Relating CYP2B6 genotype and efavirenz resistance among post-partum women living with HIV with high viremia in Uganda: a nested cross-sectional study

Study design

This was a cross-sectional study nested in “Tracing non-rEtained HIV Positive pregnant Women enrolled in option B + and ascertaining their babIeS outcomes (sTEPWISe)” study conducted between July 2017 and July 2018 [11].

Study site and population

The sTEPWISe study was conducted at HIV clinics of Kampala City Council Authority which are supported by the Infectious Diseases Institute (IDI) through CDC-PEPFAR. Study activities leveraged on routine services offered at the PMTCT clinics for newly diagnosed pregnant Women Living With HIV (WLHIV). The study aimed to trace pregnant WLHIV initiated on ART during pregnancy and disengaged from care, and were at least 6–12 weeks postpartum. Adherence to ART was self-reported and determined by clinicians as good, fair, or poor if participant reported missing < 2, 2–4, and ≥ 5 doses per month respectively. A participant was defined as disengaged from care if no clinic visit was recorded 90 days preceding database closure.

Sampling method

A random sample of retained mothers with viralogical failure (viral load ≥ 1000 copies/ml) was tested for presence of SNPs that influence plasma EFV concentration exposure and HIV resistance mutations to EFV. Seven participants were also randomly selected for EFV drug level measurement to confirm adherence status. A random sample of women disengaged from care were contacted by telephone and home visited to consent for blood draw for the aforementioned tests.

Laboratory studies

Drug resistance testing was performed at the Infectious Diseases Institute Core Laboratory, Kampala, Uganda, which is accredited by the College of American Pathologists (CAP). Viral RNA was isolated from plasma samples using the Invitrogen Purelink viral RNA/DNA kit (Thermo Fisher Scientific, Dreieich, Germany). HIV-1 genotyping was performed on extracted RNA using an in-house HIV-1 genotyping assay covering the protease (PR) and reverse transcriptase (RT) with subsequent sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Dreieich, Germany). Sequence analysis was performed using AB3730 capillary sequencer (Thermo Fisher Scientific, Dreieich, Germany).

Genotyping of the three CYP2B6 polymorphisms that predict increased EFV exposure was undertaken at the University of Liverpool. The E.Z.N.A. Blood DNA Mini Kit (Omega Bio-Tek Inc., Norcross, Georgia, USA) was used to extract genomic DNA, which was then quantified using NanoDrop (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) and stored at − 20 °C until analysis. A real-time PCR allelic discrimination assay on a DNA Engine Chromo4 system was used for genotyping (Bio-Rad Laboratories Inc., Hercules, California, USA). The PCR protocol involved denaturation of DNA at 95 °C for 10 min, 40 cycles of amplification at 95 °C for 15 s and annealing at 60◦C for 1 min. TaqMan Genotyping Master Mix and assays for CYP2B6 (516G → T [rs3745274], 983 T → C [rs28399499], and 15582C → T [rs4803419]) were obtained from Life Technologies Ltd (Paisley, Renfrewshire, UK). Allelic discrimination plots and genotype assignments were performed using Opticon Monitor, version 3.1 (Bio-Rad Laboratories Inc.).

Analysis

Drug resistance-associated mutations in protease and reverse transcriptase gene regions were interpreted using the Stanford HIV database (HIVdb) program. To predict susceptibility to EFV a resistance score was calculated and classified as susceptible, low, intermediate, and high-level following the Stanford HIVdb scoring system (http://hivdb.stanford.edu) [13] Metabolizer genotype was categorized according to the ACTG algorithm as slow, intermediate, and extensive. We used multivariate logistic model to assess association between EFV resistance and metabolizer genotypes.

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