We examined HIV-positive injection drug users from Manipur who had been identified as anti-HBc–positive during previous serosurveys conducted by the National Institute of Cholera and Enteric Diseases (2). Serum samples (stored at –80°C) taken from 63 men 18–25 years of age were available for the study. HBsAg detection was repeated with a monoclonal antibody–based Hepanostika hepatitis B surface antigen (HBsAg) kit (bioMérieux, Marcy l'Etoile, France). Anti-HCV antibody was detected using the Ortho HCV 3.0 test (Ortho-Clinical Diagnostics, Raritan, NJ, USA). We completed HBV DNA isolation, PCR amplification, genotype/subgenotype/subtype identification, recombination detection, and HBV DNA quantification with methods described earlier (7–9). We also compared nucleotide (Table A1) and deduced amino acid sequences (Table) with consensus sequence of amino acids of corresponding genotypes to detect substitutions (GenBank accession nos. DQ356432–DQ356441).

All those tested were HBsAg negative, and only 10 (15.9%) had detectable HBV DNA. Anti-HCV was detected in all but 1 sample (no. 4). Serum HBV DNA level was detectable in 5 of 10 samples (Table); the rest were below the detection limit of our assay.

HBV/C (all subtype adr except 1 adw2) was the predominant genotype, with 4 subgenotype C1 (HBV/C1) and 3 subgenotype C2 (HBV/C2) isolates. HBV/A1 adw2 was found in 1 of the isolates. Two other isolates (nos. 1 and 3, subtype adw2) indicated a possibility of intergenotypic recombination (Figure 1), but subgenotype could not be assigned for them.

BLASTN search (http://www.incogen.com/public_documents/vibe/details/NcbiBlastn.html) of sequence from sample no. 1 showed similarity to HBV/D as well as to HBV/A sequences from European countries. On the other hand, sequences from sample no. 3 showed similarity to HBV/C and HBV/A sequences from Southeast Asian countries and India. Simplot analysis confirmed recombination in these 2 isolates (Figure 2). Apart from the genotype-specific substitutions, deduced amino acid sequences did not have any remarkable escape mutant other than G145R in 2 isolates (Table).

The data from this study showed occult HBV infection in 15.9% of the injection drug users tested. The rate of HBV DNA detection (10%–45%) was considerably different in studies reported from different cohorts of injection drug users in different countries, a finding that has been attributed to coinfection with HCV or low HBV DNA levels (3). Apart from HIV, our study group had a high frequency of HCV infection and low HBV DNA levels. Undetectable HBsAg, except in 2 cases with G145R substitution, may also be a result of the above-mentioned factors.

Although the importance of occult HBV infection is not well understood, a recent study reported occult HBV to be a significant risk factor for HCC, especially among persons who were anti-HBc–positive (4). Another recent study among HIV-infected patients documented death due to liver disease in 22% who were HBV coinfected, 44% who were HCV/HBV coinfected, and 15% who were HBV coinfected and had HCC (10). Thus, the clinical relevance in our study group also needs to be followed.

Although we detected a 100% prevalence of anti-HBc in our serosurveys (2), only 1 was HBsAg-positive. Therefore, the distribution of genotypes among those who were HBsAg positive could not be determined.

Findings of HBV/C1 (prevalent in China) appear to support the history of human migration from China to northeastern India. However, we did not detect HBV/B, also prevalent in China. Further, we did detect HBV/C2, which has close similarity to strains from Southeast Asian countries. The presence of HBV/C correlated well with drug-trafficking routes and the injection drug use epidemic. The geographic proximity of Manipur to the Golden Triangle, needle sharing among injection drug users, and drug traders thus contributed to the spread of HBV through drug-trafficking routes, similar to HIV (1).

In Manipur, HIV subtypes C and Thai B are prevalent (11); these are also prevalent in the India-Myanmar and China-Myanmar border regions. The presence of similar HIV subtypes among injection drug users in Manipur supports the presence of similar HBV strains (e.g., HBV/C1, HBV/C2) and their cotransmission through drug-trafficking routes. In addition, circulation of recombinant HBV is common among injection drug users because of repeated exposure (12). The intergenotypic HBV recombinants found in this study are thus expected. HBV/C has been associated with advanced liver disease and poses a higher risk for HCC in Asians (13) than does HBV/B. However, the clinical relevance of HBV recombinants and their pathogenesis is not well understood and needs further investigation.

Poor, unemployed youths in the northeastern states of India are being recruited for drug trafficking to other regions (http://www.ipcs.org). Furthermore, national highways are associated with the prevalence of injection drug use in rural Manipur (14). As these highways connect Manipur with other parts of India, HBV/C may spread from Manipur to other parts of the country through persons who travel regularly, such as truck drivers and drug traffickers.

Presence of HBV genotypes A and D among patients from northern and western India is well documented. Recent research reported HBV/C with close similarity to Southeast Asian strains only from eastern India (9,15) and suggested injection drug users as a possible route of introduction (15). In addition, persons from northeastern India frequent Kolkata for education, employment, medical treatment, and other purposes. Studies on these mobile populations might provide further important information on the route, population at risk for infection, and changing epidemiology of these viral infections in other regions.

In conclusion, HBV/C, associated with severe liver disease in Southeastern Asia, may be emerging in the Manipur state of India through the trafficking routes of injection drugs. This genotype could spread to the general population through different modes. In light of growing information on the severity of liver disease in HBV-infected HIV/HCV patients, injection drug users should be the focus of additional education and healthcare efforts. The possibility of further spread of HIV/HBV/HCV through mobile populations to other regions of India warrants attention and further investigation.