Hepatitis C subtype distribution in chronically infected patients with mild liver fibrosis in France: the GEMHEP study.

Treatment options for Hepatitis C infection have greatly improved with direct-acting antiviral (DAA) combinations achieving high cure rates. Nevertheless, the cost of this treatment is still high and access to treatment in many countries has been preferentially reserved for patients with more severe fibrosis (F3 and F4). In this French nationwide study, we investigated the epidemiological characteristics and genotype distribution of hepatitis C virus (HCV) in treatment-naive patients with METAVIR fibrosis stages between F0 and F2 in order to identify patient profiles that became eligible for unrestricted treatment in a second period. Between 2015 and 2016 we collected data from nine French university hospitals on a total of 584 HCV positive patients with absent, mild or moderate liver fibrosis. The most represented genotypes were genotype 1b (159/584; 27.2%), followed by genotype 1a (150/584; 25.7%); genotype 3 (87/584: 14.9%); genotype 4 (80/584; 13.7%). Among genotype 4: 4a was predominantly encountered with 22 patients (27.5% of genotype 4). Genotypes 1b and 1a are currently the most frequent virus types present in treatment-naive patients with mild fibrosis in France. They can be readily cured using the available DAA. Nevertheless, non-a/non-d genotype 4 is also frequent in this population and clinical data on the efficacy of DAA on these subtypes is missing. The GEMHEP is the French group for study and evaluation of viral hepatitis on a national scale. Data collection on epidemiological and molecular aspects of viral hepatitis is performed on a regular basis in all main French teaching hospitals and serves as a basis for surveillance of these infections. Analysis and trends are regularly published on behalf of the GEMHEP group. Data collection was performed retrospectively over the 2015-2016 period, covering nine main university hospitals in France. A total of 584 hepatitis C positive patients were included in this study. Genotyping of the circulating viruses showed a high prevalence of genotypes 1b and 1a in our population. The epidemiology of hepatitis C is slowly changing in France, particularly as a consequence of the rise of 'non-a non-d' genotype 4 viruses mainly originating from African populations. More data concerning treatment efficacy of these genotypes is needed in order to guide clinical care.

Claudin-1, miR-122 and apolipoprotein E transductions improve the permissivity of SNU-182, SNU-398 and SNU-449 hepatoma cells to hepatitis C virus.

Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU-182, SNU-398 and SNU-449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin-1 (CLDN1) expression limited the viral entry process in SNU-182 and SNU-398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR-122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU-182 and SNU-398 cells but not in SNU-449 cells. Nevertheless, the supplementation of SNU-182, SNU-398 and SNU-449 cells with CLDN1, miR-122 and ApoE was not sufficient to render these cells as permissive as HuH-7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR-122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection.

Are trans-complementation systems suitable for hepatitis C virus life cycle studies?

Complementation is a naturally occurring genetic mechanism that has been studied for a number of plus-strand RNA viruses. Although trans-complementation is well documented for Flaviviridae family viruses, the first such system for hepatitis C virus (HCV) was only described in 2005. Since then, the development of a number of HCV trans-complementation models has improved our knowledge of HCV protein functions and interactions, genome replication and viral particle assembly. These models have also been used to produce defective viruses and so improvements are necessary for vaccine assays. This review provides an update on HCV trans-complementation systems, the viral mechanisms studied therewith and the production and characterization of trans-encapsidated particles.

Strong correlation between liver and serum levels of hepatitis C virus core antigen and RNA in chronically infected patients.

HCV core antigen (Ag) and HCV RNA levels were evaluated in matched liver biopsy samples and sera from 22 patients with hepatitis C infection by using the quantitative Architect HCV Ag immunoassay and a real-time RT-qPCR assay, respectively. The data showed a strong correlation between liver and serum compartments of HCV Ag levels (r = 0.80) and HCV RNA levels (r = 0.87). In summary, the serum HCV Ag and RNA levels reflect the intrahepatic values.

Genetic recombination of the hepatitis C virus: clinical implications.

Genetic recombination is a well-known feature of RNA viruses that plays a significant role in their evolution. Although recombination is well documented for Flaviviridae family viruses, the first natural recombinant strain of hepatitis C virus (HCV) was identified as recently as 2002. Since then, a few other natural inter-genotypic, intra-genotypic and intra-subtype recombinant HCV strains have been described. However, the frequency of recombination may have been underestimated because not all known HCV recombinants are screened for in routine practice. Furthermore, the choice of treatment regimen and its predictive outcome remain problematic as the therapeutic strategy for HCV infection is genotype dependent. HCV recombination also raises many questions concerning its mechanisms and effects on the epidemiological and physiopathological features of the virus. This review provides an update on recombinant HCV strains, the process that gives rise to recombinants and clinical implications of recombination.

