High prevalence of pre-treatment and acquired HIV-1 drug resistance mutations among non-citizens living with HIV in Botswana.

Background: Approximately 30,000 non-citizens are living with HIV in Botswana, all of whom as of 2020 are eligible to receive free antiretroviral treatment (ART) within the country. We assessed the prevalence of HIV-1 mutational profiles [pre-treatment drug resistance (PDR) and acquired drug resistance (ADR)] among treatment-experienced (TE) and treatment-naïve (TN) non-citizens living with HIV in Botswana. Methods: A total of 152 non-citizens living with HIV were enrolled from a migrant HIV clinic at Independence Surgery, a private practice in Botswana from 2019-2021. Viral RNA isolated from plasma samples were genotyped for HIV drug resistance (HIVDR) using Sanger sequencing. Major known HIV drug resistance mutations (DRMs) in the pol region were determined using the Stanford HIV Drug Resistance Database. The proportions of HIV DRMs amongst TE and TN non-citizens were estimated with 95% confidence intervals (95% CI) and compared between the two groups. Results: A total of 60/152 (39.5%) participants had a detectable viral load (VL) >40 copies/mL and these were included in the subsequent analyses. The median age at enrollment was 43 years (Q1, Q3: 38-48). Among individuals with VL > 40 copies/mL, 60% (36/60) were treatment-experienced with 53% (19/36) of them on Atripla. Genotyping had a 62% (37/60) success rate - 24 were TE, and 13 were TN. A total of 29 participants (78.4, 95% CI: 0.12-0.35) had major HIV DRMs, including at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) associated DRM. In TE individuals, ADR to any antiretroviral drug was 83.3% (20/24), while for PDR was 69.2% (9/13). The most frequent DRMs were nucleoside reverse transcriptase inhibitors (NRTIs) M184V (62.1%, 18/29), NNRTIs V106M (41.4%, 12/29), and K103N (34.4%, 10/29). No integrase strand transfer inhibitor-associated DRMs were reported. Conclusion: We report high rates of PDR and ADR in ART-experienced and ART-naïve non-citizens, respectively, in Botswana. Given the uncertainty of time of HIV acquisition and treatment adherence levels in this population, routine HIV-1C VL monitoring coupled with HIVDR genotyping is crucial for long-term ART success.

Fostemsavir resistance-associated polymorphisms in HIV-1 subtype C in a large cohort of treatment-naïve and treatment-experienced individuals in Botswana.

IMPORTANCE: Fostemsavir (FTR) is a newly licensed antiretroviral drug that has been shown to have activity against HIV-1. The mechanism of action of FTR is different from all currently available antiretrovirals (ARVs), and as such, it offers hope for HIV-1 suppression in those people with HIV (PWH) who harbor HIV-1 variants with drug resistance mutations to currently used ARVs. Using 6,030 HIV-1 sequences covering the HIV-1 envelope from PWH in Botswana who are antiretroviral therapy (ART) naïve as well as those who are failing ART, we explored the sequences for FTR resistance-associated polymorphisms. We found the prevalence of FTR polymorphisms to be similar in both ART-naïve and ART-experienced individuals with VF in this setting, with no prior FTR exposure. Further studies on the phenotypic impact of these polymorphisms are warranted to guide how to monitor for FTR resistance.

Archived rilpivirine-associated resistance mutations among ART-naive and virologically suppressed people living with HIV-1 subtype C in Botswana: implications for cabotegravir/rilpivirine use.

