present an unusual case of preterm premature rupture of membranes (PPROM) at 22 weeks’ gestation with subsequent intrauterine fetal death (IUFD) due to severe acute chorioamnionitis (AC). Placental subamnionic swab and tissue microbiological cultures isolated K. kingae, including fetal ear, nose, and pharyngeal swabs. Moreover, the 31-year-old mother was affected by undifferentiated connective tissue disease (UCTD) in evolution toward lupus erythematosus systemic (LES); severe neutropenia, antinuclear antibodies (ANA), and low titer of anti-neutrophil cytoplasmic antibodies (ANCA) were also present. 2. Case Description 2.1. Mother Clinical Presentation and Treatment A 31-year-old mother, gravida 2 para 1, presented at 22 weeks’ gestation to the Emergency Department of our Institution for miscarriage. The patient had been suffering from shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. Gynaecological examination revealed on speculum mild fresh blood in vagina and abdominal ultrasound (US) observed IUFD. She was admitted to the hospital, labour was induced and a stillborn female fetus was delivered. Placental microbiological samples and fetal swabs were sent to the Clinical Microbiology laboratory where cultures on different media were immediately performed. Overall, the patient had a complex clinical history. She had suffered from monoarticular juvenile idiopathic arthritis, completely resolved at 15 years of age. Six years before the current miscarriage, she had complained of bilateral gonalgia. Laboratory findings had shown severe neutropenia (250 neutrophils) and low titer of ANCA. Antinuclear antibodies (ANA) titer had been 1:160. Anti extractable nuclear antigens (ENA), anti-DNA antibodies, and antiphospholipid antibodies (lupus anticoagulant—LAC; anticardiolipin antibodies—aCL; anti-β2-glycoprotein-1—anti-β2GP-1) had been negative. Bone marrow biopsy had been reported within normal limits. In the previous six years, although the patient had always been presenting with low neutrophils, she had never contracted opportunistic infections. One year before the miscarriage, she had had her first pregnancy with a vaginally delivered male infant of 3200 g at 40 weeks + 6 days of gestational age. One month after delivery, she had severe mastitis, which required hospital admission and surgical treatment. After that, she presented with two episodes of bilateral knee arthritis, and then she was put on hydroxychloroquine 200 mg twice a day (tablets). Regarding the current miscarriage, at hospital admission, maternal laboratory findings were as follows: WBC 2.6 x 1.000/μL and C-reactive protein (CRP) 13.45 mg/dL. During the first day after IUFD, CRP worsened to 18.02 mg/dL, then progressively decreased from 16.46 (2 days), 2.7 mg/dL (3 days), 1.43 mg/dL (5 days) to 0.11 mg/dL (10 days). Blood cultures performed at admission resulted negative. Maternal medical treatment, started soon after IUFD, consisted of 2 days of meropenem, subsequently switched to piperacillin/tazobactam for 6 days. She fully recovered and was discharged after 10 days. After hematologic and rheumatologic consult, due to low neutrophil count and recent intrauterine infection, Granulocyte-Colony Stimulating Factor (G-CSF) and cyclosporine (200 mg a day) were added to hydroxychloroquine. 2.2. Fetal Autopsy and Microbiological Results Postmortem examination revealed a nonmacerated female fetus weighing 420 g and measuring 29 cm in crown-heel length. The other measurements were as follows: Crown-rump length, 19.5 cm; foot length, 3.7 cm; head, chest and abdominal circumference, 18, 16, and 15 cm, respectively. Overall, anthropometric measurements were consistent with 22 weeks’ gestation [6]. External examination showed a normal fetus with mild eyelid and nuchal oedema. On internal examination, intestinal rotation was incomplete (malrotation) with short midgut mesenteric attachment and mobile intestine. No volvulus was observed. Microscopic analysis revealed the presence of intra-alveolar, gastric and intestinal neutrophils (Figure 1); oedema, microhemorrhages and acute tubular necrosis (NTA) of the renal parenchyma were also present. The placenta was received complete, weighed 137 g, and measured 10 × 10 × 2 cm. The membranes were yellowish and opaque. Microscopically, there was necrotizing AC (Figure 2) corresponding to a maternal inflammatory response stage 3/3 and grade 2/2 [7]. Coccoid bacteria were noted at high magnification within the chorion (Figure 3). Funisitis was also observed with neutrophilic infiltrate of the umbilical vein, and the two arteries with extension to Wharton’s jelly (Figure 4). These findings were consistent with fetal inflammatory response stage 2/3 and grade 2/2 [7]. In addition, in the placental parenchyma some recent infarcts were detected, and in the decidua few spiral arteries presented initial thrombosis. Autoptical samples were cultured on Columbia blood agar (CBA), McConkey agar (MCA), mannitol salt agar (MSA) and Sabouraud agar (SAB; all these media were