Journal of Medical Virology 86:948–953 (2014)
Past and Current Hepatitis E Virus Infection in Renal Transplant Patients Tiago Hering,1 Ana Maria Passos,2 Renata Mello Perez,3 Juliana Bilar,1 Daniel Fragano,1 Celso Granato,2 Jose´ Osmar Medina-Pestana,4 and Maria Lucia Gomes Ferraz1* 1
Department Department 3 Department 4 Department 2
of of of of
Gastroenterology, Federal University of Sao Paulo, Sao Paulo, Brazil Infectious Diseases, Federal University of Sao Paulo, Sao Paulo, Brazil Internal Medicine, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Nephrology, Federal University of Sao Paulo, Sao Paulo, Brazil
The chronic course of hepatitis E virus infection in immunosuppressed patients has been recently documented; however, clinical features and factors associated with this occurrence are not well known. The aim of this study was to evaluate the prevalence of previous or current HEV infection in renal transplant patients. One hundred ninety-two kidney transplant patients were studied and classified in three groups: G1-infected with hepatitis B and/or C virus; G2-patients with elevated ALT; G3-patients with normal ALT and no hepatotropic virus infection. Demographic, epidemiologic and clinical characteristics were compared between the groups. Patients with HEV infection (previous or current) were also compared to those who tested negative for HEV. HEV infection was detected using serologic (anti-HEV IgG) and molecular (HEV RNA) methods. Anti-HEV IgG was positive in 28 (15%) while HEV RNA was positive in 20 (10%). When both markers were considered, 44 (23%) patients showed evidence of previous or current HEV infection. However, both markers were concomitantly positive in only four cases (2%). In the comparative analysis, patients infected with HBV and/or HCV showed lower frequency of anti-HEV IgG (P ¼ 0.009). There was no difference regarding demographic, epidemiologic and laboratory variable between viremic and non-viremic patients. In conclusion, past and current infection with HEV was a frequent finding among renal transplant recipients. Actively infected patients (HEV RNA positive) did not present distinct demographic and epidemiological characteristics or laboratory alterations suggestive of underlying liver damage. Therefore, infection with HEV can only be detected in immunosuppressed patients by systematic investigation of HEV RNA. J. Med. Virol. 86:948–953, 2014. # 2014 Wiley Periodicals, Inc. C 2014 WILEY PERIODICALS, INC.
KEY WORDS:
hepatitis E; HEV RNA; anti-HEV; kidney transplantation; ALT
INTRODUCTION Hepatitis E virus (HEV) is one of the five hepatotropic viruses that include hepatitis A, B, C and delta virus [Clemente-Casares et al., 2003]. Classically, infection with HEV causes a self-limited disease that resembles hepatitis A virus infection. A peculiar characteristic of HEV infection is its poor prognosis in pregnant women and patients with underlying liver disease, with mortality rates of about 20% and 75%, respectively [Dalton et al., 2007, 2008]. There are a number of studies indicating that HEV can cause chronic hepatitis and liver cirrhosis in immunosuppressed patients, including solid organ transplant recipients [Haagsma et al., 2008; Kamar et al., 2008a, b; Gerolami et al., 2009], patients undergoing chemotherapy [Peron et al., 2006; Mansuy et al., 2009; Ollier et al., 2009], and HIV-infected patients [Colson et al., 2009; Dalton et al., 2009]. It is suggested that HEV infection may progress to chronic hepatitis in up to 60% of solid organ transplant recipients [Kamar et al., 2008b]. The objective of the present study was to determine the prevalence of past or current infection with HEV in three groups of renal transplant recipients (patients chronically infected with HBV and/or HCV; patients not infected with hepatotropic viruses showing elevated levels of alanine aminotransferase Correspondence to: Maria Lucia Gomes Ferraz, Rua Diogo de Faria 561 apto 61, 04037-001 Sa˜o Paulo—SP, Brazil. E-mail: [email protected] Accepted 11 February 2014
DOI 10.1002/jmv.23915 Published online 12 March 2014 in Wiley Online Library (wileyonlinelibrary.com).
HEV Infection in Renal Transplant Patients
(ALT), and patients with no evidence of HCV or HBV infection and showing normal levels of ALT), and to compare demographic, epidemiological and clinical characteristics between patients with past or current HEV infection and patients not infected with HEV.
PATIENTS AND METHODS Renal transplant recipients seen at the Kidney Transplant Unit or referred to the Hepatitis Division of the Federal University of Sao Paulo, between January 2001 and October 2011, were studied. Patients infected with HIV and consuming more than 20 g ethanol/day were excluded. The transplant patients were divided into three groups. Group 1 consisted of patients infected with HBV and/or HCV. Infection with HCV was characterized by anti-HCV positivity (Architect anti-HCV, Abbott Laboratories, Wiesbaden, Germany) and confirmed by the detection of HCV RNA (Cobas Amplicor HCV, Roche Molecular Systems, Pleasanton, CA). Infection with HBV was characterized by HBsAg (Architect HBsAg, Abbott Laboratories) and anti-HBc positivity (Architect anti-HBc, Abbott Laboratories). Group 2 consisted of patients not infected with HCV or HBV who presented elevated levels of ALT. Group 3 consisted of patients with normal ALT levels and no evidence of infection with HBV or HCV. The following variables were evaluated in all patients: age (mean standard deviation); gender (male, female); time on dialysis (years); time after kidney transplantation (years); type of donor (cadaver or live donor); history of transfusion; immunosuppression regimen (double or triple, with or without azathioprine and/or cyclosporine); laboratory variables (ALT at the time of referral and immediate pretransplant ALT; platelet count), and infection with HBV (HBsAg positive) or HCV (HCV RNA positive). The presence of anti-HEV IgG antibodies was investigated using the recom- Well HEV IgG kit (Mikrogen, Neuried, Germany). Samples were tested for the presence of HEV RNA using nested RT-PCR with degenerate primers that target ORF 2, as previously described [Huang et al., 2002]. The purified PCR products from the positive samples were directly sequenced using an ABI Prism Big Dye Terminator Kit and an ABI 3100 Sequencer (Applied Biosystems, Foster City, CA). Human and swine HEV sequences of the same fragment were curated from public databases, and a phylogenetic tree was constructed using the neighbor-joining method with the Kimura 2-parameter model of nucleotide substitution in MEGA v. 5.0 (The Biodesign Institute, Tempe, Arizona). Statistical analyses were performed by bootstrap analysis with 1,000 pseudoreplicates. The obtained sequences are available in the GenBank database under the accession numbers JX173928 and JX173929 [Passos et al., 2013].
