Ribavirin for Hepatitis E Virus Infection After Organ Transplantation: A Large European Retrospective Multicenter Study.

MAJOR ARTICLE 1204 • cid 2020:71 (1 September) • Kamar etal Clinical Infectious Diseases Received 23 March 2019; editorial decision 20 August 2019; accepted 01 October 2019; published online December 3, 2019. aPresent affiliation: Department of Medical Microbiology, University Medical Centre Utrecht, The Netherlands Correspondence: N. Kamar, Department of Nephrology and Organ Transplantation, CHU Toulouse Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France (kamar[email protected]). Clinical Infectious Diseases® 2020;71(5):1204–11 © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: [email protected]. DOI: 10.1093/cid/ciz953 Ribavirin for Hepatitis E Virus Infection After Organ Transplantation: ALarge European Retrospective MulticenterStudy Nassim Kamar,1 Florence Abravanel,2 Patrick Behrendt,3 Jörg Hofmann,4 GeorgesPhillippe Pageaux,5 Christelle Barbet,6 Valérie Moal,7 Lionel Couzi,8 Thomas Horvatits,9 RobertA. DeMan,10 Elisabeth Cassuto,11 AhmedM. Elsharkawy,12 Annelies Riezebos-Brilman,13,a Anne Scemla,14 Sophie Hillaire,15 MhairiC. Donnelly,16 Sylvie Radenne,17 Johnny Sayegh,18 Cyril Garrouste,19 Jérôme Dumortier,20 François Glowaki,21 Marie Matignon,22 Audrey Coilly,23 Lucile Figueres,24 Christiane Mousson,25 Anne Minello,26 Sébastien Dharancy,27 JeanPhilippe Rerolle,28 Pascal Lebray,29 Isabelle Etienne,30 Peggy Perrin,31 Mira Choi,4 Olivier Marion,2 and Jacques Izopet2; for the Hepatitis E Virus Ribavirin Study Group 1Department of Nephrology, Dialysis and Organ Transplantation, Centre Hospitalier Universitaire (CHU) Rangueil, Institut National de la Santé et de la Recherche Médicale (INSERM) U1043, Institut Fédératif de Recherche Bio-médicale de Toulouse (IFR–BMT), University Paul Sabatier, Toulouse, France; 2Department of Virology, INSERM U1043, IFR–BMT, University Paul Sabatier, Toulouse, France; 3Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, and Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, German Centre for Infection Research, Hannover, Germany; 4Charité Universitätsmedizin Berlin, Department of Nephrology and Intensive Care and Institute of Virology, Labor Berlin Charité-Vivantes-GmbH, Berlin, Germany; 5Department of Hepatology, Saint Eloi Hospital, Montpellier, France; 6Department of Nephrology and Clinical Immunology, Bretonneau Hospital, University Hospital, Tours, France; 7Aix Marseille Université, Asistance Publique Hôpitaux de Marseille, Institut Pour la Recherche Pour le Développement, Microbes, Evolution, Phylogénie et Infection, Institut Hospitalo-Universitaire–Méditerranée Infection, Hôpital Conception, Centre de Néphrologie et Transplantation Rénale, Marseille, France; 8Department of Nephrology and Transplantation, CHU Bordeaux, Bordeaux, France; 9Department of Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 10Departments of Gastroenterology and Hepatology, Erasmus Medical Center, Rotterdam, The Netherlands; 11Department of Nephrology and Transplantation, CHU Nice, France; 12Liver Unit, University Hospitals Birmingham, Birmingham, United Kingdom; 13Department of Medical Microbiology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands; 14Service de néphrologie-transplantation, Hôpital Necker, Assitance publiqueHôpitaux de Paris (AP-HP), Paris et Université Paris Descartes, Paris, France; 15Department of Hepatology, Hôpital Foch, Suresnes, France; 16Department of Hepatology and Scottish Liver Transplant Unit, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; 17Department of Hepatology and Liver Transplantation, CHU de la Croix Rousse, Lyon, France; 18Department of Nephrology and Transplantation, CHU Angers, Angers, France; 19Department of Nephrology, CHU Clermont-Ferrand, Clermont-Ferrand, France; 20Department of Hepatology, Edouard Herriot Hospital, CHU Lyon, Lyon, France; 21Department of Nephrology, CHU Lille, Lille, France; 22Assistance Publique-Hôpitaux de Paris, Nephrology and Renal Transplantation Department, Groupe Hospitalier Henri-Mondor/Albert-Chenevier, Université Paris-Est-Créteil, Département Hospitalo-Universitaire Virus-Immunité-Cancer, Institut Mondor de Recherche Biomédicale, Equipe 21, INSERM U 955, Créteil, France; 23Centre Hépato-Biliaire, Hôpital