Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings

Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX

Pairwise sequence identity analysis between the optimized in-house and TRUGENE® assays.

<p>Pairwise sequence identity analysis between the optimized in-house and TRUGENE® assays.</p

Difference of mixture chromatographs generated independently by 3 different operators using the optimized in-house assay from one PT sample.

<p>Panel A shows 2 codons (37 and 41 of RT) with nucleotide base calling of AYR; Panel B shows the AWR at codon 41 (the second peaks at codon 37 were not detected in this replicate); Panel C shows ACR at codon 37 (minor T was not called by the ReCall at the cutoff of 15%) and AHR at codon 41 (almost equal height of second and third peak at the 2^nd position).</p

Genotyping efficiency and drug resistance-associated mutations identified in protease (PR) and reverse transcriptase (RT) by the optimized in-house assay from dried blood spots (DBS) PT panels.

<p>*N/A:not available; bold and underlined residues were partially discordant resistance mutations from paired DBS shipped under different temperature conditions.</p

Summary of samples used in the study, including plasma and dried blood spots (DBS).

a<p>HIV negative specimens collected from pregnant women in Tanzania used for assay specificity analysis; ^b Plasma-matched DBS samples collected from voluntary counseling and testing (VCT) sites in Ho Chi Minh City enrolled in an HIV-1 threshold survey; ^cPlasma-matched DBS samples collected from patients enrolled in the Nigeria HIVDR perspective monitoring survey at 12-15 months after commencement of first line antiretroviral therapy; ^d Not available.</p

Primers used in the optimized in-house assay.

<p>*: PRTM-F1 is a mixture of primers F1a and F1b at a ratio of 1:1 (w/w).</p

Genotyping reproducibility of replicate PCR products generated from independent RT-PCR amplification process by 3 different operators in a 5-member proficiency testing panel received from VQA.

<p>*IH1-3: tests were independently performed by 3 operators using the optimized in-house assay; ^#:TRUGENE® assay.</p

HIV-1 subtypes and circulating recombinant forms (CRFs) from samples genotyped by the optimized in-house assay.

<p>HIV-1 subtypes and circulating recombinant forms (CRFs) from samples genotyped by the optimized in-house assay.</p