Multiclade HIV-1 Infection in Pakistan

Minh-Hien T. Nguyen ^a, Fumiyuki Isami^a, M.A Memon^b, SAS, Sten H. Vermund^c, Richard Yanagihara^a, and Vivek R. Nerurkar^a

aRetrovirology Research Laboratory, Pacific Biomed Research Center, University of Hawaii at Manoa, Honolulu, Hawaii; ^bSindh AIDS Control Programme, Indoor Block, Services Hospital, Karachi, Pakistan; and ^cAIDS International Training and Research Program, Division of Geographic Medicine, University of Alabama, School of Medicine, Birmingham, Alabama.

Introduction: High-risk heterosexual sex is the predominant mode of HIV-1 transmission in Pakistan. Alarmingly high rates of HIV-1 infection have been found among commercial sex workers (CSW), injection drug users (IDU), long-distance truck drivers and women attending antenatal clinics in major cities throughout India. By contrast, HIV-1 prevalence rates among such groups in Pakistan are still low or undetectable. Internationally recognized high-risk groups such as CSW, truck drivers and IDU have remained more or less free from HIV infection. At the same time, a new category of high-risk group, namely overseas contract workers, has emerged in Pakistan with HIV-1 prevalence rates as high as 65%. Widespread multiclade HIV infection has been recognized in India and sub-Saharan Africa. No data are available on the subtypes of HIV circulating in Pakistan. Objective: The principal objective of this study was to characterize HIV-1 subtypes circulating in Pakistan, among overseas contract workers and individuals with a history of international travel. Materials and Methods: After a week-long delay in transit, buffy coat and plasma samples from 13 HIV-infected residents of Pakistan (11 Pakistani, one Kenyan and one Zairian), originally shipped on dry ice, arrived in a thoroughly thawed condition. Nevertheless, RNA and/or DNA were extracted from all specimens and HIV-1 gag, env, and nef gene sequences were amplified by PCR. Amplicons were cloned using the TA Cloning Kit and sequenced or were directly purified using QIAquick PCR Purification Kit and sequenced. Nucleotide and deduced amino acid sequences of the HIV-1 strains from Pakistan were aligned and compared with sequences of HIV-1 subtypes from various geographic regions, using programs available on the Lasergene DNASTAR software. Phylogenetic trees were generated using the maximum parsimony and neighbor-joining methods. Results and Conclusions: The presumed mode of HIV-1 acquisition among all study subjects was heterosexual intercourse. Phylogenetic analyses based on gag, env and nef genes revealed multiclade HIV-1 infection in Pakistan; subtype A (3), subtype C (7), and subtype D (3). The multiclade nature of HIV-1 infection in Pakistan has not been previously reported. The HIV-1 subtype A sequence from a Kenyan woman living in Pakistan was similar to subtype A sequences from Africa, whereas high sequence similarity was observed between subtype A sequences from two Pakistani nationals employed as overseas contract workers. Four of six Pakistani HIV-1 subtype C were closely related to subtype C strains from India. Although HIV-1 subtype D has not yet been found in India, three Pakistani nationals were infected with subtype D, suggesting introduction of subtype D from countries other than India. It is not surprising that multiclade HIV-1 infection exists in Pakistan, since all study subjects had a history of overseas employment, specifically in the United Arab Emirates and/or had traveled to India. The multiclade nature of HIV-1 infection in Pakistan is consistent with the introduction of HIV-1 via returning oversea contract workers and immigrants. Since HIV-1 subtype C is rapidly spreading in Asia and sub-Saharan Africa, more studies on subtype C virulence and strategies to incorporate subtype C in HIV-1 vaccines are warranted. [Supported by USPHS grant G12AI/RR-03061 from the Research Centers in Minority Institutions Program, National Institutes of Health]