Paul Brousse, AP-HP, INSERM U1193, Université Paris-Sud Paris-Saclay, Villejuif, France; 24Department of Nephrology and Clinical Immunology, CHU Nantes, Nantes, France; 25Department of Nephrology, CHU François Mitterrand, Dijon, France; 26Department of Hepatogastroenterology and Digestive Oncology, CHU François Mitterrand, Dijon, France; 27Hôpital Claude Huriez, Services Maladies de l’Appareil Digestif, INSERM Unité 995, Lille, France; 28Department of Nephrology, CHU Limoges, Limoges, France; 29Department of Hepatology, Pitié Salpétrière Hospital, Paris, France; 30Department of Nephrology, CHU Rouen, Rouen, France; and 31Department of Nephrology, CHU Strasbourg, Strasbourg, France (See the Editorial commentary by crum-cianflone on pages 1212–4.) Background. Ribavirin is currently recommended for treating chronic hepatitis E virus (HEV) infection. This retrospective European multicenter study aimed to assess the sustained virological response (SVR) in a large cohort of solid organ transplant (SOT) recipients with chronic HEV infection treated with ribavirin monotherapy (N=255), to identify the predictive factors for SVR, and to evaluate the impact of HEV RNA mutations on virological response. Methods. Data from 255 SOT recipients with chronic HEV infection from 30 European centers were analyzed. Ribavirin was given at the median dose of 600 (range, 29–1200) mg/day (mean, 8.6±3.6mg/kg/day) for a median duration of 3 (range, 0.25–18) months. Results. After a first course of ribavirin, the SVR rate was 81.2%. It increased to 89.8% when some patients were offered a second course of ribavirin. An increased lymphocyte count at the initiation of therapy was a predictive factor for SVR, while poor hematological tolerance of ribavirin requiring its dose reduction (28%) and blood transfusion (15.7%) were associated with more relapse after ribavirin cessation. Pretreatment HEV polymerase mutations and de novo mutations under ribavirin did not have a negative impact on HEV clearance. Anemia was the main adverse event. Conclusions. This large-scale retrospective study confirms that ribavirin is highly efficient for treating chronic HEV infection in SOT recipients and shows that the predominant HEV RNA polymerase mutations found in this study do not affect the rate of HEV clearance. Keywords. organ transplantation; hepatitis E virus; ribavirin; sustained virological response; anemia. In immunosuppressed patients, mainly in solid organ transplant (SOT) recipients, genotype 3 hepatitis E virus (HEV) can be responsible for chronic hepatitis that can lead to rapid liver fibrosis progression and cirrhosis [1–3]. The reduction of immunosuppressants targeting the T-cell response is considered as a therapeutic option [4, 5]. It leads to HEV clearance in one-third of patients with chronic HEV infection [5]; in the remaining patients, ribavirin monotherapy is recommended Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 Ribavirin for HEV Infection in SOT Recipients • cid 2020:71 (1 September) • 1205 [6]. In vitro, ribavirin inhibits HEV replication [7, 8]. In vivo, case reports and small series showed that ribavirin was efficient for treating chronic HEV infection [9–11]. A retrospective multicenter French study, which included 59 SOT recipients, confirmed this finding [12]. After a first course of ribavirin monotherapy, a sustained virological response (SVR) was observed in 78% of patients [12]. Re-treating relapses for a longer period guaranteed SVR in additional patients, thus leading to an SVR of 85% [12]. In some patients who failed to clear HEV, HEV RNA polymerase mutations were detected [13]. However, the impact of these mutations on antiviral response to ribavirin is still unknown [14, 15]. This retrospective multicenter study aims to assess the SVR in a large cohort of SOT recipients with chronic HEV infection treated with ribavirin monotherapy, to identify the predictive factors for SVR, and to evaluate the impact of HEV RNA mutations on virological response. PATIENTS AND METHODS Dutch, English, French, German, and Scottish transplant centers were asked to participate in this retrospective study; 30 of them agreed. We collected data from 280 SOT recipients with chronic autochthonous HEV infection who were treated with ribavirin monotherapy