OBJECTIVES: Pre-existing rilpivirine resistance-associated mutations (RVP-RAMs) have been found to predict HIV-1 virological failure in those switching to long-acting injectable cabotegravir/rilpivirine. We here evaluated the prevalence of archived RPV-RAMs in a cohort of people living with HIV (PWH). METHODS: We analysed near full-length HIV-1 pol sequences from proviral DNA for the presence of RPV-RAMs, which were defined according to the 2022 IAS-USA drug resistance mutation list and Stanford HIV drug resistance database. RESULTS: RPV-RAMs were identified in 757/5805 sequences, giving a prevalence of 13.0% (95% CI 12%-13.9%). Amongst the ART-naive group, 137/1281 (10.7%, 95% CI 9.1%-12.5%) had at least one RPV-RAM. Of the 4524 PWH with viral suppression on ART (VL <400 copies/mL), 620 (13.7%, 95% CI 12.7%-14.7%) had at least one RPV-RAM. E138A was the most prevalent RPV-RAM in the ART-naive group (7.9%) and the ART-suppressed group (9.3%). The rest of the mutations observed (L100I, K101E, E138G, E138K, E138Q, Y181C, H221Y, M230L, A98G, V179D, G190A, G190E and M230I) were below a prevalence of 1%. CONCLUSIONS: RPV-RAMs were present in 10.7% of ART-naive and 13.7% of ART-suppressed PWH in Botswana. The most common RPV-RAM in both groups was E138A. Since individuals with the E138A mutation may be more likely to fail cabotegravir/rilpivirine, monitoring RPV-RAMs will be crucial for effective cabotegravir/rilpivirine implementation in this setting.

HIV-1C in-House RNA-Based Genotyping Assay for Detection of Drug Resistance Mutations in Samples with Low-Level Viral Loads.

Purpose: Monitoring HIV-1 drug resistance mutations (DRM) in treated patients on combination antiretroviral therapy (cART) with a detectable HIV-1 viral load (VL) is important for the selection of appropriate cART. Currently, there is limited data on HIV DRM at low-level viremia (LLV) (VL 401-999 copies/mL) due to the use of a threshold of VL ≥1000 copies/mL for HIV DRM testing. We here assess the performance of an in-house HIV drug resistance genotyping assay using plasma for the detection of DRM at LLV. Methods: We used a total of 96 HIV plasma samples from the population-based Botswana Combination Prevention Project (BCPP). The samples were stratified by VL groups: 50 samples had LLV, defined as 401-999 copies/mL, and 46 had ≥1000 copies/mL. HIV pol (PR and RT) region was amplified and sequenced using an in-house genotyping assay with BigDye sequencing chemistry. Known HIV DRMs were identified using the Stanford HIV Drug Resistance Database. Genotyping success rate between the two groups was estimated and compared using the comparison of proportions test. Results: The overall genotyping success rate was 79% (76/96). For VL groups, the genotyping success was 72% (36/50) at LLV and 87% (40/46) at VL ≥1000 copies/mL. Among generated sequences, the overall prevalence of individuals with at least 1 major or intermediate-associated DRM was 24% (18/76). The proportions of NNRTI-, NRTI- and PI-associated resistance mutations were 28%, 24%, and 0%, respectively. The most predominant mutations detected were K103N (18%) and M184V (12%) in NNRTI- and NRTI-associated mutations, respectively. The prevalence of DRM was 17% (6/36) at LLV and 30% (12/40) at VL ≥1000 copies/mL. Conclusion: The in-house HIV genotyping assay successfully genotyped 72% of LLV samples and was able to detect 17% of DRM amongst them. Our results highlight the possibility and clinical significance of genotyping HIV among individuals with LLV.

Doravirine-associated resistance mutations in antiretroviral therapy naïve and experienced adults with HIV-1 subtype C infection in Botswana.

