Serotyping and genotyping of hepatitis C virus in Taiwanese patients with type C chronic liver disease and uraemic patients on maintenance haemodialysis

BACKGROUND: To evaluate a recombinant immunoblot hepatitis C virus (HCV) serotyping assay, which determines HCV serotypes 1, 2, and 3 by detecting type-specific antibodies to core-and NS-4-derived peptides. METHODS: Immunoreactivity of type-specific antibodies among 173 chronic hepatitis C patients and 43 haemodialysis patients in Taiwan was examined and the serotyping results were compared with genotyping by Okamoto's method. Serial specimens from 29 patients undergoing interferon-alpha therapy were also evaluated. RESULTS: Of the 205 specimens for which genotyping data were available, 51.2% were of serotype 1, 31.7% of serotype 2, 1.0% of serotype 3, 2.4% of either serotype 1 or 3, and the remaining 13.7% were untypable. The serotypable rate was significantly lower in haemodialysis patients than in chronic hepatitis C patients (70.0% vs 94.9%; P < 0.001). Serotyping of genotype 2b specimens was significantly more dependent on core peptide bands than other genotypes. Using genotyping as the reference, the overall sensitivity, specificity and concordance of the recombinant immunoblot HCV serotyping assay were 86.3%, 97.2% and 83.9%, respectively. However, the serotyping assay had significantly lower sensitivity (69.2%), specificity (77.8%) and concordance (53.8%) for genotype 2b specimens. Of nine HCV complete responders, one lost type-specific antibodies 6 months after the cessation of interferon-alpha treatment. CONCLUSIONS: These results suggest that, except for less than optimal performance with immunocompromised or genotype 2b patients, the HCV serotyping assay is a practical and useful method for HCV typing in the clinical setting in Taiwan.     

New combination test for hepatitis C virus genotype and viral load determination using Amplicor GT HCV MONITOR test v2.0

AIM: To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HOV MONITOR test v2.0 (microwell version). METHODS: The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes. RESULTS: The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test. CONCLUSION: Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.     

山东地区丙型肝炎病毒的基因型及血清学分型的研究

探索山东地区丙型肝炎病毒(HCV)的基因型及血清学分型的分布,了解HCV基因型与感染途径的关系.对96例抗HCV阳性患者的血清进行HCV RNA检测,HCV RNA阳性者,应用限制性片段长度多态性分析(RELP)进行基因分型;同时应用Murex Serotyping HCV 1-6血清学分型试剂进行血清学分型.基因非2(1b)型79例,占83.16%,2(2a)型为16例占16.84%,44份血清标本的血清学分型可分型率为90.91%,与基因分型的符合率为90.00%.不同的感染途径之间,基因型分布没有差异(P=0.15).山东地区丙型肝炎病毒流行株为基因非2(1b)和2(2a)型,非2(1b)型为优势株,基因分型与血清学分型结果基本一致,基因型与丙型肝炎的感染途径无关.     

Serotyping of hepatitis C virus in chronic type C hepatitis in Taiwan: Correlation with genotypes

To evaluate the usefulness of a new serologic assay to group hepatitis C virus (HCV), genotypes identified by this serotyping method were compared to those identified by a polymerase chain reaction (PCR) assay with type-specific primers in 71 Taiwanese patients with chronic type C hepatitis. The group-specific antibodies against different HCV genotypes were detected by using an enzyme-linked immunosorbent assay (ELISA) based on group-specific recombinant peptides (C14-1 and C14-2) within the NS4 region. Among 71 patients positive for current second-generation HCV antibodies, HCV RNA was detected in 55 patients by PCR with primers from the 5′ untranslating region, and in 52 by genotype-specific PCR. In 49 (89%) of 55 viremic patients, the results of serotyping by ELISA showed complete agreement with those determined by PCR genotyping, and none of the patients showed a group opposite to that of HCV genotype. The positive rate of group-specific antibodies (69/71;97%) was even better than that of the PCR (55/71;78%). We conclude that this new serotyping assay is highly sensitive and specific for the determination of HCV genotypes, and will be useful in future epidemiologic studies, as well for clinical application.     

