Impact of pretreatment low-abundance HIV-1 drug-resistant variants on virological failure among HIV-1/TB-co-infected individuals.

OBJECTIVES: To determine the impact of pretreatment low-abundance HIV-1 drug-resistant variants (LA-DRVs) on virological failure (VF) among HIV-1/TB-co-infected individuals treated with NNRTI first-line ART. METHODS: We conducted a case-control study of 170 adults with HIV-1/TB co-infection. Cases had at least one viral load (VL) ≥1000 RNA copies/mL after ≥6 months on NNRTI-based ART, and controls had sustained VLs <1000 copies/mL. We sequenced plasma viruses by Sanger and MiSeq next-generation sequencing (NGS). We assessed drug resistance mutations (DRMs) using the Stanford drug resistance database, and analysed NGS data for DRMs at ≥20%, 10%, 5% and 2% thresholds. We assessed the effect of pretreatment drug resistance (PDR) on VF. RESULTS: We analysed sequences from 45 cases and 125 controls. Overall prevalence of PDR detected at a ≥20% threshold was 4.7% (8/170) and was higher in cases than in controls (8.9% versus 3.2%), P = 0.210. Participants with PDR at ≥20% had almost 4-fold higher odds of VF (adjusted OR 3.7, 95% CI 0.8-18.3) compared with those without, P = 0.104. PDR prevalence increased to 18.2% (31/170) when LA-DRVs at ≥2% were included. Participants with pretreatment LA-DRVs only had 1.6-fold higher odds of VF (adjusted OR 1.6, 95% CI 0.6-4.3) compared with those without, P = 0.398. CONCLUSIONS: Pretreatment DRMs and LA-DRVs increased the odds of developing VF on NNRTI-based ART, although without statistical significance. NGS increased detection of DRMs but provided no additional benefit in identifying participants at risk of VF at lower thresholds. More studies assessing mutation thresholds predictive of VF are required to inform use of NGS in treatment decisions.

A post-partum single-dose TDF/FTC tail does not prevent the selection of NNRTI resistance in women receiving pre-partum ZDV and intrapartum single-dose nevirapine to prevent mother-to- child HIV-1 transmission.

Although the rates of vertical transmission of HIV in the developing world have improved to around 3% in countries like South Africa, resistance to antiretrovirals (ARV) used in Prevention of Mother-to-Child transmission (pMTCT) strategies may thwart such outcomes and affect the efficacy of future ARV regimens in mothers and children. This study conducted in Durban, South Africa, between 2010 and 2013 found a high rate of nevirapine (NVP) resistance among women receiving Zidovudine (AZT) from 14 weeks gestation, single dose nevirapine (sd NVP) at the onset of labor and a single dose of coformulated Tenofovir/Emtricitabine (TDF/FTC) postpartum. Using Sanger sequencing, high and intermediate levels of nevirapine (NVP) resistance were detected in 15/44 (34%) and in 1/44 (2%) of women tested, respectively. Most subjects selected the K103N mutation (22% (10/45) of all patients and 66% (10/15) of those with high-level NVP resistance). Such rate of NVP resistance is comparable to studies where only sd NVP was used. In conclusion, a post-partum single-dose TDF/FTC tail does not prevent the selection of NNRTI resistance in women receiving pre-partum ZDV and intrapartum sd NVP to prevent mother-to-child HIV-1 transmission.

HIV-1 drug resistance in adults and adolescents on protease inhibitor-based antiretroviral therapy in KwaZulu-Natal Province, South Africa.

OBJECTIVES: In low-middle-income countries, increasing levels of HIV drug resistance (HIVDR) on second-line protease inhibitor (PI)-based regimens are a cause for concern given the limited drug options for third-line antiretroviral therapy (ART). We conducted a retrospective analysis of routine HIV-1 genotyping laboratory data from KwaZulu-Natal, South Africa, to describe the frequency and patterns of HIVDR mutations and their consequent impact on standardised third-line regimens. METHODS: This was a cross-sectional analysis of all HIV-1 genotypic resistance tests conducted by the National Health Laboratory Service in KwaZulu-Natal (January 2015 to December 2016) for adults and adolescents (age ≥10 years) on second-line PI-based ART with virological failure. We assigned a third-line regimen to each record based on a national treatment algorithm and calculated the genotypic susceptibility score (GSS) for that regimen. RESULTS: Of 348 samples analysed, 287 (82.5%) had at least one drug resistance mutation (DRM) and 114 (32.8%) had at least one major PI DRM. Major PI resistance was associated with longer duration on second-line ART (aOR per 6-months = 1.11, 95% CI 1.04-1.19) and older age (aOR = 1.03, 95% CI 1.01-1.05). Of 112 patients requiring third-line ART, 12 (10.7%) had a GSS of <2 for the algorithm-assigned third-line regimen. CONCLUSION: One-third of people failing second-line ART had significant PI DRMs. A subgroup of these individuals had extensive HIVDR, where the predicted activity of third-line ART was suboptimal, highlighting the need for continuous evaluation of outcomes on third-line regimens and close monitoring for emergent HIV-1 integrase inhibitor resistance.

Trends in Pretreatment HIV-1 Drug Resistance in Antiretroviral Therapy-naive Adults in South Africa, 2000-2016: A Pooled Sequence Analysis.

