Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies

Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1,2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 2 HCV infection. A small population (n= 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients.     

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.     

Reproducibility of hepatitis C virus antibody detection with various confirmatory assays

Angiostrongylus costaricensis (classified under the more specific genus Parastrongylus by different authors) is a filiform intestinal nematode endemic to Central and South America, which afflicts primarily the pediatric population 1 to 13 years of age. Only three cases of adult infections have been previously documented outside the endemic region. To our knowledge, we report the first such case in the western United States, as well as the oldest patient yet recorded. Our patient is a 73-year-old woman living in Los Angeles who presented with an acute abdomen 4 weeks following a 5-month visit to El Salvador. Examination of the gross ileum removed at exploratory laparotomy revealed a perforation associated with a segmentally thickened intestinal wall. Microscopic examination showed eosinophilic ileitis, arterial lumens containing nematodes morphologically consistent with A costaricensis, eggs in the submucosa, and arteriolitis with thrombosis.     

Multi-measurement method comparison of three commercial hepatitis B virus DNA quantification assays

Serum specimens (327) from patients with chronic hepatitis B were evaluated for hepatitis B virus (HBV) DNA using three commercial assays--Chiron Quantiplex (CA), Digene Hybrid Capture (DA) and Abbott HBV DNA assay (AA). The HBV DNA values obtained following evaluation were used to compare the linearity, responsiveness and precision of each assay and to determine the conversion between the three different assay values. The comparison was accomplished using a new statistical approach termed the multi-measurement method (MMM). MMM is a minimal bias, non-linear regression technique that allows simultaneous multiassay performance evaluation as well as assay value interconversion. MMM analysis demonstrated that the CA was more sensitive and responsive than either the DA or the AA. Both the CA and DA were more precise than the AA. Validation of the MMM results was performed using two additional data sets of 551 and 100 specimens, respectively. MMM is a novel statistical tool that has a broad application for the generation of statistically normalized laboratory data and for interassay standardization.     

Evaluation of the automated COBAS AMPLICOR hepatitis C virus PCR system

To evaluate the reliability and feasibility of the automated Roche COBAS AMPLICOR PCR system for routine detection of hepatitis C virus (HCV) RNA, a total of 405 serum samples previously tested by an in-house nested PCR and manual Roche AMPLICOR microwell plate HCV test were examined. Complete concordance was found between the results with the HCV COBAS AMPLICOR system and the previously determined HCV RNA status. The automated HCV COBAS AMPLICOR system provides the clinical microbiology laboratory with a specific and sensitive PCR method for rapid and reliable detection of HCV RNA.     

Transmission of hepatitis C virus in allografted patients: use of viral genotyping as an epidemiological marker

One hundred and ninety-two allografted patients were tested for hepatitis C virus (HCV) RNA from 1992 to 1995 in Saint-Louis Hospital (Paris). They received blood products and intravenous immunoglobulins (IVIG) and more particularly Gammagard IVIG suspected of transmitting HCV (batches distributed in France between January 1993 and February 1994). The presence of serum HCV RNA was tested by polymerase chain reaction (PCR) in 86 patients who received Gammagard IVIG during the critical period and in 106 patients treated with IVIG other than the suspected batches of Gammagard (negative controls). HCV RNA positive sera were HCV genotyped. Ten out of 86 patients who received Gammagard IVIG during the exposed period vs 0 out of 106 negative controls were HCV RNA positive showing a higher prevalence of HCV infection in the exposed patients that in the negative controls (P = 0.001). The link between HCV transmission and IVIG infusion was reinforced by the high frequency of genotype 2b (70%) in the exposed patients because genotype 2b is an underrepresented subtype in France (< 1%).     

Influence of viraemia and genotype upon serological reactivity in screening assays for antibody to hepatitis C virus

Ganglioside GM2, expressed on the surface of some human cancers, is a promising target for immune therapy, since GM2 antibodies are cytotoxic, can be induced in humans by vaccination, and the presence of GM2 antibodies is associated with a better prognosis in melanoma patients. In our efforts to induce long-lived, cytotoxic GM2 antibodies, we investigated lipopolysaccharides (LPS) containing "GM2-like" oligosaccharides. LPS were prepared from Campylobacter jejuni serotypes O:1, O:23, or O:36 (all sharing the oligosaccharide structure GalNAcbeta1-4Gal(113NeuAc)-Hex with ganglioside GM2), and tested for their ability to induce GM2-reactive antibodies. Immunization of NZW rabbits (2 animals per vaccine) with LPS from C. jejuni serotype O:1 in Freund's adjuvant resulted in production of high-titer IgG antibodies reactive with purified bovine brain GM2 in ELISA, dot-blot immune strains and immune thin-layer chromatography, and with GM2 derived from various human tumors by immune thin-layer chromatography. These rabbit antibodies bound to cancer cell lines expressing GM2 on their cell surface, as determined by mixed hemadsorption assays, mediating strong antibody-dependent cellular cytotoxicity (ADCC) with tumor cells expressing cell-surface GM2. Antibodies induced by vaccination with C. jejuni serotype O:1 were higher-titer (IgG ELISA titer > 1:60,000) than antibodies induced by immunization with purified GM2 (IgG ELISA titer > 1:200). Immunization with LPS from C. jejuni serotype O:36 resulted in production of moderately high-titer IgM and low-titer IgG GM2 antibodies. Immunization with LPS from C. jejuni serotype O:23 did not elicit GM2-reactive antibodies. No clinical symptoms were observed in animals immunized with these LPS preparations, with purified GM2 ganglioside, or with LPS derived from C. jejuni serotype O:19 (containing a GM1-like oligosaccharide). Our results indicate that lipopolysaccharides sharing carbohydrate epitopes with gangliosides may be useful immunogens for inducing antibodies to ganglioside antigens.     