incubated at 36 °C, ambient air), chocolate agar (CA, incubated at 36 °C, 5% CO2), and Schaedler agar (SA, incubated at 36 °C, anaerobic atmosphere). After 48 hours from the fetus’s pharyngeal, nose, and ear swabs, small colonies grew on CBA and CA (better on the latter). No growth was observed on MCA. These smooth colonies were catalase negative and oxidase positive. They were identified as K. kingae using the MALDI-ToF technology (Bruker Daltonics, Germany), Biotyper OC software version 3.1. Microbiological cultures on fetal blood and tissues (lung and liver) showed no growth after 72 hours and were discarded as negative. Instead, K. kingae also grew on subamniotic swab and placental tissue cultures. If present, the microorganism grew as a pure culture in all the samples. Antimicrobial susceptibility testing was not performed. 3. Materials and Methods We searched for (Kingella kingae AND (chorioamnionitis OR genital OR amniotic OR amnios OR placenta OR placental OR funisitis OR fetus OR fetal OR pregnancy OR pregnant OR (membrane AND rupture) OR uterus OR uterine OR intrauterine)) in Pubmed (all fields, 2 results), Scopus (Title/Abstract/Keyword, 9 results) and Web of Science (Topic/Title, 2 results). No limitations were set. The bibliographic research ended on 25 December 2020. Globally, a total of 10 articles resulted from our search. Titles and Abstract of all the articles were screened. All articles were excluded as non-relevant. 4. Discussion K. kingae is a Gram-negative coccobacillus belonging to the Neisseriaceae family. It is a normal component of the oropharyngeal flora of children less than 4 years old. However, the bacterium, after having colonized and breached the epithelial surface, may disseminate in the bloodstream causing, especially in children, arthritis, osteomyelitis, and endocarditis. This latter complication may also occur in adults [1,8]. Mechanisms of virulence include biofilm formation, pili, RTX toxin production, polysaccharide capsule, and secretion of outer membrane vesicles (OMV). K. kingae is able to form biofilms, which consist of large quantities of bacteria clustered together in a polysaccharide “slime”, that tightly attaches to the mucosal surface. This kind of colonization allows bacteria to survive in a protected environment preserving them from dehydratation, immune response, and antibiotics [9,10]. K. kingae anchors to the epithelia thanks to type 4 pili, which are proteinaceous fimbriae necessary for efficient adherence to respiratory mucosa and synovia [11]. Moreover, RTX toxin expression exerts a wide-spectrum cytotoxic effect on macrophages, leukocytes, synoviocytes, and respiratory epithelial cells. This toxin plays a key role in infection spreading favouring epithelial disruption and bloodstream dissemination [12,13]. Of note, type a and b polysaccharide capsule predominate in invasive isolates [14]. A further virulence factor possessed by K. kingae is the secretion of OMV, which are small parts of the outer membrane encasing periplasm proteins that bulge away from the bacterium and then are released in the extracellular space. These OMV’s are hemolytic and leukotoxic in an in vitro model and internalized by synoviocytes inducing the synthesis of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6), likely representing the in vivo immunitary response in joint infections [15]. K. kingae has been recognized as a leading cause of septic arthritis and osteomyelitis in children less than 4 years old, usually with no underlying medical conditions [3,4]. Other rare primary manifestations in pediatric population are: Pneumonia, endocarditis, soft tissue infection, endophtalmitis, orbital cellulitis, and meningitis [1,2,16,17,18,19,20]. Being part of the HACEK group (Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae), K. kingae can cause endocarditis, affecting more frequently children and young adults [21]. Adult patients usually have predisposing conditions in order to develop invasive K. kingae disease, such as malignancies, liver cirrhosis, diabetes, sickle cell anemia, and renal transplant [1,22,23,24,25]. However, to the best of our knowledge, K. kingae infection has never been reported in pregnancy and besides as a cause of PPROM and miscarriage. Moreover, the patient was affected by UCTD in evolution toward LES with severe neutropenia, ANA, and ANCA. The 31-year-old mother presented to the hospital after having suffered shaking chills, fever (38 °C), stomatitis, sore throat, abdominal pain, and minimal vaginal bleeding for the previous three days. US revealed IUFD. Microbiological cultures on placental subamnionic swab and parenchymal tissue isolated K. kingae. The same agent grew on fetal swabs (ear, nose, and pharyngeal). Fetal tissue cultures (blood, lung and liver) resulted negative. Placental histological examination confirmed severe necrotizing chorioamnionitis and funisitis. Within the chorion, coccoid bacteria were also observed. Fetal histology showed few neutrophils within the alveoli, and gastro-intestinal