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

Abstract

A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. There were approximately 36.9 million people worldwide living with HIV at the end of 2014 (1). Despite significant advances in antiretroviral therapy (ART), the acquisition and transmission of HIV drug resistance (HIVDR) pose challenges to the continuous success of ART programs (2). The World Health Organization recommends that HIV treatment scale-up should always be accompanied by a robust assessment of drug resistance emergence and transmission (3).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). 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.(These data were presented in part at the International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative, 5 to 9 June 2012, Sitges, Spain.)We modified the 45 subtype C ASPE primers targeting the DRMs at 20 loci that are associated with resistance to commonly used antiretrovirals. These include major nucleoside reverse transcriptase inhibitor (NRTI) DRMs at eight loci (M41L, K65R, K70R, L74V, Y115F, Q151M, M184V, and K219Q/E), major non-NRTI DRMs at seven loci (L100I, K101P/E, K103N/R, V106A/M, Y181C, Y188L, and G190A), and major protease inhibitor DRMs at five loci (V32I, I47A/V, L76V, I84V, and L90M). The lengths of the modified primers were between 24 and 39 nucleotides, and the melting temperatures (T m s) were around 60°C. For a summary of all 45 modified ASPE primer sequences, see Table S1 in the supplemental material.After the assay was optimized for the detection of all of the DRMs with plasmids for several factors affecting specificity and signal output, such as Mg 2ϩ concentration, cycling parameters, annealing temperature, and ASPE primer concentrations (5), DBS samples collected from 69 subtype B-infected patients undergoing ART were used to evaluate the modified MAS assay (8). This study was approved by the ethics committee at the National Autonomous University of Honduras. The Center for Global Health at CDC approved the study protocol as a research activity involving unlinked or anonymous data or specimens collected for another purpose. Total nucleic acid extrac...

Detection of minority drug resistant mutations in Malawian HIV-1 subtype C-positive patients initiating and on first-line antiretroviral therapy

“…In fact, we were able to amplify subtype B, B/C, F and G samples using these primers which were confirmed by gel electrophoresis and successfully detect 5 of 5 subtype B samples using deep sequencing methods. 27 …”

Diagnosis of Human Immunodeficiency Virus Infection

“…For example, the multiplex allele-specific (MAS) array assay has been optimized for 20 DRM loci for subtype B (302). A study with a subtype C-specific MAS assay optimized for DBS specimens obtained from Honduras showed a high level of agreement (99.3%) of detected major DRMs compared with data generated by Sanger-based sequencing, and the low-abundance variants were verified by Roche 454 GS-based deep sequencing (303). The results from MAS assays are easy to interpret, and if optimized for known circulating viruses in a specific country or region, the assay can yield rapid DRM surveillance data to inform ART policy in the country or region.…”

HIV-1 drug resistance and resistance testing

“…The platforms in development that appear the most amenable to POC or near-POC use in LMICs are PMAs capable of detecting key DRMs at costs below $5 USD per locus (MacLeod et al, 2015; Mutsvangwa et al, 2014; Panpradist et al, 2016; Zhang et al, 2016; Zhang et al, 2013). A major challenge to the successful development of a PMA is the extreme genetic variability of HIV-1 (Clutter et al, 2016b).…”