Genotypic Drug Resistance Testing Research Articles

Comparison of Different HIV-1 Resistance Interpretation Tools for Next-Generation Sequencing in Italy.

Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia <10,000-1000 copies/mL and non-B subtypes. Mutations at an NGS frequency >10% showed fair concordance among different interpretation tools. This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.

Prevalence of integrase strand transfer inhibitor (INSTI) resistance in people living with HIV and virological failure

Background: This study aims to delineate the resistance profiles to integrase strand transfer inhibitors (INSTIs) among patients in southern Taiwan who have experienced antiretroviral therapy (ART) failure. We focus on individuals previously treated with highly active antiretroviral therapy (HAART) regimens, providing insights into the implications of INSTI resistance in a broader treatment-experienced population. Methods: Data were collected from patients failing an INSTI-containing regimen in a medical center in southern Taiwan between 2009 and 2022. Virological failure was defined as a plasma viral load &gt;1000 copies/ml. Reverse transcriptase, protease and integrase coding regions were sequenced at failure. Resistance-associated mutations included in the 2022 IAS-USA list were used. Drug resistance was analyzed using the HIV Stanford HIVDB 9.4 edition algorithm. Logistic regression analysis was used to analyze the risk factors associated with INSTI failure. Results: A total of 184 patients were enrolled for genotypic drug resistance testing due to virological failure, of whom 104 failed on nonnucleoside reverse transcriptase inhibitors, 58 on protease inhibitors, and 21 on INSTIs. For the 21 patients who failed on INSTI therapy, 6 failed on raltegravir-based treatment, 3 on elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (EVG/COBI/FTC/TAF), 2 on bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF), and 10 on abacavir/dolutegravir/lamivudine (ABC/DTG/3TC). Only 10 patients had INSTI drug resistance testing results available, of whom 40% (4/10) showed INSTI resistance at failure. Among the 7 patients who failed on second-generation INSTIs with drug resistance reports available, one harbored E157Q and another with R263K mutations, respectively. Multivariable logistic regression analysis showed that the patients with INSTI failure were less likely to have pol resistance (p=0.007, adjusted OR 0.176, 95% CI: 0.050-0.618), less previous exposure to NNRTI (p=0.003, aOR 0.063, 95% CI: 0.010-0.401), PIs (p=0.002, aOR 0.030, 95% CI: 0.003-0.272) and with long duration of HAART (p=0.018, aOR 1.02, 95% CI: 1.003-1.037) Conclusion: INSTI resistance was uncommon when used as the first-line single tablet regimen in Taiwan. The results confirmed the robustness of ABC/DTG/3TC and BIC/FTC/TAF regarding integrase resistance in cases of virological failure in routine clinical care.

Virological impact of HIV drug-resistance testing in children, adolescents, and adults failing first-line ART in Tanzania

BackgroundProspective data on the effectiveness of resistance testing in informing treatment decisions and outcomes in with first-line failure in these settings is limited. This study aimed to assess the virological impact of HIV drug-resistance testing in patients with virological failure in Tanzania. MethodsParticipants were randomly assigned to either the control or the experimental group. In addition to the standard of care, patients in the experimental group had access to genotypic drug-resistance testing, information used during treatment change, and were followed up at six-and 12-months to determine virological suppression. ResultsA total of 261 patients with a median age of 32 (14.7–44.7) years were enrolled. In the intention-to-treat analysis, at 6-months, suppression was achieved in 58 (42.3%; 95% CI, 34.1–50.1) experimental group patients versus 51 (41.1%; 95% CI, 32.5–49.8) control group patients, with a p-value of 0.4. At-12 months, suppression was achieved in 110 (80.3%; 95% CI, 73.6–87) experimental patients versus 99 (79.8%; 95% CI, 72.8–86.9) control patients, with a p-value of 0.5. In the per-protocol analysis, at 6-months, suppression was observed in 38.46% (95% CI, 27.6–49.3) experimental patients versus 38.6% (95% CI, 26.0–51.2) control patients, with a p-value of 0.5. At 12-months, suppression was observed in 79.49% (95% CI, 70.5–88.5) of experimental patients versus 75.44% (95% CI, 64.3–86.6) of control patients, with a p-value of 0.3. ConclusionConducting HIV drug-resistance testing, and switch to individualised second-line regimens did not significantly improve virological suppression in patients experiencing first-line ART failure in Tanzania.