between September 2009 and March 2018. Chronic hepatitis was defined as a persistent replication of HEV for at least 3months. Patients who had persistent increase in liver enzyme levels lasting for >3months before first screening for HEV and diagnosis and who were treated before the third month after diagnosis were considered to have chronic hepatitis. Patients who were treated during the acute HEV phase and those without a complete follow-up (ie, <6 months’ follow-up after ribavirin cessation) were excluded (n=25). Hence, data from 255 patients were analyzed. Of note, data from 57 patients were already published [12]. However, in the previous publication, HEV RNA polymerase mutations were not analyzed. For this reason, data from these patients were included for the present study. Details regarding participating centers and the number of included patients per center are presented in the Supplementary Materials. The patients’ characteristics are presented in Table 1. Data were recorded anonymously. According to French and German laws, anonymous retrospective studies do not require institutional review board (IRB) approval. In other countries, the study was approved by the IRB of each hospital. Virological Parameters As previously described [16], HEV RNA in blood was detected and quantified by real-time polymerase chain reaction (PCR) that targeted the ORF3 gene (or ORF2 gene in 1 center). Most samples were centralized and analyzed in the French reference center for HEV in Toulouse. The limit of detection for HEV RNA was 100 copies/mL. SVR was defined as undetectable HEV RNA in the serum at least 6months after ribavirin therapy was completed [17]. The detection of variants in the HEV polymerase was performed by the Sanger method by sequencing a 1100-nucleotide fragment covering the entire HEV polymerase gene. Afirst real-time PCR assay was performed using primers designed according to the HEV subtype (Supplementary File 2). Then, 3 overlapping nested PCR assays were performed to amplify the whole gene. Nested PCR products were sequenced on both strands by the dideoxy chain termination method (Prism Ready Reaction AmpliTaq Fs and BigDye terminator, Applied Table 1. Patient Characteristics Variable N=255 Age, y 51±14 Sex, male/female, no. 189/66 Type of organ transplantation, no. Kidney 153 Liver 56 Heart 14 Kidney-pancreas 10 Lung 9 Kidney-liver 6 Heart-kidney 3 Lung-kidney 2 Heart-liver 1 Pancreas 1 History of acute rejection, % 26.3 Immunosuppression at initiation of RBV, % Calcineurin inhibitor 87.7 Cyclosporine A 13.2 Tacrolimus 86.8 mTOR inhibitor 24.8 Antimetabolite 64.6 Steroids 74.1 Serum creatinine level at initiation of RBV, µmol/L 134±60 eGFR (MDRD) at initiation of RBV, mL/min/1.73 m255±23 Lymphocyte count at initiation of RBV, cells/μL1456±887 Hemoglobin level at initiation of RBV, g/dL 2.8±2 rEPO use at initiation of RBV, % 1. 6 Platelet count at initiation of RBV, cells/μL186372±22428 Anti-HEV IgG at initiation of RBV, positive/negative, no.a160/42 Anti-HEV IgM at initiation of RBV, positive/negative, no.a190/17 Serum HEV RNA at initiation of RBV, positive/negative, no. 255/0 HEV genotype 3, no.b238 3 52 3chi 90 3efg 96 Pretreatment HEV RNA polymerase mutation, no. (%)c76/112 (68) Time between transplantation and RBV therapy, mo 74±64 Time between diagnosis and RBV therapy, mo, median (range) 4 (0.5–82) Data are presented as mean ± standard deviation unless otherwise indicated. Abbreviations: eGFR, estimated glomerular filtration rate; HEV, hepatitis E virus; IgG, immunoglobulin G; IgM, immunoglobulin M; MDRD, modification in diet for renal disease; mTOR, mammalian target of rapamycin; RBV, ribavirin; rEPO, recombinant erythropoietin. aHEV serology was not done in all patients. bTwo hundred thirty-eight of the 255 patients were infected by HEV genotype 3. HEV genotyping was not done in the remaining 17 patients. cBaseline HEV RNA polymerase mutations were assessed in only 112 patients. Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 1206 • cid 2020:71 (1 September) • Kamar etal Biosystems, Paris, France) on an ABI 3130XL analyzer (Applied Biosystems, Foster City, California) using the same sense and antisense primers as used for amplification. Electrophoregraphy data were analyzed using Sequencher software version 4.8 (Gene Codes Corporation, Ann Arbor, Michigan). The HEV polymerase sequences were determined in 112 patients before ribavirin treatment initiation and in 26 patients who remained viremic after 3months of therapy or at relapse after ribavirin cessation. We failed to sequence HEV polymerase at baseline for 4 patients, as well as 1 patient in relapse. Regarding all remaining patients, no samples were available to sequence HEV polymerase. Statistical Analyses Data are presented either as mean ± standard deviation or median (range). Proportions were compared by the χ 2 test or Fisher exact test. Quantitative variables were compared by the nonparametric Friedman test for serial measurements and either Student t test or the Wilcoxon test. Independent factors associated with a sustained virological response were studied using a stepwise multivariate logistic regression model that used initial inclusion criteria that had a significance of P<.05, using Statview software (SAS Institute, Cary, North Carolina). For this purpose, patients with an SVR were compared to those without an SVR (as defined above). AP value of <.05 was considered to be statistically significant. RESULTS Ribavirin Therapy The median time between HEV diagnosis and ribavirin therapy was 3 (range, 0–82) months. The duration of ribavirin therapy varied considerably, from 0.25 to 18 (median, 3) months. Patients were given 1month of therapy or less (n=12), between 1 and 2months (n=13), 3months (n=128), 4months (n = 26), 5 months (n = 21), 6 months (n = 37), 7 months (n=4), 9months (n=1), 10months (n=5), 11months (n=1), 12months (n=2), 15months (n=2), 17months (n=1), or 18months (n=2). Ribavirin doses were given at the discretion of physicians and according to kidney function and ribavirin tolerance. The median dose of ribavirin was 600 (range, 29–1200) mg/day (mean, 8.6±3.6mg/kg/day). Most patients received 400mg/day (n=47), 600mg/day (n=72), or 800mg/ day (n=81). Thirty-one patients were given <400mg/day, and 32 patients received >800mg/day. Twenty-eight percent of patients required ribavirin dose reduction due to poor hematological tolerance. In this subgroup of patients, the mean ribavirin dose decreased from 706±221mg/day to 399±194mg/day (P<.0001). In the whole population, mean ribavirin dose decreased slightly from 614 ± 241 mg/day to 572 ± 428 mg/ day (P=.13). Conversely, it was increased in 10% of patients. Furthermore, no significant change in estimated glomerular filtration rate was observed—that is, 55±29mL/minute/1.73 m2 at the initiation of ribavirin, 55±29mL/minute/1.73 m2 at the end of therapy, and 53±27mL/minute/1.73 m2 at 6months after ribavirin cessation (P=.33). Virological Response All patients were viremic at the initiation of ribavirin. One month later, HEV RNA was assessed in 221 of 255 patients and was found to still be positive in 105 patients (47.5%). At the end of ribavirin therapy, HEV RNA was still positive in the blood in only 10 patients (3.9%). Their characteristics are presented in Supplementary Table 1. After ribavirin cessation, HEV relapsed in 38 patients. Hence, SVR was achieved after a first course of ribavirin in 207 of 255 patients (81.2%; Figure 1). The SVR rate according to the duration of ribavirin therapy is presented in Figure 2. Thirty-six of 48 patients who did not achieve SVR were offered a longer course of ribavirin (Figure 1). Of these, 18 achieved SVR after having been treated for 3months (n=1), 4months (n=1), 5months (n=2), 6months (n=10), 12months (n=2), and 15months (n=2). Seven other patients are still currently under therapy. The 11 remaining patients did not achieve SVR: 1 of them was unaccounted for during the follow-up 1month after reinitiating ribavirin, another patient was considered a nonresponder and stopped ribavirin, 4 patients were given long-term ribavirin therapy because of persisting replication (1 of them, a heart transplant recipient, died from decompensated cirrhosis related to HEV), and 5 patients relapsed after having been re-treated for 5months (n=1), 6months (n=1), 10months (n=2), and 12months (n=1) and stopped ribavirin. Interestingly, 2 of the relapsed patients undergoing the second course of ribavirin cleared the virus spontaneously a few weeks after ribavirin cessation, therefore achievingSVR. With respect