A team of authors have described a method for multiplex allele-specific assay (MAS) for HIV-1 drug resistance mutations. This assay allows for the detection of PI mutations at 5 loci, NRTI mutations at 8 loci and NNRTI mutations at 7 loci associated with resistance to Anti-retroviral drugs used in resource limited settings. Five of the mutation loci had 2 mutations targeted, therefore 2 primers specific to those mutant alleles were designed. The assay included 20 wild-type alleles and 25 mutant alleles, to give a total of 45 primers for the assay.
The primer design was based on HIV-1 Subtype C, but modifications for non-C subtypes are currently being investigated. This new method has significant implications in the Southern African context since it allows for the simultaneous detection of several resistance mutations for HIV-1 subtype C, which is predominant in this region. MAS assay can potentially reduce the costs associated with other resistance testing methods like population sequencing since equipment for suspension array technology is cheaper. Detection of mutations using MAS assay can be accomplished in a shorter time, making this method more time-efficient.
The simultaneous detection of several resistance mutations within the protease and reverse transcriptase genes with MAS assay has an advantage over current allele-specific PCR methods, in which assays are tailored to single mutation sites. MAS assay allows for the addition or removal of primers for particular known mutation sites when they need to be included or excluded in the assay. When compared to population-based sequencing which detects virus mutations in abundance of 20%, this assay can detect low-frequency mutations with a sensitivity of up to 1.6% for some mutations. The results of low-frequency mutations detected by MAS in plasma samples were confirmed by 454 sequencing.
This assay presents a methodology that is quick and easy to interpret. Therefore, it can be used in resource limited settings for surveillance and monitoring of transmitted or acquired HIV-1 drug resistance in countries where subtype C virus predominates. Mutations like K103N/R, Y181C and M184V are commonly described in South Africa for subtype C viruses, therefore this assay may prove to be useful for surveillance of these and other mutations. However, this assay cannot be used to detect new drug resistance mutations (unknown mutations) and may not be suitable for clinical diagnostics in patient management.
Blog by: Anna Salimo, Medical Scientist, Centre for HIV and STIs, National Institute of Communicable Diseases, Johannesburg, South Africa.
Manuscript title: Simultaneous detection of major drug resistance mutations in the protease and reverse transcriptase genes for HIV-1 Subtype C using a multiplex allele-specific (MAS) assay.
Authors: Zhang G, Cai F, Zhou Z, DeVos J, Wagar N, Diallo K, Zulu I, Wadonda-Kabondo N, Stringer JSA, Weidle PJ, Ndongmo CB, Sikazwe I, Sarr A, Kagoli M, Nkengasong J, Gao F and Yang Chunfu.
Journal: Journal of Clin Micro 28 August 2013, published online ahead of print.