OBJECTIVES: There are limited data on the prevalence of doravirine (DOR)-associated drug resistance mutations in people with HIV (PWH) in Botswana. This cross-sectional, retrospective study aimed to explore the prevalence of DOR-associated resistance mutations among ART-naïve and -experienced PWH in Botswana enrolled in the population-based Botswana Combination Prevention Project (BCPP). METHODS: A total of 6078 HIV-1C pol sequences were analysed for DOR-associated resistance mutations using the Stanford HIV drug resistance database, and their levels were predicted according to the Stanford DRM penalty scores and resistance interpretation. Virologic failure was defined as HIV-1 RNA load (VL) >400 copies/mL. RESULTS: Among 6078 PWH, 5999 (99%) had known ART status, and 4529/5999 (79%) were on ART at time of sampling. The suppression rate among ART-experienced was 4517/4729 (96%). The overall prevalence of any DOR-associated resistance mutations was 181/1473 (12.3% [95% confidence interval {CI}: 10.7-14.1]); by ART status: 42/212 (19.8% [95% CI: 14.7-25.4]) among ART-failing individuals (VL ≥400 copies/mL) and 139/1261 (11.0% [95% CI: 9.3-12.9]) among ART-naïve individuals (P < 0.01). Intermediate DOR-associated resistance mutations were observed in 106/1261 (7.8% [95% CI: 6.9-10.1]) in ART-naïve individuals and 29/212 (13.7% [95% CI: 9.4-8.5]) among ART-experienced participants (P < 0.01). High-level DOR-associated resistance mutations were observed in 33/1261 (2.6% [95% CI: 1.8-3.7]) among ART-naïve and 13/212 (6.1% [95% CI: 3.6-10.8]) among ART-failing PWH (P < 0.01). PWH failing ART with at least one EFV/NVP-associated resistance mutation had high prevalence 13/67 (19.4%) of high-level DOR-associated resistance mutations. CONCLUSION: DOR-associated mutations were rare (11.0%) among ART-naive PWH but present in 62.7% of Botswana individuals who failed NNRTI-based ART with at least one EFV/NVP-associated resistance mutation. Testing for HIV drug resistance should underpin the use of DOR in PWH who have taken first-generation NNRTIs.

Low-frequency HIV-1 drug resistance mutations in antiretroviral naïve individuals in Botswana.

BACKGROUND: Individuals living with human immunodeficiency virus (HIV) who experience virological failure (VF) after combination antiretroviral therapy (cART) initiation may have had low-frequency drug resistance mutations (DRMs) at cART initiation. There are no data on low-frequency DRMs among cART-naïve HIV-positive individuals in Botswana. METHODS: We evaluated the prevalence of low-frequency DRMs among cART-naïve individuals previously sequenced using Sanger sequencing. The generated pol amplicons were sequenced by next-generation sequencing. RESULTS: We observed low-frequency DRMs (detected at <20% in 33/103 (32%) of the successfully sequenced individuals, of whom four also had mutations detected at >20%. K65R was the most common low-frequency DRM detected in 8 individuals. Eighty-two of the 103 individuals had follow-up viral load data while on cART. Twenty-seven of the 82 individuals harbored low-frequency DRMs. Only 12 of 82 individuals experienced VF. The following low-frequency DRMs were observed in four individuals experiencing VF: K65R, K103N, V108I, and Y188C. No statistically significant difference was observed in the prevalence of low-frequency DRMs between individuals experiencing VF (4/12) and those not experiencing VF (23/70) (P = .97). However, individuals with non-nucleoside reverse transcriptase inhibitors-associated low-frequency DRMs were 2.68 times more likely to experience VF (odds ratio, 2.68; 95% confidential interval, 0.4-13.9) compared with those without (P = .22). CONCLUSION: Next-generation sequencing was able to detect low-frequency DRMs in this cohort in Botswana, but these DRMs did not contribute significantly to VF.

A fast-screening dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry method applied to the determination of efavirenz in human plasma samples.

We demonstrate the suitability of a fast, green, easy-to-perform, and modified sample extraction procedure, i.e., dispersive liquid-liquid microextraction (DLLME) for the determination of efavirenz (EFV) in human plasma. Data acquisition was done by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. The simplicity of the method lies in, among others, the avoidance of the use of large organic solvent volumes as mobile phases and non-volatile buffers that tend to block the plumbing in high-performance liquid chromatography (HPLC). Chromatographic and mass spectral parameters were optimized using bovine whole blood for matrix matching due to insufficient human plasma. Method validation was accomplished using the United States Food and Drug Administration (USFDA) 2018 guidelines. The calibration curve was linear with a dynamic range of 0.10-2.0 µg/mL and an R2 value of 0.9998. The within-run accuracy and precision were both less than 20% at the lower limit of quantification (LLOQ) spike level. The LLOQ was 0.027 µg/mL which compared well with some values but was also orders of magnitude better than others reported in the literature. The percent recovery was 91.5% at the LLOQ spike level. The DLLME technique was applied in human plasma samples from patients who were on treatment with EFV. The human plasma samples gave concentrations of EFV ranging between 0.14-1.00 µg/mL with three samples out of seven showing concentrations that fell within or close to the recommended therapeutic range.

