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Prevalence of hepatitis C virus (HCV) coinfection in HIV infected individuals in south India and characterization of HCV genotypes
SPD Ponamgi 1 , S Rahamathulla 1 , YN Kumar 1 , M Chandra 1 , N Lakshmi 2 , CM Habibullah 1 , MN Khaja 1
1 Center for Liver Research and Diagnostics, Deccan College of Medical Sciences and Allied Hospitals, Kanchanbagh, Hyderabad-500 058, Andhra Pradesh, India
2 Department of Biotechnology, Jawaharlal Nehru Technology University, Hyderabad-500 072, Andhra Pradesh, India
Correspondence Address:
M N Khaja
Center for Liver Research and Diagnostics, Deccan College of Medical Sciences and Allied Hospitals, Kanchanbagh, Hyderabad-500 058, Andhra Pradesh
India
Source of Support: None, Conflict of Interest: None
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.
Keywords: Coinfection, genotyping, human immunodeficiency virus, hepatitis C virus
How to cite this article:
Ponamgi S, Rahamathulla S, Kumar YN, Chandra M, Lakshmi N, Habibullah CM, Khaja MN. Prevalence of hepatitis C virus (HCV) coinfection in HIV infected individuals in south India and characterization of HCV genotypes. Indian J Med Microbiol 2009;27:12-6 How to cite this URL:
Ponamgi S, Rahamathulla S, Kumar YN, Chandra M, Lakshmi N, Habibullah CM, Khaja MN. Prevalence of hepatitis C virus (HCV) coinfection in HIV infected individuals in south India and characterization of HCV genotypes. Indian J Med Microbiol serial online 2009 cited 2009 Apr 20 ;27:12-6. Available from: Globally, a total of 39.5 million were living with HIV in 2006, of whom approximately 5.7 million (3.4-9.4 million) were in India. 2 Acquired immunodeficiency syndrome has grown more rapidly than the scientific progress of understanding how to control the main causative agent. Globally, hepatitis C virus (HCV) has infected more than 170 million people 3 and thus represents a viral pandemic seven times more widespread than infection with the HIV. It is estimated that in India approximately 1.8-2.5% of the population is presently infected by HCV 4 and about 20 million people are already having HCV infection. 5 Prolonged survival of HIV-infected patients coinfected with HCV may become an important clinical problem. In the United States and in European countries, it is estimated that approximately the prevalence of HIV/HCV coinfection in the HIV population ranges from 30 to 50%. 6 HIV and HCV show some common biological features like both are RNA viruses and both show a large heterogenicity of their viral genomes producing various genotypes. These viruses also have some differences, like HCV belongs to the Flaviviridae family and HIV to the Retroviridae family. Falviviruses have a single RNA strand whereas retroviruses have double RNA strands. The HIV-RNA, transcripted to DNA by the reverse transcriptase (RT), integrates in the infected cell's genome, constituting the integrated provirus; this integration is the cause of the irreversibility of HIV infection. In contrast, the HCV genome does not integrate into the cell's genome and the replication of the virus takes place in the liver cell's cytoplasm. This non-integration makes it easier to eradicate HCV and hence to cure the infection. These viruses share similar routes of transmission like through blood and blood products, sharing of needles to inject drugs and sexual route.
Previously, blood transfusion was a major mode of HCV transmission but now that donor blood is thoroughly screened for the virus, majority of the cases are injectable drug users. HCV is also transmitted perinatally, by improperly sterilized dialysis equipment (68% 7 of the cases) and by unprotected sex with an infected partner. Cohort studies report that men who have sex with men (MSM) and those with other sexually transmitted infections are at a greater risk of contracting HCV from unprotected sex. An estimated 20% of people with chronic HCV infection will progress to cirrhosis over a 20-50-year interval. 8 A greater proportion of HIV/HCV coinfected people may progress to cirrhosis (serious liver scarring) and liver disease than those with HCV alone. 9 HIV-infected individuals have a high probability of getting coinfected with HCV. HIV disease progression and enhanced immunosuppression has a direct bearing on the natural history and pathogenesis of these infections. Although there have been some reports of the prevalence of HCV infection and HIV infection in various populations in India, none have looked specifically at the prevalence of active HCV coinfection (i.e. HCV RNA positive) and HCV genotype and subtype in HIV-positive individuals.
The objective in this study, therefore, was to determine the prevalence of HCV antibodies in the HIV-infected Indian population. This is aimed at providing the baseline data on HIV/HCV coinfection. In order to gain a better understanding of the public health issues in these countries, we evaluated the anti-HCV antibody and genotype and subtype of HCV infection in 1487 confirmed HIV-positive individuals.
A total of 1487 confirmed status of HIV-positive samples (as per the World Health Organization strategies) were collected from different parts of South India and were anonymously tested for HCV markers. The study period was from January 2005 to February 2008.
