Diagnosis Of Hepatitis E Virus Infection Research Articles

Age and Gender Trends in the Prevalence of Markers for Hepatitis E Virus Exposure in the Heterogeneous Bulgarian Population.

The prevalence of hepatitis E virus (HEV) in the Bulgarian population remains underestimated. The aim of the present study was to evaluate age and gender trends in HEV prevalence in the heterogeneous Bulgarian population. Stored serum samples from blood donors and different patient sub-populations-kidney recipients (KR), patients with Guillain-Barre syndrome (GBS), Lyme disease (LD), patients with liver involvement and a clinical diagnosis other than viral hepatitis A and E (non-AE), hemodialysis (HD) and HIV-positive patients (HIV)-were retrospectively investigated for markers of past and recent/ongoing HEV infection. The estimated overall seroprevalence of past infection was 10.6%, ranging from 5.9% to 24.5% for the sub-populations evaluated, while the seroprevalence of recent/ongoing HEV infection was 7.5%, ranging from 2.1% to 20.4%. The analysis of the individual sub-populations showed a different prevalence with respect to sex. In regard to age, the cohort effect was preserved, as a multimodal pattern was observed only for the GBS sub-population. Molecular analysis revealed HEV 3f and 3e. The type of the population is one of the main factors on which the anti-HEV prevalence depends, highlighting the need for the development of guidelines related to the detection and diagnosis of HEV infection with regard to specific patient populations.

Monitoring of hepatitis E virus RNA during treatment for chronic hepatitis E virus infection after renal transplantation.

BackgroundRecently, chronic hepatitis E virus (HEV) infections gained increasing attention as a possible cause for elevated liver enzymes of unknown origin and liver cirrhosis in solid organ transplant recipients. Reduction of immunosuppressive therapy and/or use of antiviral drug ribavirin have been established as possible treatment strategies.MethodsThe efficacy of dose reduction of mycophenolic acid (MPA) and ribavirin therapy was retrospectively analyzed in eight renal transplant patients of our outpatient clinic who were diagnosed with HEV infection by detection of specific antibodies (immunoglobulin M and immunoglobulin G) and/or positive RNA in blood and stool. In four patients serial HEV viral loads in blood were measured.ResultsOnly one patient reached HEV clearance after reduction of immunosuppressive therapy (predominantly reduction of MPA daily dose) alone, whereas six patients were treated with ribavirin after reduction of immunosuppressive therapy due to persistent virus replication. Four of six patients reached HEV clearance after 3 months of ribavirin therapy. HEV clearance was observed after 34–42 days. Two patients, both treated with rituximab within the last 12 months before diagnosis of HEV infection, needed prolonged ribavirin therapy due to persistent viral replication.ConclusionReduction of daily dose of MPA therapy alone in transplant patients with chronic HEV infection may not be sufficient to control viral replication. HEV clearance under ribavirin therapy shows interindividual variability. Therefore, serial viral monitoring may be useful to personalize treatment duration. Rituximab therapy is a risk factor for complicated‐to‐treat chronic HEV infection.

Clinical Outcomes and Prevalence of Hepatitis E Virus (HEV) Among Non-A-C Hepatitis Patients in Egypt.

BackgroundHepatitis E virus (HEV) is an emerging infectious agent that causes acute hepatitis in developing and developed countries. Diagnosis of HEV infection has not been routinely done in Egyptian hospitals, and clinicians do not prescribe ribavirin (RBV) for acute hepatitis cases of unknown etiology (AHUE). We aimed to screen patients with AHUE for the presence of HEV markers and to determine the complications associated with HEV infection.Patients and MethodsHEV markers (anti-HEV IgM, anti-HEV IgG, and HEV RNA) were assessed in patients with AHUE (n=300) admitted to Assiut University Hospitals. RT-qPCR was used to detect the viral load and sequencing analysis was carried out to determine the genotype of the detected viruses. Phylogenetic tree was constructed to evaluate the genetic relatedness between the isolates. Laboratory parameters and the outcomes of infection were determined.ResultsAcute HEV infection (AHE) was detected in 30 out of 300 (10%) of AHUE patients. Anti-HEV IgM, HEV RNA, and anti-HEV IgG were reported in 83%, 50%, and 43% of the samples, respectively. HEV RNA load ranged from 5×102 IU/mL to 1.1×104 IU/mL. Sequencing of the isolated viruses revealed that five viruses belong to HEV-1 and one isolate belongs to HEV-3 with high homology to the virus recently isolated from the cow and goat milk in the Egyptian villages. Although previous reports showed that attenuated HEV isolates were circulating in Egypt, four out of 30 patients (13%) developed coagulopathy and hepatic encephalopathy and died due to fulminant hepatic failure (FHF) within 3–6 weeks of hospitalization. Age, malignancy, and a history of pre-existing liver diseases were a risky factor for FHF development.ConclusionAHE is common in Upper Egypt. Older patients with malignancy and/or a history of liver diseases are risky. HEV diagnosis and treatment become pivotal in Egyptian hospitals to reduce the fatality rate and they should start urgently and promptly.

