Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Advancing HIV Drug Resistance Technologies and Strategies: Insights from South Africa's Experience and Future Directions for Resource-Limited Settings.

Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.

Resistance is common in paediatric patients failing ART in South Africa.

BACKGROUND: Minimal data exist on HIV drug resistance patterns and prevalence among paediatric patients failing ART in resource-limited settings. We assessed levels of HIV drug resistance in children with virological failure. METHODS: This cross-sectional study, performed from March 2017 to March 2019 in South Africa, enrolled HIV-positive children aged ≤19 years, receiving ART through public health facilities with recent evidence suggestive of virological failure (at least one viral load ≥1000 copies/mL), across 45 randomly selected high-volume clinics from all nine provinces. Resistance genotyping was performed using next-generation sequencing technologies. Descriptive analysis taking into account survey design was used to determine outcomes. RESULTS: Among 899 participants enrolled, the adjusted proportion of HIV drug resistance among children with virological failure was 87.5% (95% CI 83.0%-90.9%). Resistance to NNRTIs was detected in 77.4% (95% CI 72.5%-81.7%) of participants, and resistance to NRTIs in 69.5% (95% CI 62.9%-75.4%) of participants. Overall, resistance to PIs was detected in 7.7% (95% CI 4.4%-13.0%) of children. CONCLUSIONS: HIV drug resistance was highly prevalent in paediatric patients failing ART in South Africa, with 9 in 10 patients harbouring resistance to NNRTIs and/or NRTIs. PI-based regimens are predicted to be highly efficacious in achieving virological suppression amongst patients failing NNRTI-based regimens. Scaling up resistance testing amongst patients would facilitate access to second- and third-line regimens in South Africa.

Performance Evaluation of Four Qualitative RT-PCR Assays for the Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019, and its rapid spread around the globe led the World Health Organization to declare it a pandemic. Laboratory diagnostics provide important information to help control virus transmission, and molecular nucleic acid amplification tests have been recognized as the gold standard for the direct detection of viral genetic material. The main aim of this study was to independently evaluate the analytical performance of four molecular assays that were designed for the detection of SARS-CoV-2 on open testing platforms under emergency use approval, namely, the COVIWOK COVID-19 RT-PCR Meril COVID-19 One-step RT-PCR Kit, the AmoyDx Novel Coronavirus (2019-nCoV) Detection Kit, the Meril COVID-19 One-step RT-PCR Kit and the NeoPlex COVID-19 Detection Kit, as alternatives to the current standard of care (SOC) assays in-country. All of the evaluated assays showed an acceptable performance, with a specificity of 100% and a sensitivity of 93.8% to 98.4%, compared to a SOC assay, with a Cohen's kappa coefficient of ≥0.9 (95% CI). In addition, the assays detected the AccuPlex reference material at 100 copies/mL, suggesting a good limit of detection. These assays provide suitable alternatives to the SOC assays that are currently available in-country, and these alternatives are acceptable for diagnostic use in South Africa. IMPORTANCE Laboratory diagnosis plays an important role in curbing the transmission of infection and reducing harmful delays in clinical and public health responses. Alternatives to the current standard of care assays for SARS-CoV-2 are important in order to overcome the challenges that are associated with global demands and supply shortages. Four molecular assays for the detection of SARS-CoV-2 that were designed for open testing platforms were evaluated in this study under emergency use approval. These assays had acceptable performance and provide suitable alternatives to the current standard of care assays that are available in-country. Their compatibilities with existing in-country amplification platforms make these assays convenient to use for diagnostic testing, both locally and globally These assays were recommended to the South African Health Products Regulatory Authority (SAHPRA) for patient care in South Africa.

Impact of rilpivirine cross-resistance on long-acting cabotegravir-rilpivirine in low and middle-income countries.

Baseline rilpivirine drug resistance mutations (DRMs) are a risk factor for virological failure in patients treated with long-acting cabotegravir and rilpivirine (CAB/RPV LA). We investigated rilpivirine cross-resistance in treatment-naive and experienced patients in South Africa. One in 10 treatment-naive patients and 74.5% of patients failing treatment presented with rilpivirine DRMs. Our data suggest targeted genotyping may be required for patients initiating CAB/RPV LA, which significantly complicates the currently used public health approach.