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The study protocol has been approved by the Institutional Ethics Committee. Statistical Analysis Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL). Continuous variables were reported as mean standard deviation (SD). Categorical variables were reported as absolute and relative frequency and analyzed by the Chi-square and Fisher exact tests. The Student t-test and Mann– Whitney test were used for comparison of numerical variables between the two groups. A level of significance of 0.05 (a ¼ 5%) was adopted. RESULTS A total of 192 renal transplant recipients were included in the study. Of these, 124 (65%) were men. The mean age was 42.6 11.9 years. Serology for anti-HEV IgG antibodies was positive in 28 (15%) patients. HEV RNA was detected in 20 (10%) patients. When the two markers were considered together, 44 (23%) patients had a past or current infection with HEV. However, simultaneous detection of the two HEV markers was observed in only 2% of cases (Table I). Sixty-one (32%) of the 192 patients belonged to group 1, 37 (19%) to group 2, and 94 (49%) to group 3. The frequency of anti-HEV and/or HEV RNA positivity in the three groups is shown in Table II. In the comparative analysis between groups, patients infected with HBV and/or HCV showed lower frequency of anti-HEV IgG (P ¼ 0.009). Table III shows the results of comparison between patients without evidence of HEV infection and patients with current or past infection with this virus. When both groups were compared, patients with past or current evidence of HEV infection showed lower prevalence of HCV infection (P ¼ 0.02) and a tendency of lower prevalence of HBV infection (P ¼ 0.09). HEV RNA-positive patients were also compared with patients who were anti-HEV positive and HEV RNA negative in order to determine the existence of differences in the variables studied between patients with active HEV infection and those with past infection (Table IV). DISCUSSION Several studies on the development of chronic hepatitis E have been published over the past 4 years [Gerolami et al., 2008; Kamar et al., 2008b; Haagsma et al., 2009; Pischke et al., 2012]. A common feature of all cases described was the presence of immunosuppression due to solid organ or bone marrow transplant, treatment of autoimmune diseases and intrinsic immunodeficiency caused by lymphomas and HIV infection. J. Med. Virol. DOI 10.1002/jmv
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Hering et al.
TABLE I. General Characteristics of the Renal Transplant Recipients Included in the Study (n ¼ 192) Characteristic Male gender (%) Age (years), mean SD Time on dialysis (years) Cadaver donor (%) Time after transplant (years), mean SD Infection with HCV (%) Infection with HBV (%) Transfusion history Immunosuppression Triple regimen Regimen including AZA Regimen including CSA Pre-transplant ALT (ULN), mean SD ALT at referral (ULN), mean SD Anti-HEV IgG positive (%) HEV RNA positive (%) Anti-HEV IgG and HEV RNA positive (%) Anti-HEV IgG or HEV RNA positive (%)
124 (65%) 42.6 11.9 3.7 3.2 67 (35%) 4.7 3.9 34 (18%) 33 (17%) 107/154 (69%) 164 (85%) 117 (61%) 111 (58%) 0.8 0.6 1.3 2.2 28 (15%) 20 (10%) 4 (2%) 44 (23%)
SD, standard deviation; AZA, azathioprine; CSA, cyclosporin A; ULN, times the upper limit of normal.
In the present study, 28 (15%) of the 192 renal transplant recipients were anti-HEV IgG positive. In the blood donors of the same region the prevalence ranged from 3.0% to 4.3% [Goncales et al., 2000; Trinta et al., 2001]. The higher prevalence of HEV infection among renal transplant recipients compared to the general population is due to a variety of factors, including parenteral exposure particularly during the period of hemodialysis. Most patients of the present study had undergone hemodialysis for a mean period of 3.7 years and had also received blood transfusion (69%). These factors increased the chance of exposure to HEV which, although transmitted predominantly by the fecal-oral route, may also be transmitted by the parenteral route. Despite ongoing controversy, parenteral transmission seems in fact to be a possible route of infection. Pourahmad et al. [2009] showed that anti-HEV IgG-positive patients underwent hemodialysis for a longer period of time than the anti-HEV IgG-negative group, a fact favoring environmental transmission of HEV similar to what is observed for HBV and HCV. Another possible explanation to this difference between the two populations is that the studies in blood donors were performed many years
before and the sensitivity of the anti-HEV immunoassay has improved during the most recent years. This fact could also have contributed to the differences observed between renal transplant patients evaluated and the rates in blood donors. In addition to the high rate of patients with past HEV infection (15% anti-HEV IgG-positive patients), 10% of the renal transplant recipients were HEV RNA positive, a finding indicating active, possibly chronic HEV infection. The establishment of a definitive diagnosis of chronic infection requires follow-up of the patients for more than 6 months and the demonstration of persistent viremia. In addition, histological analysis of a liver biopsy is necessary to confirm chronic hepatitis. However, the detection of HEV RNA in the absence of clinical and laboratory signs and symptoms of acute hepatitis strongly suggests a diagnosis of chronic infection. Several studies on solid organ transplant recipients have