Limited availability of viral load (VL) monitoring in HIV treatment programmes in sub-Saharan Africa can delay switching to second-line ART, leading to the accumulation of drug resistance mutations ...(DRMs). The objective of this study was to evaluate the accumulation of resistance to reverse transcriptase inhibitors after continued virological failure on first-line ART, among adults and children in sub-Saharan Africa.
HIV-1-positive adults and children on an NNRTI-based first-line ART were included. Retrospective VL and, if VL ≥1000 copies/mL, pol genotypic testing was performed. Among participants with continued virological failure (≥2 VL ≥1000 copies/mL), drug resistance was evaluated.
At first virological failure, DRM(s) were detected in 87% of participants: K103N (38.7%), G190A (21.8%), Y181C (20.2%), V106M (8.4%), K101E (8.4%), any E138 (7.6%) and V108I (7.6%) associated with NNRTIs, and M184V (69.7%), any thymidine analogue mutation (9.2%), K65R (5.9%) and K70R (5.0%) associated with NRTIs. New DRMs accumulated with an average rate of 1.45 (SD 2.07) DRM per year; 0.62 (SD 1.11) NNRTI DRMs and 0.84 (SD 1.38) NRTI DRMs per year, respectively. The predicted susceptibility declined significantly after continued virological failure for all reverse transcriptase inhibitors (all P < 0.001). Acquired drug resistance patterns were similar in adults and children.
Patterns of drug resistance after virological failure on first-line ART are similar in adults and children in sub-Saharan Africa. Improved VL monitoring to prevent accumulation of mutations, and new drug classes to construct fully active regimens, are required.
In order to assess the level of transmitted and/or pre-treatment antiretroviral drug resistance to HIV-1, the World Health Organization (WHO) recommends that regular surveys are conducted. This ...study's objective was to assess the frequency of HIV-1 antiretroviral drug resistance in patients initiating antiretroviral treatment (ART) in the public sector throughout South Africa.
A prospective cross-sectional survey was conducted using probability proportional to size sampling. This method ensured that samples from each province were proportionally collected, based on the number of patients receiving ART in each region. Samples were collected between March 2013 and October 2014. Pol sequences were obtained using RT-PCR and Sanger sequencing and submitted to the Stanford Calibrated Population Resistance tool v6.0.
A total of 277 sequences were available for analysis. Most participants were female (58.8%) and the median age was 34 years (IQR: 29-42). The median baseline CD4-count was 149 cells/mm3 (IQR: 62-249) and, based on self-reporting, participants had been diagnosed as HIV-positive approximately 44 days prior to sample collection (IQR: 23-179). Subtyping revealed that 98.2% were infected with HIV-1 subtype C. Overall, 25 out of 277 patients presented with ≥1 surveillance drug resistance mutation (SDRM, 9.0%, 95% CI: 6.1-13.0%). Non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations were the most numerous mutations detected (n = 23). Only two patients presented with a protease inhibitor (PI) mutation. In four patients ≥4 SDRMs were detected, which might indicate that these patients were not truly ART-naïve or were infected with a multi-resistant virus.
These results show that the level of antiretroviral drug resistance in ART-naïve South Africans has reached moderate levels, as per the WHO classification. Therefore, regular surveys of pre-treatment drug resistance levels in all regions of South Africa is highly recommended to monitor the changing levels of pre-treatment antiretroviral drug resistance.
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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.
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.
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/.
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•Rapid transition to automated SARS-CoV-2 molecular tests was challenging.•Adapted protocols for verification of assays used.•Continuous management of human resources required.•Improved workflow ...reduced time to obtaining a result.•Strategies executed increased percentage of samples reported within turnaround-time.
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.
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Tenofovir (TDF) has replaced stavudine (d4T) as the preferred nucleoside reverse transcriptase inhibitor (NRTI) in first-line regimens in South Africa, but limited information is available on the ...resistance patterns that develop after the introduction of TDF. This study investigated the antiretroviral drug resistance patterns in South African HIV-1 subtype C-infected patients failing stavudine- (d4T) and tenofovir- (TDF) based first-line regimens and assess the suitability of TDF as the preferred first-line nucleotide reverse transcriptase inhibitor (NRTI).
