Distribution of hepatitis C virus genotypes in patients with chronic hepatitis C infection in India
Background & objectives: Hepatitis C virus (HCV), an important cause of chronic hepatitis, cirrhosis and hepatoccllular carcinoma, shows a considerable genetic heterogeneity among hepatitis C virus isolates from all over the world. At least six main groups of sequence variants are recognized. The natural history of disease and response to treatment may be related to the genotype of HCV in a particular patient. Antigenic differences between genotypes also have implications for optimal design of serological sequencing and confirmatory assays for HCV. The present study was undertaken with the objective to find out various genotypes of hepatitis C virus prevalent in Indian patients with chronic hepatitis C infection.
Method: Thirty six consecutive newly diagnosed patients with chronic hepatitis C infection were included in the study. HCV RNA was extracted from the scrum by standard guanidinium thiocyanate method. Following reverse transcription and amplification, the HCV genotypes were determined by line probe assay (INNO-LiPA HCV II).
Results: Of the 36 patients, genotype 3 was found in 24 (66.6%). Of these 24 patients, 3a was seen in 5 patients (13.8%), 3b in two (5.5%) and mixed subtype 3a and 3b in 17 patients (47.2%). Genotype 1 was found in 5 patients (13.8%), with 1b in 1 and 1a in rest four cases. Two patients (5.5%) were infected with genotype 2 (subtype 2a and mixed subtype 2a, 2b respectively). One (2.7%) was infected with genotype 4 (4a). Mixed genotype infection was found in 4 patients (11.1%).
Interpretation & conclusion: The present findings showed that genotype 3 of hepatitis C virus was the most prevalent genotype in patients with chronic hepatitis C in this part of India.
Key words Genotypes – hepatitis C virus – line probe assay
Hepatitis C virus (HCV) is a hcpatotropic virus of family Flaviviridae and genus Hepacivirus having single strandard RNA of positive polarity as genomic material. A large number of genotypes have been identified among hepatitis C virus isolates from all over the world. Presently six main groups of sequence variants have been characterized corresponding to types 1-6; each group containing a number of more closely related subtypes (a, b, c, etc.)1. Hepatitis C virus can cause chronic hepatitis in about 80 per cent of cases2. Although most chronic HCV patients have mild chronic hepatitis, it is a progressive disease and can lead to cirrhosis3 or hepatocellular carcinoma4. Without treatment, 33 percent patients have an expected median time to cirrhosis of less than 20 yr3. It has been suggested that different genotypes have different clinical outcomes with regard to disease severity and response to interferon therapy6.
Very few studies have been done on the distribution of various hepatitis C virus genotypes in India7-11. Recently a study also hinted towards geographic variation in the prevalence of various HCV genotypes in India12. We took up this study to find out (he prevalence of various genotypes ofhepatitis C virus in the patients with chronic hepatitis C injection.
Material & Methods
Thirty six consecutive newly diagnosed patients with chronic hepatitis C infection (i.e., anti-HCV positive by third generation HLISA) who attended outpatient department of G.B. Pant Hospital, New Delhi during 1999-2001 were included in this study. The serum samples were taken for extraction of viral RNA and determination of viral genotypes by reverse transcriptase polymerase chain reaction (RT-PCR.) combined with line probe assay.
RNA extraction: Scrum was mixed with lysis solution containing guanidinium thiocyanate, sarcosyl and beta-mercaplocthanol. RNA was then extracted with phenol and chloroform followed by precipitation with isopropanol at -70°C13.
Nested PCR: From the RNA thus isolated, complementary DNA (cDNA) was made using avian myeloblastosis virus-reverse transcriptase (AMV-RT, Genei, Bangalore). Nested PCR was carried out using two set of primers (lnnogenetics, Belgium) from highly conserved 5′ non-coding region of the 1ICV genome, as recommended in line probe assay14. In brief, the cDNA was introduced in a reagent mixture containing an excess of dcoxyribonucleotides, outer primers and thermostable Taq DNA polymerase (Genei, Bangalore). This reagent mixture was put in thermal cycler (Perkin Elmer Gene Amp PCR System 2400, Norwalk, USA) in which the cDNA got amplified by polymerase chain reaction. This was followed by repeat PCR using inner primers having their 5′ end labeled with biotin.
HCV genotyping: The genotype of the amplified cDNA was determined using the principle of reverse hybridization in a line probe assay14 ((NNO-LiPA HCV Il kit, lnnogcnetics, Belgium). Forthisthe DNA segment obtained after nested PCR was denatured and incubated, under stringent conditions, with strips having probes corresponding to various HCV genotypes. The resulting bands were then made visible with streptavidin alkaline phosphatasc immunochemical reaction using nitroblue tetrazolium and bromo chloro indolyl phosphate (NBT/ BCIP) as substrate. The genotypes were identified using the chart provided by the manufacturer. The INNO-LiPA assay strips contain 15 probe lines to identify HCV genotypes and subtypes, according to the Simmonds classification1.