demonstrated a direct relationship between higher immunosuppression and the development of chronic infection. This premise may redefine the role of hepatitis E as an opportunistic disease in immunosuppressed patients [Colson et al., 2009; Dalton et al., 2009; le Coutre et al., 2009; Ollier et al., 2009; Kamar et al., 2010; Legrand-Abravanel et al., 2010]. In fact, among the patients studied here who had some positive HEV test, the degree of immunosuppression tended to be higher in HEV RNA-positive patients than in those with positive serology only, suggesting that the intensity of immunosuppression could influence the chronicity rate. Another interesting finding of the present study was the discrepancy between anti-HEV IgG antibody and HEV RNA positivity. Simultaneous positivity for the two markers was only observed in four (2%) of the 44 patients with serological or molecular marker of HEV infection. Anti-HEV IgG-positive and HEV RNA-negative patients corresponded to those with past and resolved HEV infection. However, most intriguing is the finding of a negative anti-HEV IgG result and a positive HEV RNA test, which is uncommon in immunocompetent patients. Within the context of an immunosuppressed population such as a renal transplant recipients, the best explanation for the lack of detectable antibodies in the viremic population would be the inability to produce elevated antibody titers as a result of the action of immunosuppressive drugs
TABLE II. Prevalence of HEV Markers in the Three Groups Studied Group 1 (n ¼ 61) Anti-HEV IgG HEV RNA Anti-HEV and/or HEV RNA
a
3 (5%) 7 (12%) 10 (16%)b
Group 2 (n ¼ 37)
Group 3(n ¼ 94)
P-value
10 (27%) 5 (14%) 13 (35%)
15 (16%) 8 (9%) 21 (22%)
0.009 0.6 0.1
Group 1: HBV or HCV; group 2: liver disease; group 3: no liver disease, no infection with HBV or HCV. a Group 1 versus group 2 (P ¼ 0.004); group 1 versus group 3 (P ¼ 0.036); group 2 versus group 3 (P ¼ 0.147). b Group 1 versus group 2 (P ¼ 0.034); group 1 versus group 3 (P ¼ 0.366); group 2 versus group 3 (P ¼ 0.13).
J. Med. Virol. DOI 10.1002/jmv
HEV Infection in Renal Transplant Patients
951
TABLE III. Comparative Analysis of Renal Transplant Recipients With and Without Current or Past HEV Infection
Male gender (%) Age (years) Time on hemodialysis (years) Time after transplant (years) Transfusion history (%) Live donor (%) Infection with HBV (%) Infection with HCV (%) Immunosuppression Triple regimen (%) Regimen including CSA (%) Regimen including AZA (%) Pre-transplant ALT (ULN) ALT at referral (ULN) Platelets/mm3
Anti-HEV and/or HEV RNA positive (n ¼ 44)
Anti-HEV and HEV RNA negative (n ¼ 148)
P-value
71 37 18 3.3 2.8 4.2 2.7 72 71 7 4
66 33 20 3.8 3.3 4.8 3.9 70 62 20 20
0.59 0.22 0.35 0.80 0.80 0.32 0.09 0.02
79 61 68 0.7 0.3 1.2 1.1 174,750
85 55 60 0.8 0.6 1.3 2.4 194,654
0.20 0.60 0.44 0.71 0.3 0.13
AZA, azathioprine; CSA, cyclosporin A; ULN, times the upper limit of normal.
[Tamura et al., 2007; Haagsma et al., 2009; Buti et al., 2010]. Another possible explanation of this finding would be the reduction of antibodies levels in the presence of active HEV infection, as observed in neutralizing antibodies. Therefore, this finding emphasizes the crucial importance to investigate the presence of HEV RNA by PCR in immunosuppressed patients whenever hepatic disease is suspected, irrespectively the serological status of HEV infection. Other reason to be considered is the reliability of the anti-HEV IgG test used. Despite the high sensitivity (96–100%) and specificity (98%) of this assay according to the manufacturer, complexity of antigenic composition, viral genetic heterogeneity and varying epidemiological patterns in different parts of the world could impair the performance of the anti-HEV assay in distinctive scenarios.
Analysis of current or past HEV infection rates in the three groups of patients (without liver disease, infected with HBV or HCV, and elevated ALT) showed a significantly lower prevalence of anti-HEV IgG antibodies in HBV or HCV infected patients when compared to the other groups (Table II). Similarly, patients with current or past HEV infection only differed from those without any evidence of HEV infection in terms of the prevalence of infection with hepatotropic viruses (Table III). In this respect, the prevalence of HBV infection tended to be lower in patients infected with HEV (P ¼ 0.09) and this difference was significant for HCV (P ¼ 0.02). This finding might be explained by a mechanism of viral interference which occurs when two or more viruses are present in the same individual. A similar phenomenon has been observed in patients infected with HBV/
TABLE IV. Comparative Analysis of Renal Transplant Recipients With Active HEV Infection and Patients With Past HEV Infection
Male gender (%) Age (years) Time on hemodialysis (years) Time after transplant (years) Transfusion history (%) Live donor (%) Infection HBV or HCV (%) Immunosuppression Triple regimen (%) Regimen including CSA (%) Regimen including AZA (%) Pre-transplant ALT (ULN) ALT at referral (ULN) Platelets/mm3
HEV RNA positive (n ¼ 20)
Anti-HEV positive and HEV RNA negative (n ¼ 24)
P-value
13 31 11 3.6 2.8 4.3 3.2 72 68 31
17 38 20 3.4 2.9 4.5 2.7 71 71 35
0.68 0.20 0.78 0.84 0.96 0.86 0.74
95 75 60 0.9 0.8 1.4 1.6 164,750
75 58 71 0.7 0.2 1.1 0.8 182,750
0.08 0.25 0.45 0.19 0.41 0.13
AZA, azathioprine; CSA, cyclosporin A; ULN, times the upper limit of normal.
J. Med. Virol. DOI 10.1002/jmv
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Hering et al.
