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88 Automated, High Throughput HIV-1 Subtyping from Small Volumes of Plasma by Taqman Real-Time PCR and Probes Specific for Subtypes A, C, and D
L. Malaza*1, M. Hoelscher2, C. Williamson1, J. K. Carr3, D. Birx3, and F. McCutchan4
1Univ. of Cape Town, South Africa; 2Univ. of Munich, Germany; 3Henry M. Jackson Fndn., Rockville, MD, USA; and 4Walter Reed Army Inst. of Res., Rockville, MD, USA
Background: As the HIV-1 pandemic has grown, the simultaneous presence of multiple HIV-1 subtypes in certain regions of the world has become common. Thus, there is a need to establish the prevalence and distribution of the recombinant forms in those populations, particularly in areas designated for vaccine trials.
Methods: Study subjects in a high risk setting in Southwest Tanzania were sampled at 2 time points 9 months apart. Viral RNA extracted and purified with a robot from 200 mul of plasma was reverse transcribed and the cDNA was amplified by PCR with highly conserved HIV-1 primer sequences in 5 genome regions. Second-round PCRs were performed with highly conserved primers in Taqman real-time PCR, which included 3 subtype-specific fluorescent probes. Recombinant strains were distinguished by different subtype determinations in different genome regions, whereas strains with the same subtype in all regions were presumed nonrecombinant.
Results: More than 450 assays were performed on samples from 55 individuals, 36 of which were followed up with a second blood draw. More than 85% of all real-time PCRs were positive and 92.1% of determinations were concordant between serial samples. Most of the discordant results were due to dual-subtype reactivity in 1 or both samples. Among the 55 individuals, 31% were infected with subtype C, 9% with subtype A, and 9% with subtype D; 51% harbored a recombinant strain. In 16 comparisons of serial samples from recombinant infections, 13 had recombinants of the same apparent structure (e.g., ACAAA in plasma 1, ACAAA in plasma 2).
Conclusions: This new approach is a powerful tool for rapid HIV-1 subtyping in populations infected exclusively with subtypes A, C, D, and their recombinants, with superior power to discriminate subtypes from recombinants compared with other screening approaches. Utilization of plasma rather than PBMC, minimal consumption of clinical samples, and high throughput are additional advantages. Application of the technique should permit much more rapid and efficient characterization of potential cohorts for HIV-1 vaccine evaluation in East Africa.
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