Human immunodeficiency virus and hepatitis C virus coinfection

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) share the same parenteral, sexual and vertical routes of transmission (McNair et al. 1992). This common epidemiology explains the high frequency of combined infections by hepatotropic viruses in HIV-infected patients. The aim of the present review is to clarify some important issues dealing with the reciprocal interactions between HIV and hepatitis C virus infections. The main topics include epidemiology, virological markers of HIV-infection, histopathology, natural course and treatment of hepatitis C in HIV-infected individuals.     

T-lymphocyte response to hepatitis C virus in different clinical courses of infection

BACKGROUND: To assess the role played by the immune response in the outcome of hepatitis C virus infection, the CD4+ T-lymphocyte response to viral antigens was studied in infected individuals with different clinical courses. METHODS: Using six recombinant proteins of hepatitis C virus, the study assessed the proliferative responses of peripheral blood mononuclear cells from 41 patients with chronic hepatitis C, 11 patients whose chronic hepatitis was successfully treated with interferon alfa and 11 healthy HCV seropositive individuals. RESULTS: (1) Sixty-five percent of hepatitis C virus-seropositive individuals had CD4+ T-cell responses to viral proteins. (2) All viral proteins were immunogenic for T cells, although NS4 was the most immunogenic. (3) There was a significant correlation between the presence of CD4+ T cell responses to Core and a benign course of infection in healthy seropositives, most of whom were viremic. CONCLUSIONS: CD4+ T-cell responses to Core, although they do not coincide with virus clearance, are associated with a benign course of infection and may be required to maintain humoral and cellular responses protective against the disease.     

Chronic hepatitis C virus positive hepatitis and antiphospholipid syndrome

The antiphospholipid syndrome, disorder in which venous and arterial thrombosis may occur, has been described in association with systemic lupus erythematosus and later in a great variety of entities, including infectious diseases. It has been described recently a high frequency of antiphospholipid syndrome in patients with chronic hepatitis C. We present a case of cirrhosis with hepatitis C virus markers and criteria for antiphospholipid syndrome, and we review the literature.     

Classifying hepatitis C virus genotypes

Current cytogenetic evaluation of solid tumors is performed on fresh tissue specimens requiring on-call tissue culture facilities. The application of cryopreservation to tumor samples prior to cytogenetic analysis allows collection of tumors to a desired sample size. We evaluated methods of cryopreservation for their effects on growth potential from 11 benign thyroids and one papillary thyroid cancer. Mitotic indices and thyroglobulin expression applying imunocytology were analyzed. Compared to fresh tumors, the revived tumor samples showed unaltered thyroglobulin expression. A statistically significant (p < 0.004) prolongation to develop mitotic activity occurred in samples received after the freezing of dispase digested tissues, but not in samples frozen as thinly cut pieces. In addition, the data show that cytogenetic analysis at the 400-band level can be achieved in cryopreserved thyroid tissues.     

Transmission modes of hepatitis C virus

SITUATION IN FRANCE: The prevalence of hepatitis C virus (HCV) infection in the French population is estimated at 1%, a level similar to that in other western countries. USUAL CONTAMINATION ROUTES: Epidemiological studies, together with gene typing, have made it possible to distinguish transmission modes. A history of intravenous drug abuse or transfusion is found in 60 to 80% of all subjects infected by the HCV. Other documented modes of contamination include hemodialysis, organ transplantation, accidental occupational-related puncture and mother-infant transmission. OTHER ROUTES: Sexual or intra-familial nonsexual transmission is uncommon and related to the length of exposure and the stage of HCV infection in the "source" subjects. Cases of HCV transmission have been reported during medical procedures. Currently the mode of of transmission is unknown in 20 to 40% of the cases.     

Assisted reproduction and hepatitis C virus infection

Medically assisted procreation poses a difficult problem when one or both partners of couple are infected with HCV. Epidemiologic and fundamental works show a low risk of HCV sexual transmission and no pregnancy complications or fetal abnormalities have been reported. However, the outcome of HCV infected children is unknown. These contrasting findings suggest that medically assisted procreation using sperm of spouse needs to be cautious. Before medically assisted procreation, testing of couples for HCV antibodies must be done and interferon therapy is required for patients with histological chronic active hepatitis and for HCV positive mothers or infected couples. In the absence of specific legislation or consensual recommendations, detailed informations must be given to the couples in order to obtain an informed consent.