Determination of HCV Genotype Using Two Antibody Assays and Genome Typing

Two serotyping assays for hepatitis C virus (Serotyping 1–6 assay; Murex, UK and RIBA Serotyping SI; Chiron, USA) were compared to a standardized genotyping assay (Inno-LiPA HCV II; Innogenetics, Belgium) using serum samples collected from 126 patients chronically infected with hepatitis C virus. Serotyping was positive in 87% and 80% of the samples tested with Murex and Chiron, respectively, and concordant with genotyping in 93% and 88%, respectively. Sequence analysis of the NS5b region of 15 samples with discrepant typing results confirmed the Inno-LiPA finding in all instances. The Murex enzyme immunoassay serotyping method was less sensitive for identifying genotype 2 (P<0.05). The concordance with genotyping of the RIBA serotyping method was lower for genotype 2 than for the other genotypes (P<0.05). Electronic Publication     

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.     

Evaluation of a prototype HCV NS5b assay for typing strains of hepatitis C virus isolated from Tunisian haemodialysis patients

Hepatitis C virus (HCV) strains isolated from 68 haemodialysis Tunisian patients exhibiting chronic infection were genotyped targeting the NS5b region of the HCV genome using a prototype assay developed by Bayer HealthCare-Diagnostics (TRUGENE NS5b HCV). The overall results were compared to those obtained with another assay of the same company based on sequencing of the 5' non-coding region (TRUGENE HCV 5'NC genotyping kit). All strains could be typed by the 5'NC typing kit, but only 62 (91; 2%) by the NS5b prototype assay. All the 62 strains typed by both methods exhibited the same pattern at the type level: 57 were type 1, 3 were type 2, and 2 were type 4. At the subtype level, eight strains that gave undetermined results by the 5'NC kit were successfully typed by the NS5b kit; eight additional strains exhibited discrepant results. The overall agreement between the two assays was 74.2% at the subtype level. In conclusion, the NS5b region appears to be much more accurate than the 5'NC region to subtype HCV strains, especially in those isolated from patients attending haemodialysis centres where the subtype distribution suggests frequent nosocomial transmissions.     

Comparison of different methods of hepatitis C virus genotyping

In order to introduce the approach of HCV genotyping in our laboratory, a comparative study of 3 molecular and 1 serological methods, was conducted on 62 HCV RNA positive sera. The molecular genotyping methods target the 5'untranslated (UTR) region of the virus genome and are based on an amplification of the viral genome, followed by partial sequencing, analyses of restriction fragment length polymorphisms (RFLP) or molecular hybridation (Inno LiPA, Innogenetics). The serological method or serotyping is based on the detection of antibodies to genotype specific epitopes derivated from the Non Structural (NS) 4 region of the viral genome (HCV 1-6 Serotyping Assay, Murex Biotech). "In house" methods, sequencing and RFLP, identified the genotype for 13 samples classified as non-typables by commercial kits Inno LiPA test and HCV 1-6 Serotyping Assay. Mixed infections revealed, especially by Inno LiPA, could not be identified by partial sequencing, which seems to detect only predominant genotype. For 4 samples, genotyping results of the methods targeting the 5'UTR were discordant with those of the serotyping of the NS4 region. Commercial kits are efficient to determine HCV genotypes, particularly in the context of antiviral therapy and patient's follow-up, sequencing remains the best alternative for more complete characterisation of viral strains and for epidemiological investigations.     

丙型肝炎病毒血清学分型应用的可行性研究

目的 探讨丙型肝炎病毒血清学分型的可行性。方法 应用EIA法对兰州地区148例抗－HCV阳性者进行血清学分型。结果 血清学方法的分型率为56．08％（83／148）,其中血清1型占50．60％（42／83）,血清2型占43．47％（36／83）,1＋2混合型占6．03％（5／83）。对其中70例HCVRNA阳性血清的基因型与血清型结果进行比较,二者完全相符。结论 丙型肝炎病毒的血清学分型具有一定的临床应用价值。     

Large-scale analysis of hepatitis C virus serological typing assay: Effectiveness and limits