A 2-log drop in viral load at 1 month is the best predictor of sustained response in HCV patients with normal ALT: a kinetic prospective study.

The most reliable predictor of a sustained virological response in patients with persistently normal ALT has not been identified. We analysed 17 patients with genotype 1 chronic HCV who underwent therapy with pegylated interferon alfa 2b and ribavirin for 48 weeks. Two patients discontinued therapy within 28 days because of side effects and the remaining 15 patients were analysed in detail. An analysis of on treatment virological response using area under the receiver operating characteristic analyses showed that a 2 log drop in HCV RNA at day 28 was the best predictor of a sustained virological response and a failure to reduce viral load by 2 logs correctly identified patients with a low (<15%) probability of achieving a sustained virological response. Introduction of this early discontinuation rule in patients with normal ALT would allow nearly half of the patients to discontinue futile therapy at an early stage.

BK polyomavirus in the urine for follow-up of kidney transplant recipients.

OBJECTIVES: After kidney transplantation, human BK polyomavirus (BKPyV) can induce a progressive disease, in three stages: viruria, viraemia, and then nephropathy after a few months of viral replication. Therapeutic intervention is recommended when BKPyV is detected in the plasma. The objective of our study was to assess urinary BKPyV nucleic acid test as a predictor for developing viraemia. METHODS: We first defined a viruria threshold based on 393 time-matched urine and plasma samples collected after kidney transplantation; to validate this threshold, we followed-up a cohort of 236 kidney transplant patients. RESULTS: A BKPyV viruria threshold of 6.71 log10 copies/mL best discriminated between plasma-positive and plasma-negative patients (sensitivity 90.9% (95% CI 86.5-95); specificity 90.3% (95% CI 86.3-94.3); area under the curve 0.953 (95% CI 0.933-0.974). In the validation cohort, the risk of developing BKPyV viraemia at 1 year was 16.5% (39/236) and rose to 90.7% (39/43) if BKPyV viruria remained above the threshold of 6.71 for more than 1 month. CONCLUSIONS: Sustained BKPyV viruria is a reliable, early marker of patients at high risk of developing BKPyV viraemia. This marker should alert the clinician early, and thus allow timely therapeutic intervention.

Prevalence of HPV genotypes determined by PCR and DNA sequencing in cervical specimens from French women with or without abnormalities.

BACKGROUND: Epidemiological data on human papillomavirus (HPV) are needed to estimate potential changes in type distribution induced by recent HPV vaccination strategies. OBJECTIVES AND STUDY DESIGN: The epidemiological distribution of HPV in 669 cervical specimens from French women with and without cytological abnormalities was evaluated using type-specific PCR or sequencing. The results were compared with those obtained using the Digene high-risk Hybrid Capture 2 (HR-HC2) assay. RESULTS: The overall prevalence of HPV was high (45.3%) in our study population. 285 of the 291 HPV-positive samples were typed. The distribution frequency concerned 34 different genotypes, with HPV16 being the most prevalent (32.6%). Other genotypes present were HPV31 (7.4%), HPV18, HPV 52 (both 6.0%), HPV6 (5.3%) and HPV66 (4.2%). The respective frequencies of all other genotypes were below 4%. The agreement with HR-HC2 was 78.8%. The distribution frequency data were also analyzed relatively to cytological and histological results. Our method enables the diagnosis of HPV infections with the additional advantage of genotyping. CONCLUSION: HPV infections in the area of France studied here involve numerous HPV types, but the high cumulative prevalences of types 16, 18, 6 and 11 (44.6% in total) would suggest a major impact of vaccination on these genotypes.

Latex enzyme immunoassay for measuring IgG antibodies to rubella virus.

A latex enzyme immunoassay (LEIA) for detecting IgG class antibody to rubella virus was compared with the latex agglutination test (LA) and a haemagglutination inhibition assay (HI). Of 243 sera tested, four discrepant results were observed among all three techniques, corresponding to borderline values. Except for one sample containing specific IgM class antibody, the difference between quantitative results from each pair of tests was always within a value corresponding to two doubling dilutions and was considered to have acceptable variation. The LEIA technique allowed 10 seroconversions to be detected, as determined by the other techniques. The LEIA required a single 1 in 8 sample dilution, took 30 minutes, and provided a useful alternative assay for the quantitation of rubella antibodies.

Solid phase reverse passive hemadsorption test for hepatitis B surface antigen.