Hepatitis E: an underestimated emerging threat.

Hepatitis E virus (HEV) is the most common cause of viral hepatitis in the world. It is estimated that millions of people are infected every year, resulting in tens of thousands of deaths. However, these estimates do not include industrialized regions and are based on studies which employ assays now known to have inferior sensitivity. As such, this is likely to represent a massive underestimate of the true global burden of disease. In the developing world, HEV causes large outbreaks and presents a significant public-health problem. Until recently HEV was thought to be uncommon in industrialized countries, and of little relevance to clinicians in these settings. We now know that this is incorrect, and that HEV is actually very common in developed regions. HEV has proved difficult to study in vitro, with reliable models only recently becoming available. Our understanding of the lifecycle of HEV is therefore incomplete. Routes of transmission vary by genotype and location: endemic regions experience large waterborne epidemics, while sporadic cases in industrialized regions are zoonotic infections likely spread via the food chain. Both acute and chronic infection has been observed, and a wide range of extrahepatic manifestations have been reported. This includes neurological, haematological and renal conditions. As the complete clinical phenotype of HEV infection is yet to be characterized, a large proportion of cases go unrecognized or misdiagnosed. In many cases HEV infection does not feature in the differential diagnosis due to a lack of knowledge and awareness of the disease amongst clinicians. In combination, these factors have contributed to an underestimation of the threat posed by HEV. Improvements are required in terms of recognition and diagnosis of HEV infection if we are to understand the natural history of the disease, improve management and reduce the burden of disease around the world.

Prevalence of hepatitis E in liver transplant recipients in Greece.

Hepatitis E virus (HEV) is a well-known cause of acute hepatitis. Immunocompromised subjects, including liver transplant recipients, are considered to be at risk for HEV infection, which occasionally follows a chronic course. The diagnosis of HEV infection in these patients must be based on HEV RNA testing, as serology has variable performance. The aim of this study was to assess the prevalence of HEV infection in liver transplant recipients in Greece by means of HEV RNA testing. Liver transplant recipients followed in the sole transplant centre in Greece were prospectively included. HEV RNA was detected by real-time RT-PCR. Positive samples were further analysed using a nested reverse transcription RT-PCR kit, which amplifies a 137-nucleotide sequence within the ORF2/ORF3 overlapping region to detect the HEV genotype and perform phylogenetic analysis. The mean age of the included patients (n = 76) was 54 years. The most common indication for liver transplantation was viral hepatitis (57%). The majority of the patients (75%) received a calcineurin inhibitor as part of their immunosuppressive regimen and had normal liver enzymes. HEV RNA was found positive in only 1/76 (1.3%) patient. Phylogenetic analysis showed that the sequence clustered into the HEV genotype 3 clade. This patient experienced an acute hepatitis flare, which nonetheless did not become chronic. The prevalence of HEV infection in liver transplant recipients in Greece is similar (1.3%) to that reported previously in other countries. Transplant physicians should be aware of this condition and its associated consequences.

Development of a novel immunoperoxidase monolayer assay for detection of swine Hepatitis E virus antibodies based on stable cell lines expressing the ORF3 protein

Hepatitis E virus (HEV) strains are classified into 4 genotypes by nucleotide sequencing. Genotypes 3 and 4 infect humans and animals via HEV-contaminated food or water. HEV RNA was detected by PCR and antibodies were detected by ELISA. Since human studies showed that HEV IgG antibodies in sera can persist for extended periods, diagnosis of HEV infection in swine or humans is mainly based on serological detection using commercial ELISA kits. However, there is no supplemental method to verify ELISA results. Hence, we developed a novel method used for mutual correction of these common processes. Here, a modified stable HepG2 cell line was transfected with pcDNA3.1-ORF3 to express the swine HEV ORF3 protein. Based on this cell line, a novel immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against HEV. The results show that this method has good specificity, sensitivity and repeatability. When used to investigate 141 porcine serum samples, the IPMA had a coincidence rate of 92.2% with a commercial ELISA kit. The established IPMA described herein is valuable as a supplemental method to ELISA and can differentiate infections by HEV and other viruses.

