Testing In Resource-limited Settings Research Articles

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ μL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ μL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ μL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar’s test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar’s test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.

Rapid Multiplexed Immunoassay for Detection of Antibodies to Kaposi's Sarcoma-Associated Herpesvirus.

Diagnosis of KSHV-infected individuals remains a challenge. KSHV prevalence is high in several populations with high prevalence of HIV, leading to increased risk of development of Kaposi’s sarcoma (KS). While current assays are reliable for detecting antibodies to KSHV, none are routinely utilized to identify individuals with KSHV infection and thus at increased risk for KS due to assay complexity, lack of access to testing, and cost, particularly in resource-limited settings. Here we describe the addition of KSHV proteins LANA and K8.1 to a previously evaluated HIV/co-infection multiplexed fluorescence immunoassay system. This study demonstrates assay performance by measuring antibody reactivity for KSHV and HIV-1 in a collection of clinical specimens from patients with biopsy-proven KS and sourced negative controls. The KSHV assay correctly identified 155 of 164 plasma samples from patients with biopsy-proven KS and 85 of 93 KSHV antibody (Ab)-negative samples for a sensitivity of 95.1% and specificity of 91.4%. Assay performance for HIV-1 detection was also assessed with 100% agreement with independently verified HIV-1 Ab-positive and Ab-negative samples. These results demonstrate good sensitivity and specificity for detection of antibody to KSHV antigens, and demonstrate the potential for multiplexed co-infection testing in resource-limited settings to identify those at increased risk for HIV-1-related complications.

Single-use, electricity-free amplification device for detection of HIV-1

Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at −20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

Point-of-Care Diagnostics for Improving Maternal Health in South Africa

Improving maternal health is a global priority, particularly in high HIV-endemic, resource-limited settings. Failure to use health care facilities due to poor access is one of the main causes of maternal deaths in South Africa. “Point-of-care” (POC) diagnostics are an innovative healthcare approach to improve healthcare access and health outcomes in remote and resource-limited settings. In this review, POC testing is defined as a diagnostic test that is carried out near patients and leads to rapid clinical decisions. We review the current and emerging POC diagnostics for maternal health, with a specific focus on the World Health Organization (WHO) quality-ASSURED (Affordability, Sensitivity, Specificity, User friendly, Rapid and robust, Equipment free and Delivered) criteria for an ideal point-of-care test in resource-limited settings. The performance of POC diagnostics, barriers and challenges related to implementing POC diagnostics for maternal health in rural and resource-limited settings are reviewed. Innovative strategies for overcoming these barriers are recommended to achieve substantial progress on improving maternal health outcomes in these settings.

Evaluation of the FACSPresto, a New Point of Care Device for the Enumeration of CD4% and Absolute CD4+ T Cell Counts in HIV Infection.

IntroductionEnumeration of CD4+ T lymphocytes is important for pre-ART disease staging and screening for opportunistic infections, however access to CD4 testing in resource limited settings is poor. Point of care (POC) technologies can facilitate improved access to CD4 testing. We evaluated the analytical performance of a novel POC device the FACSPresto compared to the FACSCalibur as a reference standard and to the PIMA, a POC device in widespread use in sub-Saharan Africa.MethodSpecimens were obtained from 253 HIV infected adults. Venous blood samples were analyzed on the FACSPresto and the FACSCalibur, in a subset of 41 samples additional analysis was done on the PIMA.ResultsThe absolute CD4 count results obtained on the FACSPresto were comparable to those on the FACSCalibur with low absolute (9.5cells/μl) and relative bias (3.2%). Bias in CD4% values was also low (1.06%) with a relative bias of 4.9%. The sensitivity was lower at a CD4 count threshold of ≤350cells/μl compared with ≤500cells/μl (84.9% vs. 92.8%) resulting in a high upward misclassification rate at low CD4 counts. Specificity at thresholds of ≤350cells/μl and ≤500cells/μl were 96.6% and 96.8% respectively. The PIMA had a high absolute (-68.6cells/μl) and relative bias (-10.5%) when compared with the FACSCalibur. At thresholds of ≤350cells/μl and ≤500cells/μl the sensitivity was 100% and 95.5% respectively; specificity was 85.7% and 84.2% respectively. The coefficients of repeatability were 4.13%, 5.29% and 9.8% respectively.DiscussionThe analytic performance of the FACSPresto against the reference standard was very good with better agreement and precision than the PIMA. The FACSPresto had comparable sensitivity at a threshold of 500 cells/μl and better specificity than the PIMA. However the FACSPresto showed reduced sensitivity at low CD4 count thresholds.ConclusionThe FACSPresto can be reliably used as a POC device for enumerating absolute CD4 count and CD4% values.

