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Injecting drug users (IDU) were enrolled from two detoxification clinics and two needle/syringe exchange programmes (NEP) in central and northwest Bangladesh. Syphilis, hepatitis C and HIV rates were highest in IDU from the NEP of central Bangladesh (23, 66.5 and 1.4%, respectively), whereas current hepatitis B infection rates were highest in IDU from the NEP of northwest Bangladesh (12%). Five HIV strains were subtype C and one E/B. The 32 base pair (bp) deletion of the CCR5 gene was not detected. During the second round of serological surveillance for HIV in Bangladesh, conducted between July 1999 and June 2000 1, 1236 injecting drug users (IDU) were tested for syphilis, hepatitis B and C (HBV and HCV) and HIV. HIV strains were subtyped and, in a subset of IDU, genotyping of the β-chemokine receptor 5 (CCR5) gene was performed to assess whether the 32 base pair (bp) deletion, which is associated with protection against HIV infection and delayed progression of illness 2 was present. Individuals injecting drugs in the past year were defined as IDU. The sample size was calculated as 380 with an estimation of the HIV prevalence rate of 1%, with a 1% precision and 95% confidence level. The first 400 IDU attending each of the three sites, two detoxification clinics in central Bangladesh and the satellite clinics of two non-government organizations conducting needle/syringe exchange programmes (NEP) in central and northwest Bangladesh, were enrolled. Information regarding the surveillance was provided through group workshops to all IDU in the NEP, and informed consent was obtained before sampling. However, IDU from detoxification clinics were enrolled passively and leftover blood drawn for syphilis and hepatitis was used for HIV testing. Serum was separated from blood collected by venepuncture into sterile, plain Vacutainers (Becton Dickinson, Rutherford, NJ, USA). Two aliquots of 500 μl whole blood, for DNA extraction, were collected each in an Eppendorf tube and an ethylenediamine tetraacetic acid-containing Vacutainer tube (Becton Dickinson). Samples were transported to the International Centre for Diarrhoeal Disease Research of Bangladesh laboratory by maintaining the cold chain where they were stored at –20°C until testing. Testing for syphilis was linked whereas that for HIV, HBV and HCV were unlinked, anonymous. Syphilis was tested by the Rapid Plasma Reagin test (Organon Teknika, Boxtel, the Netherlands) and the Treponema pallidum haemagglutination assay (Organon Teknika). Samples positive for both assays were considered to reflect active syphilis. For antibodies to HCV, sera were initially tested using an enzyme-linked immunosorbent assay (ELISA) kit (UBI HCV EIA; United Biomedical, Inc., USA). All positive samples were re-tested with a second ELISA kit (MONOLISA anti-HCV Plus Version 2, Sanofi Diagnostics Pasteur, France). Samples with discrepant results were re-tested by line immunoassay (INNO-LIA HCV Ab III update; Innogenetics NV, Ghent, Belgium). Samples positive for any two tests were considered to be positive. IgM antibodies to hepatitis B core (HBc) antigen were detected by an ELISA kit (Hepanostika, HBc IgM; Organon Teknika). An objective of the HIV surveillance system in Bangladesh is to determine the effectiveness of the overall HIV prevention programme in the country. For this, comparison of a measure of current infection over the years allows a better understanding of the field situation. Antibodies to HCV and syphilis are long lasting, whereas HBc IgM measures current infection. Therefore, HBc IgM was used as a marker for HBV infection. For HIV, samples were initially tested using an ELISA kit (Organon Teknika) and positive results were confirmed by line immunoassay (Organon Teknika). An indeterminate result by line immunoassay was considered to be negative. Standard quality control sera for HIV provided by the National Reference Centre for HIV/AIDS, India, at the Christian Medical College and Hospital, Vellore, India, were used for quality control. The chi square statistic or Fisher's exact test was used for comparing proportions. Data analyses were carried out using the Statistical Package for Social Sciences (version 8.0 for Windows; SPSS Inc., Chicago, IL, USA), Epi Info (version 6; USD, Stone Mountain, GA, USA) and