Hepatitis C virus genotypes in France: relationship with epidemiology, pathogenicity and response to interferon therapy. The GEMHEP

The aim of this study was to investigate the following in a large population of French patients with chronic hepatitis C: the geographical distribution of hepatitis C virus (HCV) genotypes; the relationship between HCV genotypes and epidemiological characteristics; severity of the disease; and response to interferon (IFN) therapy. Data from 14 tertiary referral centres, corresponding to 1872 patients with chronic hepatitis C, were prospectively collected from 1989 to 1997. HCV genotyping was performed using the line probe assay (LiPA). HCV genotypes 1b, 3, 1a, 2, 4 and a mixed infection were found in 41%, 22%, 16%, 11%, 4% and 4% of our population, respectively. HCV genotype distribution was homogeneous, except for genotype 2 that was found more frequently in the southwest than in the other regions (21% vs 9.2%) ( P =0.001). HCV distribution was associated with gender, age, and source and duration of infection. In multivariate analysis, these correlations were related to the source of infection, which was the only independent factor significantly associated with genotype ( P =0.001). Genotype 1b was significantly more common in patients with cirrhosis, but in multivariate analysis cirrhosis was independently related to older age at exposure and longer duration of infection ( P =0.001). A sustained response to IFN therapy was observed in 11% of patients infected with genotypes 1a or 1b vs 32% of those infected with genotypes 2 or 3 ( P =0.001). This study shows that HCV genotype is mainly related to the source infection, but not to the intrinsic pathogenicity of HCV, and is a strong predictor of sustained response to therapy.     

多重PCR HCV基因分型检测方法的建立及初步应用

目的建立多重PCR HCV RNA基因分型的方法,并应用于临床诊断。方法采用型特异性引物建立单管逆转录聚合酶链反应(RT-PCR)和双管引物特异性套式聚合酶链反应(nested-PCR)检测技术,分别对72例HCV RNA阳性的标本,设计HCV的C区特异性引物,采用Trizol提取RNA进行单管巢式PCR分型,1a、1b、2a、2b和3a在一管内扩增。结果统计结果表明,我国丙型肝炎流行主要以1b(Ⅱ)和2a(Ⅲ)2种基因型为主,但有部分1a、2b、3a的HCV感染者检出,其中1a占1.4%(1/72),1b占68.1%(49/72),2a占13.9%(10/72),3a占1.4%(1/72),1b/2a型混合占11.1%(8/72),未分出型或其他型者为4.2%(3/72)。结论本文建立的方法具有良好的特异性和敏感性,省时、省力,方便检测,可同时区分5种HCV基因型;我国北方地区发现有1a和3a基因型感染病例。     

Hepatitis C virus serotypes in haemodialysis patients in South-East Italy

Hepatitis C virus (HCV) infection is highly prevalent in haemodialysis patients. To date, only a few studies involving a small number of subjects have characterized HCV-infected dialysis patients by serotyping. The spread of HCV serotypes in 114 HCV-positive dialysis patients from the same geographical area was evaluated by Murex HCV serotyping assay. Serotypes were detected in 102 subjects (89.5%), with type 1 being the most frequent (37.7%), followed by types 2 (19.3%), 4 (8.8%) and 3 (7.9%). Types 5 and 6 were the least prevalent (3.5%). Ten samples (8.8%) revealed mixed infections: type 1 was detectable in all and the co-infecting HCV types were types 2, 3 and 4 in 3, 4 and 3 cases, respectively. These results suggest that the serotyping assay as an alternative method of distinguishing the major types of HCV, also for particular risk groups and especially in laboratories that lack the specific expertise to perform genotyping methods. Age-related differences in patients with type 5 compared with those with types 3 and 6 may provide evidence of a more recent spread of these latter types.     

HCV血清分型方法的建立及其评价

目的建立HCV血清分型方法,评价其在慢性丙型肝炎(丙肝)抗体阳性标本中的分型率及血清型与基因型的相关性。方法克隆表达HCVCore、非结构蛋白(non-structural protein,NS)4两个区段的型别特异性表位嵌合抗原,并应用酶联免疫竞争抑制法建立血清分型方法。分别应用酶联免疫吸附试验和聚合酶链反应法检测200例慢性丙肝患者的血清抗体和HCVRNA,并用建立的方法进行血清分型。同时采用该方法检测90例乙型肝炎患者、11例戊型肝炎患者和16例其他肝病患者的血清,评价其特异性。结果在200例慢性丙肝患者的血清标本中,基因1b型128例,2a型72例,型别特异性抗体阳性157例,分型率为78.50%,与基因型一致率为98.09%。而在另外117例乙型肝炎、戊型肝炎和其他肝病患者的血清中,均未检测出HCV型别特异性抗体。结论建立的HCV血清分型方法具有较高的分型率和特异性,可用于HCV抗体的血清学分型和预测干扰素疗效。     

