Hepatitis C virus serotype-specific core and NS4 antibodies in injecting drug users participating in the Amsterdam cohort studies

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75. 5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5' untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.     

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.     

Seroepidemiologic study of HCV genotypes in the district of Messina and Catania

Aim of the study was to identify the different hepatitis C virus (HCV) genotypes in chronic hepatitis patients living in the districts of Messina and Catania (Sicily, Italy), for epidemiological reasons but also to determine the effect of interferon therapy on therapeutic response of different viral genotypes. Genotyping was carried out by reverse-hybridization after amplification by polymerase chain reaction. The study was conducted on 271 patients with HCV-RNA positive chronic liver disease belonging to categories at risk or not for infection. HCV genotype sub-type 1b was the most common viral genotype identified.     

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.     

Hepatitis C virus infection in mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma: evidence for a pathogenetic role

We investigated the pathogenetic relevance of hepatitis C virus (HCV) infection in mixed cryoglobulinemia (MC) with or without complicating B-cell Non-Hodgkin's lymphoma (NHL) in comparison with other immunological and lymphoproliferative disorders. The following groups of patients were studied: A) 25 patients with MC in 7 cases evolved into B-cell NHL; B) 25 healthy subjects; C) 22 patients with different systemic immune diseases; D) 24 patients with chronic HCV infection without MC; E) 25 patients with B-cell idiopathic NHL. Methods used included: i) Polymerase chain reaction (PCR) for HCV RNA detection in serum and peripheral blood mononuclear cells (PBMC) (uncultured or mitogen-stimulated); ii) Branched DNA (b-DNA) for HCV RNA quantification; iii) HCV genotyping by genotype-specific primers localized in the core region and by hybridization of amplification products of the 5' untranslated region (5'UTR), obtained with universal primers, using genotype-specific probes. Serum anti-HCV and HCV RNA were detected in 88% and 73% of MC patients, respectively, and in a significantly lower percentage of healthy controls and patients with autoimmune diseases. HCV RNA concentration was significantly lower in supernatants than in corresponding whole sera (p < 0.001). Plus-strand HCV RNA was detected in 81% of peripheral blood mononuclear cell (PBMC) samples and minus-strand in the majority of fresh or mitogen stimulated cells. All MC patients with NHL had HCV RNA sequences in PBMC. HCV genotype 2a/III was detected in MC patients with a prevalence that was significantly higher than in HCV infected patients without MC. Surprisingly, HCV markers (anti-HCV and/or HCV RNA) were found in 32% of patients with idiopathic NHL. These data suggest that HCV infection is involved in the pathogenesis of MC through both direct participation in the immune complex related vasculitis and by triggering the lymphoproliferative disorder underlying the disease. This latter disorder seems to be related to HCV lymphotropism which could also be responsible for the evolution of MC to malignant lymphoma. This study also suggests that HCV infection may be involved in the pathogenesis of idiopathic B-cell NHL through a similar pathogenetic mechanism.     

Evaluation and comparison of different hepatitis C virus genotyping and serotyping assays

BACKGROUND/AIMS: Evidence that the geno/subtype of hepatitis C virus (HCV) is predictive of the response to interferon-alpha therapy suggests that typing methods are clinically useful. In the present study, HCV isolates obtained from 74 patients with chronic hepatitis C were used to evaluate three genotyping and two serotyping assays. METHODS: The reverse hybridization assay and the DNA immunoassay are based on immobilized type-specific probes for the 5'-noncoding and the core region, respectively. A third genotyping assay utilized type-specific primers for amplification of the core region. Serotyping assays detect type-specific antibodies of the nonstructural-4 region (enzyme immunoassay) or of the core and nonstructural-4 region (recombinant immunoblot assay). Gold standard geno/subtyping of HCV isolates was performed by sequence and phylogenetic analysis of the nonstructural-5B region. RESULTS: All genotyping systems amplified the respective target region of the HCV genome with high sensitivity. The reverse hybridization assay and the DNA immunoassay correctly identified HCV-1, -2, and -3. The DNA immunoassay misinterpreted all HCV-4 isolates as HCV-4 and -5 coinfection. In the type-specific amplification assay, coinfections of subtypes HCV-1a and HCV-3a with HCV-1b could not be excluded. The reverse hybridization assay misinterpreted 1/14 HCV-1a isolates as HCV-1h, and vice versa 3/36 HCV-1b isolates as HCV-1a. Furthermore, differentiation between HCV-2a and -2c was not possible using this assay. The DNA immunoassay correctly identified all HCV subtypes. The serotyping assays, recombinant immunoblot assay and enzyme immunoassay identified HCV-1, -2, and -3 in 93% and 89% of cases, respectively. HCV-4, however, could only be recognized by the enzyme immunoassay. CONCLUSIONS: The reverse hybridization assay and the DNA immunoassay specifically identified HCV genotypes 1, 2, and 3, while crossreactivity occurred in the primer-specific amplification assay. The DNA immunoassay achieved the best performance in HCV subtyping. Both serotyping systems correctly identified HCV-1, -2, and -3 in about 90% of cases, but lack the possibility of subtyping.     

