Screening, diagnosis and risks associated with Hepatitis E virus infection.

Introduction: Hepatitis E virus (HEV) is the main cause of hepatitis worldwide. Our knowledge of this single-strand positive-sense RNA virus, discovered in the 1980s, has improved greatly in recent years. Areas covered: We review the most recent information on diagnostic tools, including serological and molecular assays, the recommended diagnostic algorithm, and the clinical manifestations of HEV infections. Expert opinion: The performance of serological and molecular assays has improved greatly in recent years and the availability of a WHO standard has been invaluable for comparing the performance of molecular assays. The more efficient serological and molecular assays have led to a clearer picture of HEV epidemiology. It is now established that HEV is distributed worldwide. The European Association for the Study of the Liver (EASL) now recommends testing for anti-HEV IgM and HEV RNA. Molecular tests indicate that HEV RNA is very common in asymptomatic blood donors. The description of transfusion-transmitted HEV makes having optimal strategies essential for improving blood safety. Like other hepatitis viruses, HEV infection must be suspected whenever a patient presents with clinical or biochemical features of hepatitis. An HEV infection can also have extra-hepatic manifestations, especially neurological and renal disorders.

Multicenter quality control of hepatitis C virus protease inhibitor resistance genotyping.

Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.

Hepatitis E.

Hepatitis E virus (HEV) was discovered during the Soviet occupation of Afghanistan in the 1980s, after an outbreak of unexplained hepatitis at a military camp. A pooled faecal extract from affected soldiers was ingested by a member of the research team. He became sick, and the new virus (named HEV), was detected in his stool by electron microscopy. Subsequently, endemic HEV has been identified in many resource-poor countries. Globally, HEV is the most common cause of acute viral hepatitis. The virus was not initially thought to occur in developed countries, but recent reports have shown this notion to be mistaken. The aim of this Seminar is to describe recent discoveries regarding HEV, and how they have changed our understanding of its effect on human health worldwide.

Hepatitis E virus and the kidney in solid-organ transplant patients.

BACKGROUND: Hepatitis E virus (HEV) infection is an emerging disease in industrialized countries. Few data regarding genotype 3 HEV extrahepatic manifestations exist. METHODS: We assessed kidney function and histology in solid-organ transplant patients during HEV infection. In all, 51 cases of genotype 3 HEV infections were diagnosed (34 kidney, 14 liver, and 3 kidney-pancreas transplant patients). Of these, 43.2% were cleared of the virus spontaneously within 6 months of infection, whereas 56.8% evolved to chronic hepatitis. Twelve of these patients completed a 3-month antiviral therapy and were followed up for 6 months posttreatment. Kidney function (estimated glomerular filtration rate [eGFR] obtained by the Modification of Diet in Renal Disease equation) and proteinuria were assessed before infection, during HEV infection and during follow-up. Kidney biopsies were obtained from patients with high proteinuria and decreased eGFR levels. RESULTS: During HEV infection, there was a significant decrease in eGFR in both kidney- and liver-transplant patients. Glomerular diseases were observed in kidney biopsies obtained during the acute and chronic phases. This included membranoproliferative glomerulonephritis and relapses in IgA nephropathy. The majority of patients had cryoglobulinemia that became negative after HEV clearance. Kidney function improved and proteinuria decreased after HEV clearance. CONCLUSION: HEV-associated glomerulonephritis seems to be an HEV-related extrahepatic manifestation. Further studies are required to confirm these observations.

Hepatitis E virus antibodies in blood donors, France.

Using a validated sensitive assay, we found hepatitis E virus (HEV) IgG in 52.5% of voluntary blood donors in southwestern France. This finding suggests HEV is highly endemic to this region. The high HEV prevalence may reflect local dietary practices, such as eating uncooked pork and game products.

Evolutionary history of hepatitis C virus genotype 5a in France, a multicenter ANRS study.

The epidemic history of HCV genotype 5a is poorly documented in France, where its prevalence is very low, except in a small central area, where it accounts for 14.2% of chronic hepatitis C cases. A Bayesian coalescent phylogenetic investigation based on the E1 envelope gene and a non-structural genomic segment (NS3/4) was carried out to trace the origin of this epidemic using a large sample of genotype 5a isolates collected throughout France. The dates of documented transmissions by blood transfusion were used to calibrate five nodes in the phylogeny. The results of the E1 gene analysis showed that the best-fitting population dynamic model was the expansion growth model under a relaxed molecular clock. The rate of nucleotide substitutions and time to the most recent common ancestors (tMRCA) of genotype 5a isolates were estimated. The divergence of all the French HCV genotype 5a strains included in this study was dated to 1939 [95% HPD: 1921-1956], and the tMRCA of isolates from central France was dated to 1954 [1942-1967], which is in agreement with epidemiological data. NS3/4 analysis provided similar estimates with strongly overlapping HPD values. Phylodynamic analyses give a plausible reconstruction of the evolutionary history of HCV genotype 5a in France, suggesting the concomitant roles of transfusion, iatrogenic route and intra-familial transmission in viral diffusion.