上海地区丙型肝炎病毒基因分型的研究

目的 研究上海地区不同人群中丙型肝炎病毒基因型的分布和频率。方法 应用新改进的第二代谱线探针分析（Ｌｉｎｅ Ｐｒｏｂｅ Ａｓｓａｙ,ＬｉＰＡ）试验盒,对１０９份血清的ＨＣＶ ＲＮＡ逆转录－巢式ＰＣＲ阳性分离物进行了基因分型。结果 ８１份丙型肝炎患者ＨＣＶ分离物中７１份（８６．７％）为１ｂ型,４份（４．９％）为２ｃ型,２份（２．５％）为３ｂ份,１份（１．２％）为６ａ型,２份（２．５％）为混合型９２／     

Hepatitis C virus genotypes in Australia

The relative distribution of Australian hepatitis C virus (HCV) genotypes was determined for 500 isolates. Genotyping was performed using a commercial reverse phase hybridization assay after amplification of the 5' untranslated region of HCV by the polymerase chain reaction. Australian isolates comprised, predominantly, genotype 1 (55%) and genotype 3 (38%) with genotype 2 accounting for only 7%. Genotype 3a was the most common subtype. When the major risk groups of injecting drug users or transfusion-acquired hepatitis C were compared, there was a significantly higher incidence of genotype 1b in the transfusion-acquired group ( P < 0.03). When the age of the patients was analysed, genotype 3a was more prevalent in the 21–40-year age group than the 41–60-year age group ( P <0.05). There was no significant difference in genotype distribution between males and females. HCV genotypes 1, 2 and 3 are most often found in developed countries but the relatively high prevalence of genotype 3a in Australia is unusual.     

Relationship between genotypes of hepatitis C virus and histopathological manifestations in chronic hepatitis C patients

OBJECTIVE: The aim of this study was to assess the relationship between HCV genotype and histological liver injury. DESIGN: Prospective study on a cohort of patients with biopsy proven chronic hepatitis C. SETTING: University medical centre. PARTICIPANTS: Enrolled were 324 consecutive patients (male 197, median age 52 years, range 19-68; chronic hepatitis, 224; cirrhosis, 100). METHODS: HCV genotype was determined by the INNO LiPA assay and HCV RNA levels by the bDNA assay. The histological features were scored according to the histology activity index. RESULTS: The distribution of HCV genotypes was 1a, 4.6%; 1b, 52.4%; 2a/c, 27%; 3a, 8%; 4, 2%; mixed, 6%. Serum HCV RNA levels were similar for all genotypes. There was no difference in the distribution of HCV genotypes between patients with chronic hepatitis and those with cirrhosis. Patients with genotype 1b and those with type 2a/c showed a similar prevalence of cases of cirrhosis (33% versus 31%, respectively). In addition, in a subgroup of 102 patients with an established date of infection, the progression to cirrhosis occurred with a similar length of time for HCV type 1b and 2a/c (median 16 versus 15 years, respectively). Patients with HCV genotype 2a/c or mixed genotype showed a higher histology activity index than those with type 1b (P< 0.01), whereas there was no difference in the fibrosis score for the different genotypes. Patients with genotype 3a showed a significantly higher prevalence of steatosis compared to those infected with other genotypes. Alanine aminotransferase (ALT) values were higher in patients with HCV type 2a/c, 3a and mixed genotype than those with type 1 (P < 0.002). CONCLUSIONS: The data indicate that there is no association between a particular HCV genotype and the progression to cirrhosis, and that specific genotypes are associated with distinct histopathological and biochemical manifestations although none of them is correlated with an increase of the fibrosis stage.     

Genetic diversity of HCV among various high risk populations (IDAs,thalassemia, hemophilia,HD patients) in Iran

ObjectiveTo determine the patterns of distribution of HCV genotypes among high risk population in north of Iran.MethodsA cross-sectional study was conducted on 135 HCV RNA-positive high risk individuals including thalassemia, hemophilia, patients under hemodialysis and intravenous drug addicts. HCV genotypes were determined based on amplification with type-specific primers methods.ResultsAmong the 187 anti-HCV positive samples, only 135 (72.2%) gave HCV-RNA positvity. Over all, the most identified HCV type was genotype 3a (51.1%) followed by 1a (27.4%), 1b (8.2%). Sixteen (11.9%) out of 135 HCV RNA-positive participants have infected with more than one genotype or subtypes as follow; 1a/1b in 11 (8.2%), 2/3a in 3 (2.2%), and 1a/1b/3a in 2 (1.5%). Stratification of participants revealed that HCV subtype 3a was more prominent in thalassemia, hemophilia and HD patients but 1a and 1b were frequent in intravenous drug addicts.ConclusionsThis study is the first report on HCV genotypes among Iranian subjects with different exposure categories resided in Mazandaran, where genotype 3a was found to be the most frequent genotype in thalassemia, hemophilia, and hemodialysis patients but not in IDAs. Since the addiction age is decreasing in Iran and a lot of addicts are IDAs, it might change the subtype pattern of HCV in general population.     

