Innovative and affordable HIV-1 drug resistance testing for resource limited settings.

Masters Degree. University of KwaZulu-Natal, Durban.Background: HIV drug resistance (HIVDR) remains a major threat to achieving sustainable viral suppression on antiretroviral treatment (ART). Most countries including those in resource limited settings (RLS) have adopted use of dolutegravir (DTG), a more potent integrase strand transfer inhibitor, leading to an increase in the demand for integrase resistance testing. Current HIVDR testing methods in RLS focus on genotyping the HIV protease (PR) and reverse transcriptase (RT) genes, separate from the integrase (IN) gene. However, amplification of PR and RT separate from IN is expensive and increases the workload for HIVDR genotyping. Therefore, affordable and labour efficient methods that genotype all relevant HIV-1 genes (i.e., the PR, RT and IN genes) are required to guide clinical decisions, especially in RLS where cost is a major limiting factor. Thus, this study aimed to design an affordable in-house HIVDR genotyping method suitable for use in RLS. Methods: Remnant plasma samples were obtained from a CAPRISA 103 study and viral RNA was extracted from 500μl of plasma. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. Complimentary DNA synthesis and first-round PCR were performed followed by second-round nested PCR which was designed to amplify an ~2.9kb HIV-1 pol region (PR, RT and IN genes) using 1% gel electrophoresis. Successful second-round nested PCR products were purified using ExoSAP-IT Express PCR Product Cleanup reagent. Sanger sequencing was performed and quality of the sequences were manually edited using Geneious Prime software. HIVDR mutations were assessed using the Stanford HIV drug resistance database. HIVDR mutations using the designed method were compared to previous results obtained on the same samples. Sequence quality was also evaluated using phylogenetic analysis in Geneious software with maximum likelihood tree reconstruction using a generalized time reversible model with proportion of invariable sites and gamma distribution (GTR + I + G), and with 100 bootstrap replicates. Method cost-estimates were done by comparing costs and turn-around time to current genotyping methods. Results: Of 115 plasma samples obtained, 19 samples were not processed due to inadequate plasma volume. Of the 96 processed, we obtained sequence data for 78 (81%). Of those, 75 (96%) had at least one HIVDR mutation in the PR and RT genes, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, only 2/79 (3%) had different phenotypic predictions that affected the choice of subsequent regimens. Of 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method showed 100% concordance with EQA panel results. The overall cost per sample was estimated at ~US$43, with a turn-around time of ~15 hours. Conclusion: We successfully designed an in-house HIVDR method suitable for genotyping HIV-1C PR, RT and IN genes, at an affordable cost of US$64 and shorter turn-around time reduced from ~21 hours to ~15 hours, compared to currently available methods. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS

HIV-1 drug resistance in adults and adolescents on protease inhibitor-based antiretroviral therapy in KwaZulu-Natal Province, South Africa

Objectives: In low–middle-income countries, increasing levels of HIV drug resistance (HIVDR) on second-line protease inhibitor (PI)-based regimens are a cause for concern given the limited drug options for third-line antiretroviral therapy (ART). We conducted a retrospective analysis of routine HIV-1 genotyping laboratory data from KwaZulu-Natal, South Africa, to describe the frequency and patterns of HIVDR mutations and their consequent impact on standardised third-line regimens. Methods: This was a cross-sectional analysis of all HIV-1 genotypic resistance tests conducted by the National Health Laboratory Service in KwaZulu-Natal (January 2015 to December 2016) for adults and adolescents (age ≥10 years) on second-line PI-based ART with virological failure. We assigned a third-line regimen to each record based on a national treatment algorithm and calculated the genotypic susceptibility score (GSS) for that regimen. Results: Of 348 samples analysed, 287 (82.5%) had at least one drug resistance mutation (DRM) and 114 (32.8%) had at least one major PI DRM. Major PI resistance was associated with longer duration on second-line ART (aOR per 6-months = 1.11, 95% CI 1.04–1.19) and older age (aOR = 1.03, 95% CI 1.01–1.05). Of 112 patients requiring third-line ART, 12 (10.7%) had a GSS of <2 for the algorithm-assigned third-line regimen. Conclusion: One-third of people failing second-line ART had significant PI DRMs. A subgroup of these individuals had extensive HIVDR, where the predicted activity of third-line ART was suboptimal, highlighting the need for continuous evaluation of outcomes on third-line regimens and close monitoring for emergent HIV-1 integrase inhibitor resistance

Alphaviruses Detected in Mosquitoes in the North-Eastern Regions of South Africa, 2014 to 2018

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa.publishersversionpublishe

Potential mosquito vectors for Shuni virus, South Africa, 2014–2018

Shuni virus is associated with neurologic and febrile illness in animals and humans. To determine potential vectors, we collected mosquitoes in South Africa and detected the virus in species of the genera Mansonia, Culex, Aedes, and Anopheles. These mosquitoes may be associated with Shuni virus outbreaks in Africa and emergence in other regions

Alphaviruses detected in mosquitoes in the North-Eastern regions of South Africa, 2014 to 2018

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa