Detection of HIV Type 1envSubtypes A, B, C, and E in Asia Using Dried Blood Spots: A New Surveillance Tool for Molecular Epidemiology

Abstract

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.

Multicenter Evaluation of Use of Dried Blood and Plasma Spot Specimens in Quantitative Assays for Human Immunodeficiency Virus RNA: Measurement, Precision, and RNA Stability

“…The 903 filter paper is inexpensive, available worldwide, and commonly used for assays for the screening of newborns (Guthrie cards) and has been used before for the detection of HIV antibodies (2,15), DNA (5)(6)(7)(9)(10)(11)(12), and RNA (8,14,17). The spotted filter papers were allowed to dry overnight at room temperature, placed in individual ziplock bags containing a silica desiccant (MultiSorb Technology, Inc., Buffalo, N.Y.), and then frozen at Ϫ30 or Ϫ70°C or stored at room temperature.…”

Regional Clustering of Shared Neutralization Determinants on Primary Isolates of Clade C Human Immunodeficiency Virus Type 1 from South Africa

“…Group M is further divided into 10 phylogenically related genetic subtypes (clades A, B, C, D, F1, F2, G, H, J, and K) that, together with a growing number of circulating intersubtype recombinant forms, comprise the majority of HIV-1 variants in the world today. Clade C is emerging as most prevalent, being common in India (15,16,31,41) and the southern African countries of Botswana, Zimbabwe, Malawi, Mozambique, and South Africa (7,8,25,26,60,64,79,81). Clade B is dominant in North America and Western Europe and has been a major focus for vaccine development (27).…”

Worldwide molecular epidemiology of HIV

“…20,69,77,83 Subtype C has been reported in Malaysia and Southwest China, which may reflect the virus spreading via links with India where this subtype predominate. 20,25,110,136 In Tanzania (Africa) 3 subtype C shows 50% prevalence followed by subtypes A and B. Recent epicenter of HIV subtype C has been verified in Southern Africa involving Botswana, Zimbabwe, Malawi, Zambia, Namibia, South Africa and it has spread to India, Nepal and China.…”

“…Other Thai subtype named B B is identif ied by HLGPGQAW and PLGPGQAW octameric motifs also present in Myanmar and China. 20 In Brazil subtype B strains (or B Br ) present predominantly GPGGAF motif that circulate also in other South American countries including Argentina, Uruguay and Molecular epidemiology of HIV Requejo HIZ Venezuela. 13 Another subtype B with V3 loop motif GPGRTW was recently described in Venezuela.…”