Genotype 3 was observed in 24 of the 36 patients (66.6%). Of these 24 patients, 5 showed infection with subtype 3a (13.8%), 2 had subtype 3b (5.5%) and 17 showed both 3a and 3b infection (47.2%). Genotype I was seen in 5 patients comprising 13.8 per cent of all cases – one of these had subtype I b and four had subtype Ia.
Two patients (5.5%) showed infection with genotype 2 (one each with subtype 2a and mixed subtype 2a, 2b), and one (2.7%) was infected with genotype 4 (subtype 4a). Four showed mixed genotype infection ( 11.1 %). One of these patients had infection with genotype I b, 4a, 5a; another had coexistent 2b, 4a, 5a; one patient showed mixed infection with Ia, 4e, 5a and the last had co-infection with 3a, 3b and genotype 2.
With the discovery of hepatitis C virus by Choo et al15′, a large number of patients belonging to the nonA, non-B hepatitis could be etiologically defined and definitely categorized. The virus was identified by cDNA cloning and sequencing of RNA genome. A comparison of HCV genome sequences from various geographical regions of the world has shown substantial heterogeneity of nucleotide sequences within several regions of viral genome. On the basis of these gcnomic differences HCV has been classified into various genotypes. Six major genotypes with several subtypes were identified and a nomenclature for these was given following a consensus proposal’. Subsequently several new genotypes and subtypes have been reported from different parts of the world. In one study”16 atleast 12 genotypes were predicted by sequence analysis of the putative El gene of isolates collected worldwide. But currently, the existence of six major HCV types is generally accepted, and scientific debate is ongoing about whether types 7-11 are distinct types or subtypes of types 3 and 617.
There are very few studies7-11 reporting the presence of various HCV genotypes in India. HCV types Ia, Ib, 2a, 3a, 3b, and 3g have been identified in the earlier studies from northern and western lndia7,12 and genotype 1 predominated over genotype 3 in southern India8. Recently a study on samples from many parts of India also found high prevalence of genotype 3′-.
Our analysis of HCV infection in 36 patients with chronic hepatitis C demonstrated genotype 3 as the predominant genotype, most of our cases with genotype 3 infection showed presence of mixed subtype 3a and 3b. Genotype 1 was the second most common genotype, with most cases showing infection by HCV subtype 1a. Other genotypes seen included genotype 2,4 and 5. The earlier studies7-12 as well as the present study indicated that genotype 3 was the most prevalent genotype in north India.
From other parts of the world studies reveal that genotype 3 is prevalent in South East Asia whereas genotype 1 is common in USA and Western Europe’*. These geographical differences may help in predicting the origin of HCV virus.
The remarkable heterogeneity of virus is the major limitation for developing a vaccine for HCV infection. It has been suggested that the degree of sequence variability of HCV is sufficient to significantly alter the antigenic and biologic properties of the virus’^. Antigenic differences between genotypes have implications for optimal design of serological sequencing and confirmatory assays for HCV. Diminished sensitivity to genotype 3 was seen in recombinant immunoblot assay-2 (RIBA-2f and to a lesser extent in R1BA-3-‘, and this might also be reflected in the sensitivity of screening ELlSAs^. Future tests will need to incorporate antigens from various genotypes to ensure an optimal sensitivity for all, especially genotype 3, which has been reported as the commonest genotype in our country as well as South East Asian countries.
The severity of disease, its progression, and response to therapy may vary according to the genotype23-25. A number of studies have reported that severe liver disease occurs in relation to type 1 infection (especially type lby-^ and that cirrhotic patients infected with HCV type Ib carry a significantly higher risk of developing hepatocellular carcinoma as compared to those infected with other HCV types^. But the results of various studies are conflicting23″25. H is known that genotype 1 is the second most common genotype reported from varions parts of our country, as also seen in the present study. Thus knowledge on the distribution of various genotypes in our country is essential for its prognostic implications in chronic hepatitis C infection.
In conclusion, genotype 3 was found to be the most prevalent genotype in patients with chronic hepatitis C in north India.
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Sompal Singh, Veena Malhotra & Shiv Kumar Sarin*
Departments of Pathology & * Gastroenterology, G. B. Pant Hospital, New Delhi, India
Received April 22, 2003
Reprint requests’. Dr Vccna Malhotra, Director, Professor & Head, Department ofPathology, G.B, Pant Hospital Jawahar LaI Nehru Marg, New Delhi 110002. India
Copyright Indian Council of Medical Research Apr 2004
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