The HCV (hepatitis C virus) Serotyping 1–6 Assay™ (Murex Laboratories) was evaluated on 303 French HCV-infected patients. Serological typing results were compared to the genotypes obtained from sequence analyses of the 5′ noncoding regions of the virus genome from 46 HCV-infected patients, and assay specificity was found to be high (97.6%). The serological typing assay, run in 257 consecutive HCV-infected patients, yielded an assay sensitivity lower (70.6%) than that previously reported. This finding was attributed mainly to nonreactive sera from human immunodeficiency virus (HIV)-positive patients (P < 0.001) and perhaps reflected cryoglobulin positivity in others. No anti-type 6 reactivity was detected, and the overall serological type distribution values for types 1 to 5 were 67.3, 7.9, 16.4, 6.6, and 0.9%, respectively. A higher prevalence of type 4 was noted among HIV-infected patients (P < 0.001). In addition, serotype 2 was significantly more frequent in cryoglobulinemia positive than in cryoglobulinemia-negative patients (P < 0.05). Although an initial high level (7%) of mixed serological typing reactivities was found, after predilution of serum only two mixed infections could be confirmed (0.9%). It is suggested, therefore, that mixed reactivities have to be interpreted carefully and retested with prediluted serum, particularly when the optical density of the reactivity is >2.5 or remains >0.4 after competition with all type-specific peptides. The high specificity and relatively good sensitivity even in immunocompromised patients obtained with this assay indicate that it can be used routinely. Because response to treatment is linked to HCV type, this assay could be used to identify HCV serotype to guide therapeutic decisions. J. Med. Virol. 55:18–23, 1998. © 1998 Wiley-Liss, Inc.     

Subtyping of human immunodeficiency virus type 1 strains by using antibodies specific for the third variable domain (V3) of gp120: results may be affected by divergent V3 sequences.

Human immunodeficiency virus type 1 serotype C was found in 545 of 712 Ethiopian patients by peptide enzyme immunoassay. Serotyping failed in 146 samples due to the absence of V3 antibodies or multiple reactivities. In 6 of 34 samples, discordant results were obtained by serotyping and genotyping, possibly due to divergent V3 sequences.     

Usefulness of simultaneous screening for HIV‐specific and HCV‐specific antibodies and HBsAg by a capillary‐based multiplex rapid diagnostic test to strengthen linkage‐to‐care in sub‐Saharan patients attending sexually transmitted infection clinic

Adult outpatients attending the main sexually transmitted infection clinic of Bangui, Central African Republic, were prospectively subjected to a multiplex rapid diagnostic test for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). In group I (n = 208) of patients already followed for HIV, 6 (2.9%) were unexpectedly negative, thus corresponding to false positive for HIV by the national HIV algorithm; hepatitis B surface antigen and HCV positivities were high (18.7% and 4.3%, respectively). In group II (n = 71) of patients with unknown HIV status, at least 1 chronic viral disease was diagnosed in 26 (36.6%) patients, including 5 (7.1%) HIV, 17 (23.9%) HBV, and 3 (4.2%) HCV infections.     

Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: Full‐length sequences of two new subtype 6w strains and a recombinant form_2b6w

Human immunodeficiency virus type 1 (HIV‐1) circulating recombinant form (CRF) 07_BC strain has caused serious outbreaks among injection drug users in Taiwan since 2004. The objective of this study was to conduct a molecular epidemiological study of HCV genotypes in intravenous drug users in Taiwan. Blood samples and questionnaires from 591 intravenous drug users infected with HIV‐1 were collected nationwide. In total, 180 samples were selected for HCV genotyping using multiplex PCR and phylogenetic analysis of the core, E1 and NS5B regions. The Inno‐Lipa assay was used to confirm multiple infections with different genotypes. Eighty percent had a single infection with subtype 1b being the most common subtype (24%), 12% had double infections and two had triple infections. In addition, three recombinant forms (RFs)‐2a1a, 3a1b, and 2b6w were identified. Phylogenetic analyses showed that the 3a, 6a, and 6n strains were clustered with strains present in Thailand and mainland China. Full‐length sequence analysis showed that two 6w strains shared 89.4–90.2% sequence homology with the 6(r) strain from the Guangdong Province, China. Bootscan analysis revealed that the recombination breakpoint of RF_2b6w was located at the NS2‐NS3 junction. In summary, the distribution of HCV genotypes among Taiwanese intravenous drug users was complex and more than 12% of the drug users were infected with more than one genotype of HCV. J. Med. Virol. 82:57–68, 2010. © 2009 Wiley‐Liss, Inc.     