Ribavirin for Chronic Hepatitis E Virus Infection in Transplant Recipients

BackgroundThere is no established therapy for hepatitis E virus (HEV) infection. The aim of this retrospective, multicenter case series was to assess the effects of ribavirin as monotherapy for solid-organ transplant recipients with prolonged HEV viremia.MethodsWe examined the records of 59 patients who had received a solid-organ transplant (37 kidney-transplant recipients, 10 liver-transplant recipients, 5 heart-transplant recipients, 5 kidney and pancreas–transplant recipients, and 2 lung-transplant recipients). Ribavirin therapy was initiated a median of 9 months (range, 1 to 82) after the diagnosis of HEV infection at a median dose of 600 mg per day (range, 29 to 1200), which was equivalent to 8.1 mg per kilogram of body weight per day (range, 0.6 to 16.3). Patients received ribavirin for a median of 3 months (range, 1 to 18); 66% of the patients received ribavirin for 3 months or less.ResultsAll the patients had HEV viremia when ribavirin was initiated (all 54 in whom genotyping was performed had HEV genotype 3). At the end of therapy, HEV clearance was observed in 95% of the patients. A recurrence of HEV replication occurred in 10 patients after ribavirin was stopped. A sustained virologic response, defined as an undetectable serum HEV RNA level at least 6 months after cessation of ribavirin therapy, occurred in 46 of the 59 patients (78%). A sustained virologic response was also observed in 4 patients who had a recurrence and were re-treated for a longer period. A higher lymphocyte count when ribavirin therapy was initiated was associated with a greater likelihood of a sustained virologic response. Anemia was the main identified side effect and required a reduction in ribavirin dose in 29% of the patients, the use of erythropoietin in 54%, and blood transfusions in 12%.ConclusionsThis retrospective, multicenter study showed that ribavirin as monotherapy may be effective in the treatment of chronic HEV infection; a 3-month course seemed to be an appropriate duration of therapy for most patients.

Diagnosis of HEV infection by serological and real-time PCR assays: a study on acute non-A-C hepatitis collected from 2004 to 2010 in Italy

BackgroundThe impact of hepatitis E in developed countries, like Italy, still requires a clear definition. In the present study, we evaluated HEV infection in patients with acute non-A-C hepatitis by an approach comparing data from Real-time PCR and serological assays.MethodsIn a first analysis, sera from 52 patients hospitalized with a diagnosis of acute viral non-A-C hepatitis in Italy were tested by in-house Real-Time PCR assay for identification of Hepatitis E Virus (HEV) RNA and by anti-HEV IgM and IgG assays. In a subsequent analysis, selected samples were evaluated by additional IgM tests to confirm diagnosis.ResultsAmong the 52 samples, 21 showed positive results for all three markers (IgM, IgG and HEV RNA). One patient showed HEV RNA as single marker. Uncertain results were found in 8 samples while the remaining 22 were negative for all markers. Further analysis of the 8 undefined samples by additional IgM tests confirmed HEV infection in 1 patient. Overall, acute HEV infections were reliably identified in 23 (44.2%) out of 52 patients.ConclusionsIn the present paper, we performed a study evaluating HEV infection in 52 sporadic non-A-C acute hepatitis cases. All samples were collected from 2004 to 2010 in Italy. By a diagnostic strategy based on genomic and serological assays we identified HEV infections in 23 out of 52 patients (44.2%), a percentage higher than previous estimates. Thus, the actual impact of HEV infections in Italy needs to be further evaluated on a national scale by a diagnostic strategy based on multiple and last generation assays.

Hepatitis E virus infection as a cause of graft hepatitis in liver transplant recipients

Hepatitis E virus (HEV) infection induces self-limiting liver disease in immunocompetent individuals. Cases of chronic hepatitis E have recently been identified in organ transplant recipients. We questioned if chronic hepatitis E plays a role in graft hepatitis after liver transplantation in a low endemic area. Two hundred twenty-six liver transplant recipients, 129 nontransplanted patients with chronic liver disease, and 108 healthy controls were tested for HEV antibodies. HEV RNA was investigated in all sera from transplanted patients. HEV antibodies were detected in 1 healthy control (1%), 4 patients with chronic liver disease (3%), and 10 liver transplant recipients (4%). Three liver transplant patients also tested positive for HEV RNA. Two of them developed persistent viremia with HEV genotype 3. The patients were anti-HEV immunoglobulin G-negative and HEV RNA-negative before transplantation and had an episode of acute hepatitis 5 or 7 months after transplantation, which led to advanced liver fibrosis after 22 months in 1 patient. Seroconversion to anti-HEV occurred not before 4 months after the first detection of HEV RNA. The possibility of reverse zoonotic transmission was experimentally confirmed by the infection of 5 pigs with a patient's serum. The pigs showed histological inflammation in the liver, and HEV RNA was detectable in different organs, including muscle. In conclusion, the prevalence of HEV infection in Central European liver transplant recipients is low; however, chronic hepatitis E may occur and needs to be considered in the differential diagnosis of graft hepatitis. The diagnosis of HEV infection should be based on HEV RNA determination in immunosuppressed patients. We suggest that immunocompromised individuals should avoid eating uncooked meat and contact with possibly HEV-infected animals.