Hepatitis E virus infection without reactivation in solid-organ transplant recipients, France.

Infections with hepatitis E virus (HEV) in solid-organ transplant recipients can lead to chronic hepatitis. However, the incidence of de novo HEV infections after transplantation and risk for reactivation in patients with antibodies against HEV before transplantation are unknown. Pretransplant prevalence of these antibodies in 700 solid-organ transplant recipients at Toulouse University Hospital in France was 14.1%. We found no HEV reactivation among patients with antibodies against HEV at the first annual checkup or by measuring liver enzyme activities and HEV RNA. In contrast, we found 34 locally acquired HEV infections among patients with no antibodies against HEV, 47% of whom had a chronic infection, resulting in an incidence of 3.2/100 person-years. Independent risk factors for HEV infection were an age <52 years at transplantation and receiving a liver transplant. Effective prophylactic measures that include those for potential zoonotic infections should reduce the risk for HEV transmission in this population.

Characteristics of autochthonous hepatitis E virus infection in solid-organ transplant recipients in France.

BACKGROUND: Hepatitis E virus (HEV) infections can lead to chronic hepatitis in immunocompromised patients. We have investigated the risk factors for HEV infection among solid-organ transplant recipients and the characteristics of these infections. METHODS: We performed serological tests, quantified the virus, and genotyped the virus in plasma samples. We performed a case-control study with HEV-infected patients and control participants matched for sex and age who were recruited from a population of solid-organ transplant recipients with no markers of HEV infection. RESULTS: We investigated 38 consecutive cases of HEV genotype 3 infection. Twenty-two (58%) of these 38 patients developed a chronic infection. The acute-phase aminotransferase levels were higher in the patients who cleared the virus than in those who developed chronic infections. The anti-HEV immunoglobulin G and immunoglobulin M profiles and HEV RNA concentration in patients who cleared the virus were similar to those in patients who developed a chronic infection. A logistic regression analysis of 37 case patients and 148 control participants indicated that the only factor independently associated with HEV infection was the consumption of game meat (68% of case patients vs 47% of control participants; odds ratio, 2.32; 95% confidence interval, 1.04-5.15). CONCLUSION: Immunocompromised patients should avoid eating insufficiently cooked game meat or pork products so as to reduce the risk of HEV infection and chronic liver disease.

New NS5B polymerase inhibitors for hepatitis C.

IMPORTANCE OF THE FIELD: The current treatment of chronic hepatitis C based on the combination of pegylated interferon and ribavirin is effective in only 50% of patients. Specific targeted antiviral therapies represent a promising approach to eradicate the infection. AREAS COVERED IN THIS REVIEW: This review focuses on progress towards the development of the hepatitis C virus (HCV) polymerase inhibitors that have entered clinical development in recent years. WHAT THE READER WILL GAIN: Nucleos(t)ide analogues target the active site of the HCV polymerase and acts as chain terminators. They have similar activity against all genotypes and the virus has a high genetic barrier to drug resistance. Non-nucleoside inhibitors achieve polymerase inhibition by binding to one of the at least four allosteric enzyme sites. Most of them have a genotype-specific activity and they may select rapidly drug-resistant variants if HCV replication is not completely suppressed. Nonetheless, they provide additional options for addressing the needs of infected patients. TAKE HOME MESSAGE: NS5B polymerase inhibitors will form an integral part of more effective anti-HCV therapy, in combination with interferon or with other directly acting antiviral agents.

Influence of the HCV subtype on the virological response to pegylated interferon and ribavirin therapy.