Limited HIV-1 Subtype C nef 3'PPT Variation in Combination Antiretroviral Therapy Naïve and Experienced People Living with HIV in Botswana.

Dolutegravir (DTG) is a potent anti-HIV drug that is used to treat HIV globally. There have been reports of mutations in the HIV-1 3'-polypurine tract (3'PPT) of the nef gene, contributing to DTG failure; however, there are limited 'real-world' data on this. In addition, there is a knowledge gap on the variability of 3'PPT residues in patients receiving combination antiretroviral therapy (cART) with and without viral load (VL) suppression. HIV-1 subtype C (HIV-1C) whole-genome sequences from cART naïve and experienced individuals were generated using next-generation sequencing. The nef gene sequences were trimmed from the generated whole-genome sequences using standard bioinformatics tools. In addition, we generated separate integrase and nef gene sequences by Sanger sequencing of plasma samples from individuals with virologic failure (VF) while on a DTG/raltegravir (RAL)-based cART. Analysis of 3'PPT residues was performed, and comparison of proportions computed using Pearson's chi-square test with p-values < 0.05 was considered statistically significant. A total of 6009 HIV-1C full genome sequences were generated and had a median log10 HIV-1 VL (Q1, Q3) copies/mL of 1.60 (1.60, 2.60). A total of 12 matching integrase and nef gene sequences from therapy-experienced participants failing DTG/ RAL-based cART were generated. HIV-1C 3'PPT nef gene sequences from therapy-experienced patients failing DTG cART (n = 12), cART naïve individuals (n = 1263), and individuals on cART with and without virological suppression (n = 4696) all had a highly conserved 3'PPT motif with no statistically significant differences identified. Our study confirms the high conservation of the HIV-1 nef gene 3'PPT motif in 'real-world' patients and showed no differences in the motif according to VL suppression or INSTI-based cART failure. Future studies should explore other HIV-1 regions outside of the pol gene for associations with DTG failure.

HIV-1 Subtype C Drug Resistance Mutations in Heavily Treated Patients Failing Integrase Strand Transfer Inhibitor-Based Regimens in Botswana.

There are limited real-world mutational and virological outcomes data of treatment-experienced persons diagnosed with HIV-1 subtype C (HIV-1 C) who are failing Integrase Strand Transfer Inhibitor-based regimens. Requisition forms sent for HIV-1 genotypic resistance testing (GRT) between May 2015 and September 2019 were reviewed and participants experiencing virologic failure while on dolutegravir (DTG) or raltegravir (RAL) cART at sampling recruited. Sanger sequencing of the HIV-1 Pol gene was performed from residual plasma samples and drug resistance mutational (DRM) analysis performed using the Stanford University HIV drug resistance database. 40 HIV-1C integrase sequences were generated from 34 individuals, 24 of whom were on DTG cART, three on RAL cART and seven on an unknown (DTG or RAL)-anchored cART at time of GRT. 11/34 (32%) individuals had DRMs to DTG and other integrase inhibitors. 7/11 (64%) patients had exposure to a RAL-based cART at the time of sampling. Out of the 11 individuals with DRMs, one (9%) had 2-class, 6 (55%) had 3-class, and 4 (36%) had 4-class multidrug-resistant HIV-1C. 7/11 individuals (64%) are currently virologically suppressed. Of the four individuals not virologically suppressed, three had extensive DRMs involving 4-classes of ARV drugs and one individual has demised. Resistance to DTG occurs more often in patients exposed to RAL cART. Individuals with 4-class DRMs plus integrase T97 and E157Q mutations appear to have worse outcomes. There is a need for frequent VL monitoring and GRT amongst treatment-experienced HIV-1C diagnosed individuals.

Association of CYP2B6 Genetic Variation with Efavirenz and Nevirapine Drug Resistance in HIV-1 Patients from Botswana.

