Assessment of minority frequency pretreatment HIV drug-resistant variants in pregnant women and associations with virologic non-suppression at term.

OBJECTIVE: To assess in ART-naïve pregnant women randomized to efavirenz- versus raltegravir-based ART (IMPAACT P1081) whether pretreatment drug resistance (PDR) with minority frequency variants (<20% of individual's viral quasispecies) affects antiretroviral treatment (ART)-suppression at term. DESIGN: A case-control study design compared PDR minority variants in cases with virologic non-suppression (plasma HIV RNA >200 copies/mL) at delivery to randomly selected ART-suppressed controls. METHODS: HIV pol genotypes were derived from pretreatment plasma specimens by Illumina sequencing. Resistance mutations were assessed using the HIV Stanford Database, and the proportion of cases versus controls with PDR to their ART regimens was compared. RESULTS: PDR was observed in 7 participants (11.3%; 95% CI 4.7, 21.9) and did not differ between 21 cases and 41 controls (4.8% vs 14.6%, p = 0.4061). PDR detected only as minority variants was less common (3.2%; 95% CI 0.2, 11.7) and also did not differ between groups (0% vs. 4.9%; p = 0.5447). Cases' median plasma HIV RNA at delivery was 347c/mL, with most (n = 19/22) showing progressive diminution of viral load but not ≤200c/mL. Among cases with viral rebound (n = 3/22), none had PDR detected. Virologic non-suppression at term was associated with higher plasma HIV RNA at study entry (p<0.0001), a shorter duration of ART prior to delivery (p<0.0001), and randomization to efavirenz- (versus raltegravir-) based ART (p = 0.0085). CONCLUSIONS: We observed a moderate frequency of PDR that did not significantly contribute to virologic non-suppression at term. Rather, higher pretreatment plasma HIV RNA, randomization to efavirenz-based ART, and shorter duration of ART were associated with non-suppression. These findings support early prenatal care engagement of pregnant women and initiation of integrase inhibitor-based ART due to its association with more rapid suppression of plasma RNA levels. Furthermore, because minority variants appeared infrequent in ART-naïve pregnant women and inconsequential to ART-suppression, testing for minority variants may be unwarranted.

Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed. We developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. The extracted RNA is then submitted to RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV virologic failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). In contrived samples, the in-house RNA extraction workflow achieved a detection limit of 200-copies per mL for HIV RNA in plasma and 100-copies per mL for SARS-CoV-2 RNA in NS. Similar detection limits were observed for HIV and SARS-CoV-2 in pooled plasma/NS contrived samples. When comparing in-house with standard extraction methods, we found high agreement (>0.91) between input and measured RNA copies for HIV LTR in contrived plasma; SARS-CoV-2 N1/N2 in contrived NS; and LTR, N1, and N2 in pooled plasma/NS samples. We further evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-positive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 combined plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house RNA extraction to those using a commercial kit (standard extraction method). The in-house extraction and standard extraction of clinical specimens were positively correlated: plasma HIV VL (R2 of 0.81) and NS SARS-CoV-2 VL (R2 of 0.95 and 0.99 for N1 and N2 genes, respectively); and pooled plasma/NS HIV VL (R2 of 0.71) and SARS-CoV-2 VL (R2 of 1 both for N1 and N2 genes). Our low-cost molecular test workflow ($1.85 per pooled sample extraction) for HIV RNA and SARS-CoV-2 RNA could serve as an alternative to current standard assays ($12 per pooled sample extraction) for laboratories in low-resource settings.

Cost-effectiveness analysis of pre-ART HIV drug resistance testing in Kenyan women.

BACKGROUND: The prevalence of pre-treatment drug resistance (PDR) to non-nucleoside reverse-transcriptase inhibitor (NNRTI) agents is increasing in sub-Saharan Africa, which may decrease the effectiveness of efavirenz-based antiretroviral therapy (ART) programs. However, due to recent safety concerns, there has been hesitancy to replace efavirenz-based ART with dolutegravir in women of reproductive potential. Our objective was to evaluate whether PDR testing for women not initiating dolutegravir-based ART would be a cost-effective strategy to address the challenges posed by PDR. METHODS: We developed an HIV drug resistance model that simulates the emergence and transmission of resistance mutations, calibrated to the Kenyan epidemic. We modeled three care strategies for PDR testing among women not initiating dolutegravir-based ART: no PDR testing, PDR testing with a low-cost point mutation assay, known as oligonucleotide ligation assay (OLA), and PDR testing with consensus sequencing. Using a health sector perspective, this model was used to evaluate the health outcomes, lifetime costs, and cost-effectiveness under each strategy over a 15-year time horizon starting in 2019. FINDINGS: OLA and CS PDR testing were projected to have incremental cost-effectiveness ratios (ICER) of $10,741/QALY gained and $134,396/QALY gained, respectively, which are not cost-effective by national income standards. Viral suppression rates among women at 12 months after ART initiation were 87·8%, 89·0%, and 89·3% with no testing, OLA testing, and CS testing, respectively. PDR testing with OLA and CS were associated with a 0.5% and 0.6% reduction in incidence rate compared to no PDR testing. Initial PDR prevalence among women was 13.1% in 2019. By 2034, this prevalence was 17·6%, 17·4%, and 17·3% with no testing, OLA testing, and CS testing, respectively. INTERPRETATION: PDR testing for women is unlikely to be cost-effective in Kenya whether one uses a low-cost assay, such as OLA, or consensus sequencing. FUNDING: National Institutes of Health, Gilead Sciences.

Implementation of a point mutation assay for HIV drug resistance testing in Kenya.

OBJECTIVES: An increasing prevalence of HIV pretreatment drug resistance (PDR) has been observed in Africa, which could decrease the effectiveness of antiretroviral therapy (ART) programs. We describe our experiences, the costs and challenges of implementing an oligonucleotide ligation assay (OLA) for management of PDR in Nairobi, Kenya. DESIGN: An observational report of the implementation of OLA in a Kenyan laboratory for a randomized clinical trial evaluating whether onsite use of OLA in individuals initiating ART would decrease rates of virologic failure. METHODS: Compared detection of mutations and proportion of mutants in participants' viral quasispecies by OLA in Kenya vs. Seattle. Reviewed records of laboratory workflow and performance of OLA. Calculated the costs of laboratory set-up and of performing the OLA based on equipment purchase receipts and supplies and labor utilization, respectively. RESULTS: OLA was performed on 492 trial participants. Weekly batch-testing of median of seven (range: 2-13) specimens provided test results to Kenyan clinicians within 10-14 days of sample collection at a cost of US$ 42 per person tested. Cost of laboratory setup was US$ 32 594. Challenges included an unreliable local supply chain for reagents and the need for an experienced molecular biologist to supervise OLA performance. CONCLUSION: OLA was successfully implemented in a Kenyan research laboratory. Cost was twice that projected because of fewer than predicted specimens per batch because of slow enrollment. OLA is a potential simple, low-cost method for PDR testing in resource-limited settings (RLS). Ongoing work to develop a simplified kit could improve future implementation of OLA in RLS.

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation.

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.