HIV-1C in-House RNA-Based Genotyping Assay for Detection of Drug Resistance Mutations in Samples with Low-Level Viral Loads

Abstract

Monitoring HIV-1 drug resistance mutations (DRM) in treated patients on combination antiretroviral therapy (cART) with a detectable HIV-1 viral load (VL) is important for the selection of appropriate cART. Currently, there is limited data on HIV DRM at low-level viremia (LLV) (VL 401-999 copies/mL) due to the use of a threshold of VL ≥1000 copies/mL for HIV DRM testing. We here assess the performance of an in-house HIV drug resistance genotyping assay using plasma for the detection of DRM at LLV. Methods: We used a total of 96 HIV plasma samples from the population-based Botswana Combination Prevention Project (BCPP). The samples were stratified by VL groups: 50 samples had LLV, defined as 401-999 copies/mL, and 46 had ≥1000 copies/mL. HIV pol (PR and RT) region was amplified and sequenced using an in-house genotyping assay with BigDye sequencing chemistry. Known HIV DRMs were identified using the Stanford HIV Drug Resistance Database. Genotyping success rate between the two groups was estimated and compared using the comparison of proportions test. Results:The overall genotyping success rate was 79% (76/96). For VL groups, the genotyping success was 72% (36/50) at LLV and 87% (40/46) at VL ≥1000 copies/mL. Among generated sequences, the overall prevalence of individuals with at least 1 major or intermediate-associated DRM was 24% (18/76). The proportions of NNRTI-, NRTI-and PI-associated resistance mutations were 28%, 24%, and 0%, respectively. The most predominant mutations detected were K103N (18%) and M184V (12%) in NNRTI-and NRTIassociated mutations, respectively. The prevalence of DRM was 17% (6/36) at LLV and 30% (12/40) at VL ≥1000 copies/mL. Conclusion:The in-house HIV genotyping assay successfully genotyped 72% of LLV samples and was able to detect 17% of DRM amongst them. Our results highlight the possibility and clinical significance of genotyping HIV among individuals with LLV.

High prevalence of pre-treatment and acquired HIV-1 drug resistance mutations among non-citizens living with HIV in Botswana

“…Using in-house genotypic resistance testing ( Seatla et al, 2019 ; Bareng et al, 2022 ), the extracted RNA was amplified by nested Polymerase chain reaction (PCR) to obtain the HIV-1 pol which comprised the entire protease (PR) gene (codons 1-99) and reverse transcriptase (RT) gene (codons 1-560). The RT-PCR was done using the following primers ( Rowley et al, 2016 ; Bareng et al, 2022 ).…”

“…Both one-step RT-PCR and nested PCR amplifications were carried out in the Applied Biosystems 2,720 Thermal Cycler. The RT-PCR was performed using Roche one-step transcriptor enzyme while the nested PCR was done using Phusion High Fidelity enzyme ( Rowley et al, 2016 ; Bareng et al, 2022 ; Kelentse et al, 2023 ).…”

“…Both one-step RT-PCR and nested PCR amplifications were carried out using Applied Biosystems 2,720 Thermal Cycler. Primers used for the first and second rounds were adopted from the inhouse protocol, ( Seatla et al, 2019 ; Bareng et al, 2022 ) without modification.…”

Clinical significance and management of low‐level HIV viremia in the era of integrase strand transfer inhibitors

“…However, if the VL falls below 1000 copies/mL, the amplification success rate for drug resistance testing by genotyping will be low. In contrast, studies identified drug‐resistant mutations (DRMs) even in LLV, and the presence of DRMs in LLV strongly predicted subsequent VF [23, 24]. HIV RNA genotyping resistance testing (GRT) may not be a reliable option for PLWH with LLV due to the possibility of results being inaccurate with HIV RNA <500 copies/mL or < 1000 copies/mL [13, 25].…”

“…DHHS guidelines suggest GRT in viral levels above 1000 copies/mL and warn that GRT taken within 500–1000 copies/mL may be unsuccessful [13]. DRMs have been reported at variable percentages in different studies [23]. Results from a multicentre cohort study in Europe found that of 1654 individuals experiencing LLV, 140 (8%) underwent a registered resistance test within 90 days of the first VL recorded during LLV, and of these 49 (35%) had DRMs ≥1 [20].…”