HCV and HIV/HGV, with demonstration of the inhibition of one virus by another [Wang et al., 1999; Sagnelli et al., 2000; Alves-Sousa et al., 2012]. It is important to note that this difference in the prevalence of HEV infection among patients infected with other hepatotropic viruses was observed only in the comparative analysis of anti-HEV positivity, suggesting that the infection could have a protective effect on acquiring HEV virus or an interference on the antibodies production against HEV. In order to determine the factors associated to progression to chronic infection after the exposure to HEV virus, HEV RNA-positive renal transplant recipients were compared to anti-HEV IgG-positive/ HEV RNA-negative patients. Comparative analysis showed no differences between groups, except for the finding that immunosuppression tended to be more intense in viremic patients (higher frequency of triple regimens). No difference was observed regarding demographic, clinical or laboratory variables. Elevated levels of aminotransferases are frequently observed after kidney transplantation as a result of viral infections or the action of immunosuppressive drugs. However, normal liver enzyme levels have been demonstrated in immunosuppressed patients with chronic hepatitis caused by HEV, as observed in the present study. This fact, together with the frequent occurrence of negative serology, makes the diagnosis difficult and the chronic hepatitis E can only be identified based on the detection of HEV RNA and altered histology [Kamar et al., 2008b]. In conclusion, past and current infection with HEV was a frequent finding among renal transplant recipients. Actively infected patients (HEV RNA positive) did not present distinct demographic and epidemiological characteristics or laboratory alterations suggestive of underlying liver damage. Therefore, infection with HEV can only be detected in renal transplant patients by systematic investigation of HEV RNA. ACKNOWLEDGMENTS The authors thank Christini Emori, Silvia Uehara, Ivonete Silva, Roberto Jose´ Carvalho-Filho, and Antonio Eduardo Silva for the valuable contribution to this study. REFERENCES Alves-Sousa VK, Komninakis SC, Baggio-Zappia GL, Barbosa AJ, Mantovani NP, Diaz RS, Abrao P, Lanzara GA, Granato CF. 2012. Short communication: Evaluation of GB virus C/hepatitis G viral load among HIV type 1-coinfected patients in Sao Paulo, Brazil. AIDS Res Hum Retroviruses 28:1301–1304. Buti M, Cabrera C, Jardi R, Castells L, Esteban R. 2010. Are recipients of solid organ transplantation a high-risk population for hepatitis E virus infection? Liver Transplant 16:106–107. Clemente-Casares P, Pina S, Buti M, Jardi R, MartIn M, Bofill-Mas S, Girones R. 2003. Hepatitis E virus epidemiology in industrialized countries. Emerg Infect Dis 9:448–454. Colson P, Kaba M, Moreau J, Brouqui P. 2009. Hepatitis E in an HIV-infected patient. J Clin Virol 45:269–271. Dalton HR, Thurairajah PH, Fellows HJ, Hussaini HS, Mitchell J, Bendall R, Banks M, Ijaz S, Teo CG, Levine DF. 2007.
J. Med. Virol. DOI 10.1002/jmv
Autochthonous hepatitis E in southwest England. J Viral Hepat 14:304–309. Dalton HR, Bendall R, Ijaz S, Banks M. 2008. Hepatitis E: An emerging infection in developed countries. Lancet Infect Dis 8:698–709. Dalton HR, Bendall RP, Keane FE, Tedder RS, Ijaz S. 2009. Persistent carriage of hepatitis E virus in patients with HIV infection. N Engl J Med 361:1025–1027. Gerolami R, Moal V, Colson P. 2008. Chronic hepatitis E with cirrhosis in a kidney-transplant recipient. N Engl J Med 358:859–860. Gerolami R, Moal V, Picard C, Colson P. 2009. Hepatitis E virus as an emerging cause of chronic liver disease in organ transplant recipients. J Hepatol 50:622–624. Goncales NS, Pinho JR, Moreira RC, Saraceni CP, Spina AM, Stucchi RB, Filho AD, Magna LA, Goncales Junior FL. 2000. Hepatitis E virus immunoglobulin G antibodies in different populations in Campinas, Brazil. Clin Diagn Lab Immunol 7:813–816. Haagsma EB, van den Berg AP, Porte RJ, Benne CA, Vennema H, Reimerink JH, Koopmans MP. 2008. Chronic hepatitis E virus infection in liver transplant recipients. Liver Transplant 14:547– 553. Haagsma EB, Niesters HG, van den Berg AP, Riezebos-Brilman A, Porte RJ, Vennema H, Reimerink JH, Koopmans MP. 2009. Prevalence of hepatitis E virus infection in liver transplant recipients. Liver Transplant 15:1225–1228. Huang FF, Haqshenas G, Guenette DK, Halbur PG, Schommer SK, Pierson FW, Toth TE, Meng XJ. 2002. Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol 40:1326–1332. Kamar N, Mansuy JM, Cointault O, Selves J, Abravanel F, Danjoux M, Otal P, Esposito L, Durand D, Izopet J, Rostaing L. 2008a. Hepatitis E virus-related cirrhosis in kidney- and kidney-pancreas-transplant recipients. Am J Transplant 8:1744–1748. Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, Cointault O, Esposito L, Abravanel F, Danjoux M, Durand D, Vinel JP, Izopet J, Rostaing L. 2008b. