Transmission dynamics of hepatitis C virus among intra venous drug users in the border state of Manipur, India

https://doi.org/10.1016/j.meegid.2014.03.008Get rights and content

Highlights

  • 91.20% of the study population were HCV viremic.

  • Eight different HCV subtypes were detected from north-eastern state of Manipur.

  • Emergence of new genotype 6 has a direct evolutionary link with the strains of Vietnam and South-China.

Abstract

Intra venous drug users (IVDUs) are at high risk for hepatitis C virus (HCV) infection owing to their high rate of drug abuses. The north-eastern part of India has a high prevalence of IVDUs with Manipur being the worst hit state. The aim of the study was to document the molecular epidemiology, the patterns of HCV transmission, genomic variation and recombination events within HCV genome among IVDUs of Manipur, India. 91 anti-HCV sero-reactive blood samples were collected from IVDUs in Manipur. The samples were processed for RNA extraction, nested RT-PCR, sequencing and quantitative viral RNA estimation. Phylogeographic analysis of the sequenced core and NS5B regions of HCV genome was performed to determine the probable transmission route and recombinant HCV strains. 83 out of 91 anti-HCV seropositive samples were RNA positive (91.20%) based on 5′UTR of HCV genome by nested RT-PCR. Of the RNA positive samples, 73 paired partial core and NS5B gene were sequenced. Three major genotype and eight subtypes were detected while no recombinant strains were found. Individuals with genotype 1 had the mean viral load (5.94 ± 0.705 log10IU/ml) followed by genotype 3 (4.91 ± 0.49 log10IU/ml) and 6 (3.96 ± 0.32 log10IU/ml). The viral load was statistically significant among the male individuals at 4.822 ± 1.36 log10IU/ml compared to 4.767 ± 0.49 log10IU/ml for females (t = 3.249, p < 0.005). The phylogeographic results indicated 3b, 6h originated from Vietnam, 1a had Indian origin, 3a, 6k originated from southern China while 1b originated from Myanmar, respectively. The incidence of eight different subtypes in Manipur reflects the transmission of these strains from the “Golden Triangle” drug trafficking regions. Sequence analysis confirmed the transmission routes of HCV, which is linked to China and Vietnam for the newly emergent genotype 6 in north-eastern India.

Introduction

Hepatitis C virus (HCV) is a single-stranded positive-sense enveloped RNA virus with a genome size of ∼9.6 kb. HCV belongs to the genus Hepacivirus of the Flaviviridae family. This is the main causative agent of liver related disorders including hepatocellular carcinoma (HCC) (Reed and Rice, 2000, Chen et al., 2002). Among the 185 million infected people worldwide, self-clearance occurs within 20–40% of the patients (Chen et al., 2002, Negro, 2013). Chronic carriers are at risk of liver cirrhosis and HCC (Lindenbach and Rice, 2005). There is no vaccine available against HCV because of high viral genomic variations and rapid mutation rates leading to swarms of closely related viral quasispecies to escape adaptive immunity (Abrignani et al., 1999, Prince and Shata, 2001). HCV has seven genotypes (Smith et al., 2014) with more than 67 subtypes, the distribution of HCV genotypes and subtypes is geographically limited. The most common genotypes found throughout the world include genotypes 1 and 3 while others are limited to specific regions of the world. For example, genotype 4 is restricted to the Middle East and Central Africa, genotype 5 in South Africa and genotype 6 in China and South East Asia (Adinolfi et al., 2001).

In India, national surveillance data suggests 1.1–1.68 million intra venous drug users (IVDUs) resides in India (Hagan and Des Jarlais, 2000, Aceijas and Rhodes, 2007, Mathers et al., 2008, Sharma et al., 2009) but their status of HCV remains unexplored (National AIDS control organization, 2006, Aceijas and Rhodes, 2007, Sharma et al., 2009). Manipur, a north-eastern state of India is the most affected state by IVDU-related epidemic according to national surveillance programme and had the highest prevalence of HIV in India (HIV Sentinel surveillance and HIV estimation in India, 2007).

The population of Manipur is comprised of the ethnic tribes Meiteis, Paite, Thadou, Gangte, Kukis, Nagas and other similar ones living predominantly within the rural belts (Medhi et al., 2011). Within the state, IVDUs are scattered throughout the nine districts and are predominant within the local tribes. Manipur is one of the main transit points for drug trafficking in India. Drugs enter India through the notorious “Golden Triangle” a specific region north of Chiang Rai where the borders of Thailand, Myanmar and Laos meet. The state of Manipur has a porous border with Myanmar and it is widely believed that the start of trade from Moreh in 1994 led to an increase in drug flow. These trafficked drugs find their way to other parts of the country as well as the Middle East countries and Europe. The National AIDS Control Organization (2006), India reported that IVDUs with infections in Manipur were found mainly within the age group of early twenties and mid-thirties. Porous borders of north-eastern states also provide drug addicts and trafficker’s access to China and other regions of Southeast Asia. These drug paddlers are supposed to be responsible for the variable transmission dynamics of these HCV (Zhang et al., 2011).