to the 12 remaining patients who relapsed after the first course of ribavirin and who have not been re-treated, immunosuppression was stopped and dialysis initiated in 1 patient, which allowed him to achieve SVR. Another patient cleared the virus spontaneously 2months after HEV relapse and before being re-treated. He achieved SVR. Eight patients were still viremic and were not offered additional therapy. Finally, 2 patients died from cardiovascular disease and lung cancer while viremic. Hence, SVR was achieved in 22 patients who relapsed after the first course of ribavirin. Consequently, overall, SVR was observed in 229 of the 255 patients (89.8%). Predictive Factors forSVR Results of univariate analysis are presented in Table 2. Interestingly, the detection of HEV RNA in the blood at week 4 after the initiation of therapy was not associated with more frequent relapses after ribavirin cessation. The following variables were included in a multivariate analysis model: recipients’ Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 Ribavirin for HEV Infection in SOT Recipients • cid 2020:71 (1 September) • 1207 sex, alanine aminotransferase level, total lymphocyte count, anti-HEV immunoglobulin G, and the use of tacrolimus (vs cyclosporine A) at the initiation of ribavirin, as well as the use of recombinant erythropoietin (rEPO) during therapy, ribavirin dose reduction, and the need of blood transfusion under therapy. High lymphocyte count at the initiation of therapy was identified Figure 2. Sustained virological response rate according to the duration of ribavirin therapy. Figure 1. Outcomes of solid organ transplant recipients treated with ribavirin. Abbreviations: IS, immunosuppressants; SVR,sustained virological response. Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 1208 • cid 2020:71 (1 September) • Kamar etal Table 2. Predictive Factors for Sustained Virological Response (N=255) Variable Patients With SVR (n=207) Patients Without SVR (n=48) P Value Univariate analysis Age, y 52±14 49±13 .15 Sex, male/female, n 159/48 30/18 .05 Liver transplant recipient, yes/no, n 51/156 12/36 >.99 Rejection history, % 25.1 31.2 .46 CNIs at RBV initiation, % 87.3 89.4 .81 Tacrolimus at RBV initiation, % 84 97 .02 mTOR inhibitor at RBV initiation, % 21.2 18.8 .84 Antimetabolite at RBV initiation, % 70.2 68.1 .86 Steroids at RBV initiation, % 73.4 78.7 .58 AST level at RBV initiation, IU/La68 (19–1448) 79 (23–480) .41 ALT level at RBV initiation, IU/La120 (10–1733) 100 (32–373) .03 γGT level at RBV initiation, IU/La122 (19–914) 129 (22–1015) .04 Bilirubin level at RBV initiation, µmol/La11 (2–186) 13 (4–102) .16 Creatinine level at RBV initiation, µmol/L 136±62 129.5±49 .52 eGFR (MDRD) at RBV initiation, mL/min/1.73 m254±22 57±26 .45 Hemoglobin level at RBV initiation, g/dL 12.9±1.9 12.4±2 .08 Lymphocyte count at RBV initiation, cells/μL1529±895 1095±758 .02 Tacrolimus trough level at RBV initiation, ng/mL 6.3±2.8 7.2±29 .07 Platelet count at RBV initiation, cells/μL185160±104539 191550±149320 .7 Time between transplantation and RBV, mo 76±66 62±50 .18 Time between diagnosis and RBV, moa4 (0.5–82) 6 (0.5–50) .49 Duration of RBV therapy, moa3 (0.25–18) 3 (0.75–12) .92 Duration of RBV therapy <3 mo, yes/no, n 18/189 7/41 .27 Duration of RBV therapy ≤3 mo, yes/no, n 124/83 29/19 .99 Duration of RBV therapy 3 mo, yes/no, n 106/101 22/26 .52 Duration of RBV therapy >3 and <6 mo, yes/no, n 40/167 7/41 .54 Duration of RBV therapy ≤6 mo, yes/no, n 194/13 43/5 .35 Duration of RBV therapy of 6 mo, yes/no, n 30/177 7/41 .99 Duration of RBV therapy ≥6 mo, yes/no, n 43/164 12/36 .56 Initial RBV dose, mg/d a600 (29–1200) 600 (200–1000) .47 Initial RBV dose, mg/kg/d 8.6±3.7 8.6±3.5 .97 Cumulative RBV dose, mg 71 280±53 828 67 626±44 743 .67 RBV trough level at month 1 2.14±1.38 (n=54) 2.25±1.26 (n=19) .75 RBV dose <400mg/d at EOT, % 20.9 22.5 .83 RBV dose reduction, % 24.3 46.8 .004 Use of rEPO during RBV therapy, % 41.9 61.7 .02 Blood transfusion, % 10.9 36.2 <.0001 Hemoglobin level at EOT, g/dL 11.7±2.3 9.8±2.3 .001 Positive anti-HEV IgG at RBV initiation, yes/no, nb135/26 25/16 .002 Positive anti-HEV IgM at RBV initiation, yes/no, nb156/12 34/5 .32 HEV RNA concentration at RBV initiation, IU/mLa599 500 (200–166 000 000) 1 444 000 (200–37 000 000) .96 HEV genotype 3chi/3efg, n 68/81 22/15 .14 Positive HEV RNA at month 1, yes/no, nb82/95 23/21 .5 Pretreatment HEV polymerase mutations, yes/no, nb57/25 19/11 .65 Multivariate analysis OR (95% CI) P Value Sex, male 1008 (.35–2.9) .99 ALT at baseline 2.22 (.65–7.58) .19 Cyclosporine A(vs tacrolimus) … … .99 Anti-HEV IgG at baseline (positive) 1. 