Virological Findings and Treatment Outcomes of Cases That Developed Dolutegravir Resistance in Malawi's National HIV Treatment Program.

Millions of Africans are on dolutegravir-based antiretroviral therapy (ART), but few detailed descriptions of dolutegravir resistance and its clinical management exist. We reviewed HIV drug resistance (HIVDR) testing application forms submitted between June 2019 and October 2022, data from the national HIVDR database, and genotypic test results. We obtained standardized ART outcomes and virological results of cases with dolutegravir resistance, and explored associations with dolutegravir resistance among individuals with successful integrase sequencing. All cases were on two nucleoside reverse transcriptase inhibitors (NRTIs)/dolutegravir, and had confirmed virological failure, generally with prolonged viremia. Among 89 samples with successful integrase sequencing, 24 showed dolutegravir resistance. Dolutegravir resistance-associated mutations included R263K (16/24), E138K (7/24), and G118R (6/24). In multivariable logistic regression analysis, older age and the presence of high-level NRTI resistance were significantly associated with dolutegravir resistance. After treatment modification recommendations, four individuals (17%) with dolutegravir resistance died, one self-discontinued ART, one defaulted, and one transferred out. Of the 17 remaining individuals, 12 had follow-up VL results, and 11 (92%) were <1000 copies/mL. Twenty-four cases with dolutegravir resistance among 89 individuals with confirmed virological failure suggests a considerable prevalence in the Malawi HIV program. Successful management of dolutegravir resistance was possible, but early mortality was high. More research on the management of treatment-experienced individuals with dolutegravir resistance is needed.

Minimal Cross-resistance to Tenofovir in Children and Adolescents Failing ART Makes Them Eligible for Tenofovir-Lamivudine-Dolutegravir Treatment.

BACKGROUND: Fixed-dose combination of dolutegravir (DTG) with tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) likely improves adherence and has a favorable resistance profile. We evaluated predicted efficacy of TLD (TDF-3TC-DTG) in children and adolescents failing abacavir (ABC), zidovudine (AZT), or TDF containing regimens. METHODS: Drug resistance mutations were analyzed in a retrospective dataset of individuals <19 years of age, failing ABC (n = 293) AZT (n = 288) or TDF (n = 69) based treatment. Pol sequences were submitted to Stanford HIVdb v8.9. Genotypic susceptibility scores were calculated for various DTG-containing regimens. RESULTS: Genotypes were assessed for 650 individuals with a median age of 14 years (IQR 10-17 years). More individuals failed a protease inhibitor (PI)-based (78.3%) than a non-nucleoside reverse transcriptase inhibitors (NNRTI)-based (21.7%) regimen. Most individuals in the AZT group (n = 288; 94.4%) failed a PI-based regimen, compared with 71.0% and 64.2% in the TDF (n = 69) and ABC group (n = 293). Genotypic sensitivity scores <2 to TLD were observed in 8.5% and 9.4% of ABC- and AZT-exposed individuals, compared with 23.2% in the TDF group. The M184V mutation was most often detected in the ABC group (70.6%) versus 60.0% and 52.4% in TDF and AZT groups. The presence of K65R was rare (n = 13, 2.0%) and reduced TLD susceptibility was commonly caused by accumulation of nucleoside reverse transcriptase inhibitor (NRTI) mutations. CONCLUSIONS: Cross-resistance to TDF was limited, further reducing concerns about use of transition to TLD in children and adolescents. The NADIA trial has subsequently shown that patients failing a TDF/3TC/EFV regimen can safely be transitioned to a TLD regimen but we do not have data for patients failing an ABC/3TC/NNRTI or PI regimens. Frequent virological monitoring is recommended after switch to DTG, especially in children continuing ABC in the backbone. Clinical studies correlating predicted resistance with clinical outcomes, especially in settings without access to genotyping, are required.