A solid phase reverse passive hemadsorption test (SP-RPHAd) for hepatitis B surface antigen detection is described. It was compared with a commercial reverse passive hemagglutination assay (Hepatest, Wellcome, U.K.). SP-RPHAd is four-fold less expensive than Hepatest and undiluted sera can be used instead of eight-fold diluted sera without risk of non-specific hemagglutination. Thus, the threshold of detection is lowered to about 5 ng/ml by SP-RPHAd.

A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection.

A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.

Determining hepatitis C genotype by analyzing the sequence of the NS5b region.

Assays to determine the hepatitis C virus (HCV) genotype have recently become useful for clinical decision making and may be suitable for epidemiological investigations, such as identifying HCV outbreaks in a given population. Molecular assays are the most common diagnostic tools for HCV genotyping. This study compares two genome typing assays, one, the Trugene 5'NC genotyping kit, uses the sequence of the 5' non-coding (5'NC) region and the other, a non-commercial assay, uses the non-structural 5b (NS5b) region. Serum samples from 203 chronically HCV-infected patients were tested. The 5'NC and the NS5b assays were both very effective in identifying the genotype (99 and 98.5%) and the results with the two methods were always concordant for the genotype. The NS5b analysis permitted the identification of the subtype in all samples, whereas the 5'NC region assay did not in 33% of samples. The NS5b analysis showed that one patient had a mixed infection with HCV subtypes 1a and 2c, while the 5'NC assay did not. It is concluded that phylogenetic analysis using both the 5'NC and the NS5b regions are reliable and convenient methods for HCV typing in clinical practice. But analysis of the NS5b region may be more useful for tracing the source of an HCV infection.

Direct blotting electrophoresis for sequencing and genotyping hepatitis C virus.

Many methods have been used to differentiate the hepatitis C virus (HCV) genotypes based on, for example, type specific primers, probes and restriction fragment length polymorphism. However, determination of the nucleotide sequence remains the reference. Therefore, a simple non-radioactive cycle sequencing technique was developed for clinical tests. PCR-amplified products of the 5' non-coding region (from position -274 to -31) were sequenced using a 5' digoxygenin-labeled primer. After denaturation, the samples were loaded on a direct blotting electrophoresis system (GATC 1500). Sequencing products were blotted onto a nylon membrane during the electrophoresis. The DNA fragments were then UV-cross-linked, incubated with phosphatase-labeled anti-digoxygenin antibody and stained with a precipitating substrate. Reading the sequence of six samples were possible within 2 days. In 41 different samples, five different genotypes were found by sequence analysis from position -245 to -69, of which 17 were type 1a, 7 type 1b, 5 type 2a, 8 type 3a, 3 type 4 and 1 type 5. These results agreed with those obtained by reverse hybridization assay. Direct blotting electrophoresis offered a good non-radioactive method of performing clinical sequencing on a medium scale, with a minimum of investment.

[Seroprevalence of viral hepatitis A in at the Amiens University Hospital]. / Séroprévalence de l'hépatite virale A au CHU d'Amiens.

OBJECTIVES AND METHODS: The epidemiology of viral hepatitis A has been evolved in the past few years, resulting in an increasing number of people without immunity to this virus. Health care workers are usually considered to be a group at risk of contamination by hepatitis A. A sero-epidemiologic study was performed in 525 members of the Pediatry, Gastroenterology, Internal medicine, Digestive radiology, kitchen and maintenance department staffs in the Amiens University Hospital. The aim of this study was to describe the epidemiology of hepatitis A and to estimate the level of occupational hazard it represents in the hospital. RESULTS: Age, low education level, country of origin in an endemic region and more than 2 siblings or children were significantly associated with the presence of anti-HAV antibodies. The prevalence of 50% was similar to that observed in other hospitals, but lower than that found in the general population. Seroprevalence was not higher in departments exposed to stools (Pediatry, Digestive endoscopy and laboratories) than in others. A higher rate of seroprevalence was observed in kitchen and maintenance staffs than in medical, laboratory and Radiology staffs, in Internal medicine than in the Gastroenterology Department, and in the laboratory than in Radiology Department. These differences disappeared after adjustment for extraprofessional parameters which appeared to be most important for hepatitis A epidemiology. CONCLUSIONS: The hospital occupational hazard for hepatitis A virus did not seem higher than that observed in the general population.

Hepatitis C Virus p7 membrane protein quasispecies variability in chronically infected patients treated with interferon and ribavirin, with or without amantadine.