Chronic hepatitis E in the immunosuppressed: A new source of trouble?

Chronic hepatitis E virus infection in liver transplant recipients. Haagsma EB, van den Berg AP, Porte RJ, Benne CA, Vennema H, Reimerink JH, Koopmans MP. Hepatitis E virus (HEV) infection is known to run a self-limiting course. Sporadic cases of acute hepatitis due to infection with HEV genotype 3, present in pig populations, are increasingly recognized. Zoonotic transmission seems infrequent. The entity of unexplained chronic hepatitis after liver transplantation has been recognized. Detection of HEV in 2 liver transplant recipients triggered a review of these cases. Freeze-stored sera were available for retrospective analysis. HEV antibodies were determined. For virus detection and identification, a fragment of the gene encoding the major capsid protein (open reading frame 2) was amplified by reverse-transcription polymerase chain reaction and sequenced to identify the genotype. Two months after liver transplantation, case A developed unexplained chronic hepatitis, which developed into cirrhosis. Retransplantation followed 7 years later, after which chronic hepatitis recurred. In retrospect, HEV RNA was present in serum 3 weeks after the first transplantation and remained present afterwards. HEV RNA was also present in retransplant liver tissue. HEV antibodies appeared late after retransplantation. Case B developed unexplained chronic hepatitis 7 years after transplantation. Retransplantation was needed 5 years later, after which no signs of hepatitis recurred. In retrospect, the period of chronic hepatitis up to the retransplantation coincided with HEV RNA in serum. In case B, antibodies developed, the viral load was much lower than in case A, and the virus seemed to be cleared after retransplantation. Genotyping in both cases revealed 2 unique strains of genotype 3. In conclusion, chronic HEV infection may develop in immunosuppressed patients, who may then serve as long-term carriers of the virus. We hypothesize that HEV may be the cause of chronic hepatitis in liver transplant recipients. (c) 2008 AASLD. [Abstract reproduced by permission of Liver Transpl 2008;14:547–553]. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. 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. Hepatitis E virus (HEV) is considered an agent responsible for acute hepatitis that does not progress to chronic hepatitis. We identified 14 cases of acute HEV infection in three patients receiving liver transplants, nine receiving kidney transplants, and two receiving kidney and pancreas transplants. All patients were positive for serum HEV RNA. Chronic hepatitis developed in eight patients, as confirmed by persistently elevated aminotransferase levels, serum HEV RNA, and histologic features of chronic hepatitis. The time from transplantation to diagnosis was significantly shorter and the total counts of lymphocytes and of CD2, CD3, and CD4 T cells were significantly lower in patients in whom chronic disease developed. [Abstract reproduced by permission of N Engl J Med 2008;358:811–817].

Comparative Studies on the Diagnosis of Hepatitis E Virus Antibodies with ORF-2 and ORF-3 Proteins Expressed in Insect Cells.

Hepatitis E is a worldwide health problem, especially in developing countries. The virus genome contains three different open reading frames (ORFs): ORF-1, which is believed to encode nonstructural proteins, and ORF-2 and ORF-3, which are believed to encode structural proteins. Presently, serologic tests for the detection of human antibodies to hepatitis E virus (HEV) infection are primarily based on the ORF-2 structural protein expressed in Escherichia coli, insect cells or synthetic peptides. We report here the comparative studies on the diagnosis of HEV infection with full-length ORF-2 and ORF-3 proteins expressed in insect cells. We found that 31 of 74 (42%) sera were positive for IgM antibody to HEV (anti-HEV) using the ORF-2 protein as an antigen, as compared to 6 of 74 sera (8%) using the ORF-3 protein as an antigen (p < 0.001). Similarly, 49 of 74 sera (66% were positive for IgG anti-HEV utilizing the ORF-2 protein versus 12 of 74 sera (16%) when the ORF-3 protein was used (p < 0.001). These results suggest that the recombinant ORF-2 protein is more sensitive as a diagnostic antigen for detecting antibodies to HEV in both acute-phase and convalescent-phase sera than ORF-3 protein. Copyright 1996 S. Karger AG, Basel