Guidance for SARS-CoV-2 RNA-Based Molecular Assay Analytical Performance Evaluations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is frequently diagnosed through detection of viral RNA using nucleic acid amplification testing (NAAT) assays that are usually used in centralized settings. Following the publication of the SARS-CoV-2 genetic sequence, multiple diagnostic assays were launched in 2020. These assays require evaluation beyond manufacturer self-reported performance to determine whether they are suitable for use, meet country acceptance criteria, and are compatible with existing in-country platforms. In order to meet the demand for testing services, rapid yet robust assay performance evaluations are required. In our setting, these evaluation protocols required the use of residual patient specimens and reference materials, as typical clinical trials are time-consuming and limited by cost and the cyclical nature of SARS-CoV-2 infection. This protocol is designed to assist in the rapid and robust evaluation of nucleic acid-based assays for the detection of SARS-CoV-2 using limited specimens, reference materials, and test kits. While it is specific for RNA-based assays, it can be adapted for fully automated analyses. The preparation and processing of evaluation panels is described, followed by methods for analytical precision analysis and data visualization. Assay robustness and scalability are briefly discussed as these can be critical for implementation. This protocol is designed to be flexible and alternative options are provided throughout the text where possible.

Long-term HIV treatment outcomes and associated factors in sub-Saharan Africa: multicountry longitudinal cohort analysis.

OBJECTIVE: In a multicountry prospective cohort of persons with HIV from six countries between 2007 and 2015, we evaluated long-term outcomes of first-line non-nucleoside reverse-transcriptase inhibitor-based antiretroviral therapy (ART), and risk factors for loss-to-follow-up, mortality, virological failure, and incomplete CD4 + T-cell recovery. METHODS: We calculated cumulative incidence of lost-to-follow-up, death, virological failure (VL ≥ 1000 cps/ml) and incomplete CD4 + T-cell recovery (<500 cells/µl) at successive years, using Kaplan-Meier and Cox regression. RESULTS: Of 2735 participants, 58.0% were female, median age was 37 (interquartile range [IQR] 32-43) years, and median pre-ART CD4 + T-cell count was 135 (IQR 63-205)/µl. Total follow-up time was 7208 person-years (median 24.3 months, IQR 18.7-58.3). Deaths by any cause and loss to follow-up occurred mostly during the first year of ART (84%, 201/240 and 56%, 199/353, respectively). During their first 6 years of ART, 71% (95% confidence interval [CI] 69.0-73.7) were retained on first-line, and among those 90-93% sustained viral suppression (<1000 cps/ml); CD4 + T-cell recovery was incomplete in 60% (220/363) of participants. The risk factors associated with poor outcomes during long-term ART were: for loss-to-follow-up, recent VL ≥1000 cps/ml, recent CD4 + T-cell count ≤50 cells/µl, age <30 years, being underweight; for mortality, recent CD4 + T-cell count ≤50 cells/µl; and, for virological failure, age <40 years, recent CD4 + T-cell count ≤200 cells/µl, poor adherence, male sex, and low-level viremia. CONCLUSION: To achieve long-term ART success towards the UNAIDS targets, early ART initiation is crucial, coupled with careful monitoring and retention support, particularly in the first year of ART. Male and youth-centred care delivery models are needed to improve outcomes for those vulnerable groups.

Dolutegravir Resistance in Malawi's National HIV Treatment Program.

Dolutegravir HIV drug resistance (HIVDR) data from Africa remain sparse. We reviewed HIVDR results of Malawians on dolutegravir-based antiretroviral therapy (November 2020-September 2021). Of 6462 eligible clients, 33 samples were submitted to South Africa, 27 were sequenced successfully, and 8 (30%) had dolutegravir HIVDR. Malawi urgently requires adequate HIVDR testing capacity.

Rapid Evaluation of the Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus Tests.