Virological assays
The sera were screened for anti-HCV antibodies using a third generation ELISA kit (Ortho 3.0; Ortho Clinical Diagnostics, Raritan, NJ, USA). The Ortho HCV 3.0 ELISA test is a qualitative assay, each microwell being coated with a combination of recombinant HCV antigens c22-3, c200 and NS5. The amino acid sequence of the three HCV recombinant proteins are c22-3 AA # 2-120, c200 AA # 1192-1931, NS5 AA # 2045-2995. The assay was carried out as per the standard test procedure mentioned by the manufacturer.
Nucleic acid extraction
HCV RNA was isolated from the serum by the guanidinium isothiocyanate (GITC)-acid-phenol method. 10 Briefly, 200 Before RT PCR the RNA was denatured by heating at 95 o C for 2 min, followed by rapid chilling. Amplification by PCR was carried out essentially by the method of Das et al . 11 Briefly, the 50 non-coding region amplification was carried out with 25 pmole each of the primers, 10X Taq buffer (Mg 2+ free), 2.5 mM MgCl 2 , 200 mM dNTPs, 25 units of ribonuclease inhibitor (RNAsin), 4 units of AMV reverse transcriptase (Promega, Madison, WI, USA), 1 unit of Taq DNA polymerase and RNA template and was made up to a volume of 50 L. The RT-PCR step was carried out in a single tube using a programmable thermocycler (MJ Research, MA, USA) at 42 o C for 1 h, followed by 95 o C for 2 min, 35 cycles at 94 o C for 30 s, 50 o C for 45 s, 72 o C for 1 min and a final extension at 72 o C for 5 min. First-round PCR was performed using the forward primer 5'-ACTGTCTTCACGCAGAAAGCGTCTAGCCAT-3' and the reverse primer 5'-CGAGACCTCCCGGGGCACTCGCAAGCACCC-3'; 10 PCR product was re-amplified with internal primers (forward primer 5'-ACGCAGAAAGCGTCTAGCC-ATGGCGTTAGT- 3' and reverse primer 5'-TCCCGGGGCACTCGCA-AGCACCCTATCAGG-3') for another 35 cycles under the same conditions. A negative control, a positive control and a water blank were tested during RNA extraction, reverse transcription and amplification for quality control and to exclude false-positive results in the PCR due to cross contamination. PCR products were analysed on 2% agarose gels followed by staining with ethidium bromide and visualized under a UV trans-illuminator. A 100-bp ladder (Promega) was used as a size marker. Detection of a 256 bp PCR product indicated that the sample was positive for HCV Figure 1 .
Restriction fragment length polymorphism (RFLP) analysis of the 5' UTR amplified product
The amplified product obtained by RT-PCR was subjected to RFLP analysis by digestion with Hae III , Hinf I and Bst NI enzymes ( New England Biolabs, Beverly, MA, USA) . The size of the undigested amplicon is 256 bp, which is cleaved separately with these three enzymes. After treatment, the restriction fragments are separated by agarose gel electrophoresis and visualized in gel doc. The restriction patterns of the samples are compared with the predicted patterns to determine the genotype. For generating the above patterns, representative full-length sequences of various genotypes HCV-1 (1a); HCV-J (1b); HC-G9 (1c); HC-J6 (2a); HC-J8 (2b); Bebel (2c); NZIL (3a); TR (3b) were selected and the electrophoretypes predicted using RESTRI and DIGEST programs of PCGENE software package. The Hae III and Mva I enzymes were originally used for typing of the HCV isolates from India. 11
NS5 typing
An expected size of DNA fragments for each genotype is as follows: 1a, 3356 bp; 1b, 143 bp; 2a, 240bp; 2b, 309bp; 3a, 143bp; 3b, 201 bp.
Primer sequences used in the study
1a: 5'-GAGTCACTGAGAGCGSACATCCGTACG-3'
1b: 5'-AGGCCACTGCGGCCTGTCGAGCTGCGAA-3'
2a: 5'-TATGTTCAACAGCAAGGGCCAGA-3'
2b: 5'-GGCTTGTTCCCTGCCTCAAGAGGCCA-3'
3a: 5'-CTCGGACCCTGACTTTCT-3'
3b: 5'-CCGCGCTAGCGGCGTCTTGC-3'
CA: 5'-CCTGGTCATAGCCTCCGTGAA-3' (anti-sense primer for all genotypes).
Additional information:
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Hepatitis C Virus Core Genotype - 3a Recombinant ,
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BiomedExperts: An in vitro model of hepatitis C virus genotype 3a
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Comparative Hepatology , Full text , Distribution of hepatitis C
Virology Journal , Full text , Separation of Hepatitis C genotype
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