7 (.6–4.8) .31 rEPO during therapy (Y) 1.41 (.49–4.03) .52 Higher lymphocyte count at baseline 1. 0 01 (1–1.02) .04 RBV dose reduction (Y) 0.34 (.14–.84) .02 Transfusion during therapy (Y) 0.3 (.1–.84) .02 Data are presented as mean ± standard deviation unless otherwise indicated. Numbers are bold correspond to statistically significant differences. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CI, confidence interval; CNI, calcineurin inhibitor; eGFR, estimated glomerular filtration rate; EOT, end of therapy; γGT, gamma-glutamyl transpeptidase; HEV, hepatitis E virus; IgG, immunoglobulin G; IgM, immunoglobulin M; MDRD, modification in diet for renal disease; mTOR, mammalian target of rapamycin; N, no; OR, odds ratio; RBV, ribavirin; rEPO, recombinant erythropoietin; SVR, sustained virological response; Y, yes. aMedian (range). bHEV serology at baseline, quantitative HEV RNA concentration at baseline, pretreatment HEV polymerase mutations, and HEV RNA at month 1 were not obtained for all patients. Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 Ribavirin for HEV Infection in SOT Recipients • cid 2020:71 (1 September) • 1209 as an independent predictive factor that was associated with SVR. Conversely, ribavirin dose reduction and blood transfusions were independent predictive factors associated with no achievement of SVR (Table 2). Effect of HEV RNA Mutations on Virological Response HEV RNA mutations were assessed in 112 patients before the initiation of ribavirin. HEV polymerase mutations were detected in 76 patients (67.9%). V1479I was the most common mutation detected (n=75), followed by G1634R (n=10). The following mutations and combinations were observed: V1479I alone (n=64), V1479I and G134R (n=9), V1479I and Y1320H (n = 1), V1479I and D138G/N (n = 1), and G1634R alone (n=1). The presence of pretreatment mutation did not impact SVR. Pretreatment mutations were observed in 57 of 82 patients (69.5%) who achieved SVR and 19 of 30 patients (63.3%) who did not (P=.65). The type of mutation did not have any impact on SVR (Table 3). Among 19 patients who had pretreatment mutations and who did not achieve SVR, 17 patients were re-treated with ribavirin: 13 achieved SVR and 4 are still under therapy. The 2 remaining patients were not re-treated: 1 of them spontaneously cleared the virus before being re-treated. HEV polymerase mutations were assessed in 26 patients who did not achieve SVR after the first course of ribavirin. Twenty patients had de novo mutations: 5 did not have any mutation before therapy whereas all 15 remaining patients already had other mutations before therapy. Among these 20 patients who developed de novo mutations under therapy, 16 patients were re-treated with ribavirin: 12 achieved SVR and 4 are still under therapy. The remaining 4 patients were not re-treated. Ribavirin Tolerance Anemia was the main side effect observed under ribavirin therapy. Under ribavirin therapy, rEPO was required in 45.6% of patients. At initiation of therapy, 21.6% were already receiving rEPO at the median dose of 12 000 (range, 1000– 120000) IU/week. At the end of therapy, 38% were given rEPO (P<.0001, compared to baseline) at the median dose of 20000 (range, 1000–320000) IU/week (P=.0003, compared to baseline). Afew patients required only transient use of rEPO. Blood transfusions were required for 15.7% of patients. In patients who required increased doses of rEPO, the introduction of rEPO, or blood transfusion, ribavirin was initiated at a slightly lower dose (581±243mg/day) compared to those who did not require any intervention (636±238mg/day) (P=.07). However, at baseline, they had a significantly lower eGFR rate (47.5±19.3 vs 60.6±22mL/minute/1.73 m2, P<.0001), had a significantly lower hemoglobin level (12.5±1.6g/dL vs 13.1±2.25g/dL, P = .009), and more frequently received often mycophenolic acid (79.3% vs 57.7%, P=.0005). DISCUSSION Ribavirin monotherapy was found to be efficient for