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 358:811–817. Kamar N, Abravanel F, Selves J, Garrouste C, Esposito L, Lavayssiere L, Cointault O, Ribes D, Cardeau I, Nogier MB, Mansuy JM, Muscari F, Peron JM, Izopet J, Rostaing L. 2010. Influence of immunosuppressive therapy on the natural history of genotype 3 hepatitis-E virus infection after organ transplantation. Transplantation 89:353–360. le Coutre P, Meisel H, Hofmann J, Rocken C, Vuong GL, Neuburger S, Hemmati PG, Dorken B, Arnold R. 2009. Reactivation of hepatitis E infection in a patient with acute lymphoblastic leukaemia after allogeneic stem cell transplantation. Gut 58:699–702. Legrand-Abravanel F, Kamar N, Sandres-Saune K, Garrouste C, Dubois M, Mansuy JM, Muscari F, Sallusto F, Rostaing L, Izopet J. 2010. Characteristics of autochthonous hepatitis E virus infection in solid-organ transplant recipients in France. J Infect Dis 202:835–844. Mansuy JM, Huynh A, Abravanel F, Recher C, Peron JM, Izopet J. 2009. Molecular evidence of patient-to-patient transmission of hepatitis E virus in a hematology ward. Clin Infect Dis 48: 373–374. Ollier L, Tieulie N, Sanderson F, Heudier P, Giordanengo V, Fuzibet JG, Nicand E. 2009. Chronic hepatitis after hepatitis E virus infection in a patient with non-Hodgkin lymphoma taking rituximab. Ann Intern Med 150:430–431. Passos AM, Heringer TP, Medina-Pestana JO, Ferraz ML, Granato CF. 2013. First report and molecular characterization of hepatitis E virus infection in renal transplant recipients in Brazil. J Med Virol 85:615–619. Peron JM, Mansuy JM, Recher C, Bureau C, Poirson H, Alric L, Izopet J, Vinel JP. 2006. Prolonged hepatitis E in an immunocompromised patient. J Gastroenterol Hepatol 21:1223–1224. Pischke S, Stiefel P, Franz B, Bremer B, Suneetha PV, Heim A, Ganzenmueller T, Schlue J, Horn-Wichmann R, Raupach R, Darnedde M, Scheibner Y, Taubert R, Haverich A, Manns MP, Wedemeyer H, Bara CL. 2012. Chronic Hepatitis E in Heart Transplant Recipients. Am J Transplant 12:3128–3133.
HEV Infection in Renal Transplant Patients Pourahmad M, Sotoodeh AR, Nasiri H. 2009. Hepatitis E virus infection in hemodialysis patients: A seroepidemiological survey in Jahrom, Southern Iran. Hepat Mon 9:232–235. Sagnelli E, Coppola N, Scolastico C, Filippini P, Santantonio T, Stroffolini T, Piccinino F. 2000. Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis. Hepatology 32:1106–1110. Tamura A, Shimizu YK, Tanaka T, Kuroda K, Arakawa Y, Takahashi K, Mishiro S, Shimizu K, Moriyama M. 2007.
953 Persistent infection of hepatitis E virus transmitted by blood transfusion in a patient with T-cell lymphoma. Hepatol Res 37:113–120. Trinta KS, Liberto MI, de Paula VS, Yoshida CF, Gaspar AM. 2001. Hepatitis E virus infection in selected Brazilian populations. Mem Inst Oswaldo Cruz 96:25–29. Wang YM, Ng WC, Lo SK. 1999. Suppression of hepatitis C virus by hepatitis B virus in coinfected patients at the National University Hospital of Singapore. J Gastroenterol 34:481–485.
Past and Current Hepatitis E Virus Infection in Renal Transplant Patients Tiago Hering,1 Ana Maria Passos,2 Renata Mello Perez,3 Juliana Bilar,1 Daniel Fragano,1 Celso Granato,2 Jose´ Osmar Medina-Pestana,4 and Maria Lucia Gomes Ferraz1* 1
Department Department 3 Department 4 Department 2
of of of of
Gastroenterology, Federal University of Sao Paulo, Sao Paulo, Brazil Infectious Diseases, Federal University of Sao Paulo, Sao Paulo, Brazil Internal Medicine, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Nephrology, Federal University of Sao Paulo, Sao Paulo, Brazil
The chronic course of hepatitis E virus infection in immunosuppressed patients has been recently documented; however, clinical features and factors associated with this occurrence are not well known. The aim of this study was to evaluate the prevalence of previous or current HEV infection in renal transplant patients. One hundred ninety-two kidney transplant patients were studied and classified in three groups: G1-infected with hepatitis B and/or C virus; G2-patients with elevated ALT; G3-patients with normal ALT and no hepatotropic virus infection. Demographic, epidemiologic and clinical characteristics were compared between the groups. Patients with HEV infection (previous or current) were also compared to those who tested negative for HEV. HEV infection was detected using serologic (anti-HEV IgG) and molecular (HEV RNA) methods. Anti-HEV IgG was positive in 28 (15%) while HEV RNA was positive in 20 (10%). When both markers were considered, 44 (23%) patients showed evidence of previous or current HEV infection. However, both markers were concomitantly positive in only four cases (2%). In the comparative analysis, patients infected with HBV and/or HCV showed lower frequency of anti-HEV IgG (P ¼ 0.009). There was no difference regarding demographic, epidemiologic and laboratory variable between viremic and non-viremic patients. In conclusion, past and current infection with HEV was a frequent finding among renal transplant recipients. Actively infected patients (HEV RNA positive) did not present distinct demographic and epidemiological characteristics or laboratory alterations suggestive of underlying liver damage. Therefore, infection with HEV can only be detected in immunosuppressed patients by systematic investigation of HEV RNA. J. Med. Virol. 86:948–953, 2014. # 2014 Wiley Periodicals, Inc. C 2014 WILEY PERIODICALS, INC.