Resistance patterns of HIV-1 from 160 adult patients virologically failing TDF- (n = 80) and d4T- (n = 80) based first-line regimens were retrospectively analyzed. The pol gene was sequenced using an in-house protocol and mutations were analysed using the IAS-USA 2014 Drug Resistance Mutation list.
Compared to d4T-exposed patients (n = 7), patients failing on a TDF-containing regimen (n = 43) were almost 5 times more likely to present with a K65R mutation (aRR 4.86 95% CI 2.29 - 10.34). Y115F was absent in the d4T group, and detected in 13.8% (n = 11) of TDF-exposed patients, p = 0.0007. Virus from 9 of the 11 patients (82.0%) who developed the Y115F mutation also developed K65R. Intermediate or high-level resistance to most NRTIs was common in the TDF-treatment group, but these patients twice more likely to remain susceptible to AZT as compared to those exposed to d4T (aRR 2.09 95% CI 1.13 - 3.90).
The frequency of the TDF induced K65R mutation was higher in our setting compared to non-subtype C dominated countries. However, despite the higher frequency of cross-resistance to NRTIs, most patients remained susceptible to AZT, which is reflected in the South African treatment guidelines that recommend AZT as an essential component of second-line regimens.
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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.
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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
and Xpert
Xpress CoV-2/Flu/RSV
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
test and included 50 genotyped specimens. The Xpert
Xpress CoV-2/Flu/RSV
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
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
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
and Xpert
Xpress CoV-2/Flu/RSV
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.
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Introduction Dolutegravir (DTG), an integrase strand transfer inhibitor (INSTI)-based HIV-1 therapy, is widely recommended in first-line and second-line regimens. 1 , 2 Integrase strand transfer ...inhibitor resistance mutations associated with DTG-containing regimens have been well described, most often occurring after DTG monotherapy or in INSTI-experienced patients. 3 Although rare, emergence of these mutations has also been described in patients on DTG-containing triple-drug regimens 4 and INSTI-naïve patients. 5 , 6 The R263K mutation is commonly associated with the emergence of DTG resistance but reduces viral fitness and DNA integration. 7 , 8 Here we describe a case of very slow viral decline (~42 months) in a treatment-experienced, INSTI-naïve patient on a DTG-based triple therapy regimen. Variable Description Age 43 Gender Male Diagnosis 2012 Samples (time after DTG initiation) and viral load (copies/mL) S1 (~16 months): 1420 S2 (~20 months): 1290 S3 (~24 months): 1128 Duration of detectable viraemia ~42 months Previous ART regimens and viral load range (copies/mL) FTC/TDF/EFV (2012–2015): 29 271–173 455 3TC/AZT/LPV/r (2015–2018): 14 269–149 000 Study period ART regimen and viral load range (copies/mL) 3TC/AZT/DTG (January 2018 – August 2019): 313–149 000 3TC/AZT/DRV/r (August 2019 – November 2019): 650 3TC/AZT/DTG (November 2019 onward): 50–193 ART, antiretroviral treatment; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; EFV, efavirenz; AZT, zidovudine; DRV, darunavir; DTG, dolutegravir; LPV, lopinavir; r, ritonavir; PBMCs, peripheral blood mononuclear cells. Sample (time after DTG initiation) Sequence source Number of sequences obtained Drug resistance mutations NRTI NNRTI S1 (~16 months) Plasma RNA 8 A62V, K65R, M184V L100I, K103N Buffy coat DNA 24 None None S2 (~20 months) Plasma RNA 12 A62V, K65R, M184V L100I, K103N S3 (~24 months) Plasma RNA 22 A62V, K65R, M184V L100I, K103N PBMC DNA 24 None None PBMC RNA 21 None K103N NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PBMC, peripheral blood mononuclear cell; DTG, dolutegravir. Reported clinical risk factors associated with emergence of DTG resistance include poor treatment adherence, drug interactions and HIV factors such as a high baseline viral load. 6 In this case we report good adherence but the high viral load (> 100 000 copies/mL) prior to DTG initiation could be a contributing risk factor, even though INSTI resistance was only detected after 24 months.
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