The Xpert® Xpress SARS-CoV-2 and Xpert® Xpress SARS-CoV-2/Flu/RSV tests were rapidly developed and widely used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. In response to emerging genetic variability, a new SARS-CoV-2 target (RNA-dependent RNA-polymerase) has been added to both tests: Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus test. A rapid evaluation of both tests was performed in South Africa, using residual respiratory specimens. Residual respiratory specimens (n = 125) were used to evaluate the Xpert® Xpress CoV-2 plus test and included 50 genotyped specimens. The Xpert® Xpress CoV-2/Flu/RSV plus test was assessed using 45 genotyped SARS-CoV-2 specimens, 10 influenza A, 10 influenza B and 20 respiratory syncytial virus specimens. Results were compared to in-country standard-of-care tests. Genotyped specimens tested the performance of the test under pressure from circulating SARS-CoV-2 variants of concern. Reference material was included to assess the test limits and linearity. The Xpert® Xpress CoV-2 plus test performance compared to reference results across residual respiratory specimens was good (positive percentage agreement (PPA) = 95.2%, negative percentage agreement (NPA) = 95.0%) The Xpert® Xpress CoV-2/Flu/RSV plus test showed good performance across all residual respiratory specimens (PPA = 100%, NPA = 98.3%). All genotyped variants of concern were detected by both tests. The Xpert® Xpress CoV-2 plus and Xpert® Xpress CoV-2/Flu/RSV plus tests can be used to diagnose SARS-CoV-2, and to diagnose and differentiate SARS-CoV-2, influenza A, influenza B and respiratory syncytial virus, respectively. The NPA was lower than the recommended 99%, but was influenced by the low number of negative specimens tested. The variants of concern assessed did not affect test performance. It is recommended that sites perform their own assessments compared to in-country standard-of-care tests.

Feasibility and clinical relevance of HIV-1 drug resistance testing in patients with low-level viraemia in South Africa.

OBJECTIVES: To determine the feasibility of HIV genotyping at low-level viraemia (LLV) using an in-house assay in a South African population and the prevalence, as well as the clinical relevance, of drug resistance (HIVDR) in this population. METHODS: We conducted an observational, retrospective, cohort study on patient samples with LLV referred for routine HIVDR testing at a public sector Johannesburg laboratory from August 2017 to October 2018. Genotyping was performed using a nested RT-PCR assay and Sanger sequencing. The genotyping success rate was evaluated for different viraemia categories. Sequences were loaded onto the Stanford HIVdb genotypic resistance tool (version 8.7) for drug resistance interpretation. RESULTS: Plasma samples from 159 HIV-1-infected, treatment-experienced adults with LLV (5-999 copies/mL) were analysed. The in-house assay performed well with an overall success rate of 78.6% (125/159, 95% CI 71.6-84.3). The prevalence of drug resistance mutations in the LLV cohort was 79.2% (99/125, 95% CI 71.2-85.4) with most patients (n = 109, 68.6%) on a PI-based regimen at the time of genotyping. Of 125 sequences obtained, 73.6% (92/125) had ≥1 NRTI mutation while 70.4% (88/125) had ≥1 NNRTI mutation. Major PI mutations, including M46I and V82A, were detected in 7.2% (9/125) of patients. CONCLUSIONS: Current South African virological failure guidelines may keep patients on failing regimens for longer than necessary. Our data suggest that genotyping at LLV is feasible and implementation could result in earlier identification and referral of patients requiring third-line regimens.

Preparing for the next pandemic: Lessons from rapid scale-up of SARS-CoV-2 testing in a South African high-throughput automated HIV molecular laboratory.

Africa's readiness to respond to the SARS-COV-2 pandemic was tested due to reliance on rapid turn-around-time of polymerase chain reaction results for clinical management, isolation and quarantine decisions. The NHLS HIV Molecular Laboratory in Johannesburg, South Africa, is one of the largest automated HIV molecular laboratories worldwide. Despite its extensive molecular capacity and experience in managing high volumes acquired from a large HIV program, significant challenges were encountered during its rapid transition to large scale SARS-CoV-2 testing. We describe the strategies employed to manage these challenges that resulted in a 30% improvement in SARS-CoV-2 test turn-around-time during the first wave peak during which approximately 25000 samples were tested per month, and further improvement during the second wave peak, with 81% within targeted turn-around-time.

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

INTRODUCTION: Dolutegravir 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. METHODS: A 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 DISCUSSION: Less 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. CONCLUSIONS: Although 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.

