Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana

Abstract

Background: HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs). Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure. Methods: A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results: Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants. There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion: The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Advancing HIV Drug Resistance Technologies and Strategies: Insights from South Africa’s Experience and Future Directions for Resource-Limited Settings

“…Another interesting technology is the pan-degenerate amplification and adaptation (PANDAA) technology, which is based on quantitative PCR and uses extremely degenerate primers that target specific mutation sites and can cope with high HIV diversity next to the mutation site [ 53 ]. This technology has been tested in several settings [ 54 , 55 ] and shows high specificity and sensitivity. However, the need for a quantitative PCR instrument remains a challenge for POC applications.…”

Point-of-Care Tests for HIV Drug Resistance Monitoring: Advances and Potentials

“…In a traditional qPCR reaction, the binding of probes that are not in perfect complementation to the template are unstable and can produce false-negative results. In contrast, the PANDAA assay addresses high sequence variability through normalization of probe-binding regions, as seen in Figure 1 B. PANDAA primers have two main features: (1) a pan-degenerate region (PDR) containing degenerate bases to account for nucleotide variability and (2) an adaptor region (AR) that matches the probe-binding regions flanking the mutation of interest [ 24 ].…”

“…The results demonstrated that PANDAA rendered excellent sensitivity (95~98%) and specificity (83~100%), although the readouts fluctuated among assays targeting different HIVDRMs. Maraupala et al conducted another study to test the diagnostic accuracy of PANDAA against Sanger in a cohort of patients from Botswana to use the assay as an alternative approach for rapid HIVDR test, and high concordance was observed between the data obtained from the two compared assays [ 24 ]. This positions PANDAA as a promising assay for HIVDR, although further refinement is required to meet the ASSURED criteria [ 7 ].…”

Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana

“…Figshare: Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana; https://doi.org/10.6084/m9.figshare.12644930 23 .…”