The hepatitis C virus genotype is considered to be the most important baseline predictor of a sustained virological response in patients with chronic hepatitis C treated with pegylated interferon and ribavirin. The influence of the subtype on the sustained virological response was investigated in patients infected with genotypes 1, 4, 5, or 6. This study was done on 597 patients with chronic hepatitis C who were given pegylated interferon and ribavirin for 48 weeks. The overall rate of sustained virological response in the 597 patients was 37.8%. Univariate analysis indicated that the sustained virological response of patients infected with subtype 1b (39%) tended to be higher than that of patients infected with subtype 1a (30.6%; P = 0.06) and it was similar to those patients infected with subtypes 4a (51.3%; P = 0.12) or 4d (51.7%; P = 0.16). Multivariate analysis indicated that five factors were independently associated with sustained virological response: the age (OR 0.97; 95% CI = 0.95-0.99), absence of cirrhosis (OR: 2.92; 95% CI = 1.7-5.0; P < 0.01), absence of HIV co-infection (OR: 2.08; 95% CI = 1.2-3.5; P < 0.01), low baseline plasma HCV RNA concentration (OR: 1.74; 95% CI = 1.2-2.6; P < 0.01), and the subtype 1b (OR: 1.61; 95% CI = 1.0-2.5; P = 0.04) or subtypes 4a and 4d (OR: 2.03; 95% CI = 1.1-3.8; P = 0.03). In conclusion, among difficult-to-treat genotypes, the subtype 1a is associated with a lower response to anti-HCV therapy than subtypes 1b, 4a, and 4d.

Naturally occurring substitutions conferring resistance to hepatitis C virus polymerase inhibitors in treatment-naive patients infected with genotypes 1-5.

BACKGROUND: The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B, is essential for virus RNA replication. It is thus an attractive therapeutic target. Several compound nucleoside analogues, non-nucleoside inhibitors and cyclosporine analogues are being developed to inhibit NS5B activity. However, nucleotide changes in the NS5B gene can confer resistance to them. METHODS: We investigated the prevalence of known substitutions conferring resistance in HCV polymerase in 124 treatment-naive French patients infected with HCV genotypes 1, 2, 3, 4 or 5 by sequencing the NS5B gene. RESULTS: None of the 124 HCV NS5B sequences analysed contained substitutions conferring resistance to nucleoside analogues; however, NS5B polymerases containing substitutions conferring resistance to non-nucleoside inhibitors were frequent within genotype 1 strains (17%) and very common in non-genotype 1 strains. Similarly, substitutions conferring resistance to cyclosporine analogues were more prevalent within the various genotypes. CONCLUSIONS: Naturally occurring substitutions conferring resistance to NS5B inhibitors are common in treatment-naive patients infected with HCV genotype 1, 2, 3, 4 or 5. Their influence on treatment outcome should be assessed.

Good performance of immunoglobulin M assays in diagnosing genotype 3 hepatitis E virus infections.

We have evaluated three anti-hepatitis E virus (anti-HEV) immunoglobulin M (IgM) assays, the EIAgen HEV IgM assay (Adaltis), the HEV IgM enzyme-linked immunosorbent assay 3.0, and the Assure HEV IgM rapid test (MP Diagnostics), for the routine detection of acute genotype 3 HEV. Their sensitivities were fairly good (90%, 88%, and 82%), and their specificities were excellent (100%, 99.5%, and 100%).

Hepatitis E virus genotype 3 diversity, France.

We characterized 42 hepatitis E virus (HEV) genotype 3 strains from infected patients in France in 3 parts of the genome and sequenced the full-length HEV genotype 3f genome found in Europe. These strains are closely related to swine strains in Europe, which suggests zoonotic transmission of HEV in France.

Laboratory evaluation of the UniCel DxI 800 analyser (Beckman Coulter) for detecting HBV and HCV serological markers.

BACKGROUND: Due to their high prevalence, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections need accurate and rapid diagnosis tools. OBJECTIVES: Technical performances of the UniCel DxI 800 analyser (Beckman Coulter) and a comparison with the Vitros ECi (Ortho Clinical Diagnostics) were performed for five serological markers: HBsAg, total anti-HBc, anti-HBc IgM, anti-HBs and anti-HCV. STUDY DESIGN: Reproducibility was determined by repeated tests on the manufacturers' controls. The performance of the UniCel DxI 800 was assessed by testing negative and positive samples previously analysed with the Vitros ECi. The accuracy and linearity of anti-HBs assay were evaluated using the WHO international standard (W1042). RESULTS: The intra-assay and inter-assay coefficients of variation were: 0.8% and 4.4% for HBsAg, 2.4% and 6.2% for anti-HBc, 5% and 8.7% for anti-HBc IgM, 2.1% and 5.1% for anti-HBs and 3.7% and 7.4% for anti-HCV. The two methods were concordant: 100% agreement for the five markers except for the negative HBsAg sera (99%). The anti-HBs results correlated well with the Vitros ECi (r=0.925 with p<0.0001) and the WHO standard (r2=0.9996). Throughput was 216 tests/h. CONCLUSION: The high throughput, specificity and sensitivity make UniCel DxI 800 assays useful for routine diagnoses of HCV and HBV infections.

Molecular epidemiology and interferon susceptibility of the natural recombinant hepatitis C virus strain RF1_2k/1b.