Hepatitis C virus infection among HIV-1 infected individuals from northern Mexico

Aims: The prevalence of hepatitis C virus (HCV) infection, risk factors and HCV genotypes in 140 HIV-1 infected individuals from northern Mexico was determined. Methods: Hepatitis C infection was confirmed by the detection of anti-HCV antibodies and HCV-RNA in sera, and genotyping was performed by the InnoLiPA-HCV genotype assay. Results: Seventeen (12.1%) out of 140 HIV-infected individuals were found to be HCV-positive. Coinfected individuals were more likely to be male (87%). The most frequent genotype was 1a (41%), followed by 1b (29.4%), 2a/c (17.6%), 2b (5.9%) and 3 (5.9%). Serum transaminase concentrations (AST and ALT) were higher in coinfected patients. Among the risk factors for coinfection: sexual transmission was the most frequently observed (men who have sex with men (MSM); 64.7% and bisexual behavior; 64.7%) followed by intravenous drug users (IVDU) (53%). There was no association of the HCV genotypes with the age and risk factors for HIV-1 and HCV infection observed in the studied patients. Conclusion: The results suggest that the prevalence of HIV-1/HCV coinfection in Mexico is lower than in other American countries.     

Virological characteristics of HCV infection in Japanese haemophiliacs

It has been found that almost all haemophiliacs treated with pooled concentrates of clotting factor VIII or IX before 1985/6 have been infected with hepatitis C virus (HCV). In order to clarify the characteristics of HCV infection in Japanese haemophiliacs, we investigated the HCV genotype and HCV-RNA level in 80 patients with haemophilia who had been confirmed to be positive by a second-generation HCV antibody test. HCV-RNA was detected in 60 (75.0%) individuals and various HCV genotypes were found. Although 80% (48/60) of the patients had genotype 1b, the frequency of each genotype was quite different from that in HCV-infected non haemophiliac Japanese. Particularly, multiple HCV genotypes were observed in 27 (46.7%) patients. The mean (± SD) level of HCV-RNA was 5.3 × 10⁵ ±  1.1 × 10⁶ copies mL⁻¹. The viral load in patients with genotype 2a was significantly less common than those with genotype 1a ( P = 0.0007), genotype 1b ( P = 0.0009) and combined genotype 1a/1b ( P = 0.0019). In patients co-infected with human immunodeficiency virus (HIV), the HCV-RNA level was significantly higher ( P = 0.05) than in those without co-infection. However, there was no significant difference ( P = 0.25) in the HCV-RNA level with HCV/HIV co-infection among the 40 patients with group 1 genotypes. We conclude that this biased distribution of HCV genotypes in Japanese haemophiliacs reflects their specific mode of HCV infection. Moreover, these results suggest that super-infection with HIV does not greatly influence the HCV load in patients with no marked immunological deterioration.     

HCV serotypes in Brazilian patients.

OBJECTIVE: To investigate the prevalence of the different types of hepatitis C virus (HCV) in a population of chronic HCV carriers using the Murex HCV serotyping 1-6 assay. METHODS: All serum samples from these patients had a positive nested PCR HCV reaction. The sera were submitted to ELISA, modified, for the identification of antibodies against HCV serotypes 1, 2, 3, 4, 5 and 6 (Murex HCV serotyping 1-6 assay). RESULTS: The viral serotype was identified in 166 (75.8%) of the 219 patients, 108 (65.11%) males and 58 (34.9%) females. Patient age ranged from 12 to 73 years, with a mean of 41.1 years. The form of acquisition of the disease most frequently reported was blood transfusion. The results showed a predominance of type 1 (70.0%), followed by type 3 (22.3%) and type 2 (4.2%). CONCLUSION: Samples presenting low and very close optical density readings may lead to discrepant diagnoses concerning HCV serotypes and should be confirmed by genotyping. The serotyping can be useful in clinical practice and can be of help in establishing the prognosis of the disease, also favoring epidemiologic studies independently of the technology required for genotyping tests.     

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

Summary.  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.