Increased prevalence of pretreatment drug resistance mutations in treatment-naïve people living with HIV-1 in Henan Province, China (2022/23)

Human deficiency virus type 1 (HIV-1) harboring drug resistance mutations (DRMs) before the initiation of antiretroviral therapy (ART) poses a serious threat to the efficacy of current ART regimens. Currently, the prevalence of pre-treatment drug resistance mutations (PDRMs) including transmitted DRMs (TDRMs) is not completely clear. Understanding this prevalence better should offer valuable data for clinical- and government-level decision-making. To closely monitor the PDRM trend in treatment-naïve people living with HIV/AIDS (PLWHA) in Henan Province, China, plasma samples from the patients seeking treatments at our hospital from January 2022 to February 2023 were collected for genotypic drug resistance testing. From the 645 patients whose samples were collected, partial pol and integrase gene sequences were obtained from 637 patients. Subtyping analysis indicated that the top-three most common subtypes, in descending order, were CRF07_BC (41.76%, 266/637), CRF01_AE (28.26%, 180/637), and B (20.41%, 130/637). PDRMs were observed in 5.18% (33/637), 6.28% (40/637), 0.31% (2/637), and 2.83% (18/637) cases for nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), and integrase strand transfer inhibitors (INSTIs), respectively; all these medications contributed to an overall PDRM prevalence of 11.93% (76/637). On analyzing individual PDRMs, we noted that the most commonly observed mutation(s) were K103S/N (3.77%, 24/637), M184I/V (3.14%, 20/637), followed by K65R (1.26%, 8/637), and V106A/M (1.10%, 7/637). PDRM prevalence in ART-naïve PLWHA of Henan Province is high and increased compared with that noted in previous years. However, evidence of cluster-linked outbreaks of PDRMs is lacking, suggesting that measures such as education about adherence and improved treatment strategies with a low incidence of failure can effectively reduce PDRM prevalence.

HIV Drug Resistance Patterns and Characteristics Associated with Clinically Significant Drug Resistance among Children with Virologic Failure on Antiretroviral Treatment in Kenya: Findings from the Opt4Kids Randomized Controlled Trial.

Increasing HIV drug resistance (DR) among children with HIV (CHIV) on antiretroviral treatment (ART) is concerning. CHIV ages 1-14 years enrolled from March 2019 to December 2020 from five facilities in Kisumu County, Kenya, were included. Children were randomized 1:1 to control (standard-of-care) or intervention (point-of-care viral load (POC VL) testing every three months with targeted genotypic drug resistance testing (DRT) for virologic failure (VF) (≥1000 copies/mL)). A multidisciplinary committee reviewed CHIV with DRT results and offered treatment recommendations. We describe DR mutations and present logistic regression models to identify factors associated with clinically significant DR. We enrolled 704 children in the study; the median age was 9 years (interquartile range (IQR) 7, 12), 344 (49%) were female, and the median time on ART was 5 years (IQR 3, 8). During the study period, 106 (15%) children had DRT results (84 intervention and 22 control). DRT detected mutations associated with DR in all participants tested, with 93 (88%) having major mutations, including 51 (54%) with dual-class resistance. A history of VF in the prior 2 years (adjusted odds ratio (aOR) 11.1; 95% confidence interval (CI) 6.3, 20.0) and less than 2 years on ART at enrollment (aOR 2.2; 95% CI 1.1, 4.4) were associated with increased odds of major DR. DR is highly prevalent among CHIV on ART with VF in Kenya. Factors associated with drug resistance may be used to determine which children should be prioritized for DRT.

Treatment-emergent reverse transcriptase resistance during antiretroviral therapy with bictegravir, tenofovir alafenamide, and emtricitabine: A case series.