treating chronic genotype 3 and 4 HEV infection [12]. Most results were obtained in SOT recipients, mainly from a retrospective multicenter French study that included 59 patients [12]. In the present study, we aimed to assess the efficiency and safety of ribavirin in a larger cohort of SOT recipients and to assess the impact of HEV polymerase mutations on virological response. Our findings were 4-fold: (1) After a first course of ribavirin, the SVR rate was 81.2%; this rate increased to 89.8% when some patients were offered a second course of ribavirin. (2) Increased lymphocyte count at initiation of therapy was a predictive factor for SVR, while poor hematological tolerance of ribavirin requiring its dose reduction and blood transfusion were associated with more frequent relapses after ribavirin cessation. (3) Pretreatment HEV polymerase mutations and de novo mutations under ribavirin did not have a negative impact on HEV clearance. (4) Anemia was the main adverseevent. Table 3. Hepatitis E Virus Polymerase Mutations Mutation Patients Who Achieved SVR After First RBV Course (n=57) Patients Who Did Not Achieve SVR After First RBV Course (n=19) P Value Pretreatment combinations .39 V1479I 49 15 V1479I+G1634R 6 3 V1479I+Y1320 H 1 0 V1479I+D1384G/N 0 1 G1634R 1 0 Pretreatment mutations V1479I 56 19 .99 G1634R 7 3 .7 Y1320 H 1 0 >.99 D138G/N 0 1 .25 De novo mutations after therapy (n=20) K1383N 1 G1634R 3 D1384N or G 2 K1383N+D1384G 2 K1383N+K1398R 1 K1383N+G1634R 1 K1383N+Y1587Y 1 D1384G+K1398R 1 D1384G+ G1634R 2 K1383N+D1384G+G1634R 2 Y1320H+D1384G+G1634R 1 K1383N+Y1587Y/F  +G1634G/R 1 K1383N+D1384G+Y1587F +G1634R 1 K1383N+K1398R+Y1587F +G1634R 1 Abbreviations: RBV, ribavirin; SVR, sustained virological response. Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 1210 • cid 2020:71 (1 September) • Kamar etal In the present study, in a large cohort, the SVR rate was 81.2%, and was quite similar to the one reported previously (78%) [12]. In addition, similar to our previous study, treating relapsed patients for a longer period resulted in the achievement of SVR in at least 50% of reported cases. Unfortunately, treatment failure under ribavirin can be observed. Debing etal have previously detected G1634R mutations in the HEV RNA polymerase of 2 patients who failed to clear HEV with ribavirin therapy [13]. Later on, in case reports and small case series, other mutations such as K1383N, D1384G, V1479I, and Y1587F were identified [15, 18, 19]. They were detected in some patients before ribavirin therapy or appeared under ribavirin in relapsed patients [14, 15, 18]. In the present study, HEV polymerase mutations were assessed—not in all, but in a large number of patients. As previously reported by Lhomme etal in a series that included 63 patients, pretreatment G1634R mutation did not impact SVR [14]. However, other mutations in the polymerase could play a role [20]. In the present study, these mutations, analyzed alone or in combination, did not have any impact on virological response. Furthermore, the majority of relapsed patients and partial responders to ribavirin (77%) developed de novo mutations after a first course of ribavirin therapy. Most of them achieved SVR after re-treatment. Hence, HEV RNA polymerase mutations do not seem to have an impact on HEV clearance. This is in line with in vitro data according to which mutations may improve ribavirin antiviral activity, but they may also increase HEV replication [15]. In nonresponders or subjects who relapsed after re-treatment, no existing therapy seems relevant [21], except pegylated interferon, which can only be used in liver transplantation [22] because it increases the risk of acute rejection [23]. Sofosbuvir has shown a synergistic effect with ribavirin in vitro [24]. However, this was not confirmed in vivo [25]. Similar to what has been done in some patients in the current study, it has been suggested that maintaining ribavirin long-term may have a beneficial effect on preventing the progression on liver fibrosis [26]. The main issue is the hematological tolerance of ribavirin. The reduction of ribavirin doses and blood transfusion were independent predictive factors of non-SVR. In a previous study, we did not observe a relationship between ribavirin levels and SVR [27]. In the present