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 80c ) taken from 63 men 1825 years of age were available for the study .
hbsag detection was repeated with a monoclonal antibody  based hepanostika hepatitis b surface antigen ( hbsag ) kit ( biomrieux , 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 ( 79 ) .
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 .
hbv , hepatitis b virus ; idu , injection drug user ; nd , not detectable by the assay .  genotype - specific sites .  subtype - specific sites .
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 .
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 .
phylogenetic relationships among the sequences of s gene from hepatitis b virus strains isolated in this study ( shown with prefix " idu " ) compared with reference sequences from genbank ( accession nos . are shown ) .
percentage of bootstrap replications supporting the clusters ( > 75% ) are also shown at the nodes .
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 .
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 location of recombination events in isolates idu1 ( a ) and idu3 ( b ) , determined by using the bootscanning program of simplot .
possible tree topologies ( a , b , and c ) are shown ( c ) .
the phylogenetically informative sites and the tree topologies supported at each of those sites are indicated over each plot . in the tree topologies ,
q , a , and h indicate query sequences ( idu1 or idu3 ) , genotype a , and genotype h ( outgroup ) consensus sequences , respectively .
s indicates consensus sequence of genotype d and genotype c for idu1 and idu3 , respectively .
possible crossover points are indicated by solid triangles . a sliding window size of 60 bp , step size of 10 bp , kimura-2 parameter , 1,000 bootstrap replicates , and neighbor - joining method were used for the analysis of recombination .
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 ) .
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 .
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 ) .
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 .
 prevalence of hepatitis b genotype c in injection drug users in the northeastern indian state of manipur , neighboring the " golden triangle , " correlates well with overland drug - trafficking routes , the injection drug use epidemic , and the spread of hiv . 
 further spread to other regions of india through mobile populations is possible . 
The Study
Conclusions