Bictegravir/tenofovir alafenamide/emtricitabine (BIC/TAF/FTC) is a complete regimen for the treatment of HIV with a high barrier to resistance and few reported cases of treatment failure. We present three cases of treatment-emergent resistance to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) in patients with suboptimal treatment adherence and assess whether the resistance-associated mutations were present before BIC/TAF/FTC initiation or emerged during therapy. We used genotypic drug resistance testing by Sanger sequencing to identify emergent resistance mutations in plasma viral load specimens collected after combination antiretroviral therapy initiation in all participants. Additionally, we performed ultra-deep sequencing by Illumina MiSeq on the earliest available plasma HIV-1 viral load specimen and on any available specimens closest in time to the initiation of BIC/TAF/FTC therapy to identify low-abundance resistance mutations present in the viral quasispecies. All three participants developed NRTI resistance after prolonged exposure and incomplete adherence to BIC/TAF/FTC. The T69N, K70E, M184I, and/or T215I mutations identified in clinical samples at the time of virological failure were not present on deep sequencing of either baseline samples or samples collected before BIC/TAF/FTC initiation. Despite a generally high genetic barrier to resistance, NRTI resistance-associated mutations may emerge during therapy with BIC/TAF/FTC in the setting of suboptimal adherence.

Highly Ambiguous HIV-1 Pol Positions Encoding Multiple Amino Acids Usually Result from Antiviral or Immune Selection Pressure.

HIV-1 pol nucleotide ambiguities encoding amino acid mixtures occur commonly during population-based genotypic drug resistance testing. However, few studies have addressed the validity of sequences with fully ambiguous codons (FACs) containing codons translatable to more than four amino acids. We identified 839 published HIV-1 pol sequences with 846 FACs at 131 positions and determined their distribution relative to 215 HLA-associated pol positions (HAPs) and 84 drug-resistance positions. Among HIV-1 reverse transcriptase (RT) and protease sequences from antiretroviral therapy (ART)-naive and -experienced persons, there was a strong correlation between the likelihood a position was a FAC and that it was an HAP (Spearman's correlation coefficient rho >0.40; p < 1e-6). Among HIV-1 RT sequences from ART-experienced persons, there was a correlation between the likelihood that a position was a FAC and that it was a drug-resistance position (rho = 0.2; p = 8e-4). In the context of population-based genotypic resistance testing, FACs usually result from antiviral or immune selection pressure.

High-level resistance to non-nucleos(t)ide reverse transcriptase inhibitor based first-line antiretroviral therapy in Ghana; A 2017 study.

Expanding access to effective antiretroviral therapy (ART) is a major tool for management of Human Immunodeficiency Virus (HIV) infection. However, rising levels of HIV drug-resistance have significantly hampered the anticipated success of ART in persons living with HIV (PLWH), particularly those from Africa. Though great strides have been made in Ghana toward achieving the UNAIDS “95-95-95” target, a substantial number of PLWH receiving ART have not attained viral suppression. This study investigated patterns of drug resistance mutations in ART naïve as well as ART-experienced PLWH receiving first-line regimen drugs from Ghana. In a cross-sectional study, blood samples were collected from HIV-1 infected adults (≥18 years) attending HIV/AIDS clinic at the Eastern Regional Hospital, Koforidua, Ghana from September to October 2017. Viral RNA isolated from plasma were subjected to genotypic drug resistance testing for Protease Inhibitors (PI), Reverse Transcriptase Inhibitors (RTI), and Integrase Strand Transfer Inhibitors (INSTI). A total of 95 (84 ART experienced, 11 ART naïve) HIV-1 infected participants were sampled in this study. Sixty percent (50/84) of the ART-experienced participants were controlling viremia (viral load < 1,000 copies/ml). Of the 95 patient samples, 32, 34, and 33 were successfully sequenced for protease, reverse-transcriptase, and integrase regions, respectively. The dominant HIV-1 subtypes detected were CRF02_AG (70%), and A3 (10%). Major drug resistance associated mutations were only detected for reverse transcriptase inhibitors. The predominant drug resistance mutations were against nucleos(t)ide reverse transcriptase inhibitors (NRTI)—M184V/I and non-nucleos(t)ide reverse transcriptase inhibitors (NNRTI)—K103N. In the ART-experienced group, M184V/I and K103N were detected in 54% (15/28) and 46% (13/28) of individuals, respectively. Both mutations were each detected in 33% (2/6) of ART naïve individuals. Multiclass resistance to NRTI and NNRTI was detected in 57% of ART-experienced individuals and two ART naïve individuals. This study reports high-level resistance to NNRTI-based antiretroviral therapy in PLWH in Ghana. However, the absence of major PI and INSTI associated-mutations is a good signal that the current WHO recommendation of Dolutegravir in combination with an NRTI backbone will yield maximum benefits as first-line regimen for PLWH in Ghana.