KEY WORDS:
hepatitis E; HEV RNA; anti-HEV; kidney transplantation; ALT
INTRODUCTION Hepatitis E virus (HEV) is one of the five hepatotropic viruses that include hepatitis A, B, C and delta virus [Clemente-Casares et al., 2003]. Classically, infection with HEV causes a self-limited disease that resembles hepatitis A virus infection. A peculiar characteristic of HEV infection is its poor prognosis in pregnant women and patients with underlying liver disease, with mortality rates of about 20% and 75%, respectively [Dalton et al., 2007, 2008]. There are a number of studies indicating that HEV can cause chronic hepatitis and liver cirrhosis in immunosuppressed patients, including solid organ transplant recipients [Haagsma et al., 2008; Kamar et al., 2008a, b; Gerolami et al., 2009], patients undergoing chemotherapy [Peron et al., 2006; Mansuy et al., 2009; Ollier et al., 2009], and HIV-infected patients [Colson et al., 2009; Dalton et al., 2009]. It is suggested that HEV infection may progress to chronic hepatitis in up to 60% of solid organ transplant recipients [Kamar et al., 2008b]. The objective of the present study was to determine the prevalence of past or current infection with HEV in three groups of renal transplant recipients (patients chronically infected with HBV and/or HCV; patients not infected with hepatotropic viruses showing elevated levels of alanine aminotransferase Correspondence to: Maria Lucia Gomes Ferraz, Rua Diogo de Faria 561 apto 61, 04037-001 Sa˜o Paulo—SP, Brazil. E-mail: [email protected] Accepted 11 February 2014
DOI 10.1002/jmv.23915 Published online 12 March 2014 in Wiley Online Library (wileyonlinelibrary.com).
HEV Infection in Renal Transplant Patients
(ALT), and patients with no evidence of HCV or HBV infection and showing normal levels of ALT), and to compare demographic, epidemiological and clinical characteristics between patients with past or current HEV infection and patients not infected with HEV.
PATIENTS AND METHODS Renal transplant recipients seen at the Kidney Transplant Unit or referred to the Hepatitis Division of the Federal University of Sao Paulo, between January 2001 and October 2011, were studied. Patients infected with HIV and consuming more than 20 g ethanol/day were excluded. The transplant patients were divided into three groups. Group 1 consisted of patients infected with HBV and/or HCV. Infection with HCV was characterized by anti-HCV positivity (Architect anti-HCV, Abbott Laboratories, Wiesbaden, Germany) and confirmed by the detection of HCV RNA (Cobas Amplicor HCV, Roche Molecular Systems, Pleasanton, CA). Infection with HBV was characterized by HBsAg (Architect HBsAg, Abbott Laboratories) and anti-HBc positivity (Architect anti-HBc, Abbott Laboratories). Group 2 consisted of patients not infected with HCV or HBV who presented elevated levels of ALT. Group 3 consisted of patients with normal ALT levels and no evidence of infection with HBV or HCV. The following variables were evaluated in all patients: age (mean standard deviation); gender (male, female); time on dialysis (years); time after kidney transplantation (years); type of donor (cadaver or live donor); history of transfusion; immunosuppression regimen (double or triple, with or without azathioprine and/or cyclosporine); laboratory variables (ALT at the time of referral and immediate pretransplant ALT; platelet count), and infection with HBV (HBsAg positive) or HCV (HCV RNA positive). The presence of anti-HEV IgG antibodies was investigated using the recom- Well HEV IgG kit (Mikrogen, Neuried, Germany). Samples were tested for the presence of HEV RNA using nested RT-PCR with degenerate primers that target ORF 2, as previously described [Huang et al., 2002]. The purified PCR products from the positive samples were directly sequenced using an ABI Prism Big Dye Terminator Kit and an ABI 3100 Sequencer (Applied Biosystems, Foster City, CA). Human and swine HEV sequences of the same fragment were curated from public databases, and a phylogenetic tree was constructed using the neighbor-joining method with the Kimura 2-parameter model of nucleotide substitution in MEGA v. 5.0 (The Biodesign Institute, Tempe, Arizona). Statistical analyses were performed by bootstrap analysis with 1,000 pseudoreplicates. The obtained sequences are available in the GenBank database under the accession numbers JX173928 and JX173929 [Passos et al., 2013].
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The study protocol has been approved by the Institutional Ethics Committee. Statistical Analysis Statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL). Continuous variables were reported as mean standard deviation (SD). Categorical variables were reported as absolute and relative frequency and analyzed by the Chi-square and Fisher exact tests. The Student t-test and Mann– Whitney test were used for comparison of numerical variables between the two groups. A level of significance of 0.05 (a ¼ 5%) was adopted. RESULTS A total of 192 renal transplant recipients were included in the study. Of these, 124 (65%) were men. The mean age was 42.6 11.9 years. Serology for anti-HEV IgG antibodies was positive in 28 (15%) patients. HEV RNA was detected in 20 (10%) patients. When the two markers were considered together, 44 (23%) patients had a past or current infection with HEV. However, simultaneous detection of the two HEV markers was observed in only 2% of cases (Table I). Sixty-one (32%) of the 192 patients belonged to group 1, 37 (19%) to group 2, and 94 (49%) to group 3. The frequency of anti-HEV and/or HEV RNA positivity in the three groups is shown in Table II. In the comparative analysis between groups, patients infected with HBV and/or HCV showed lower frequency of anti-HEV IgG (P ¼ 0.009). Table III shows the results of comparison between patients without evidence of HEV infection and patients with current or past infection with this virus. When both groups were compared, patients with past or current evidence of HEV infection showed lower prevalence of HCV infection (P ¼ 0.02) and a tendency of lower prevalence of HBV infection (P ¼ 0.09). HEV RNA-positive patients were also compared with patients who were anti-HEV positive and HEV RNA negative in order to determine the existence of differences in the variables studied between patients with active HEV infection and those with past infection (Table IV). DISCUSSION Several studies on the development of chronic hepatitis E have been published over the past 4 years [Gerolami et al., 2008; Kamar et al., 2008b; Haagsma et al., 2009; Pischke et al., 2012]. A common feature of all cases described was the presence of immunosuppression due to solid organ or bone marrow transplant, treatment of autoimmune diseases and intrinsic immunodeficiency caused by lymphomas and HIV infection. J. Med. Virol. DOI 10.1002/jmv
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TABLE I. General Characteristics of the Renal Transplant Recipients Included in the Study (n ¼ 192) Characteristic Male gender (%) Age (years), mean SD Time on dialysis (years) Cadaver donor (%) Time after transplant (years), mean SD Infection with HCV (%) Infection with HBV (%) Transfusion history Immunosuppression Triple regimen Regimen including AZA Regimen including CSA Pre-transplant ALT (ULN), mean SD ALT at referral (ULN), mean SD Anti-HEV IgG positive (%) HEV RNA positive (%) Anti-HEV IgG and HEV RNA positive (%) Anti-HEV IgG or HEV RNA positive (%)
124 (65%) 42.6 11.9 3.7 3.2 67 (35%) 4.7 3.9 34 (18%) 33 (17%) 107/154 (69%) 164 (85%) 117 (61%) 111 (58%) 0.8 0.6 1.3 2.2 28 (15%) 20 (10%) 4 (2%) 44 (23%)
SD, standard deviation; AZA, azathioprine; CSA, cyclosporin A; ULN, times the upper limit of normal.
