Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

Abstract

High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5= end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.

Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

“…The CDC supplies WHO-designated and CDC-supported President's Emergency Plan for AIDS Relief (PEPFAR) Genotyping Laboratories with the ATCC HIV-1 Drug Resistance Genotyping kit (46) for drug resistance testing. Many experienced genotyping laboratories have developed their own in-house amplification and sequencing protocols (11,(47)(48)(49)(50)(51)(52)(53)(54)(55)(56), including identification of minor viral variants that are normally missed by commercial genotyping kits (57-61). All of these approaches generally include smaller and more restricted regions for testing HIV-1 drug resistance.…”

Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

“…Low-abundance mutations in patient specimens were detected by the MAS assay but missed by Sanger sequencing. The major advantages of the MAS assay are straightforward result interpretation and high throughput (5). Unlike common sequencing methods, allele-specific assays directly detect each targeted mutation.…”

“…Applying suspension array technology (4), we developed a multiplex allele-specific (MAS) assay that simultaneously detects major HIV drug resistance mutations (DRMs) at 20 loci in subtype C viruses (5). Because of significant genetic variation between different HIV-1 subtypes, not all allele-specific primer extension (ASPE) primers designed on the basis of subtype C sequences will work for other subtypes (5,6).…”

“…Because of significant genetic variation between different HIV-1 subtypes, not all allele-specific primer extension (ASPE) primers designed on the basis of subtype C sequences will work for other subtypes (5,6). Since subtype B is dominant in many countries in the Americas, Europe, Asia, and Africa (7), we modified the original subtype C MAS assay to detect DRMs in subtype B viruses and evaluated the performance of the MAS assay with dried blood spots (DBS) collected from patients on ART in this study.…”

“…The nested PCR products were then analyzed by the MAS assay, and the results were compared to Sanger sequencing results (9). To verify the additional low-abundance alleles detected by the MAS assay, selected DBS samples with low-abundance alleles were analyzed by next-generation sequencing with Roche 454 GS-FLX Titanium sequencing kit XLR70 (Roche Applied Science, Indianapolis, IN, USA) (5).…”