Resource and infrastructure challenges on the RESIST-2 Trial: an implementation study of drug resistance genotype-based algorithmic ART switches in HIV-2-infected adults in Senegal

Background Second-line treatment of HIV-2 in resource-limited settings (RLS) is complicated by a lack of controlled trial data, limited availability of HIV-2-active antiretroviral drugs, and inadequate access to drug resistance testing. We conducted an implementation trial of a dried blood spot- (DBS) based, drug resistance genotype-informed antiretroviral therapy (ART) switching algorithm for HIV-2-infected patients in Senegal. Methods HIV-2-infected adults initiating or receiving ART through the Senegalese national AIDS program were invited to participate in this single-arm trial. DBS from participants with virologic failure (defined as viral load (VL) > 250 copies/mL after > 6 months on the current ART regimen) were shipped to Seattle for genotypic drug resistance testing. Participants with evidence of drug resistance in protease or reverse transcriptase were switched to new regimens according to a pre-specified algorithm. Participant clinical and immuno-virologic outcomes were assessed, as were implementation challenges. Results We enrolled 152 participants. Ten were initiating ART. The remainder were ART-experienced, with 91.0% virologically suppressed (< 50 copies/mL). Problems with viral load testing capability resulted in obtaining VL results for only 227 of 613 (37.0%) participant-visits. Six of 115 participants (5.2%) with VL available after > 6 months on current ART regimen experienced virologic failure, with per-protocol genotypic testing attempted. One additional test was performed for a participant with a VL of 222 copies/mL. Genotypes from three participants showed no evidence of major drug resistance mutations, two showed nucleoside reverse transcriptase inhibitor (NRTI) resistance, one showed both NRTI and protease inhibitor resistance, and one test failed. No integrase inhibitor resistance was observed. Five of six successfully-tested participants switched to the correct regimen or received additional adherence counseling according to the algorithm; the sixth was lost to follow-up. Follow-up VL testing was available for two participants; both of these were virally suppressed (< 10 copies/mL). The trial was terminated early due to the COVID-19 pandemic (which prevented further VL and genotypic testing), planned rollout of dolutegravir-based 1st-line ART, and funding. Conclusions The RESIST-2 trial demonstrated that a DBS-based genotypic test can be used to help inform second-line ART decisions as part of a programmatic algorithm in RLS, albeit with significant implementation challenges. Trial registration ClinicalTrials.govNCT03394196. Registered on January 9, 2018.

Evidence of virological failure in patients on second-line anti-retroviral therapy in Southwestern Nigeria: An indication for HIV drug resistance testing

