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Chronic Infection With Camelid Hepatitis E Virus in a Liver Transplant Recipient Who Regularly Consumes Camel Meat and Milk

2015; Elsevier BV; Volume: 150; Issue: 2 Linguagem: Inglês

10.1053/j.gastro.2015.10.048

1528-0012

Guan Huei Lee, Boon Huan Tan, Esmeralda C. Teo, Seng Gee Lim, Yock‐Young Dan, Aileen Wee, Pauline Aw, Yuan Zhu, Martin L. Hibberd, Chee‐Kiat Tan, Michael A. Purdy, Chong–Gee Teo,

Liver Diseases and Immunity

There have been increasing reports of food-borne zoonotic transmission of hepatitis E virus (HEV) genotype 3, which causes chronic infections in immunosuppressed patients. We performed phylogenetic analyses of the HEV sequence (partial and full-length) from 1 patient from the Middle East who underwent liver transplantation, and compared it with other orthohepevirus A sequences. We found the patient to be infected by camelid HEV. This patient regularly consumed camel meat and milk, therefore camelid HEV, which is genotype 7, might infect human beings. Our finding links consumption of camel-derived food products to post-transplantation hepatitis E, which, if detected at early stages, can be cured with antiviral therapy and reduced administration of immunosuppressive agents. There have been increasing reports of food-borne zoonotic transmission of hepatitis E virus (HEV) genotype 3, which causes chronic infections in immunosuppressed patients. We performed phylogenetic analyses of the HEV sequence (partial and full-length) from 1 patient from the Middle East who underwent liver transplantation, and compared it with other orthohepevirus A sequences. We found the patient to be infected by camelid HEV. This patient regularly consumed camel meat and milk, therefore camelid HEV, which is genotype 7, might infect human beings. Our finding links consumption of camel-derived food products to post-transplantation hepatitis E, which, if detected at early stages, can be cured with antiviral therapy and reduced administration of immunosuppressive agents. See Covering the Cover synopsis on page 291. See Covering the Cover synopsis on page 291. A 55-year-old Somalian man from the United Arab Emirates underwent living-donor liver transplantation in Singapore in 2010 for hepatitis B cirrhosis and hepatocellular carcinoma. After recuperating well, he returned to the United Arab Emirates 3 months later with normal liver enzyme levels. At month 17 after transplantation, he returned to Singapore with the following abnormal serum liver test results: alanine aminotransferase level of 118 U/L, aspartate aminotransferase level of 104 U/L, alkaline phosphatase level of 132 U/L, and γ-glutamyltransferase level of 324 U/L. Total bilirubin level was normal. He denied alcohol consumption, noncompliance or alternative use of medication, or contact with persons with viral hepatitis. His serum hepatitis B virus–DNA level was less than 13.5 IU/mL. Serologic tests for active infection with human immunodeficiency virus, hepatitis A virus, hepatitis C virus, cytomegalovirus, and Epstein–Barr virus were negative. Tacrolimus level was less than 2.0 ug/L. Liver radiology was unremarkable. The initial differential diagnosis included mild acute cellular rejection and drug-induced liver injury from ezetimibe and alfuzosin. Both these drugs were discontinued and the dosages of the immunosuppressants were increased. However, moderate transaminitis persisted (Figure 1A), leading to suspicion that an infection may be involved. Liver biopsy specimens at 17, 19, and 20 months after transplant showed increasing interface hepatitis with mild lobular necroinflammation (METAVIR scores A2, A2, and A3, respectively), with F2 grade portal and focal septal fibrosis (Figure 1B). Acute cellular rejection–like features and central perivenulitis with hepatocytic dropout were seen in the biopsy specimens at 19 and 20 months, respectively. At 22 month after transplant, anti–hepatitis E virus (HEV) IgM but not IgG was detected in serum. After contemporary reports were published of chronic hepatitis E in solid-organ transplant patients,1Kamar N. Selves J. Mansuy J.M. et al.Hepatitis E virus and chronic hepatitis in organ-transplant recipients.N Engl J Med. 2008; 358: 811-817Crossref PubMed Scopus (1032) Google Scholar polymerase chain reaction (PCR) for HEV RNA in serum was conducted, which confirmed its presence. Infection by HEV belonging to genotype 