Paper # 572 
Evaluation of an Affordable Assay for Drug-resistance Genotyping of All Major HIV-1 Subtypes
Susan Aitken*1,2, A Kliphuis2,3, C Wallis1, M L Chu2, W Stevens1,4, T Rinke de Wit3, R Schuurman2, and the Consortium to Develop an Affordable Resistance Test for Africa
1Univ of the Witwatersrand, Johannesburg, South Africa; 2Univ Med Ctr Utrecht, The Netherlands; 3Ctr for Poverty-related Communicable Diseases, PharmAccess Fndn, Academic Med Ctr, Amsterdam, The Netherlands; and 4Natl Hlth Lab Svc, Johannesburg, South Africa
Background: Current commercial kits for HIV-1 drug-resistance genotyping are
expensive and in some cases feature reduced detection sensitivity of HIV-1
non-B subtypes, thus limiting application in resource-constrained settings. The
aim of our research was the development and evaluation of an affordable,
subtype-independent genotyping assay for use in monitoring HIV drug resistance
in Africa.
Methods: A
subtype-independent, nested polymerase chain reaction (PCR) encompassing the
entire protease and the reverse transcriptase up to amino acid 321 of HIV-1 was
designed to detect all group M subtypes of HIV-1. The nested PCR was evaluated
using a panel of reference viruses for subtypes A, B, C, D, A/E, F, G, and H.
In addition, the analytical sensitivity of the nested PCR and a set of
sequencing primers compatible with all subtypes were determined. This assay was
evaluated on 155 plasma samples from HIV-infected females from Africa harboring various viral loads. Sequence reactions were
analyzed on an ABI3100 automated sequencer and SeqScape data
analysis software (Applied Biosystems).
Results: All
major HIV-1 subtypes could be detected in the nested PCR with an analytical
sensitivity of 500 RNA copies/mL. Application of the genotyping assay on 155
primarily African clinical samples comprising of subtypes A (27%), B (8%), C
(42%), D (11%), AE (6%), and AG (6%), with a viral load range from 434 to 30,700,000
(median 365,000) RNA copies/mL was 94% successful: 84% were genotyped using
the primary set of 6 sequencing primers, and an additional 10% using 1 or 2
second choice primers. Of the 155 sample, 7 were unable to give a full
bi-directional sequence in the protease region, and 2 in the reverse
transcriptase region.
Conclusions: We have
developed an affordable genotyping assay for HIV drug resistance. The assay can
be applied to all group M HIV-1 subtypes with adequate analytical sensitivity
and is potentially suitable for use in resource-constrained settings. Due to
the sensitivity of this assay, it has potential application in combination with
dried fluid spot samples.
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