Overview of the Analytes Applied in Genotypic HIV Drug Resistance Testing.

The close monitoring of HIV drug resistance using genotypic HIV drug resistance testing (HIVDRT) has become essential for effective HIV/AIDS management at both individual and population levels. Over the years, a broad spectrum of analytes or specimens have been applied or attempted in HIVDRT; however, the suitability and performance of these analytes in HIVDRT and the clinical relevance of the results from them may vary significantly. This article provides a focused overview of the performance, strengths, and weaknesses of various analytes while used in HIVDRT, which may inform the optimal analytes selection in different application contexts.

Spectrum of Activity of Raltegravir and Dolutegravir Against Novel Treatment-Associated Mutations in HIV-2 Integrase: A Phenotypic Analysis Using an Expanded Panel of Site-Directed Mutants.

Integrase inhibitors (INIs) are a key component of antiretroviral therapy for human immunodeficiency virus-1 (HIV-1) and HIV-2 infection. Although INI resistance pathways are well-defined for HIV-1, mutations that emerge in HIV-2 in response to INIs are incompletely characterized. We performed systematic searches of GenBank and HIV-2 drug resistance literature to identify treatment-associated mutations for phenotypic evaluation. We then constructed a library of 95 mutants of HIV-2ROD9 that contained single or multiple amino acid changes in the integrase protein. Each variant was tested for susceptibility to raltegravir and dolutegravir using a single-cycle indicator cell assay. We observed extensive cross-resistance between raltegravir and dolutegravir in HIV-2ROD9. HIV-2-specific integrase mutations Q91R, E92A, A153G, and H157Q/S, which have not been previously characterized, significantly increased the half maximum effective concentration (EC50) for raltegravir when introduced into 1 or more mutational backgrounds; mutations E92A/Q, T97A, and G140A/S conferred similar enhancements of dolutegravir resistance. HIV-2ROD9 variants encoding G118R alone, or insertions of residues SREGK or SREGR at position 231, were resistant to both INIs. Our analysis demonstrates the contributions of novel INI-associated mutations to raltegravir and dolutegravir resistance in HIV-2. These findings should help to improve algorithms for genotypic drug resistance testing in HIV-2-infected individuals.

Drug Resistance, Rather than Low Tenofovir Levels in Blood or Urine, Is Associated with Tenofovir, Emtricitabine, and Efavirenz Failure in Resource-Limited Settings.

The high cost of viral load (VL) testing limits its use for antiretroviral therapy (ART) adherence support. A low-cost lateral flow urine tenofovir (TFV) rapid assay predicts pre-exposure prophylaxis breakthroughs, but has not yet been investigated in HIV treatment. We therefore evaluated its utility in a pilot cross-sectional study of TFV-containing ART recipients at an increased risk of virologic failure (VF). Participants who had a treatment interruption ≥30 days or had ≥1 episode of viremia (VL ≥400 copies/mL) in the previous year were recruited from a public health setting in Cape Town, South Africa. Self-reported adherence data were collected, the urine TFV assay performed, and concurrent TFV-diphosphate analyzed in dried blood spots. VL testing was done concurrently and, if viremic, genotypic HIV drug resistance testing was performed. Of 48 participants, 18 (37.5%) had VL (>400 copies/mL) at the time of the study, including 16 of 39 receiving efavirenz (EFV), 2 of 6 receiving protease inhibitors, and 0 of 3 receiving dolutegravir. Resistance testing succeeded in 17/18, of which 14 had significant mutations compromising ≥2 agents of the current EFV-based regimen. Of these 14, all had detected urine TFV. Urine TFV was undetectable in two out of three without regimen-relevant resistance; p = .02. In participants on EFV-based regimens returning to care, VF was largely due to viral resistance, where detectable urine TFV had 100% sensitivity (14/14 participants) in predicting resistance. Conversely, when undetectable, the urine-based assay could be used to preclude participants with poor adherence from undergoing costly HIV drug resistance testing.

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

BackgroundSecond-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.MethodsHIV-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.ResultsWe 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.ConclusionsThe 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 registrationClinicalTrials.govNCT03394196. Registered on January 9, 2018.