The Next Phase for Point-of-Care Testing in Resource-Limited Settings

In this essay we ask the question, what tests are clinically important in low-resource settings but not widely available in point-of-care (POC) formats? A survey of laboratory test offerings in Kampala, Uganda revealed three tests (and our three wishes) commonly offered in high complexity laboratories but rarely found in point of care laboratories: viral hepatitis immunoassays/liver enzymes, renal function tests, and lipid profiles. The focus of the POC industry on infectious disease in emerging nations was understandable when the burden of disease was primarily infectious, but due to the demographic transition occurring in sub-Saharan Africa and other resource-poor regions, there is a now a co-burden of communicable and noncommunicable disease. An agenda for POC testing of noncommunicable disease in these settings is not yet mature and should be informed by local test utilization patterns.

Rapid diagnostic point of care tests in resource limited settings

Abstract: One of the definitions of diagnostic point of care testing is provision of laboratory testing at or near the site of patient care. It has the potential to minimise the time to obtain the result of the test, which expedites the diagnosis and initiation of the treatment especially in resource-limited settings where health care infrastructure is weak and access to quality and timely medical care represents a challenge. It is estimated, that introduction of rapid, laboratory-independent diagnostic tests for four diseases (syphilis, malaria, tuberculosis and bacterial pneumonia) in developing countries could prevent more than 1.2 million deaths annually. In resource-limited settings, laboratory medicine is still one of the most neglected pillars of the health care. In such settings, primary health care services largely depend on diagnostic point of care testing; therefore the benefits need to overweight the costs. To optimise the diagnostic point-of-care, there is a need for strict evaluation focused on relevant clinical outcomes and operational costs and these evaluations differ from the conventional tests. However, there is no consistent, standardized approach to assess the point-of-care testing technologies in resource-limited settings. Diagnostic point-of-care testing possesses significant importance in infections like TB or HIV, because it eliminates the long turn-around times and delays and the resulting loss of patients from the testing and therapy pathway. An ideal rapid diagnostic point-of-care test used in resource-limited settings should fulfil the following criteria: allowing a quick clinical decision, can be used by a health care worker (possibly by a nonprofessional), affordable, rapid, acceptable test efficacy, and cost effectiveness.

Developing a Screening Algorithm for Type II Diabetes Mellitus in the Resource-Limited Setting of Rural Tanzania

BackgroundNoncommunicable diseases are on pace to outnumber infectious disease as the leading cause of death in sub-Saharan Africa, yet many questions remain unanswered with concern toward effective methods of screening for type II diabetes mellitus (DM) in this resource-limited setting. We aim to design a screening algorithm for type II DM that optimizes sensitivity and specificity of identifying individuals with undiagnosed DM, as well as affordability to health systems and individuals. MethodsBaseline demographic and clinical data, including hemoglobin A1c (HbA1c), were collected from 713 participants using probability sampling of the general population. We used these data, along with model parameters obtained from the literature, to mathematically model 8 purposed DM screening algorithms, while optimizing the sensitivity and specificity using Monte Carlo and Latin Hypercube simulation. ResultsAn algorithm that combines risk assessment and measurement of fasting blood glucose was found to be superior for the most resource-limited settings (sensitivity 68%, sensitivity 99% and cost per patient having DM identified as $2.94). Incorporating HbA1c testing improves the sensitivity to 75.62%, but raises the cost per DM case identified to $6.04. The preferred algorithms are heavily biased to diagnose those with more severe cases of DM. ConclusionsUsing basic risk assessment tools and fasting blood sugar testing in lieu of HbA1c testing in resource-limited settings could allow for significantly more feasible DM screening programs with reasonable sensitivity and specificity.

C-reactive protein point-of-care testing and antibiotic prescribing for acute respiratory tract infections in rural primary health centres of North Ethiopia: a cross-sectional study.

Unjustified antibiotic prescribing for acute upper respiratory infections (URTIs) is probably more common in poor-resource settings where physicians are scarce. Introducing C-reactive protein (CRP) point-of-care testing in such settings could reduce the misuse of antibiotics, which could avert antibiotic resistance. However, information useful for the applicability of CRP test in resource-limited settings is lacking. This study aimed to elicit the frequency of antibiotic prescribing and distribution of CRP levels in remote, rural settings in Ethiopia. We included 414 patients with acute URTIs from four health centres. Health professionals recorded the clinical features of the patients, but the laboratory professionals measured the CRP levels of all patients at the point of care. The most prominent respiratory causes for consultation were acute URTIs combined (44.4%), and lower respiratory tract infections—pneumonia (29.71%) and acute bronchitis (25.84%). The CRP distribution was <20 mg/l, 20–99 mg/l and 100 mg/l or more in 66.6%, 27.9% and 5.5% of the patients, respectively. The CRP levels were significantly different among these clinical diagnoses (X2=114.3, P<0.001, d.f.=4). A wide range of antibiotics was administered for 87.8% of the patients, regardless of the diagnostic or prognostic nature of their diseases. Antibiotic prescribing for acute URTIs in the rural areas of Ethiopia is unduly high, with high proportions of mild, self-limiting illness, mostly URTIs. Implementation of CRP point-of-care testing in such resource-constrained settings, with low- or middle-grade healthcare professionals, could help reconcile the inappropriate use of antibiotics by withholding from patients who do not benefit from antibiotic treatment.

Scale-up of Routine Viral Load Testing in Resource-Poor Settings: Current and Future Implementation Challenges.

Despite immense progress in antiretroviral therapy (ART) scale-up, many people still lack access to basic standards of care, with our ability to meet the Joint United Nations Programme on HIV/AIDS 90-90-90 treatment targets for HIV/AIDS dependent on dramatic improvements in diagnostics. The World Health Organization recommends routine monitoring of ART effectiveness using viral load (VL) testing at 6 months and every 12 months, to monitor treatment adherence and minimize failure, and will publish its VL toolkit later this year. However, the cost and complexity of VL is preventing scale-up beyond developed countries and there is a lack of awareness among clinicians as to the long-term patient benefits and its role in prolonging the longevity of treatment programs. With developments in this diagnostic field rapidly evolving-including the recent improvements for accurately using dried blood spots and the imminent appearance to the market of point-of-care technologies offering decentralized diagnosis-we describe current barriers to VL testing in resource-limited settings. Effective scale-up can be achieved through health system and laboratory system strengthening and test price reductions, as well as tackling multiple programmatic and funding challenges.

Still Far From 90-90-90: Virologic Outcomes of Children on Antiretroviral Therapy in Nurse-led Clinics in Rural Lesotho.

This survey assessed virologic outcomes of children on antiretroviral therapy and potential predictors in 10 nurse-led clinics in Lesotho. Viral suppression was achieved in 72% of the 191 children. No predictors for virologic outcome were found, underlining the need for routine viral load testing in resource-limited settings to achieve 90-90-90.

Assessment of colorimetric amplification methods in a paper-based immunoassay for diagnosis of malaria.

Colorimetric detection methods that produce results readable by eye are important for diagnostic tests in resource-limited settings. In this work, we have compared three main types of colorimetric methods - enzymatic reactions, silver deposition catalyzed by gold nanoparticles, and polymerization-based amplification - in a paper-based immunoassay for detection of Plasmodium falciparum histidine-rich protein 2, a biomarker of malarial infection. We kept the binding events in the immunoassay constant in order to isolate the effect of the detection method on the outcome of the test. We have highlighted that the optimal readout time in a test can vary significantly - ranging from immediately after addition of a visualization agent to 25 minutes after addition of a visualization agent - depending on the colorimetric method being used, and accurate time keeping is essential to prevent false positives in methods where substantial color develops over time in negative tests. We have also shown that the choice of a colorimetric method impacts the calculated limit-of-detection, the ease of visual perception of the readout, and the total cost of the assay, and therefore directly impacts the feasibility and the ease-of-use of a test in field settings.

Genital Shedding of Resistant Human Immunodeficiency Virus-1 Among Women Diagnosed With Treatment Failure by Clinical and Immunologic Monitoring.