High levels of HIV drug resistance among adults failing second-line antiretroviral therapy in Namibia.

To support optimal third-line antiretroviral therapy (ART) selection in Namibia, we investigated the prevalence of HIV drug resistance (HIVDR) at time of failure of second-line ART. A cross-sectional study was conducted between August 2016 and February 2017. HIV-infected people ≥15 years of age with confirmed virological failure while receiving ritonavir-boosted protease inhibitor (PI/r)-based second-line ART were identified at 15 high-volume ART clinics representing over >70% of the total population receiving second-line ART. HIVDR genotyping of dried blood spots obtained from these individuals was performed using standard population sequencing methods. The Stanford HIVDR algorithm was used to identify sequences with predicted resistance; genotypic susceptibility scores for potential third-line regimens were calculated. Two hundred thirty-eight individuals were enrolled; 57.6% were female. The median age and duration on PI/r-based ART at time of enrolment were 37 years and 3.46 years, respectively. 97.5% received lopinavir/ritonavir-based regimens. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and PI/r resistance was 50.6%, 63.1%, and 13.1%, respectively. No significant association was observed between HIVDR prevalence and age or sex. This study demonstrates high levels of NRTI and NNRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART in Namibia. Findings underscore the need for objective and inexpensive measures of adherence to identify those in need of intensive adherence counselling, routine viral load monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs in Namibia.

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.

INTRODUCTION: When 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. METHODS: We 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. RESULTS: Samples 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. CONCLUSIONS: PI-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.

Resistance in patients failing integrase strand transfer inhibitors: a call to replace raltegravir with dolutegravir in third-line treatment in South Africa.

Data on integrase resistance patterns in low- and middle-income countries (LMICs) is scarce. We assessed genotypic drug resistance in 43 patients with virological failure on integrase strand transfer inhibitors (INSTIs) containing regimens as part of the third-line treatment program in South Africa. Of the raltegravir (RAL) exposed patients 20/34 (59%) had ≥1 major INSTI mutation, including two (6%) with dolutegravir (DTG) cross-resistance. DTG resistance was detected in one out of four DTG-exposed patients. Replacing RAL with DTG may reduce the risk of INSTI mutations. We recommend DTG drug resistance monitoring when DTG is introduced at a larger scale in LMICs.

PhyloPi: An affordable, purpose built phylogenetic pipeline for the HIV drug resistance testing facility.

INTRODUCTION: Phylogenetic analysis plays a crucial role in quality control in the HIV drug resistance testing laboratory. If previous patient sequence data is available sample swaps can be detected and investigated. As Antiretroviral treatment coverage is increasing in many developing countries, so is the need for HIV drug resistance testing. In countries with multiple languages, transcription errors are easily made with patient identifiers. Here a self-contained blastn integrated phylogenetic pipeline can be especially useful. Even though our pipeline can run on any unix based system, a Raspberry Pi 3 is used here as a very affordable and integrated solution. PERFORMANCE BENCHMARKS: The computational capability of this single board computer is demonstrated as well as the utility thereof in the HIV drug resistance laboratory. Benchmarking analysis against a large public database shows excellent time performance with minimal user intervention. This pipeline also contains utilities to find previous sequences as well as phylogenetic analysis and a graphical sequence mapping utility against the pol area of the HIV HXB2 reference genome. Sequence data from the Los Alamos HIV database was analyzed for inter- and intra-patient diversity and logistic regression was conducted on the calculated genetic distances. These findings show that allowable clustering and genetic distance between viral sequences from different patients is very dependent on subtype as well as the area of the viral genome being analyzed. AVAILABILITY: The Raspberry Pi image for PhyloPi, source code of the pipeline, sequence data, bash-, python- and R-scripts for the logistic regression, benchmarking as well as helper scripts are available at http://scholar.ufs.ac.za:8080/xmlui/handle/11660/7638 and https://github.com/ArmandBester/phylopi. The PhyloPi image and the source code are published under the GPLv3 license. A demo version of the PhyloPi pipeline is available at http://phylopi.hpc.ufs.ac.za/.