Detection and clinical features of hepatitis C virus type 6 infections in blood donors from Hong Kong

The genotype distribution of hepatitis C virus (HCV) was investigated in 212 viraemic blood donors from Hong Kong. A subset of the samples was investigated using three different genotyping assays to establish the accuracy of each in this population. These assays were restriction fragment length polymorphism (RFLP) of amplified 5' noncoding region (5'NCR) sequences, RFLP of the core region, and a serotyping assay using peptides from two antigenic regions of NS4. Genotypes detected in Hong Kong blood donors were 1a (6.2%), 1b (58.8%), 2a (1.4%), 2b (1.4%), 3a (1.9%), and 6a (27.0%). All genotyping assays produced concordant results. No evidence was obtained for the presence of type 6 group variants recently identified in Southeast Asia, other than type 6a. A serotyping assay based upon the detection of type-specific antibody to epitopes in NS4 produced similar results to the genotyping assays (98% concordance), but a reduced sensitivity (75%) compared with genotyping methods. Sequence variation in NS4 was not the cause of the reduced rate of detection of type 6 antibody in this population. Eighty-four percent donors infected with type 6a were male, compared to 75% donors infected with type 1b. The median alanine transaminase (ALT) level in type 6 infected donors was lower than in type 1b, (43.8 and 51.1 U/l, respectively) although these values were not statistically significant (P = 0.094). There was no significant difference between the ages of donors infected with types 1b and 6a. Risk factors for HCV infection in the blood donors included blood transfusion, intravenous drug abuse, and tattooing. A significantly greater number of donors infected with HCV-6a reported a history of drug abuse (66%) than donors infected with HCV-1b (7%).     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

This study comprised 100 persons with antibodies to hepatitis C virus (HCV), including 77 intravenous drug users (IVDUs). They were tested with serological HCV typing assays (Murex HCV serotyping 1-6 assay; Chiron RIBA HCV Serotyping SIA). Patients with a positive polymerase chain reaction (PCR) for HCV (n = 66) were tested with genotyping molecular assays (Inno-Lipa HCV II test; Sorin GEN-ETI-K HCV typing assay). Comparison of the results of these tests showed that (a) 92% of samples could be typed by one test at least; 44% could be typed by all four tests; 88% could be typed by one serological test at least and 66% by one molecular test at least; (b) 81% of the samples successfully tested with both serological tests gave comparable results; 95% of the samples successfully tested with both molecular tests gave comparable results; (c) when serological and molecular tests yielded different results, sequences in the 5'-non-coding (5' NC) or E1 regions always confirmed the results of the molecular tests; (d) in case of discrepancy between the results of the molecular tests the E1 region sequences confirmed the Sorin test results. It is concluded that the molecular tests compared gave similar results. The fact that the Murex serological test gave comparable results in more than 80% of cases indicates that it is an alternative to the molecular tests for routine diagnosis. However, comparison of the results of this test with those obtained in patients consulting a hepatology department showed that it gave the best results in a population of patients not exposed repeatedly to HCV.     

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%).     

Hepatitis C virus genotype in anti-HCV-positive hemodialysed patients

We investigated the influence of hepatitis C virus (HCV) genotypes on the clinical course of HCV infection in a haemodialysis population. In June 1991, a 4 year prospective follow-up programme was implemented in 184 consecutive haemodialysis patients. Alanine aminotransferase (ALT) and gamma glutamine transferase (GGT) were performed every 2 months. When HCV antibody (Ab) (by second-generation ELISA) was positive, it was confirmed by RIBA 2 and HCV RNA amplification by PCR. The pattern of nucleotide sequence variability in the 5' non-coding region was categorized according to Simmonds' genotype classification. Risk factors including blood transfusions were evaluated. The levels of hepatic enzymes in HCV Ab-positive patients were retrospectively studied over a mean period of 11.8 years. ALT and GGT levels were assigned a score for every year of infection (0 = normal, 1 = fluctuating 2 = high levels). Fifty-two patients were HCV Ab reactive (30.4%), eight were RIBA undetermined and 44 were RIBA positive; 40 of these were HCV RNA positive (91%). Twelve patients were HCV RNA negative, suggesting that they had recovered from the infection. Four genotypes were identified: 1b [26 patients (65%)], 1a (one patient), 2 [12 patients (30%)] and 3 (one patient). The genotype distribution was not different from that found in patients with chronic hepatitis C and normal renal function of the same geographical area. Genotype 1b accounted for 75% of the cases before 1985 and an equal prevalence of the two major genotypes was observed after 1985. Patients infected with HCV subtype 1 had normal mean ALT levels, but higher levels in the follow-up period (28 +/- 15.6 IU/l) and higher ALT and GGT personal scores in the retrospective study. Genotype 1 patients had higher mean ALT levels after 6 months. HCV RNA-negative patients had lower ALT levels after 24 months. RIBA pattern could differentiate the patients. Patients with genotype 1 received a higher number of transfusions, while only 50% of HCV RNA-negative patients had been transfused. Our data suggest a worse course of HCV infection in haemodialysis patients infected with HCV subtype 1, but the severity of HCV infection can only be assessed by histology. Transaminases are only loosely correlated with severity.     