Epidemiological and phylogenetic analysis for non-B subtypes of human immunodeficiency virus type 1 in Busan, Korea

Recent data on non-B subtypes’ epidemiology among patients infected with human immunodeficiency virus-1 (HIV-1) in Korea are lacking. We aimed to assess the changing trends in the epidemiology of non-B subtypes of HIV-1 in Korea using phyloepidemiological analyses. We analyzed the demographic records and sequencing data obtained from genotypic drug resistance tests between 2005 and 2019 from 517 patients infected with HIV attending a tertiary care hospital in Busan, Korea. Subtyping and phylogenetic analyses with reference sequences were performed. Additionally, transmission clusters were identified via maximum-likelihood trees. Non-B subtypes accounted for 21.3% of the 517 sequences. CRF01_AE (52.7%) was the most common non-B subtype, followed by CRF02_AG (16.4%), A1 (11.8%), and C (5.5%). The prevalence of non-B subtypes decreased from 36.4 to 13.4% by 2009, while it increased to 27.4% between 2015 and 2019. Among patients with non-B subtypes, the proportion of overseas sailors decreased from 66.7 to 7.5%; contrarily, the proportion of men-who-have-sex-with-men (MSM) increased from 0 to 46.9% over the study period. We identified 8 transmission clusters involving non-B subtypes, with sizes ranging from 2 to 4 patients, including 3 clusters containing MSM. Our results highlight the changes in the epidemiological trends of non-B subtypes of HIV-1 in Korea.

Is there a role for doravirine in African HIV treatment programmes? A large observational resistance study in South Africa.

IntroductionDolutegravir has replaced efavirenz in most low‐ and middle‐income countries, due to better tolerability and formidable resistance profile, but dolutegravir side effects suggest alternatives are needed. We evaluated doravirine resistance in South Africa as a first step to assess whether doravirine may replace dolutegravir.MethodsA retrospective dataset was analysed for predicted doravirine susceptibility, including sequences obtained from three patient groups. First, data from 277 patients initiating antiretroviral treatment (ART) were collected between February 2013 and October 2014 as part of a national survey. Second, data from 788 patients experiencing NNRTI‐based ART failure were obtained between February 2013 and October 2014 as part of a national survey. Third, data derived from 584 patients who had genotypic drug resistance testing requested after NNRT‐based failure as part of individual patient management between January 2016 and December 2019. Pol sequences were generated using validated population‐based in‐house genotyping and submitted to Stanford HIVdb v8.9.Results and discussionLess than 5% of patients initiating ART presented with genotypic doravirine resistance, whereas most patients experiencing NNRTI‐based ART failure presented with predicted intermediate (41.0%) or high‐level resistance (43.8%) to doravirine. High‐level resistance to doravirine was commonly predicted by the presence of at least three DRMs (79.7%). The predicted resistance profile to doravirine in ART‐naïve patients is promising, but less so in those experiencing failure to first‐generation NNRTIs. Accumulation of NNRTI DRMs seems to be an important factor in the poor resistance prediction for doravirine.ConclusionsAlthough doravirine is approved as initial therapy in patients who are ART‐naïve, it is currently recommended to obtain a genotype prior to the initiation of ART. Clinical studies are needed to ascertain whether predicted resistance profiles in ART naïve and NNRTI‐treated patients translate into poor clinical outcomes, especially in settings where genotypic resistance testing is not available.

HIV-2 Drug Resistance Genotyping from Dried Blood Spots.

The 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.

Laboratory Optimization Tweaks for Sanger Sequencing in a Resource-Limited Setting.

Despite various challenges that hinder the implementation of high-tech molecular methods in resource-limited settings, we have been able to implement and achieve International Organization for Standardization 15189:2012 accreditation for genotypic HIV drug resistance testing in our facility. At the Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Nigeria has recorded a high sequencing success rate and good quality sequence data. This was achieved by optimizing laboratory processes from 2008 to the current date. We have optimized sample preparation, RT-PCR, several post-PCR processes, and the cycle sequencing to improve the sensitivity of amplification even with limited plasma samples and low viral copy numbers. The optimized workflow maximizes output, minimizes reagent wastage, and achieves substantial cost savings without compromising the quality of the sequence data. Our performance at our last external quality assurance program is a testimonial to the efficiency of the workflow. For the 5-sample panel, each with 67-68 mutation points evaluated, we scored 100% for all 5 specimens. Our optimized laboratory workflow is thus documented to support laboratories and to help researchers achieve excellent results the first time and eliminate contamination while minimizing the wastage of costly sequencing reagents.