A clinical study was carried out to compare the response rate of two groups of non-responder (NR) hepatitis C virus (HCV) genotype 1 chronically infected patients treated with interferon and ribavirin, with or without amantadine. The viral load decreased more markedly in the group treated by tritherapy including amantadine, but the response rate at the end of treatment was not significantly different between bitherapy and tritherapy. As amantadine could have an antiviral effect on the ion channel activity of the p7 HCV protein, the p7 quasispecies was characterized by cloning and sequencing. Sequence data were analyzed to determine the pattern and significance of p7 genetic heterogeneity and a possible relationship with therapy. Subtype differences were confirmed between p7 HCV genotypes 1a and 1b, and quasispecies analysis showed a reduction of genetic diversity in subtype 1a, but not 1b, during tritherapy. However, the absence of changes at numerous positions, as well as the conservative changes at other positions, indicated the high conservation of the p7 structure. Residue His-17, proposed to interact with amantadine, was fully conserved in both subtypes 1a and 1b, independently of amantadine administration. In conclusion, although the analysis of the p7 sequences revealed a selective pressure during therapy, no specific residues appeared to be linked to the effect of amantadine on viral decline. These results suggest that the potential antiviral effect of amantadine might be non-specific and related to a reduction in endosomal acidification and therefore reduced viral entry of HCV via its pH-dependent pathway.

Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: The GEMHEP GenoCII Study.

This cross-sectional study aimed to investigate, during a short period between 2000 and 2001, in a large population of patients with chronic hepatitis C, the epidemiological characteristics of hepatitis C virus (HCV) genotypes in France. Data from 26 referral centres, corresponding to 1769 patients with chronic hepatitis C were collected consecutively during a 6-month period. HCV genotyping in the 5'-non-coding region (NCR) was performed in each center using the line probe assay (LiPA, in 63% of cases), sequencing (25%) or primer-specific polymerase chain reaction (PCR) (12%). HCV genotypes 1a, 1b, 2, 3, 4, 5, non-subtyped 1 and mixed infection were found in 18, 27, 9, 21, 9, 3, 11 and 1% of our population, respectively. HCV genotype distribution was associated with gender, age, source and duration of infection, alanine aminotransferase (ALT) levels, cirrhosis, alcohol consumption, hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection. In multivariate analysis, only the source of infection was the independent factor significantly associated with genotype (P = 0.0001). In conclusion, this study shows a changing pattern of HCV genotypes in France, with i.v. drug abuse as the major risk factor, an increase of genotype 4, and to a lesser extent 1a and 5, and a decrease of genotypes 1b and 2. The modification of the HCV genotype pattern in France in the next 10 years may require new therapeutic strategies, and further survey studies.

Microparticle enzyme immunoassay for determination of immunoglobulin G antibodies to human cytomegalovirus.

A microparticle enzyme immunoassay (LEIA) that uses passive latex agglutination was used to detect cytomegalovirus immunoglobulin G antibodies in 495 serum samples. LEIA was in excellent concordance with latex agglutination (97%) and an enzyme-linked immunosorbent assay (98.3%). The sensitivity and specificity of LEIA compared with those of the enzyme-linked immunosorbent assay were 100 and 92.5%, respectively.

Heterophile IgM, IgA, and IgE antibodies in infectious mononucleosis.

Fifty infectious mononucleosis (IM) and 150 non-infectious mononucleosis (non-IM) sera were tested by a hemadsorption immunocapture test (HIT) for the detection of heterophile antibodies of IgM, IgA, and IgE classes. The specificity of Paul-Bunnell (PB) antibodies was ascertained by a differential absorption test. IgM PB-antibodies were demonstrated in 100% of IM sera, IgA in 92%, and IgE in 88%. PB antibodies were present only in IM sera; but other heterophile antibodies of IgM (68%), IgA (6.7%), and IgE (9.3%) classes were demonstrated in non-IM sera. IgA and IgE heterophile antibodies were thus present, especially in IM sera. HIT was found to be more sensitive than Paul-Bunnell Davidsohn test (PBD test) and suitable for the diagnosis of infectious mononucleosis.

Etude épidémiologique de gastroentérites infantiles d'origine virale dans la région amienoise.

A systematic survey was carried on stools from 130 children suffering of acute gastroenteritis. Electron microscopy, enzymo-linked immunosorbent assay (ELISA) and counter electrophoresis were employed. This survey allowed to the detection by electron microscopy of Rotavirus (40 cases), Coronaviruses (3 cases), Astroviruses (2 cases), Adenoviruses (2 cases) and Small Round Viruses (1 case). Serological tests (complement fixation, ELISA and counter electrophoresis) done with 86 sera showed a good correlation with results obtained with electron microscopy.