Multiple infections by different HCV genotypes may be of great clinico-pathological interest. Individuals who are resistant to standard therapy and having high probability of multiple infections (such as hemophiliacs, dialysis, thalassemics, and IVDUs) can give rise to recombinant HCV strains. Recently both intergenotypic and intragenotypic recombinant HCV strains have been identified from different geographic locations in the world, as observed in Russia (2k/1b), Peru (1a/1b), Vietnam (2/6), Philippines (2b/1b), and France (2/5) (Colina et al., 2004, Cristina and Colina, 2006, Noppornpanth et al., 2006, Kageyama et al., 2006, Lee et al., 2010). The crossover points of natural intergenotypic and intragenotypic recombinants were found within the NS2/NS3 or NS5A regions (Kalinina et al., 2004, Moreno et al., 2006, Legrand-Abravanel et al., 2007). Hence, for proper genotyping, one must sequence both the 5′- and 3′-regions including part of the structural genes core/E1 and the non-structural gene NS5B of the HCV genome.

In India, genotype 3 is most common, with subtype 3a being predominant, followed by genotype 1. The distribution of HCV genotypes has a north–south divide with genotype 1 being the predominant form in the south whereas genotype 3 is common in the north, east and west of India (Saha et al., 2000, Mukhopadhya, 2008).

Here, we report the pattern and characterization of genotypes circulating among the IVDUs population of Manipur. The phylogenetic, phylogeographic relationship and recombinant HCV strains among the viral types prevalent in Manipur were correlated with the adjoining regions of China, Myanmar, Thailand and Vietnam.

Section snippets

Material and methods

The study is in accordance with the Helsinki declaration and International Conference on Harmonisation Good Clinical Practices guidelines, 1961. All inclusions were approved by the Institutional Ethical Committee and written consent was obtained from each individual who participated in the study.

Prevalence of active HCV infection

Active HCV infection was determined by the presence of viral RNA in serum. Of the 91 anti-HCV sero-reactive samples, 83 (91.20%) were RNA positive based on the nested RT-PCR of 5′UTR of HCV genome. We successfully amplified 73 paired partial core and NS5B genes for phylogenetic analysis from the HCV RNA positive samples by nested RT-PCR (Table 1).

Viral load estimation

The mean values of viral load were significantly higher among genotypes 1 (4.5–7.4 log10IU/ml) and 3 (4.0–5.4 log10IU/ml) compared to genotype 6 (3.5–4.2

Discussion

The analysis of HCV genotype and subtype distributions among IVDUs of Manipur amalgamates useful information about the transmission dynamics, porous borders, drug trafficking, and high variability of the genotypes in this part of India. The phylogeographic analysis shows how Manipur is a vulnerable point of HCV transmission with its adjoining countries.

The movement of infected individuals as drug users and traffickers renders the spread of HCV difficult to control (Cochrane et al., 2002). The

Conclusion

This is the first comprehensive report describing the genotypic transmission of HCV within the IVDUs of Manipur. Common practices associated with IVDUs like needles sharing and other social networking’s are the major factors associated with this high prevalence. Hence adequate measures must be taken on a national level to prevent the further spread of this dreadful disease.

Acknowledgement

We would like to acknowledge the funding agencies Department of Biotechnology (DBT), New Delhi, India and University Grants Commission (UGC), New Delhi, India for providing the fellowship of Kallol Saha and Rushna Firdaus. We also acknowledge Kelli Jackson and Mansun Law from The Scripps Research Institute, La Jolla, California USA for the English proofreading of the manuscript.

References (47)

  • R. Colina et al.

    Evidence of intratypic recombination in natural populations of hepatits C virus

    J. Gen. Virol.

    (2004)
  • J. Cristina et al.

    Evidence of structural genomic region recombination in hepatitis C Virus

    Virol. J.

    (2006)
  • A.J. Drummond et al.

    BEAST: Bayesian evolutionary analysis by sampling trees

    BMC Evol. Biol.

    (2007)
  • R. Firdaus et al.

    Rapid immunoassay alone is insufficient for the detection of hepatitis C virus infection among high-risk population

    J. Viral. Hep.

    (2013)
  • H. Hagan et al.

    HCV and HIV coinfection among Injection drug users

    Mt. Sinai J. Med.

    (2000)
  • M. Hasegawa et al.

    Dating of the human-ape splitting by a molecular clock of mitochondrial DNA

    J. Mol. Evol.

    (1985)
  • HIV Sentinel surveillance and HIV estimation in India 2007. A technical brief. National AIDS Control Organization....
  • S. Kageyama et al.

    A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Phillipines

    J. Med. Virol.

    (2006)
  • O. Kalinina et al.

    Full length open reading frame of a recombinant hepatitis C virus strain from St Petersberg: proposed mechanism for its formation

    J. Gen. Virol.

    (2004)
  • Y.M. Lee et al.

    Molecular epidemiology of HCV genotypes among Injection drug users in Taiwan: Full length sequences of two new subtypes 6w strains and a recombinant form 2b6w (Taiwan)

    J. Med. Virol.

    (2010)
  • F. Legrand-Abravanel et al.

    A new natural intergenotypic (2/5) recombinant of hepatitis C virus

    J. Virol.

    (2007)
  • B.D. Lindenbach et al.

    Unravelling hepatitis C virus replication from genome to function

    Nature

    (2005)
  • G.C. Luna

    Use and abuse of amphetamine-type stimulants in United States

    Pan. Am. J. Public. Health.

    (2001)
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