Comparison of two hepatitis C virus typing assays in a Tunisian population

In patients with hepatitis C, it is necessary to determine the genotype of the hepatitis C virus (HCV) to tailor treatment schedules. HCV-positive sera from 60 chronically infected patients were analyzed by two methods: serotyping and genotyping, to evaluate the suitability of serotyping method for routine determination of HCV genotype; 47 men and 13 women were included in this study, nine were renal transplant patients and five were hemodialysis patients. Anti-HCV antibodies were detected by Elisa (HCV Ab III, Innogenetics SA) and confirmed by immunoblot assay (INNO-LIA-HCV Ab III update, Innogenetics SA). Genotyping analysis was performed by a line probe assay (Inno-LiPA-HCV, Innogenetics SA) and serotypes were determined by an Elisa-based serotyping assay (Murex HCV serotyping 1-6 HCO2, Murex SA) which detect type specific antibodies against NS4-derived epitopes. Among 60 patients positive for anti-HCV antibodies and confirmed by immunoblot assay only 90% show a strong reactivity with NS4. We found the following genotype distribution : 1b (73.3%), 4 (10%), 1a (5%), 3a (5%), 4c/4d (5%) and 1 not classified (1.7%). The most prevalent serotype was type 1 (60%) followed by serotype 6 (20%), 4 (8.3%), 2 (1.7%) and 10% were no type specific antibodies. The sensitivity of the serotyping and the genotyping assays was 90% with a total concordance of 68.3%. Thirteen samples revealed discrepant results with genotype : 1b (4), 1a (3), 3a (3), 4 (2) and 4c/4d (1). This study indicates that the serotyping assay is less specific than genotyping. However, the test is rapid, relatively easy to perform and represent a reliable alternative in laboratories that lack the specific expertise to typing the HCV by molecular methods.     

Natural history of compensated viral cirrhosis in a cohort of patients with HIV infection

BACKGROUND: The natural history of initially compensated cirrhosis in patients with HIV and concurrent hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection is poorly defined. This study was designed to investigate the incidence and type of liver-related complications and mortality in coinfected cirrhotic patients. METHODS: We retrospectively identified a cohort of patients coinfected with HIV and HCV or HBV and initially compensated viral cirrhosis. Time to decompensation and mortality from liver-related causes were recorded. RESULTS: Between 1999 and 2004, 392 HIV-infected patients underwent a follow-up of > or =6 months. Sixty-nine patients (17.6%) with initially compensated cirrhosis were identified (7 HBV positive, 59 HCV positive, and 3 positive for both HBV and HCV). The most frequent complication was ascites. The mortality was 71.3 per 1000 person-years (95% confidence interval [CI], 47 to 108) in HIV-infected patients with HBV and/or HCV compensated cirrhosis, 8 (95% CI, 4 to 16) in HIV/HCV-coinfected patients without cirrhosis, and 6.5 (95% CI, 2.7 to 15.5) in HIV-monoinfected patients. After the first event of decompensation, the survival rate was 48% at 1 year and 18.1% at 3 years. Treatment with HAART after the first event of decompensation was associated with an increased survival rate (61.1% and 26.2% at 1 and 3 years, respectively, vs. 26.7% and 0%; P < 0.0001). CONCLUSIONS: These results indicate significant morbidity and mortality during the 6 years after the diagnosis of compensated cirrhosis due to HBV and/or HCV in HIV-infected patients, identifying ascites as the most frequent complication.     