Increased HIV-1 pretreatment drug resistance with consistent clade homogeneity among ART-naive HIV-1 infected individuals in Ethiopia

BackgroundThe development of pretreatment drug resistance (PDR) is becoming an obstacle to the success of antiretroviral therapy (ART). Besides, data from developing settings including Ethiopia is still limited. Therefore, this study was aimed to assess HIV-1 genetic diversity and PDR mutations among ART-naive recently diagnosed HIV-1 infected individuals in Addis Ababa, Ethiopia.MethodsInstitutional based cross-sectional study was conducted from June to December 2018 in Addis Ababa among ART-naive recently diagnosed individuals. Partial HIV-1 pol region covering the entire protease (PR) and partial reverse transcriptase (RT) regions of 51 samples were amplified and sequenced using an in-house assay. Drug resistance mutations were examined using calibrated population resistance (CPR) tool version 6.0 from the Stanford HIV drug resistance database and the International Antiviral Society-USA (IAS-USA) 2019 mutation list.ResultsAccording to both algorithms used, 9.8% (5/51) of analyzed samples had at least one PDR Mutation. PDR mutations to Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) were the most frequently detected (7.8% and 9.8%, according to the CPR tool and IAS-USA algorithm, respectively). The most frequently observed NNRTIs-associated mutations common to both algorithms were K103N (2%), Y188L (2%), K101E (2%), and V106A (2%), while E138A (2%) was observed according to IAS-USA only. Y115F and M184V (mutations that confer resistance to NRTIs) dual mutations were detected according to both criteria in a single study participant (2%). PDR mutation to protease inhibitors was found to be low (only G73S; 2% according to the CPR tool). Phylogenetic analysis showed that 98% (50/51) of the study participants were infected with HIV-1C virus while one individual (2%) was infected with HIV-1A1 virus.ConclusionsThis study showed an increased level of PDR and persistence HIV-1C clade homogeneity after 15 years of the rollout of ART and 3 decades of HIV-1C circulation in Ethiopia, respectively. Therefore, we recommend routine baseline genotypic drug resistance testing for all newly diagnosed HIV infected patients before initiating treatment. This will aid the selection of appropriate therapy in achieving the 90% of patients having an undetectable viral load in consonance with the UN target.

Drug level testing as a strategy to determine eligibility for drug resistance testing after failure of ART: a retrospective analysis of South African adult patients on second-line ART.

abstract IntroductionWhen protease inhibitor (PI)‐based second‐line ART fails, guidelines recommend drug resistance testing and individualized third‐line treatment. However, PI‐resistant viral strains are rare and drug resistance testing is costly. We investigated whether less costly PI‐exposure testing can be used to select those patients who would benefit most from drug resistance testing.MethodsWe performed a retrospective analysis of South African adults living with HIV experiencing failure of ritonavir‐boosted‐lopinavir (LPV/r)‐based second‐line ART for whom drug resistance testing results were available. We included patients who received plasma‐based drug resistance testing at a central South African reference laboratory in 2017 and patients who received dried blood spots (DBS)‐based drug resistance testing at a rural South African clinic between 2009 and 2017. PI‐exposure testing was performed on remnant plasma or DBS using liquid chromatography mass spectrometry (LCMS). Additionally, a low‐cost immunoassay was used on plasma. Population genotypic drug resistance testing of the pol region was performed on plasma and DBS using standard clinical protocols.ResultsSamples from 544 patients (494 plasma samples and 50 DBS) were included. Median age was 41.0 years (IQR: 33.3 to 48.5) and 58.6% were women. Median HIV‐RNA load was 4.9 log10 copies/mL (4.3 to 5.4). Prevalence of resistance to the NRTI‐backbone was 70.6% (349/494) in plasma samples and 56.0% (28/50) in DBS. Major PI‐resistance mutations conferring high‐level resistance to LPV/r were observed in 26.7% (132/494) of plasma samples and 12% (6/50) of DBS. PI‐exposure testing revealed undetectable LPV levels in 47.0% (232/494) of plasma samples and in 60.0% (30/50) of DBS. In pooled analysis of plasma and DBS samples, detectable LPV levels had a sensitivity of 90% (84% to 94%) and a negative predictive failure of 95% (91% to 97%) for the presence of major LPV/r resistance.ConclusionsPI‐exposure testing revealed non‐adherence in half of patients experiencing failure on second‐line ART and accurately predicted the presence or absence of clinically relevant PI resistance. PI‐exposure testing constitutes a novel screening strategy in patients with virological failure of ART that can differentiate between different underlying causes of therapy failure and may allow for more effective use of limited resources available for drug resistance testing.