BACKGROUND: South Africa has the largest public antiretroviral therapy (ART) programme in the world. We assessed temporal trends in pretreatment HIV-1 drug resistance (PDR) in ART-naïve adults from South Africa. METHODS: We included datasets from studies conducted between 2000 and 2016, with HIV-1 pol sequences from more than ten ART-naïve adults. We analysed sequences for the presence of 101 drug resistance mutations. We pooled sequences by sampling year and performed a sequence-level analysis using a generalized linear mixed model, including the dataset as a random effect. FINDINGS: We identified 38 datasets, and retrieved 6880 HIV-1 pol sequences for analysis. The pooled annual prevalence of PDR remained below 5% until 2009, then increased to a peak of 11·9% (95% confidence interval (CI) 9·2-15·0) in 2015. The pooled annual prevalence of non-nucleoside reverse-transcriptase inhibitor (NNRTI) PDR remained below 5% until 2011, then increased to 10.0% (95% CI 8.4-11.8) by 2014. Between 2000 and 2016, there was a 1.18-fold (95% CI 1.13-1.23) annual increase in NNRTI PDR (p < 0.001), and a 1.10-fold (95% CI 1.05-1.16) annual increase in nucleoside reverse-transcriptase inhibitor PDR (p = 0.001). INTERPRETATION: Increasing PDR in South Africa presents a threat to the efforts to end the HIV/AIDS epidemic. These findings support the recent decision to modify the standard first-line ART regimen, but also highlights the need for broader public health action to prevent the further emergence and transmission of drug-resistant HIV. SOURCE OF FUNDING: This research project was funded by the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package. DISCLAIMER: The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of CDC.

Primary HIV-1 Drug Resistant Minority Variants.

Primary HIV drug resistant mutations are mutations that occur in an HIV-infected individual prior to the initiation of antiretroviral therapy (ART). These mutations may arise by de novo mutagenesis or result from transmission. Drug resistant mutations (DRMs) may reduce the effectiveness of ART leading to inadequate virological outcomes. Currently, Sanger sequencing is the standard method for detection of DRMs to inform treatment decisions, but does not detect minor variant mutations. Drug resistant minority variants (DRMVs) can be detected by next generation sequencing (NGS). However, several challenges including cost of infrastructure and the need for complex data analysis bioinformatics tools remain major setbacks for NGS use. More importantly, the clinical impact of DRMVs on ART is not well understood, underscoring the importance for understanding whether the levels of primary DRMVs for different mutations impact on the effectiveness of ART and the rationale for inclusion in routine diagnostics. Understanding the impact of primary DRMVs will help inform how NGS may be utilized in the future for pre-emptive clinical ART decision making.

HIV-1 Drug Resistance by Ultra-Deep Sequencing Following Short Course Zidovudine, Single-Dose Nevirapine, and Single-Dose Tenofovir with Emtricitabine for Prevention of Mother-to-Child Transmission.

Antiretroviral drug resistance following pMTCT strategies remains a significant problem. With rapid advancements in next generation sequencing technologies, there is more focus on HIV drug-resistant variants of low frequency, or the so-called minority variants. In South Africa, AZT monotherapy for pMTCT, similar to World Health Organization option A, has been used since 2008. In 2010, a single dose of co-formulated TDF/FTC was included in the strategy for prevention of resistance conferred by single-dose nevirapine (sd NVP). The study was conducted in KwaZulu-Natal, South Africa, among pMTCT participants who received AZT monotherapy from 14 weeks of gestation, intrapartum AZT and sd NVP, and postpartum sd TDF/FTC. Twenty-six specimens collected at 6 weeks post-delivery were successfully sequenced using 454 ultra-deep sequencing. Non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance was detected in 17 of 26 (65%) patients, 2 (7%) had Thymidine analogue mutations, and 3 (11%) had K65R. Of the 17 patients with NNRTI resistance, 11 (65%) had high-level NNRTI resistance, whereas 6 (35%) had intermediate NNRTI resistance. The levels of NNRTI resistance are much higher than would be expected, given the inclusion of antepartum AZT and postpartum TDF/FTC. This high level of NNRTI resistance could impact future NNRTI-containing treatment for a large proportion of pMTCT-exposed women. The detection of Thymidine analogue mutations highlights the need to understand the clinical impact of these on AZT-containing antiretroviral treatment in women exposed to AZT monotherapy.

Minority HIV-1 drug-resistant mutations and prevention of mother-to-child transmission: perspectives for resource-limited countries.

The detection and clinical significance of HIV-1 minority drug-resistant variants is a major topic of current HIV research. Whereas much attention has been placed on the clinical impact of minority drug-resistant variants in patients initiating antiretroviral therapy, their possible influence on the effectiveness of antiretroviral therapy following prevention of mother-to-child transmission strategies in resource-limited settings remains largely unexplored. This review outlines the clinical significance and detection of minority drug-resistant variants, focusing primarily on studies of minority variants in the context of prevention of mother-to-child transmission and their possible influence on current regimens, especially those available in resource-limited countries. The clinical impact of minority nevirapine-resistant variants that arise in the context of prevention of mother-to-child transmission, for example, is an important factor to consider when these women initiate antiretroviral therapy that may include nevirapine or efavirenz. Minority nonnucleoside reverse transcriptase inhibitor-resistant variants have been associated with treatment failure in women exposed to single-dose nevirapine. In countries like South Africa, with its longstanding use of single-dose nevirapine, this question is relevant as it is for other resource-limited countries where single-dose nevirapine is used. In the same context, various other minority drug-resistant variants (e.g. Y181C, K65R and thymidine analogue mutations etc.) are discussed. The field of next generation sequencing is very dynamic, with rapid improvements on present technologies and the introduction of novel technologies as discussed in this review. As the impact of minority drug-resistant variants in the setting of prevention of mother-to-child transmission becomes more evident, guidelines for this, especially in resource-limited countries, will need revision in order to optimize the clinical benefit from future antiretroviral therapy.

Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.

BACKGROUND: The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs. METHODS: A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser. RESULTS: Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454. CONCLUSIONS: In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.