Background. The accumulation of human immunodeficiency virus (HIV) resistance mutations can compromise treatment outcomes and promote transmission of drug-resistant virus. We conducted a study to determine the duration and evolution of genotypic drug resistance in the female genital tract among HIV-1-infected women failing first-line therapy. Methods. Treatment failure was diagnosed based on World Health Organization (WHO) clinical or immunologic criteria, and second-line therapy was initiated. Stored plasma and genital samples were tested to determine the presence and timing of virologic failure and emergence of drug resistance. The median duration of genital shedding of genotypically resistant virus prior to regimen switch was estimated. Results. Nineteen of 184 women were diagnosed with treatment failure, of whom 12 (63.2%) had confirmed virologic failure at the switch date. All 12 women with virologic failure (viral load, 5855-1 086 500 copies/mL) had dual-class resistance in plasma. Seven of the 12 (58.3%) had genital HIV-1 RNA levels high enough to amplify (673-116 494 copies/swab), all with dual-class resistance. The median time from detection of resistance in stored samples to regimen switch was 895 days (95% confidence interval [CI], 130-1414 days) for plasma and 629 days (95% CI, 341-984 days) for genital tract secretions. Conclusions. Among women diagnosed with treatment failure using WHO clinical or immunologic criteria, over half had virologic failure confirmed in stored samples. Resistant HIV-1 RNA was shed in the genital tract at detectable levels for ≈1.7 years before failure diagnosis, with steady accumulation of mutations. These findings add urgency to the ongoing scale-up of viral load testing in resource-limited settings.

3D-printed supercapacitor-powered electrochemiluminescent protein immunoarray.

Herein we report a low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) protein immunoarray fabricated by an inexpensive 3-dimensional (3D) printer. The immunosensor detects three cancer biomarker proteins in serum within 35 min. The 3D-printed device employs hand screen printed carbon sensors with gravity flow for sample/reagent delivery and washing. Prostate cancer biomarker proteins, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA) and platelet factor-4 (PF-4) in serum were captured on the antibody-coated carbon sensors followed by delivery of detection-antibody-coated Ru(bpy)3(2+) (RuBPY)-doped silica nanoparticles in a sandwich immunoassay. ECL light was initiated from RuBPY in the silica nanoparticles by electrochemical oxidation with tripropylamine (TPrA) co-reactant using supercapacitor power and ECL was captured with a CCD camera. The supercapacitor was rapidly photo-recharged between assays using an inexpensive solar cell. Detection limits were 300-500f gmL(-1) for the 3 proteins in undiluted calf serum. Assays of 6 prostate cancer patient serum samples gave good correlation with conventional single protein ELISAs. This technology could provide sensitive onsite cancer diagnostic tests in resource-limited settings with the need for only moderate-level training.

Electrochemical Detection in Stacked Paper Networks.

Paper-based electrochemical biosensors are a promising technology that enables rapid, quantitative measurements on an inexpensive platform. However, the control of liquids in paper networks is generally limited to a single sample delivery step. Here, we propose a simple method to automate the loading and delivery of liquid samples to sensing electrodes on paper networks by stacking multiple layers of paper. Using these stacked paper devices (SPDs), we demonstrate a unique strategy to fully immerse planar electrodes by aqueous liquids via capillary flow. Amperometric measurements of xanthine oxidase revealed that electrochemical sensors on four-layer SPDs generated detection signals up to 75% higher compared with those on single-layer paper devices. Furthermore, measurements could be performed with minimal user involvement and completed within 30 min. Due to its simplicity, enhanced automation, and capability for quantitative measurements, stacked paper electrochemical biosensors can be useful tools for point-of-care testing in resource-limited settings.

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.

Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution. To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture. Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded. One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay. The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting.

Monitoring the quality of HIV-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings.

HIV-1 viral load (VL) levels are used for monitoring disease progression and antiretroviral therapy outcomes in HIV-infected patients. To assess the performance of laboratories conducting HIV-1 VL testing in resource-limited settings, the U.S. Centers for Disease Control and Prevention implemented a voluntary, free-of-charge, external quality assurance program using dried tube specimens (DTSs). Between 2010 and 2012, DTS proficiency testing (PT) panels consisting of 5 specimens were distributed at ambient temperature to participants. The results from the participants (n≥6) using the same assay were grouped, analyzed, and graded as acceptable within a group mean±3 standard deviations. Mean proficiency scores were calculated by dividing the combined PT scores by the number of testing cycles using a linear regression model. Between 2010 and 2012, the number of participants enrolled increased from 32 in 16 countries to 114 in 44 countries. A total of 78.2% of the participants reported results using 10 different VL assays. The rates of reporting of acceptable results by the participants were 96.6% for the Abbott assay, 96.3% for the Roche Cobas assay, 94.5% for the Roche Amplicor assay, 93.0% for the Biocentric assay, and 89.3% for the NucliSens assay. The overall mean proficiency scores improved over time (P=0.024). DTSs are a good alternative specimen type to plasma specimens for VL PT programs, as they do not require cold chain transportation and can be used on PCR-based assays. Our data suggest that the CDC HIV-1 VL PT program using DTSs positively impacts the testing performance of the participants, which might translate into better and more accurate VL testing services for patients.

