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

Abstract

Purpose

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 NRTI-associated 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.

Keywords:

• in-house genotyping
• low-level viremia
• samples
• HIV-1C drug resistance testing

Data Sharing Statement

All relevant data are within the paper, including the figures and tables. The sequences obtained in our study were submitted to National Center for Biotechnology Information (NCBI) GenBank and the accession numbers are OP922678 – OP922753.

Ethics Approval

The BCPP study was approved by the Institutional Review Boards (IRBs) at the US Centers for Disease Control and Prevention and the Botswana Health Research and Development Committee and is registered at ClinicalTrials.gov (NCT01965470). The study was conducted according to the principle expressed in the declaration of Helsinki. All BCPP participants provided written informed consent for their samples to be used in future studies.

Acknowledgments

We thank all the study participants. We thank the BCPP study team for their contribution to this study. We thank the Ministry of Health and Wellness, Botswana Harvard AIDS Institute Partnership, CDC Botswana, and US CDC for their excellent support and contributions to the BCPP study. We also thank Katlego Selelo, Pearl Kaumba and Charlene Raphaka for their assistance with the laboratory work and Lendsey Melton for his valuable edits.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in relation to this work.

Additional information

Funding

The BCPP Impact Evaluation was funded by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC, cooperative agreements U01 GH000447 and U2G GH001911). OTB was supported by the Fogarty International Center at the US National Institutes of Health (D43 TW009610). SM is partially supported by the Fogarty International Center at the US National Institutes of Health (1 K43 TW 012350-01). SG and WTC were partially supported by H3ABioNet. H3ABioNet is supported by the National Institutes of Health Common Fund (U41 HG006941). H3ABioNet is an initiative of the Human Health and Heredity in Africa Consortium (H3Africa) program of the African Academy of Science (AAS). SM, RM, DD & OTB were supported by the Trials of Excellence in Southern Africa (TESA III), which is part of the EDCTP2 program supported by the European Union (CSA2020NoE-3104 TESAIII CSA2020NoE). SG, SM, WTC and NOM are partly supported through the Sub-Saharan African Network for TB/HIV Research Excellence (SANTHE 2.0) from the Bill and Melinda Gates Foundation (INV-033558). PANGEA-HIV is funded primarily by the Bill and Melinda Gates Foundation. The views expressed in this publication are those of the authors and not necessarily those of the funding agencies. The funders had no role in the study design, data collection, decision to publish, or preparation of this manuscript.