A validated in-house assay for HIV drug resistance mutation surveillance from dried blood spot specimens.

Despite increasing scale-up of antiretroviral therapy (ART) coverage, challenges related to adherence and HIV drug resistance (HIVDR) remain. The high cost of HIVDR surveillance is a persistent challenge with implementation in resource-constrained settings. Dried blood spot (DBS) specimens have been demonstrated to be a feasible alternative to plasma or serum for HIVDR genotyping and are more suitable for lower resource settings. There is a need for affordable HIVDR genotyping assays which can amplify HIV-1 sequences from DBS specimens, particularly those with low viral loads, at a low cost. Here, we present an in-house assay capable of reliably amplifying HIV-1 protease and partial reverse transcriptase genes from DBS specimens, which covers the complete World Health Organization 2009 list of drug resistance mutations under surveillance. DBS specimens were prepared using whole blood spiked with HIV-1 at concentrations of 10,000, 5000, 1000, and 500 copies/mL (n=30 for each concentration). Specimens were tested in triplicate. A two-step approach was used consisting of cDNA synthesis followed by nested PCR. The limit of detection of the assay was calculated to be approximately 5000 (95% CI: 3200-10,700) copies/mL for the protease gene and 3600 (95% CI: 2200-10,000) copies/mL for reverse transcriptase. The assay was observed to be most sensitive with higher viral load specimens (97.8% [95% CI: 92.2-99.7]) for both protease and reverse transcriptase at 10,000 copies/mL with performance decreasing with the use of specimens with lower viral loads (46.7% [36.1-57.5] and 60.0% [49.1-70.2] at 500 copies/mL for protease and reverse transcriptase, respectively). Ultimately, this assay presents a promising opportunity for use in resource-constrained settings. Future work should involve validation under field conditions including sub-optimal storage conditions and preparation of DBS with fingerprick blood in order to accurately reflect real-world collection scenarios.

HIV acquisition prior to entry into formal sex work: inference from next-generation viral sequencing.

OBJECTIVE: To infer the timing of HIV acquisition in relation to self-reported events in the sexual life course of adolescent girls and young women (AGYW) who self-identify as female sex workers (FSW) in Mombasa, Kenya. DESIGN: Next-generation viral sequencing of samples of AGYW living with HIV in the Transitions study, a cross-sectional bio-behavioural survey of AGYW aged 14-24 years in Mombasa, Kenya. METHOD: Dried blood spot specimens were collected from study participants ( n  = 37, all FSW). A portion of the HIV pol gene was sequenced using an in-house next-generation sequencing assay for HIV drug resistance mutation genotyping. Estimated time since infection (ETI) was inferred using the HIV EVO web-based tool ( https://hiv.biozentrum.unibas.ch/ETI/ ), and data on self-reported events were obtained from the survey. RESULTS: The median ETI among FSW was 3.4 (interquartile range = 1.7, 6.3) years, with a median ETI of 1.5 years prior to entry into formal sex work. We estimated that 74.1% (95% confidence interval = 53.7-88.9%) of participants living with HIV and who self-identified as FSW likely acquired HIV prior to self-identification as a sex worker. CONCLUSIONS: Findings suggest a large fraction of prevalent HIV infection among AGYW engaged in sex work stems from acquisition prior to entry into formal sex work. Current HIV prevention programs tailored for sex workers may miss key opportunities for HIV prevention as they are designed to reach women after entry into formal sex work, signaling a need for tailored programs to reach high-risk AGYW earlier on in their sexual life course.