In the present study, 28 (15%) of the 192 renal transplant recipients were anti-HEV IgG positive. In the blood donors of the same region the prevalence ranged from 3.0% to 4.3% [Goncales et al., 2000; Trinta et al., 2001]. The higher prevalence of HEV infection among renal transplant recipients compared to the general population is due to a variety of factors, including parenteral exposure particularly during the period of hemodialysis. Most patients of the present study had undergone hemodialysis for a mean period of 3.7 years and had also received blood transfusion (69%). These factors increased the chance of exposure to HEV which, although transmitted predominantly by the fecal-oral route, may also be transmitted by the parenteral route. Despite ongoing controversy, parenteral transmission seems in fact to be a possible route of infection. Pourahmad et al. [2009] showed that anti-HEV IgG-positive patients underwent hemodialysis for a longer period of time than the anti-HEV IgG-negative group, a fact favoring environmental transmission of HEV similar to what is observed for HBV and HCV. Another possible explanation to this difference between the two populations is that the studies in blood donors were performed many years
before and the sensitivity of the anti-HEV immunoassay has improved during the most recent years. This fact could also have contributed to the differences observed between renal transplant patients evaluated and the rates in blood donors. In addition to the high rate of patients with past HEV infection (15% anti-HEV IgG-positive patients), 10% of the renal transplant recipients were HEV RNA positive, a finding indicating active, possibly chronic HEV infection. The establishment of a definitive diagnosis of chronic infection requires follow-up of the patients for more than 6 months and the demonstration of persistent viremia. In addition, histological analysis of a liver biopsy is necessary to confirm chronic hepatitis. However, the detection of HEV RNA in the absence of clinical and laboratory signs and symptoms of acute hepatitis strongly suggests a diagnosis of chronic infection. Several studies on solid organ transplant recipients have demonstrated a direct relationship between higher immunosuppression and the development of chronic infection. This premise may redefine the role of hepatitis E as an opportunistic disease in immunosuppressed patients [Colson et al., 2009; Dalton et al., 2009; le Coutre et al., 2009; Ollier et al., 2009; Kamar et al., 2010; Legrand-Abravanel et al., 2010]. In fact, among the patients studied here who had some positive HEV test, the degree of immunosuppression tended to be higher in HEV RNA-positive patients than in those with positive serology only, suggesting that the intensity of immunosuppression could influence the chronicity rate. Another interesting finding of the present study was the discrepancy between anti-HEV IgG antibody and HEV RNA positivity. Simultaneous positivity for the two markers was only observed in four (2%) of the 44 patients with serological or molecular marker of HEV infection. Anti-HEV IgG-positive and HEV RNA-negative patients corresponded to those with past and resolved HEV infection. However, most intriguing is the finding of a negative anti-HEV IgG result and a positive HEV RNA test, which is uncommon in immunocompetent patients. Within the context of an immunosuppressed population such as a renal transplant recipients, the best explanation for the lack of detectable antibodies in the viremic population would be the inability to produce elevated antibody titers as a result of the action of immunosuppressive drugs
TABLE II. Prevalence of HEV Markers in the Three Groups Studied Group 1 (n ¼ 61) Anti-HEV IgG HEV RNA Anti-HEV and/or HEV RNA
a
3 (5%) 7 (12%) 10 (16%)b
Group 2 (n ¼ 37)
Group 3(n ¼ 94)
P-value
10 (27%) 5 (14%) 13 (35%)
15 (16%) 8 (9%) 21 (22%)
0.009 0.6 0.1
Group 1: HBV or HCV; group 2: liver disease; group 3: no liver disease, no infection with HBV or HCV. a Group 1 versus group 2 (P ¼ 0.004); group 1 versus group 3 (P ¼ 0.036); group 2 versus group 3 (P ¼ 0.147). b Group 1 versus group 2 (P ¼ 0.034); group 1 versus group 3 (P ¼ 0.366); group 2 versus group 3 (P ¼ 0.13).
J. Med. Virol. DOI 10.1002/jmv
HEV Infection in Renal Transplant Patients
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TABLE III. Comparative Analysis of Renal Transplant Recipients With and Without Current or Past HEV Infection
Male gender (%) Age (years) Time on hemodialysis (years) Time after transplant (years) Transfusion history (%) Live donor (%) Infection with HBV (%) Infection with HCV (%) Immunosuppression Triple regimen (%) Regimen including CSA (%) Regimen including AZA (%) Pre-transplant ALT (ULN) ALT at referral (ULN) Platelets/mm3
Anti-HEV and/or HEV RNA positive (n ¼ 44)
Anti-HEV and HEV RNA negative (n ¼ 148)
P-value
71 37 18 3.3 2.8 4.2 2.7 72 71 7 4
66 33 20 3.8 3.3 4.8 3.9 70 62 20 20
0.59 0.22 0.35 0.80 0.80 0.32 0.09 0.02
79 61 68 0.7 0.3 1.2 1.1 174,750
85 55 60 0.8 0.6 1.3 2.4 194,654
0.20 0.60 0.44 0.71 0.3 0.13
AZA, azathioprine; CSA, cyclosporin A; ULN, times the upper limit of normal.