慢性丙型肝炎患者HCV血清学分型研究

目的探讨HCV基因分型与血清学分型的关系。方法对来自14家医院的104例已知HCV基因型的慢性丙型肝炎患者血清，经ELISA方法，用Murex HCV Serotyping 1-6 Assay血清分型试剂进行HCV的血清学分型。结果104例血清中的86（82，69％）例可分出血清型，检出血清型病毒株91株，病毒株检出率为78．4％。血清型与基因型的总符合率为62，1％，血清型1型、2型和3型的符合率分别为69，4％、51，2％和70．0％，以2b基因型的符合率和漏检率最低（54．5％）。结论HCV血清型特异性受病毒基因型的影响，与基因型存在一定的差异。     

Impact of human immunodeficiency virus infection on the histological features of chronic hepatitis C: a case-control study. The MULTIVIRC group

Hepatitis C virus (HCV) is frequently encountered in human immunodeficiency virus (HIV)-infected patients because of common routes of transmission. Previous studies suggested that HIV infection impaired the natural course of chronic hepatitis C, with a more rapid progression to cirrhosis. However, these studies did not assess the HIV infection impact on chronic hepatitis C by taking into account the risk factors for liver fibrosis progression: alcohol, sex, age at the contamination, and duration of HCV infection. We studied liver biopsy specimens of 2 groups of 58 patients that were infected by both HCV and HIV or by HCV alone. The 2 groups were matched according those risk factors, and liver biopsy responses were evaluated with the METAVIR items. The METAVIR activity was higher in HIV-positive than HIV-negative patients. Cirrhosis was more frequent: (1) in HIV-positive patients with CD4 < or = 200 cells/microL (45%) than in HIV-negative patients (10%) (P = .003), (2) in HIV-positive patients with CD4 < or = 200 cells/microL (45%) than in HIV-positive patients with CD4 > 200 cells/microL (17%) (P = .04). These differences, which were linked to HIV status, might be related to the enhanced HCV replication during HIV infection or other immune mechanisms that need further studies.     

The unique HCV genotype distribution and the discovery of a novel subtype 6u among IDUs co-infected with HIV-1 in Yunnan, China

The Yunnan province is the epicenter of HIV-1 epidemics in China and a center for drug trafficking to the other parts of the world. In six prefectures of this province, a total of 132 IDUs were recruited to determine the sero-prevalence of HCV and HIV-1 and the positive rates were 93.94% and 68.18%, respectively (P<0.001). Co-infection with HCV and HIV-1 was found among 89 IDUs, of whom several HCV fragments were amplified and sequenced. Sequences of the HCV 5'NCR-C and NS5B region were determined from 82 IDUs. Phylogenetic analyses showed consistent genotyping among 80 IDUs. Among them HCV genotypes 1a, 1b, 3a, 3b, 6a, 6n, and a tentatively assigned novel 6u subtype were found in 1 (1.25%), 16 (20%), 19 (23.75%), 24 (30%), 4 (5%), 9 (11.25%) and 7 (8.75%) individuals, respectively. In two IDUs, genotyping results were discordant, suggesting mixed HCV infections or recombination. The proportion of patients with HCV 1b tended to decrease from the north to south and from the east to west in this province. Genotype 3 and 6 strains were more frequent in the southern prefectures. The novel subtype 6u strains were only detected in Dehong which borders Myanmar. Our findings showed a unique pattern of HCV genotype distribution, which is similar to that in the southeastern Asian countries but distinct from that among the general population in China. Routes of drug trafficking and the resulting high prevalence of HIV-1 infection may have contributed to this pattern of HCV genotype distribution.     

Development of an improved genotyping assay for the detection of hepatitis C virus genotypes and subtypes in Pakistan

A new genotyping system was established for the specific detection of HCV genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a–h, 5a and 6a during the course of this study. The system is based on entire core region and a part of 5′ noncoding region (5′NCR) with genotype-specific primers. Genotype-specific primers were designed on the basis of 114 HCV isolates. Serum samples with known genotypes were used as positive controls to validate the assay developed and to generate PCR band patterns. Band patterns generated from the clinical serum samples from HCV patients were compared to the patterns produced from these control samples. In addition, the type-specific bands were sequenced from the test patients and control clinical samples to validate further the test results. To determine sensitivity and specificity of the assay, a total 260 samples were analyzed simultaneously by this HCV genotyping method and that developed by Ohno and Murex HCV Serotyping 1–6 Assay. The system showed 79.2% concordance with Ohno's system and 65.38% with serotyping system. Samples with discordant results were sequenced and their genotypes were determined by molecular evolutionary analysis. The data indicate that the method described in this study may offer better sensitivity and specificity for the detection directly of HCV genotypes present at low levels in HCV patient samples.     