Stata (version 5.0; Stata Corporation, TX, USA). Whole blood samples from HIV-positive IDU were transported on dry ice to the Centre for HIV Research, University of Edinburgh, UK, for determination of HIV subtype by viral sequencing as described elsewhere 3,4. For genotyping of the CCR5 gene, a fragment of the CCR5 gene encompassing the 32 bp deletion was amplified by polymerase chain reaction from genomic DNA and analysed on a 2% agarose gel as described before 5. Genomic DNA was obtained from the whole blood of 101 randomly selected IDU from the NEP of central Bangladesh. All except five IDU were men. The prevalence of syphilis, hepatitis and HIV in IDU is shown in Table 1. For HCV, of the 607 IDU who tested positive in the first ELISA, 596 were positive by at least two tests. More syphilis and HCV-positive IDU were found in the NEP of central Bangladesh (Table 1) than in that of northwest Bangladesh (P ≤ 0.001 and 0.044, respectively) and the detoxification clinics (P ≤ 0.001 for both). Acute HBV infection rates were higher in IDU from the NEP of northwest Bangladesh than from that of central Bangladesh (P = 0.037) or the detoxification clinics (P ≤ 0.001). For HIV, 12 IDU were positive by the first test, of whom seven were confirmed positive (Table 1). Six were from the NEP of central Bangladesh and one was from the detoxification clinic. HIV-positive individuals from the NEP but not from the detoxification clinic were co-infected with HCV. A greater risk of infections through the blood-borne and sexual routes exists in IDU in central Bangladesh, as shown by behaviour surveillance 1. It is therefore not clear why the acute HBV rates are higher in the IDU from northwest Bangladesh.Table 1: Prevalence of syphilis, hepatitis and HIV. Of the seven samples positive for HIV, DNA sequences were obtained in both the env and gag regions for six samples. Five of these were subtype C in both regions of the genome. The sixth isolate was a subtype E (A/E) env and a subtype B gag. The virus sequences obtained were compared with those from other countries. Such comparisons showed that for subtype C viruses, sequences in gag from five samples and from env in four samples were most closely associated with sequences obtained from India, although none of these associations were well supported by bootstrap re-sampling. The fifth isolate did not associate with any particular isolate in the env region. Four of the five subtype C samples clustered together in both the env and gag regions, with bootstrap values between 90 and 100%, suggesting a transmission group. The isolate with subtype E/B, in the env region of the genome showed an association with subtype E sequences from Thailand, although this association was not significant. In the gag region, sequences from this subtype associated with a subtype B isolate from China, supported by 80% bootstrap replicates. Subtype C viruses are to be expected as they are the dominant strain in India 6 and have also been reported from Bangladesh 7,8. Subtype E/B recombinants have been reported from IDU in Thailand 9. Genotyping of the CCR5 gene showed that none of the 101 IDU tested had the 32 bp deletion. This is not surprising as this deletion is rare in Indians 5. Therefore, protection against HIV infection through this mechanism is unlikely in Bangladeshis. Therefore, despite the present low HIV prevalence in IDU in Bangladesh, high syphilis and HCV rates, risky behaviour, transmission from neighbouring countries and a lack of genetic protection against HIV infection suggest that IDU are at risk of an HIV epidemic. Acknowledgements The authors are grateful to the following institutions for their active participation in providing access to the IDU: CARE, Bangladesh; Marie Stopes Clinic Society; Central Drug Treatment and Rehabilitation Centre and Mukti Lawrence Foundation. They are also grateful to the Surveillance Advisory Committee for their support and advice. The International Centre for Diarrhoeal Disease Research of Bangladesh acknowledges with gratitude the commitment of Department for International Development to the Centre's research efforts. Tasnim Azima Jozef Bogaertsa David L. Yirrellb Akhil C. Banerjeac Mohammed S. Sarkera Giasuddin Ahmeda Mian M. M. Amind Abu S. M. M. Rahmane Abu M. Z. Hussainf
Azim et al. (Tue,) studied this question.