Introduction to the special section on attachment and psychopathology: 2. Overview of the field of attachment

An improved system for amplification of hepatitis C virus genome (HCV) was developed based on a multiplex nested polymerase chain reaction format. Two sets of oligonucleotide primers were used simultaneously. One was derived from the conserved sequences in the 5' non-coding region of the viral genome which can bind to the viral genome of all genotypes. The other set of primers was designed from a sequence in the nonstructural-5 region of HCV. HCV genotypes 1 and 3 can be differentiated by the banding patterns of amplified DNA products. All of 39 samples containing the HCV genotype 1 could be amplified with primers in the 5' non-coding region only, whereas 92% of those with genotype 3 could be amplified by both primer sets. In addition, HCV RNA can be detected in 81% of 84 anti-HCV-positive blood donors and in 0% of 34 anti-HCV-negative cases. Of the HCV RNA-positive specimens, 69% showed genotype 1-like patterns while 31% showed genotype 3-like patterns. The detection rate of HCV RNA in this study was much higher than that in our previous report due to the improvement of new primers which can detect all genotypes of the virus. In conclusion, this improved amplification system is a sensitive method for rapid identification of HCV RNA in clinical specimens that can simultaneously differentiate the two most common genotypes of HCV found in Thailand.     

Long-term effect of botulinum toxin on impairment and functional health in cervical dystonia

Two methods for genotyping hepatitis C virus (DNA enzyme immunoassay [DEIA] and line probe assay [Inno-LiPA HCV I and II]) were compared on 120 samples and of these 87% were assigned to the same subtype by both assays. There were 15 subtyping discrepancies which involved 5% of type 1 isolates and 90% of type 2 isolates. Amplified products from the core and 5' untranslated regions (UTR) were sequenced to resolve conflicts. Type 1 discordant samples had a guanosine at position -99 in the 5' UTR, a characteristic of genotype 1b, and a core region typical of subtype 1a. The eight isolates classified as 2a/2c by LiPA and as subtype 2c by DEIA belonged to type 2.     

HIV type 1 subtypes in Malaysia, determined with serologic assays: 1992-1996

We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.     

Rapamycin: distribution, pharmacokinetics and therapeutic range investigations: an update

The prevalence of hepatitis C virus (HCV) infection increases with advancing age, but the disease has been poorly studied in the elderly. A population-based study was therefore carried out to investigate the prevalence of HCV infection and the severity of HCV-related chronic liver disease in the elderly. One thousand and sixty-three people (> or = 60 years of age) were screened for antibodies to HCV (anti-HCV) and for possible abnormalities of common liver function tests. Positive subjects and sex and age-matched anti-HCV-negative controls were recalled 12 months later for measurements of liver enzymes, confirmatory testing of anti-HCV, HCV RNA analysis and HCV genotyping. All subjects answered a specific questionnaire concerning medical history and possible risk factors. Forty-four subjects were positive for anit-HCV, the prevalence being 4.1%. Thirty-five positive subjects and 35 controls were investigated further. Risk factors for acquiring HCV were found to be: blood transfusion, surgical intervention and the use of non-disposable syringes. Abnormal alanine aminotransferase levels were found in 13 patients (37.1%). HCV RNA genotyping showed type 1b in three (15.8%), type 2a in 13 (68.4%) and not classified in three (15.8%) patients. There was no relationship between abnormalities of serum aminotransferase, the rate of HCV RNA positivity and HCV genotypes. Ultrasound abnormalities were present in 13 (37.1%) patients. In this elderly population the relatively high prevalence of HCV infection was thought to be caused by previous parenteral exposure. The low incidence of liver disease could be related to the prevalence of HCV genotype 2a in the majority of these patients, and hints at the possibility of an HCV carrier state in elderly individuals.