BACKGROUND: Hepatitis C virus (HCV) genotype is an important determinant of virological response to antiviral therapies. Currently, there are no data available on the molecular epidemiology and interferon susceptibility of the natural intergenotypic recombinant RF1_2k/1b (RF1) strain. METHODS: Genotyping and RF1-PCR screening were performed on samples from 604 HCV RNA-positive individuals from 7 countries. uPA/SCID mice carrying human hepatocytes (chimeric mice) were infected with the RF1_2k/1b strain, and the susceptibility of the strain to interferon and ribavirin was compared with the susceptibilities of 2 different strains of genotype B, used as references. RESULTS: Six new RF1 cases were identified in this study; 5 (2%) of 281 in Russia and 1 (1%) of 90 in Uzbekistan. Phylogenetic analyses based on Core/E1 and NS5b indicated that all RF1 representatives share a common evolutionary ancestor. Infection with RF1 was established in chimeric mice. Reduction of RF1 viral load was observed in response to 3 injections of 3 microg/kg pegylated-interferon alpha-2a alone or in combination with 50 mg/kg of ribavirin (0.5 or 1.4 log-copies/mL). CONCLUSIONS: All identified RF1-type strains appear to be introduced from a single source, suggesting that intergenotypic recombination in HCV is sporadic and not associated with cocirculation of different genotypes in a population. The RF1 strain in this study was responsive to interferon in vivo.

Serum concentrations of ribavirin and pegylated interferon and viral responses in patients infected with HIV and HCV.

The hepatitis C virus (HCV) infects a substantial proportion of patients infected with human immunodeficiency virus (HIV). Patients infected with both HCV and HIV respond poorly to anti-HCV treatment with pegylated interferon alpha and ribavirin. But few data are available on the influence of ribavirin and interferon concentrations on treatment outcome for these patients. This study investigated the relationship between the serum pegylated interferon and ribavirin concentrations 3 and 6 months after treatment initiation, and treatment outcome in 35 HCV-HIV coinfected patients. The pegylated interferon and ribavirin concentrations at months 3 and 6 were similar. The pegylated interferon concentrations at 3 months in responders and nonresponders were similar. However, responders tended to have higher ribavirin concentrations (2,322 ng/ml) than nonresponders (1,833 ng/ml; P = 0.08). Responders infected with HCV genotype 1 or 4 had higher ribavirin concentrations (2,672 ng/ml) than did similarly infected nonresponders (1,758 ng/ml; P = 0.04). ROC curve analysis showed that a ribavirin concentration of 2,300 ng/ml was the best threshold for predicting a nonresponse (ROC area = 0.80 +/- 0.12). Thus ribavirin concentrations influence treatment outcome in HIV patients infected with HCV genotype 1 or 4. Monitoring ribavirin concentrations could help adapt ribavirin concentrations and improve the sustained virological response.

High prevalence of anti-hepatitis E virus antibodies in blood donors from South West France.

Cases of autochthonous acute hepatitis E occur in most industrialized countries and are frequent in the South West of France. The prevalence of anti-hepatitis E virus (HEV) IgG antibodies in blood donors in this area was determined. A total of 529 samples from rural and urban blood donors were tested. The overall prevalence was 16.6%, 19.1% of rural donors and 14.2% of urban donors had anti-HEV antibodies (P = 0.13). The antibodies were widely distributed among all age groups and the sex ratio of the anti-HEV positive blood donors was 1.12 (P = 0.57). Hunting was the only pastime or profession associated with a high prevalence of anti-HEV antibodies (P = 0.038). The frequency of anti-HEV antibodies in blood donors could reflect active autochthonous transmission in this area of France. As the risk factors for HEV infection in industrialized countries are still unknown, further studies are needed to clarify the epidemiology of HEV infection in the Midi-Pyrénées region.

Full-length genome sequences of hepatitis C virus subtype 4f.

Hepatitis C virus genotype 4 (HCV-4) is very common in central Africa, prevalent in the Middle East, and is becoming increasingly frequent among southern Europeans. We have determined the complete nucleotide sequences of HCV-4f strains and investigated their phylogenetic relationships with other genotypes. We amplified the complete genome of two HCV subtype 4f strains, IFBT84 and IFBT88. The HCV-4f strains have a total of 9181 and 9304 nt, respectively, including the 5' untranslated region followed by a single open reading frame. There was no evidence of genomic recombination in the IFBT84 and IFBT88 strains. The sequences of the HCV-4f strain genomes are closer to those of HCV-1b than to genotypes 2, 3, 5 and 6. It is important to know the full-length sequences of HCV-4 subtypes in order to classify them correctly and to obtain more detailed knowledge about HCV epidemiology and sensitivity to interferon.