lumen, respectively. K. kingae was then considered the etiologic agent responsible for the miscarriage. While other members of the genus Kingella such as K. negevensis and K. denitrificans are known to cause bacterial vaginosis, chorioamnionitis, and pediatric vaginitis [26,27,28]; K. kingae infections of the urogenital tract are extremely rare [29]. However, AC due to K. kingae has never been described. The main microorganisms responsible for AC usually are group B Streptococcus, Fusobacterium nucleatum, Peptostreptococcus, Escherichia coli, Bacteroides species, Ureaplasma urealyticum, and Listeria monocytogenes [30,31]. As K. kingae is not part of the lower genital tract flora, in the case described, an ascending infection was unlikely. The bacterium probably reached the amniotic cavity through the hematogenous pathway from the oropharynx. In fact, the mother presented with sore throat and stomatitis. Although reported in children, K. kingae invasive disease has been associated with recent or concomitant virus infections like coxsackievirus, herpes simplex, varicella zoster, and rhinovirus in the oropharynx and upper respiratory tract [32,33,34,35]. Therefore, a concomitant viral infection in those locations may represent a cofactor in the pathophysiology of invasive K. kingae, probably favoring epithelial breaching and altering the local immune response [36]. In the case we described, it is impossible to differentiate a condition of maternal oropharyngeal carrier exacerbated by a concomitant viral infection or a K. kingae primary oropharyngeal infection turned out to be invasive. Notwithstanding, the mother was affected by UCTD in evolution toward LES with severe neutropenia. To date, K. kingae infection has never been described in this kind of peculiar setting. Only few cases have been observed in a granulocytopenic host [22], LES [37,38,39,40], rheumatoid arthritis [41], and acquired immunodeficiency syndrome (AIDS) [42,43,44,45,46]. Besides, recognition of K. kingae intrauterine infection allowed modifying maternal therapeutic plan, as G-CSF and cyclosporine were added to hydroxychloroquine. In our specific case, placental cultures and fetal swabs revealed K. kingae infection, but the microorganism was not retrieved from fetal blood and tissues. This may be explained as a low bacterial load or a possible specimen type inhibition. In fact, it is known that recovery of K. kingae from bacteriological solid media may be difficult and nucleic acid amplification is advisable [1]. In our case, polymerase chain reaction (PCR) on the fetal tissues was not performed as infection was clearly demonstrated by neutrophils within the alveoli and in the lumen of gastrointestinal tract. Usually, in the event of miscarriage due to chorioamnionitis, identification of the correct etiologic agent is of paramount importance as maternal medical treatment can be adjusted accordingly. Placental subamniotic swab and parenchymal cultures as well as fetal blood and tissues microbiological studies should always be carried out as recommended by perinatal autopsy protocols [47,48]. Author Contributions M.P.B.; carried out the pathological diagnosis and written the manuscript—original draft preparation, review and editing, A.P.; provided the histological pictures and searched for the bibliograhy, G.D.D.; provided the draft preparation and writing, G.C. and G.P.; clinically managed the patient, G.R., G.B., M.B., C.Z., and E.C.; carried out the microbiological diagnosis. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Institutional Review Board Statement Our investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. According to Italian legislation, Ethical Approval for a single case is not required, as long as the data are kept anonymous and the investigations performed do not imply genetic results. Informed Consent Statement The current Italian legislation neither requires the family’s consent or ethical approval for a single case, as long as the data are strictly kept anonymous. As summoning the mother was not possible because it would interfere with the grieving process, patient’s consent was completely waived, according to the Italian Authority of Privacy and Data Protection (“Garante della Privacy”: GDPR nr 679/2016; 9/2016 and recent law addition number 424/ 19th of July 2018; http://www.garanteprivacy.it). Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fetal lung: A few neutrophils within the alveoli (blue arrows; Hematoxylin Eosin (HE) staining 20×). Figure 2 Amniochorial membranes: Severe chorioamnionitis with amnion necrosis (blue arrow; HE staining 10×). Figure 3 Chorion: Abundant coccoid bacteria within the chorionic stroma (HE staining 40×). Figure 4 Umbilical artery acute vasculitis: Neutrophils within the arterial wall (blue star) with extension to Warthon’s jelly (red star) (HE staining, 4×). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.	Fatal	ReactionOutcome	CC BY	33557386	20,145,774	2021-02-04

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