Background: In sub-Saharan Africa where genotypic anti-retroviral (ARV) drug resistance testing is rarely performed and poor adherence is blamed for the inability to achieve viral suppression and treatment failure, programmatic approaches to preventing and handling these are essential. This study was aimed at assessing the virological outcomes among HIV patients receiving second-line anti-retroviral therapy (ART) in Southwestern Nigeria.Methodology: This was a 5-year observational retrospective study of randomly selected people living with HIV (PLWHIV) who have been switched to second-line ART for at least six months before the commencement of the study in multiple comprehensive ART sites across the three levels of care, in Ondo and Ekiti States, Southwestern Nigeria, from January 2015 to December 2019. Quantitative viral load analysis was done using polymerase chain reaction (PCR) assay. Data were analyzed using the Statistical Package for Social Sciences (SPSS) version 24.0.Results: A total of 249 (71 males and 178 females) subjects eligible for the study were recruited using simple random sampling technique. The mean age (± SD) of the subjects was 44.21 ± 11.45 years. The mean number of years the patients have been on ART regimen was 7.92 ± 2.68 years. The mean number of years the patients were on first line ART regimen before being switched to second line was 4.27 ± 2.63 years. Patients with viral load <1000 RNA copies/ml (suppressed viral load) were 216 (86.7%) out of which 113 (45.4%) had viral load <20 RNA copies/ml while 33 (13.3%) had viral load >1000 RNA copies/ml (unsuppressed viral load or virological failure).Conclusion: About 13% of the patients on second line ART had unsuppressed viral load of more than 1000 RNA copies/ml indicating virological failure. Thus, critical factors such as poor adherence to ART and drug resistance chiefly contributing to virological failure have to be routinely checked. Keywords: suppression, ART, resistance, virological, failure, Nigeria French title: Preuve d'échec virologique chez les patients sous traitement antirétroviral de deuxième intention dans le sud-ouest du Nigeria: une indication pour le test de résistance aux médicaments contre le VIH Contexte: En Afrique subsaharienne, où les tests génotypiques de résistance aux antirétroviraux (ARV) sont rarement effectués et où une mauvaise observance est imputée à l'incapacité d'obtenir la suppression virale et l'échec du traitement, des approches programmatiques pour les prévenir et les gérer sont essentielles. Cette étude visait à évaluer les résultats virologiques chez les patients VIH recevant un traitement antirétroviral (TAR) de deuxième intention dans le sud-ouest du Nigeria. Méthodologie: Il s'agissait d'une étude rétrospective d'observation de 5 ans portant sur des personnes vivant avec le VIH (PVVIH) sélectionnées au hasard et passées à un TAR de deuxième intention pendant au moins six mois avant le début de l'étude dans plusieurs sites de TAR complets aux trois niveaux. de soins, dans les États d'Ondo et d'Ekiti, dans le sud-ouest du Nigéria, de janvier 2015 à décembre 2019. L'analyse quantitative de la charge virale a été effectuée à l'aide d'un test de réaction en chaîne par polymérase (PCR). Les données ont été analysées à l'aide du logiciel Paquet statistique pour les sciences sociales (SPSS) version 24.0. Résultats: Un total de 249 (71 hommes et 178 femmes) sujets éligibles à l'étude ont été recrutés à l'aide d'une technique d'échantillonnage aléatoire simple. L'âge moyen (± ET) des sujets était de 44,21±11,45 ans. Le nombre moyen d'années pendant lesquelles les patients ont été sous traitement antirétroviral était de 7,92±2,68 ans. Le nombre moyen d'années pendant lesquelles les patients étaient sous traitement antirétroviral de première ligne avant de passer en deuxième ligne était de 4,27 ± 2,63 ans. Les patients avec une charge virale <1000 copies d'ARN/ml (charge virale supprimée) étaient 216 (86,7%) dont 113 (45,4%) avaient une charge virale <20 copies d'ARN/ml tandis que 33 (13,3%) avaient une charge virale >1000 ARN copies/ml (charge virale non supprimée ou échec virologique). Conclusion: Environ 13 % des patients sous TAR de deuxième ligne avaient une charge virale non supprimée de plus de 1000 copies d'ARN/ml indiquant un échec virologique. Ainsi, les facteurs critiques tels qu'une mauvaise adhésion au TARV et la résistance aux médicaments contribuant principalement à l'échec virologique doivent être systématiquement vérifiés. Mots clés: suppression, TAR, résistance, virologique, échec, Nigeria

Next Generation Sequencing for HIV-1 Drug Resistance Testing—A Special Issue Walkthrough

Drug resistance remains a global challenge in the fight against the HIV pandemic [...]

Evaluation of the HIV-1 drug resistance to elsulfavirine and the effectiveness of it among Russian treatment-naïve patients