[Tamura et al., 2007; Haagsma et al., 2009; Buti et al., 2010]. Another possible explanation of this finding would be the reduction of antibodies levels in the presence of active HEV infection, as observed in neutralizing antibodies. Therefore, this finding emphasizes the crucial importance to investigate the presence of HEV RNA by PCR in immunosuppressed patients whenever hepatic disease is suspected, irrespectively the serological status of HEV infection. Other reason to be considered is the reliability of the anti-HEV IgG test used. Despite the high sensitivity (96–100%) and specificity (98%) of this assay according to the manufacturer, complexity of antigenic composition, viral genetic heterogeneity and varying epidemiological patterns in different parts of the world could impair the performance of the anti-HEV assay in distinctive scenarios.
Analysis of current or past HEV infection rates in the three groups of patients (without liver disease, infected with HBV or HCV, and elevated ALT) showed a significantly lower prevalence of anti-HEV IgG antibodies in HBV or HCV infected patients when compared to the other groups (Table II). Similarly, patients with current or past HEV infection only differed from those without any evidence of HEV infection in terms of the prevalence of infection with hepatotropic viruses (Table III). In this respect, the prevalence of HBV infection tended to be lower in patients infected with HEV (P ¼ 0.09) and this difference was significant for HCV (P ¼ 0.02). This finding might be explained by a mechanism of viral interference which occurs when two or more viruses are present in the same individual. A similar phenomenon has been observed in patients infected with HBV/
TABLE IV. Comparative Analysis of Renal Transplant Recipients With Active HEV Infection and Patients With Past HEV Infection
Male gender (%) Age (years) Time on hemodialysis (years) Time after transplant (years) Transfusion history (%) Live donor (%) Infection HBV or HCV (%) Immunosuppression Triple regimen (%) Regimen including CSA (%) Regimen including AZA (%) Pre-transplant ALT (ULN) ALT at referral (ULN) Platelets/mm3
HEV RNA positive (n ¼ 20)
Anti-HEV positive and HEV RNA negative (n ¼ 24)
P-value
13 31 11 3.6 2.8 4.3 3.2 72 68 31
17 38 20 3.4 2.9 4.5 2.7 71 71 35
0.68 0.20 0.78 0.84 0.96 0.86 0.74
95 75 60 0.9 0.8 1.4 1.6 164,750
75 58 71 0.7 0.2 1.1 0.8 182,750
0.08 0.25 0.45 0.19 0.41 0.13
AZA, azathioprine; CSA, cyclosporin A; ULN, times the upper limit of normal.
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HCV and HIV/HGV, with demonstration of the inhibition of one virus by another [Wang et al., 1999; Sagnelli et al., 2000; Alves-Sousa et al., 2012]. It is important to note that this difference in the prevalence of HEV infection among patients infected with other hepatotropic viruses was observed only in the comparative analysis of anti-HEV positivity, suggesting that the infection could have a protective effect on acquiring HEV virus or an interference on the antibodies production against HEV. In order to determine the factors associated to progression to chronic infection after the exposure to HEV virus, HEV RNA-positive renal transplant recipients were compared to anti-HEV IgG-positive/ HEV RNA-negative patients. Comparative analysis showed no differences between groups, except for the finding that immunosuppression tended to be more intense in viremic patients (higher frequency of triple regimens). No difference was observed regarding demographic, clinical or laboratory variables. Elevated levels of aminotransferases are frequently observed after kidney transplantation as a result of viral infections or the action of immunosuppressive drugs. However, normal liver enzyme levels have been demonstrated in immunosuppressed patients with chronic hepatitis caused by HEV, as observed in the present study. This fact, together with the frequent occurrence of negative serology, makes the diagnosis difficult and the chronic hepatitis E can only be identified based on the detection of HEV RNA and altered histology [Kamar et al., 2008b]. In conclusion, past and current infection with HEV was a frequent finding among renal transplant recipients. Actively infected patients (HEV RNA positive) did not present distinct demographic and epidemiological characteristics or laboratory alterations suggestive of underlying liver damage. Therefore, infection with HEV can only be detected in renal transplant patients by systematic investigation of HEV RNA. ACKNOWLEDGMENTS The authors thank Christini Emori, Silvia Uehara, Ivonete Silva, Roberto Jose´ Carvalho-Filho, and Antonio Eduardo Silva for the valuable contribution to this study. REFERENCES Alves-Sousa VK, Komninakis SC, Baggio-Zappia GL, Barbosa AJ, Mantovani NP, Diaz RS, Abrao P, Lanzara GA, Granato CF. 2012. Short communication: Evaluation of GB virus C/hepatitis G viral load among HIV type 1-coinfected patients in Sao Paulo, Brazil. AIDS Res Hum Retroviruses 28:1301–1304. Buti M, Cabrera C, Jardi R, Castells L, Esteban R. 2010. Are recipients of solid organ transplantation a high-risk population for hepatitis E virus infection? Liver Transplant 16:106–107. Clemente-Casares P, Pina S, Buti M, Jardi R, MartIn M, Bofill-Mas S, Girones R. 2003. Hepatitis E virus epidemiology in industrialized countries. Emerg Infect Dis 9:448–454. Colson P, Kaba M, Moreau J, Brouqui P. 2009. Hepatitis E in an HIV-infected patient. J Clin Virol 45:269–271. Dalton HR, Thurairajah PH, Fellows HJ, Hussaini HS, Mitchell J, Bendall R, Banks M, Ijaz S, Teo CG, Levine DF. 2007.
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