study, ribavirin levels were not assessed in most patients. Therefore, we were not able to assess their impact on SVR. However, as previously reported, we found that a high lymphocyte count at baseline was a predictive factor for SVR [12]. This finding is in line with previous reports suggesting that HEV persists in immunosuppressed patients [28]. Abravanel etal had previously observed that patients with persistent HEV RNA shedding in the stools despite undetectable HEV RNA in the blood at the end of scheduled duration of ribavirin therapy were at increased risk of relapsing after ribavirin cessation [29]. Very recently, Marion etal found that in patients who tested positive for HEV in the stools at the end of therapy, prolonging the duration of ribavirin therapy significantly improved the SVR rate [30]. In the present study, HEV RNA was not systematically assessed in the stools at the end of therapy. In our previous multicenter study, in univariate analysis, we found that positive HEV RNA in the blood after 1month of therapy was associated with SVR [12]. This was not confirmed in the present study. Finally, in a small case series that included 35 patients, we observed that a decrease of HEV RNA concentration ≥0.5 log at day 7 after starting ribavirin was an independent predictive factor for SVR [27]. In the present study, data regarding HEV RNA at day 7 were not collected. Due to its retrospective nature, our study has several limitations. The duration of ribavirin varied considerably across different centers. We cannot exclude that in some patients the duration of ribavirin was determined according to their immunological status or to the evolution of viral load under ribavirin, especially those who were given >6months of therapy. However, only 5% of patients received ribavirin for >6months. In addition, the SVR rate did not differ between patients who were initially given ≤3months or >3months. Because HEV RNA was not collected at day 7, and since it was not assessed in the stools at the end of scheduled therapy, we cannot recommend an optimal duration of therapy. However, based on recent results by Marion etal [30], we suggest a 3-month course of therapy after which, in case of persistent HEV RNA in the stools, we suggest prolonging therapy for 3 more months. Another limitation of this study is the lack of assessment of HEV RNA polymerase mutations in all patients. However, it has been done in 44% of them, and no impact of these mutations on virological response was observed. Finally, ribavirin trough levels were not systematically assessed in all patients. Further studies are required to better determine the optimal dose of ribavirin. In summary, this large-scale retrospective European study confirms that ribavirin is highly efficient for treating chronic HEV infection in transplant recipients. Re-treating relapsed patients for a longer period allows clearing the virus. The predominant HEV RNA polymerase mutations found in this study do not affect the rate of HEV clearance. Finally, there is still an unmet need for those patients who fail to clear the virus with ribavirin. SupplementaryData Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Hepatitis E Virus Ribavirin Study Group. J.Bellière, O.Cointault, A.Del Bello, L.Espostio, A.L. Hebral, L.Lavayssière, S.Lhomme, J.M. Mansuy (Toulouse); H. Wedemeyer (Hannover); P. Nickel (Berlin); M. Bismuth (Montpellier); K. Stefic, M. Büchler, L. D’Alteroche (Tours); P. Colson (Marseille); S. Bufton (Birmingham); C. Ramière (Lyon); P. Trimoulet (Bordeaux); S. Pischke (Hamburg); E. Todesco; R. Sberro Soussan; C.Legendre, V.Mallet (Paris); I.Johannessen, K.Simpson (Edinburgh). Downloaded from https://academic.oup.com/cid/article/71/5/1204/5650877 by GBF Gesellschaft fuer Biotechnologische Forschung mbH, -Bibliothekuser on 13 June 2022 Ribavirin for HEV Infection in SOT Recipients • cid 2020:71 (1 September) • 1211 Author contributions. N.K.designed the study, collected the data, did the statistical analyses, and wrote the manuscript. O.M.collected the data. F.A.and J.I.did the virological workup and reviewed the manuscript. All other authors provided patients from different centers and reviewed the manuscript. Potential conflicts of interest. 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