Genotyping and subtyping of non-B HIV-1 clinical  samples using a DNA Chip technology .

J N Telles¹, R Gonzalez¹, C Hebrard¹, P Breyton, G Cartet, A Troesch¹, G Vernet¹. ¹bioMerieux S.A., Venissieux (France).

Background: Our objective was to evaluate the DNA Chip technology for HIV-1 genotyping and subtype identification on non B clinical samples. Material and Methods: Viral RNA is extracted from 30 HIV-1 infected patient plasma (subtypes A to G), and amplifications covering gag cleavage sites to the end of the reverse transcriptase, gp41 and integrase are generated. Amplicons are fragmented and labeled using a proprietary technology. Labeled amplicons are purified and hybridised on an HIV-1 DNA Chip. The hybridization is performed on the fluidic station developed by Affymetrix. The Chip is designed with sequences covering all known resistance mutations in the Protease, the whole reverse transciptase, integrase, gp41 , gag cleavage sites and are representative of all group M subtypes. Data are analysed by proprietary software: a resistance mutation profile and a subtype identification for each patient are generated. The genotypes and subtypes are compared to the results obtained by a  sequencing method. Results: Our results showed more than 95% of correct mutation identification in protease, reverse transcriptase, gp41 and integrase compared with DNA sequencing, The subtype in pol gene was correctly identified in more than 90% of the samples. Conclusion: Our data showed that the HIV-1 DNA Chip® assay we developed could successfully perform genotyping in non B clinical samples (subtypes A to G). The chip was also to able to correctly identify the pol subtype in most of  the samples. We are now evaluating the ability of the chip to efficiently subtype in the gp41 region. Having a subtype identification in both pol and env would be a valuable tool to identify the recombinant forms of HIV-1.