PURPOSE: CYP2B6 liver enzyme metabolizes the two non-nucleoside reverse transcriptase inhibitors Efavirenz (EFV) and Nevirapine (NVP) used in the antiretroviral therapy (ART) regimens for HIV-infected individuals. Polymorphisms of the CYP2B6 gene influence drug levels in plasma and possibly virological outcomes. The aim of this study was to explore the potential impact of CYP2B6 genotype and haplotype variation on the risk of developing EFV/NVP drug resistance mutations (DRMs) in HIV-1 patients receiving EFV-/NVP-containing regimens in Botswana. PATIENTS AND METHODS: Participants were a sub-sample of a larger study (Tshepo study) conducted in Gaborone, Botswana, among HIV-infected individuals taking EFV/NVP containing ART. Study samples were retrieved and assigned to cases (with DRMs) and controls (without DRMs). Four single-nucleotide polymorphisms (SNPs) in the CYP2B6 gene (-82T>C; 516G>T; 785A>G; 983T>C) were genotyped, the haplotypes reconstructed, and the metabolic score assigned. The possible association between drug resistance and several independent factors (baseline characteristics and CYP2B6 genotypes) was assessed by Binary Logistic Regression (BLR) analysis. EFV/NVP resistance status and CYP2B6 haplotypes were also analyzed using Z-test, chi-square and Fisher's exact test statistics. RESULTS: Two hundred and twenty-seven samples were analysed (40 with DRMs, 187 without DRMs). BLR analysis showed an association between EFV/NVP resistance and CYP2B6 516G allele (OR: 2.26; 95% CI: 1.27-4.01; P=0.005). Moreover, haplotype analysis revealed that the proportion of EFV/NVP-resistant infections was higher among CYP2B6 fast than extensive/slow metabolizers (30.8% vs 16.8%; P=0.035), with the 516G allele more represented in the haplotypes of fast than extensive/slow metabolizers (100.0% vs 53.8%; P<0.001). CONCLUSION: We demonstrated that the CYP2B6 516G allele, and even more when combined in fast metabolic haplotypes, is associated with the presence of EFV/NVP resistance, strengthening the need to assess the CYP2B6 genetic profiles in HIV-infected patients in order to improve the virologic outcomes of NNRTI containing ART.

Differences in human immunodeficiency virus-1C viral load and drug resistance mutation between plasma and cerebrospinal fluid in patients with human immunodeficiency virus-associated cryptococcal meningitis in Botswana.

To determine effects of cryptococcal meningitis (CM) on human immunodeficiency virus (HIV)-1C cerebrospinal fluid (CSF) viral escape, CSF/plasma viral discordance, and drug resistance mutation (DRM) discordance between CSF and plasma compartments, we compared CSF and plasma viral load (VL) and DRMs in individuals with HIV-associated CM in Botswana.This cross-sectional study utilized 45 paired CSF/plasma samples from participants in a CM treatment trial (2014-2016). HIV-1 VL was determined and HIV-1 protease and reverse transcriptase genotyping performed. DRMs were determined using the Stanford HIV database. CSF viral escape was defined as HIV-1 ribonucleic acid ≥0.5 log10 higher in CSF than plasma and VL discordance as CSF VL > plasma VL.HIV-1 VL was successfully measured in 39/45 pairs, with insufficient sample volume in 6; 34/39 (87.2%) participants had detectable HIV-1 in plasma and CSF, median 5.1 (interquartile range: 4.7-5.7) and 4.6 (interquartile range:3.7-4.9) log10 copies/mL, respectively (P≤.001). CSF viral escape was present in 1/34 (2.9%) and VL discordance in 6/34 (17.6%). Discordance was not associated with CD4 count, antiretroviral status, fungal burden, CSF lymphocyte percentage nor mental status. Twenty-six of 45 (57.8%) CSF/plasma pairs were successfully sequenced. HIV-1 DRM discordance was found in 3/26 (11.5%); 1 had I84IT and another had M46MI in CSF only. The third had K101E in plasma and V106 M in CSF.Our findings suggest that HIV-1 escape and DRM discordance may occur at lower rates in participants with advanced HIV-disease and CM compared to those with HIV associated neurocognitive impairment.

In Silico Prediction of Human Leukocytes Antigen (HLA) Class II Binding Hepatitis B Virus (HBV) Peptides in Botswana.