Addressing Barriers to the Development and Adoption of Rapid Diagnostic Tests in Global Health.

Immunochromatographic rapid diagnostic tests (RDTs) have demonstrated significant potential for use as point-of-care diagnostic tests in resource-limited settings. Most notably, RDTs for malaria have reached an unparalleled level of technological maturity and market penetration, and are now considered an important complement to standard microscopic methods of malaria diagnosis. However, the technical development of RDTs for other infectious diseases, and their uptake within the global health community as a core diagnostic modality, has been hindered by a number of extant challenges. These range from technical and biological issues, such as the need for better affinity agents and biomarkers of disease, to social, infrastructural, regulatory and economic barriers, which have all served to slow their adoption and diminish their impact. In order for the immunochromatographic RDT format to be successfully adapted to other disease targets, to see widespread distribution, and to improve clinical outcomes for patients on a global scale, these challenges must be identified and addressed, and the global health community must be engaged in championing the broader use of RDTs.

Expanding access to HIV viral load testing: a systematic review of RNA stability in EDTA tubes and PPT beyond current time and temperature thresholds.

BackgroundHIV viral load (VL) testing is the gold standard for antiretroviral treatment monitoring, but many barriers exist to VL testing in resource-limited settings, including storage and transport limitations for whole blood and plasma. Data from various studies indicate that HIV RNA is stable beyond current recommendations. We conducted a systematic review to assess stability data of HIV RNA in whole blood and plasma across times and temperatures.Methods and FindingsUsing a pre-defined protocol, five databases were searched for studies where blood samples from HIV patients were stored at time and temperature points that exceeded manufacturer recommendations. RNA stability, the primary outcome, was measured by the difference in means compared to samples stored within established thresholds. RNA stability was defined as ≤0.5 log degradation. The search identified 10,716 titles, of which nine full-text articles were included for review. HIV RNA maintained stability in EDTA whole blood and plasma at all measured time points up to 168 hours when stored at 4°C, while stability was detected at 72 hours (95% confidence) in whole blood at 25°C, with data points before and beyond 72 hours suggesting stability but not reaching statistical significance. For EDTA plasma stored at 30°C, stability was maintained up to 48 hours (95% confidence), with OLS linear regression estimates up to 127 hours, suggesting stability. Overall, quality of studies was moderate. Limitations included small sample sizes, few studies meeting inclusion criteria, and no studies examining RNA stability in low viremia (<3,000 copies/mL) environments.ConclusionsWhole blood and plasma samples in EDTA may remain stable under conditions exceeding current manufacturer recommendations for HIV VL testing. However, given the limited number of studies addressing this question, especially at low levels of viremia, additional evaluations on HIV RNA stability in EDTA tubes and PPT in field conditions are needed.

High level of HIV-1 resistance in patients failing long-term first-line antiretroviral therapy in Mali.

In resource-limited settings, few data are available on virological failure after long-term first-line antiretroviral therapy. This study characterized the genotypic resistance patterns at the time of failure after at least 36 months of a first-line regimen in Mali, West Africa. Plasma samples from 84 patients who were receiving first-line antiretroviral treatment and with an HIV-1 RNA viral load (VL) >1000 copies/mL were analysed. Genotypic resistance testing was performed and HIV-1 drug resistance was interpreted according to the latest version of the National Agency for HIV and Hepatitis Research algorithm. At the time of resistance testing, patients had been treated for a median of 60 months (IQR 36-132 months) and had a median CD4 cell count of 292 cells/mm(3) (IQR 6-1319 cells/mm(3)), a median HIV-1 RNA level of 28266 copies/mL (IQR 1000-2 93 495 copies/mL) and a median genotypic susceptibility score of 1 (IQR 1-4). The prevalence of nucleoside reverse transcriptase inhibitor (NRTI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations was 78% and 82%, respectively. Viruses were resistant to at least one drug in 92% of cases. Although etravirine and rilpivirine were not used in the first-line regimens, viruses were resistant to etravirine in 34% of cases and to rilpivirine in 49% of cases. The treatment duration, median number of NRTI and NNRTI mutations and some reverse transcriptase mutations (T215Y/F/N, L210W, L74I, M41L and H221Y) were associated with the VL at virological failure. This study demonstrated a high level of resistance to NRTIs and NNRTIs, compromising second-generation NNRTIs, for patients who stayed on long-term first-line regimens. It is crucial to expand the accessibility of virological testing in resource-limited settings to limit the expansion of resistance and preserve second-line treatment efficacy.