3 was presumed. The patient was started on oral ribavirin 600 mg/d for 12 weeks, together with reduction of immunosuppression. Liver test results rapidly normalized (Figure 1A). Eight weeks after therapy (24 months after transplant), HEV became undetectable. A retrospective analysis of a stored plasma sample collected at month 19 after transplant showed a HEV RNA concentration of 8.34 × 106 IU/mL, and the presence of both anti-HEV IgM and IgG. To date, HEV responsible for chronic hepatitis in organ transplant patients has been reported to be transmissible from pigs and boars, and less extensively from deer.2Teo C.G. Much meat, much malady: changing perceptions of the epidemiology of hepatitis E.Clin Microbiol Infect. 2010; 16: 24-32Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Because the patient is a Muslim, the route of HEV transmission remained a puzzle. The mystery was solved when the patient’s subgenomic HEV sequence amplified from positions 6394 to 7040 in open reading frame 2 was characterized using MEGA6,3Tamura K. Stecher G. Peterson D. et al.MEGA6: molecular evolutionary genetics analysis version 6.0.Mol Biol Evol. 2013; 30: 2725-2729Crossref PubMed Scopus (31412) Google Scholar which showed that the patient’s HEV sequence belongs to camelid HEV. Not only did it cluster with recently reported camelid HEV sequences,4Woo P.C. Lau S.K. Teng J.L. et al.New hepatitis E virus genotype in camels, the Middle East.Emerg Infect Dis. 2014; 20: 1044-1048Crossref PubMed Scopus (174) Google Scholar but the entire cluster showed a bootstrap value of 1.0 (Supplementary Materials and Methods section). This finding was confirmed by phylogenetic analysis of the full-length HEV genomic sequence after capillary sequencing (Figure 2). The patient owns a camel farm and has regular direct contact with camels. A dietary history was re-taken, showing his regular and continuing consumption of camel meat and milk. This report shows that camelid HEV, which belongs to genotype 7,5Purdy MA, Smith DB, Simmonds P, et al. ICTV taxonomic proposal 2014.008a-hV.A.v6.Hepeviridae. New classification scheme for Hepeviridae. Available from: http://www.ictvonline.org/proposals-14/2014.008a-hV.A.v6.Hepeviridae.pdf. Accessed September 22, 2015.Google Scholar can infect human beings. The protracted clinical course but favorable response to ribavirin are similar to infection of organ transplant recipients from porcine HEV belonging to genotype 3.6Zhou X. de Man R.A. de Knegt R.J. et al.Epidemiology and management of chronic hepatitis E infection in solid organ transplantation: a comprehensive literature review.Rev Med Virol. 2013; 23: 295-304Crossref PubMed Scopus (57) Google Scholar Unlike patients with chronic genotype 3 infection,7Kamar N. Bendall R.P. Peron J.M. et al.Hepatitis E virus and neurologic disorders.Emerg Infect Dis. 2011; 17: 173-179Crossref PubMed Scopus (246) Google Scholar no extrahepatic features were observed. Zoonotic HEV infection from camels is of concern, particularly in desert areas in the Middle East and Africa where HEV infection is prevalent.8Kumar R.M. Uduman S. Rana S. et al.Sero-prevalence and mother-to-infant transmission of hepatitis E virus among pregnant women in the United Arab Emirates.Eur J Obstet Gynecol Reprod Biol. 2001; 100: 9-15Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Camels are often in close contact with human beings, and camel-derived food products are readily available.9Faye B. The camel today: assets and potentials.Anthropozoologica. 2014; 49: 167-176Crossref Scopus (41) Google Scholar Organ transplant recipients there, who already may be avoiding food of porcine origin, should be cautioned against consumption of such products, and hepatitis E should be considered in the differential diagnosis of transaminitis, so that appropriate treatment can be administered promptly. How extensively camelid HEV infects and contributes to ill health in the general population merits further studies. Serologic diagnosis of HEV was performed by assaying plasma for the presence of anti-HEV IgM and IgG with a commercially available enzyme immunoassay (Mikrogen GmbH, Neuried, Germany). Real-time PCR detection of HEV RNA was performed using the RealStar HEV RT-PCR kit (Altona Diagnostics, Hamburg, Germany) according to the manufacturer's instructions. The lowest detection limit for this assay is 100 IU/mL. A total of 202 HEV orthohepevirus A sequences covering open reading frame 2, positions 6394 to 7040 (reference KJ496143), were retrieved from GenBank. The sequences were aligned in Clustal X1Larkin M.A. Blackshields G. Brown N.P. et al.Clustal W and Clustal X version 2.0.Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22516) Google Scholar and modified as required manually. The best substitution model was determined using modeltest in MEGA6.2Tamura K. Stecher G. Peterson D. et al.MEGA6: molecular evolutionary genetics analysis version 6.0.Mol Biol Evol. 2013; 30: 2725-2729Crossref PubMed Scopus (33504) Google Scholar By using this information, an unrooted maximum likelihood tree was created in MEGA6 using the Tamura Nei substitution model with 4 gamma rate categories and invariant sites. Bootstrap analysis was performed using 1000 replicates. The sequence from the patient (KT336568) clustered with camelid HEV sequences KJ496143 and KJ496144, and the bootstrap value for this clade, was 0.999 (Supplementary Figure 1). Primers were designed based on the reference sequences KJ496143 and KJ496144 (Supplementary Table 1). Viral genome amplification was performed as described by Aw et al.3Aw P.P.K. Sessions de P.F. Wilm A. et al.Next-generation whole genome sequencing of Dengue virus.in: Padmanabhan R. Vasudevan S.G. Dengue. Springer, New York2014: 175-195Crossref Scopus (13) Google Scholar Complementary DNA synthesis was performed with the Maxima H Minus First Strand Complementary DNA Synthesis Kit (ThermoFisher Scientific, Pittsburgh, PA) using primer HEV_f3R1. The entire HEV genome was PCR-amplified in 19 fragments using the PfuUltra II Fusion HS DNA Polymerase (Agilent Technologies, Santa Clara, CA). PCR products were viewed on agarose gel and purified using the Qiagen Gel Extraction Kit (Qiagen, Hilden, Germany). Samples were quantitated using the Agilent Bioanalyser and Nanodrop (ThermoFisher Scientific) before capillary sequencing. Capillary sequencing data were aligned to reference sequence KJ496144 using SeqScape Software v3.0 (ThermoFisher Scientific). Evolutionary analyses with bootstrap 1000 were conducted using MEGA62Tamura K. Stecher G. Peterson D. et al.MEGA6: molecular evolutionary genetics analysis version 6.0.Mol Biol Evol. 2013; 30: 2725-2729Crossref PubMed Scopus (33504) Google Scholar (Figure 2).Supplementary Table 1Sequences of Primers Used to Amplify Patient’s HEV GenomeForwardPrimer sequence (5′-3′)ReversePrimer sequence (5′-3′)HEV_f1F1GTAAAACGACGGCCAG GGTAGACCACGTACGTTGCTHEV_RaAGTATGGCAACATCATGGTTRTHEV_f1F2GTAAAACGACGGCCAG ACCACGTACGTTGCTCGCGTHEV_RaAGTATGGCAACATCATGGTTRTHEV_FaAYAACCATGATGTTGCCATACTHEV_RcTGACCCAGCCACCGCATGAHEV_FcTCATGCGGTGGCTGGGTCAHEV_f1R1TCCCAGATATGATTCGGCGAHEV_f2F1TCGCCGAATCATATCTGGGAHEV_ReTSAGATAAAGCTCATCCCCTHEV_FcTCATGCGGTGGCTGGGTCAHEV_ReTSAGATAAAGCTCATCCCCTHEV_FeAGGGGATGAGCTTTATCTSAHEV_RhTCAGCTGTGGCAATGATWGTAHEV_FhTACWATCATTGCCACAGCTGAHEV_RiATGGTGGGAATAAACTTAGCTAHEV_FlTATGGCTATTATAGCGCATTGTHEV_RmATGGTGTACCCATGGGCGAHEV_FmTCGCCCATGGGTACACCATHEV_RoTCGAAGTRATTGAATTCATATCAAHEV_f1F1GTAAAACGACGGCCAG GGTAGACCACGTACGTTGCTHEV_RcTGACCCAGCCACCGCATGAHEV_FcTCATGCGGTGGCTGGGTCAHEV_RiATGGTGGGAATAAACTTAGCTAHEV_FiTAGCTAAGTTTATTCCCACCATHEV_RlACAATGCGCTATAATAGCCATAHEV_FrAAGACCATCAATCAGTAYTCTAHEV_f3R1CAGGAAACAGCTATGACTTTTCAGGGAGCGCGAAACGHEV_FlTATGGCTATTATAGCGCATTGTHEV_RnATGGGTTGGTTGGATGAATATAHEV_FnTATATTCATCCAACCAACCCATHEV_RoTCGAAGTRATTGAATTCATATCAAHEV_FoTTGATATGAATTCAATYACTTCGAHEV_RqACGAAATCAATTCTGTCGGGAHEV_FqTCCCGACAGAATTGATTTCGTHEV_RrTAGARTACTGATTGATGGTCTTHEV_f1F1GTAAAACGACGGCCAGGGTAGACCACGTACGTTGCTHEV_RuTCGGACCCTCTCAATAACCANOTE. The GenBank accession number for the complete genotype 7 sequences from patient's plasma is KT818608. Open table in a new tab NOTE. The GenBank accession number for the complete genotype 7 sequences from patient's plasma is KT818608. Covering the CoverGastroenterologyVol. 150Issue 2PreviewHepatitis E virus (HEV) infection is considered to be among the most common causes for enterically transmitted acute hepatitis in developing countries. In developed countries, sporadic HEV infection is associated with exposure to domestic animals or consumption of raw or undercooked pork or game meat. Foodborne zoonotic transmission of HEV, predominantly HEV genotype 3, has been reported to cause chronic hepatitis in immunocompromised patients, such as organ transplant patients and has been associated with extrahepatic, predominantly neurological, manifestations. Full-Text PDF