Affordable in-house antiretroviral drug resistance assay with good performance in non-subtype B HIV-1

Abstract

The introduction of antiretroviral therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. This has led to a demand for affordable HIV-1 drug resistance assays, since treatment failure due to development of drug resistance is common. This study developed and evaluated an affordable “in–house” genotyping assay to monitor HIV-1 drug resistance in Africa, particularly South Africa. An “in-house” assay using automated RNA extraction, and subtype C specific PCR and sequencing primers was developed and successfully evaluated 396 patient samples (viral load ranges 1,000->1.6million RNA copies/ml). The “in-house” assay was validated by comparing sequence data and drug resistance profiles from 90 patient and 10 external quality control samples to data from the ViroSeqTM HIV-1 Genotyping kit. The “in-house” assay was more efficient, amplifying all 100 samples, compared to 91 samples using Viroseq. The “in house” sequences were 99.2%) homologous to the ViroSeq sequences, and identical drug resistance mutation profiles were observed in 96 samples. Furthermore, the “in-house” assay genotyped 260 of 295 samples from seven African sites, where 47% were non-subtype C. Overall, the newly validated “in-house” drug resistance assay is suited for use in Africa as it overcomes the obstacle of subtype diversity.

Performance evaluation of an in-house human immunodeficiency virus type-1 protease-reverse transcriptase genotyping assay in Cameroon

“…The capacity to identify these subtypes was greater compared to other in-house antiretroviral drug resistance assays used previously in limited-resource settings. These previous in-house assays were restricted either to the sequencing of four pure subtypes (A1, B, C, D) and two known CRFs (CRF01_AE and CRF02_AG) in South Africa [25] or to three pure subtypes (B, C, D) in the case of the Indian study [24]. Therefore, the results indicate a highly successful sequencing performance using the in-house protocol in a context of high HIV-1 genetic diversity, as is found in sub-Saharan Africa [42,43].…”

“…Interestingly, it should be considered that viral dynamics (due to the high error rate of RT), evolution over time (due to the high levels of HIV-1 replication), and the large spread of non-B subtypes might influence the overall performance of the existing genotyping tools [22]. Improved sensitivity of in-house sequencing assays compared to these commercially available kits has been reported by several laboratories, especially for some non-B subtypes [23][24][25]. These established in-house genotyping assays were designed in order to overcome cost and subtype-specific constraints [26,27].…”

“…These established in-house genotyping assays were designed in order to overcome cost and subtype-specific constraints [26,27]. However, the performance of these in-house assays has not been validated against internationally approved reference systems [28,29], except for two recent ones, developed for subtypes B, C, and D circulating in India, and subtype C circulating in West and South Africa [24,25].…”

A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

“…The size of the amplicon is roughly half the length of those generated using commercial and in-house HIV-1 drug resistance genotyping assays (11,15,16). By amplifying as little as possible of the RT using highly fine-tuned primer combinations, focusing on the region encompassing all of the relevant HIV-1 drug resistance mutations, it was possible to achieve a genotyping success rate of 94.8% for clinical plasma samples with Ն1.00Eϩ3 RNA copies/ml in a single-round RT-PCR.…”

“…From a reagent perspective, using the described assay would result in a Ͼ75% savings compared to using a commercial assay such as the ViroSeq genotyping system version 2.0 (Celera Diagnostics, USA) and an approximate 40% savings compared to using our current in-house assay (11). Furthermore, the quicker laboratory protocol and shorter sequence to be analyzed result in a decrease in the labor required compared to currently available methods (11,14,15).…”