Performance comparison of next generation sequencing analysis pipelines for HIV-1 drug resistance testing

Next generation sequencing (NGS) is a trending new standard for genotypic HIV-1 drug resistance (HIVDR) testing. Many NGS HIVDR data analysis pipelines have been independently developed, each with variable outputs and data management protocols. Standardization of such analytical methods and comparison of available pipelines are lacking, yet may impact subsequent HIVDR interpretation and other downstream applications. Here we compared the performance of five NGS HIVDR pipelines using proficiency panel samples from NIAID Virology Quality Assurance (VQA) program. Ten VQA panel specimens were genotyped by each of six international laboratories using their own in-house NGS assays. Raw NGS data were then processed using each of the five different pipelines including HyDRA, MiCall, PASeq, Hivmmer and DEEPGEN. All pipelines detected amino acid variants (AAVs) at full range of frequencies (1~100%) and demonstrated good linearity as compared to the reference frequency values. While the sensitivity in detecting low abundance AAVs, with frequencies between 1~20%, is less a concern for all pipelines, their specificity dramatically decreased at AAV frequencies <2%, suggesting that 2% threshold may be a more reliable reporting threshold for ensured specificity in AAV calling and reporting. More variations were observed among the pipelines when low abundance AAVs are concerned, likely due to differences in their NGS read quality control strategies. Findings from this study highlight the need for standardized strategies for NGS HIVDR data analysis, especially for the detection of minority HIVDR variants.

The progress towards achieving the UNAIDS ambitious viral suppression target among adults living with HIV in South-Western Nigeria

Background: In sub-Saharan Africa where genotypic 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 &amp; handling these are thus essential. Hypothesis tested was antiretroviral therapy adherence effect on viral load outcome. This study was aimed at determining and monitoring HIV/AIDS disease progression using viral load to provide prognostic information and evaluate patients for viral suppression using the World Health Organization (WHO) guideline strategies.&#x0D; Methods: This study was an observational study of subjects living with HIV already initiated on antiretroviral therapy for at least six months, enrolled in health facilities across Ondo State, South-Western Nigeria, during a 12-month observation period starting October 2018 till September 2019. Quantitative viral load analysis was done using Polymerase Chain Reaction, Roche Cobas Taqman 96 Analyzer. All data were statistically analyzed, using Statistical Package for the Social Sciences (SPSS), with multiple comparisons done using Post Hoc Bonferonni test. Results: A total of 8124 (1947 males &amp; 6177 females) subjects eligible for the study were recruited. Most of them are in the age range of 35 – 39 years, with a mean age of 42.02 ± 10.88 years. 7162 (88.2%) &amp; 1771 (21.8%) of the subjects had viral suppression of &lt;1000 RNA copies per ml and &lt;20 RNA copies per ml respectively. The unsuppressed subjects went through enhanced adherence counselling (EAC) for three months and viral load test repeated thereafter. 192 patients who had completed the three sessions of EAC and repeated viral load increased the entire suppression numbers to 7339 (90.3%) &amp; 1824 (22.5%) &lt;1000 RNA copies per ml and &lt;20 RNA copies per ml respectively during the period of observation. ART adherence has significant effect on viral load outcome from the study hypothesis tested.&#x0D; Conclusion: Current ART regimen &amp; HIV treatment enhanced adherence counseling are key to the achieving viral suppression, thus, routine viral load monitoring will ultimately help in HIV/AIDS disease progression follow up and reduce treatment failure tendencies. This will help more patients stay on first line regimen and prolong their life expectancy, indicating that the UNAIDS last 90 target is achievable.