The aim of the study: to analyze the prevalence of resistance mutations to elsulfavirine and to evaluate the effectiveness of it among HIV-infected treatment-naïve patients in real clinical practice.Materials and methods. The study included 578 patients with HIV infection, which divided into 3 groups. The first group is 354 HIV-infected treatment-naïve patients for whom HIV-1 nucleotide sequences were obtained as part of routine drug resistance testing. The second study group included 111 HIV-infected treatment-naïve patients, tested for drug resistance before the antiretroviral therapy containing elsulfavirine. The third study group included 113 HIV-infected treatment-naïve patients, each of whom was assigned a treatment regimen containing elsulfavirine without prior drug resistance testing. The observation period for patients of the second and third groups who received treatment was 24 weeks. To assess the effectiveness of antiretroviral therapy in patients, viral load, CD4+ T-cell counts, and adherence to therapy were assessed. HIV-1 subtypes and drug resistance mutations were determined using the Stanford HIV Resistance Database (v. 8.9-1). To clarify the results of subtyping, phylogenetic analysis of nucleotide sequences was carried out using the MEGA program (v. 6.0).Results. The prevalence of mutations associated with decreased susceptibility to elsulfavirine among HIV-infected treatment-naïve patients was 1.7% and 4.5% for the first and second groups of patients, respectively. All of the patients have only single resistance mutations which, according to the results of preclinical studies, cannot cause drug resistance. The use of elsulfavirine in real clinical practice among treatment-naïve patients has demonstrated good virological and immunological efficacy of the drug. As a result of 24 weeks of therapy in patients of the second group, no treatment ineffectiveness, and the development of drug resistance were observed. Among the patients of the third group, 6 patients (5.3%) have the virological failure of therapy associated with the resistance to the used drugs. All patients with virological failure had a resistance mutation profile associated with a high level of drug resistance to one of the drugs in the treatment regimen, lamivudine. Additionally, 1 patient had a combination of mutations that reduce susceptibility to elsulfavirine, and 4 patients had mutations that can reduce susceptibility to elsulfavirine in combination with other mutations.Conclusion. The low prevalence of mutations associated with a decrease in susceptibility to elsulfavirine and the absence of combinations of mutations make it possible to predict the successful use of this drug for Russian treatment-naïve patients. Reported cases of virological failure of antiretroviral therapy are difficult to interpret in the context of elsulfavirine due to the lack of an exact list of mutations and their combinations, and associations with the degree of resistance to it. This study describes for the first time the mutation profiles in patients with virological failure of therapy containing elsulfavirine and demonstrates the necessity of the further study of drug resistance profile to drug in vitro and in vivo.

HIV-2 Drug Resistance Genotyping from Dried Blood Spots

ABSTRACTThe treatment of HIV-2 in resource-limited settings (RLS) is complicated by the limited availability of HIV-2-active antiretroviral drugs and inadequate access to HIV-2 viral load and drug resistance testing. Dried blood spots (DBS)-based drug resistance testing, widely studied for HIV-1, has not been reported for HIV-2 and could present an opportunity to improve care for HIV-2-infected individuals. We selected 150 DBS specimens from ongoing studies of antiretroviral therapy (ART) for HIV-2 infection in Senegal and subjected them to genotypic drug resistance testing. Total nucleic acid was extracted from DBS, reverse transcribed, PCR amplified, and analyzed by population-based Sanger sequencing, and major drug resistance-associated mutations (RAM) were identified. Parallel samples from plasma and peripheral blood mononuclear cells (PBMC) were also genotyped. We obtained 58 protease/reverse transcriptase genotypes. Plasma viral load was significantly correlated with genotyping success (P < 0.001); DBS samples with corresponding plasma viral load >250 copies/ml had a success rate of 86.8%. In paired DBS-plasma genotypes, 83.8% of RAM found in plasma were also found in DBS, and replicate DBS genotyping revealed that a single test detected 86.7% of known RAM. These findings demonstrate that DBS-based genotypic drug resistance testing for HIV-2 is feasible and can be deployed in RLS with limited infrastructure.

Dry Panels Supporting External Quality Assessment Programs for Next Generation Sequencing-Based HIV Drug Resistance Testing

External quality assessment (EQA) is a keystone element in the validation and implementation of next generation sequencing (NGS)-based HIV drug resistance testing (DRT). Software validation and evaluation is a critical element in NGS EQA programs. While the development, sharing, and adoption of wet lab protocols is coupled with the increasing access to NGS technology worldwide, rendering it easy to produce NGS data for HIV-DRT, bioinformatic data analysis remains a bottleneck for most of the diagnostic laboratories. Several computational tools have been made available, via free or commercial sources, to automate the conversion of raw NGS data into an actionable clinical report. Although different software platforms yield equivalent results when identical raw NGS datasets are analyzed for variations at higher abundance, discrepancies arise when variations at lower frequencies are considered. This implies that validation and performance assessment of the bioinformatics tools applied in NGS HIV-DRT is critical, and the origins of the observed discrepancies should be determined. Well-characterized reference NGS datasets with ground truth on the genotype composition at all examined loci and the exact frequencies of HIV variations they may harbor, so-called dry panels, would be essential in such cases. The strategic design and construction of such panels are challenging but imperative tasks in support of EQA programs for NGS-based HIV-DRT and the validation of relevant bioinformatics tools. Here, we present criteria that can guide the design of such dry panels, which were discussed in the Second International Winnipeg Symposium themed for EQA strategies for NGS HIVDR assays.

Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations

Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.