Analysis of the 5' noncoding region versus the NS5b region in genotyping hepatitis C virus isolates from blood donors in France

The 5' noncoding region (5' NCR) of the hepatitis C virus (HCV) has become the standard for genotyping even though several reports show that its use can result in classification errors. The purpose of this study was to perform genotyping based on sequence analysis of the NS5b region in a set of 357 HCV strains isolated from blood donors in France in 2002 and 2003. Results were compared with those previously obtained using 5' NCR analysis, and HCV subtype distribution was reevaluated. Twenty-six of 120 strains (approximately 22%) initially identified as genotype 1b by 5' NCR region sequence analysis were reclassified as genotype 1a by NS5b region sequence analysis. Similarly, 14 of 23 strains (approximately 61%) initially identified as 2a/2c were reclassified as non-2a and non-2c subtypes, and 12 of 22 strains (approximately 45%) initially identified as 4c/4d subtypes were reclassified as non-4c and non-4d subtypes. Sequence analysis of the NS5b region also revealed 5 putative new subtype 2 variants and 2 putative new subtype 4 variants. Although these findings demonstrated full agreement between 5' NCR and NS5b sequence analysis with regard to type classification, genotyping based on phylogenetic analysis of the NS5b region is more accurate for subtype determination than genotyping based on analysis of the 5' NCR. Sequence analysis of the NS5b region is mandatory for epidemiologic studies.     

PREVALENCE OF HEPATITIS C VIRUS (HCV) COINFECTION IN HIV INFECTED INDIVIDUALS IN SOUTH INDIA AND CHARACTERIZATION OF HCV GENOTYPES

Purpose: To determine anti-HCV antibodies and genomic subtype of HCV in 1487 confirmed human immunodeficiency virus (HIV) positive samples. Methods: A total of 1487 confirmed HIV-positive samples were tested for anti-HCV antibodies by using a third generation ELISA kit (Ortho 3.0) and by RT PCR for HCV. HIV and HCV coinfected samples were selected for HCV genotyping by RFLP and subtyping with NS5-type specific primers. Results: A total of 1487 HIV-infected serum samples were screened for HCV infection, of which, a 1443 (97.04%) were negative and 45 (3.02%) were coinfected. HIV–HCV coinfection was predominant in the age group 41–50 years (51.1%). HCV genotyping and subtyping was done for the 45 HCV RNA-positive specimens of which genotype 1 was observed in 31 (68.8%) and genotype 3 was observed in 14 (31.1%) subjects. Further subtyping analysis showed the genotype 1b in 23 (51.1%), 1a in eight (17.7%), 3a in 10 (22.2%) and 3b in four (8.8%) subjects. Conclusion: HIV and HCV seroprevalence is higher in South India, and the most prevalent genotype in coinfection was genotype 1b.     

Genomic characterization of hepatitis C virus isolates from Argentina

Thirty-three Argentinian patients infected with hepatitis C virus (HCV) were studied for viral genotyping. The patients included 10 hemophiliac and 4 polytransfused children and 19 adults: 3 polytransfused, 7 dialyzed and 9 sporadic cases. Core-based genotyping permitted the classification of 31 samples. Genotypes II, I and V were the most frequent: 21 (63.6%), 16 (48.4%) and 10 (30.3%) of the 33 patients, respectively. Only one polytransfused patient carried genotype IV. Genotype II was detected in 7 out of 9 sporadic cases. Thirteen patients (39.3%) were coinfected with two genotypes, and 2 others were coinfected with three genotypes. The remaining 2 samples which could not be typed were characterized following the restriction fragment length polymorphism (RFLP) method, and were classified as type 1. One of these had two consecutive transitional mutations in the 5′ untranslated region (5′ UTR). © 1995 Wiley-Liss, Inc.