Hepatitis B virus (HBV) is the primary cause of liver-related malignancies worldwide, and there is no effective cure for chronic HBV infection (CHB) currently. Strong immunological responses induced by T cells are associated with HBV clearance during acute infection; however, the repertoire of epitopes (epi) presented by major histocompatibility complexes (MHCs) to elicit these responses in various African populations is not well understood. In silico approaches were used to map and investigate 15-mers HBV peptides restricted to 9 HLA class II alleles with high population coverage in Botswana. Sequences from 44 HBV genotype A and 48 genotype D surface genes (PreS/S) from Botswana were used. Of the 1819 epi bindings predicted, 20.2% were strong binders (SB), and none of the putative epi bind to all the 9 alleles suggesting that multi-epitope, genotype-based, population-based vaccines will be more effective against HBV infections as opposed to previously proposed broad potency epitope-vaccines which were assumed to work for all alleles. In total, there were 297 unique epi predicted from the 3 proteins and amongst, S regions had the highest number of epi (n = 186). Epitope-densities (Depi) between genotypes A and D were similar. A number of mutations that hindered HLA-peptide binding were observed. We also identified antigenic and genotype-specific peptides with characteristics that are well suited for the development of sensitive diagnostic kits. This study identified candidate peptides that can be used for developing multi-epitope vaccines and highly sensitive diagnostic kits against HBV infection in an African population. Our results suggest that viral variability may hinder HBV peptide-MHC binding, required to initiate a cascade of immunological responses against infection.

Use of a mutation-specific genotyping method to assess for HIV-1 drug resistance in antiretroviral-naïve HIV-1 Subtype C-infected patients in Botswana.

Background: HIV-1 drug resistance poses a major threat to the success of antiretroviral therapy. The high costs of available HIV drug resistance assays prohibit their routine usage in resource-limited settings. Pan-degenerate amplification and adaptation (PANDAA), a focused genotyping approach based on quantitative PCR (qPCR), promises a fast and cost-effective way to detect HIV drug resistance mutations (HIVDRMs).  Given the high cost of current genotyping methods, we sought to use PANDAA for screening key HIVDRMs in antiretroviral-naïve individuals at codons 103, 106 and 184 of the HIV-1 reverse transcriptase gene. Mutations selected at these positions have been shown to be the most common driver mutations in treatment failure.  Methods: A total of 103 samples from antiretroviral-naïve individuals previously genotyped by Sanger population sequencing were used to assess and verify the performance of PANDAA. PANDAA samples were run on the ABI 7500 Sequence Detection System to genotype the K103N, V106M and M184V HIVDRMs. In addition, the cost per sample and reaction times were compared. Results: Sanger population sequencing and PANDAA detected K103N mutation in three (2.9%) out of 103 participants.  There was no evidence of baseline V106M and M184V mutations observed in our study. To genotype the six HIVDRMs it costs approximately 40 USD using PANDAA, while the reagents cost per test for Sanger population sequencing is approximately 100 USD per sample. PANDAA was performed quicker compared to Sanger sequencing, 2 hours for PANDAA versus 15 hours for Sanger sequencing. Conclusion: The performance of PANDAA and Sanger population sequencing demonstrated complete concordance. PANDAA could improve patient management by providing quick and relatively cheap access to drug-resistance information.

Comparison of an in-house 'home-brew' and commercial ViroSeq integrase genotyping assays on HIV-1 subtype C samples.

BACKGROUND: Roll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals. METHODS: We used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time. RESULTS: Among 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C. CONCLUSIONS: The IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

Four-class drug-resistant HIV-1 subtype C in a treatment experienced individual on dolutegravir-based antiretroviral therapy in Botswana.

: There are limited data on the effectiveness of dolutegravir (DTG)-based combination antiretroviral therapy (ART) in real-life settings in southern Africa where HIV-1 subtype C predominates. We report a patient infected with HIV-1 subtype C on DTG-based ART previously exposed to raltegravir who developed multidrug resistance mutations to four antiretroviral classes. There is need for drug resistance monitoring and clinical vigilance to ensure effectiveness of HIV treatment programs even in the era of DTG-based ART.