Detection of HIV type 1 env subtypes A, B, C, and E in Asia using dried blood spots: a new surveillance tool for molecular epidemiology

Global surveillance of HIV-1 subtypes for genetic characterization is hampered by the biohazard of processing and the difficulties of shipping whole blood or cells from many developing country regions. We developed a technique for the direct automated sequencing of viral DNA from dried blood spot (DBS) specimens collected on absorbent paper, which can be mailed unrefrigerated in sturdy paper envelopes with low biohazard risk. DBS were collected nonrandomly from HIV-1-infected, mostly asymptomatic, patients in five Asian countries in 1991, and shipped via airmail or hand carried without refrigeration to Bangkok, and then transshipped to North America for processing. After more than 2 years of storage, including 6 months at ambient temperatures, proviral DNA in the DBS was amplified by nested PCR, and a 389-nucleotide segment of the C2-V3 env gene region was sequenced, from which 287 base pairs were aligned and subtyped by phylogenetic analysis with neighbor-joining and other methods. From southern India, there were 25 infections with subtype C and 2 with subtype A. From Myanmar (Burma), we identified the first subtype E infection, as well as six subtype BB, a distinct cluster within subtype B that was first discovered in Thailand and that has now appeared in China, Malaysia, and Japan. From southwest China, one BB was identified, while a "classical" B typical of North American and European strains was found in Indonesia. From Thailand, five DBS of ambiguous serotype were identified as three B, one BB, and one E. A blinded control serotype E specimen was correctly identified, but a serotype BB control was not tested. Most HIV-1 in southern India appears to be env subtype C, with rare A, as others have reported in western and northern India. The subtypes BB and E in Myanmar, and the BB in China, suggest epidemiological linkage with these subtypes in neighboring Thailand. DBS are a practical, economical technique for conducting large-scale molecular epidemiological surveillance to track the global distribution and spread of HIV-1 variants.