Host genetic variation at a locus near CHD1L impacts HIV sequence diversity in a South African population.

IMPORTANCE: It has been previously shown that genetic variants near CHD1L on chromosome 1 are associated with reduced HIV VL in African populations. However, the impact of these variants on viral diversity and how they restrict viral replication are unknown. We report on a regional association analysis in a South African population and show evidence of selective pressure by variants near CHD1L on HIV RT and gag. Our findings provide further insight into how genetic variability at this locus contributes to host control of HIV in a South African population.

Affordable drug resistance genotyping of HIV-1 reverse transcriptase, protease and integrase genes, for resource limited settings.

BACKGROUND: As use of dolutegravir (DTG) becomes more common in resource limited settings (RLS), the demand for integrase resistance testing is increasing. Affordable methods for genotyping all relevant HIV-1 pol genes (i.e., protease (PR), reverse transcriptase (RT) and integrase (IN)) are required to guide choice of future antiretroviral therapy (ART). We designed an in-house HIV-1 drug resistance (HIVDR) genotyping method that is affordable and suitable for use in RLS. METHODS: We obtained remnant plasma samples from CAPRISA 103 study and amplified HIV-1 PR, RT and IN genes, using an innovative PCR assay. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. We genotyped samples by Sanger sequencing and assessed HIVDR mutations using the Stanford HIV drug resistance database. We compared drug resistance mutations with previous genotypes and calculated method cost-estimates. RESULTS: From 96 samples processed, we obtained sequence data for 78 (81%), of which 75 (96%) had a least one HIVDR mutation, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, 18/78 (23%) had at least one discordant mutation, but only 2/78 (3%) resulted in different phenotypic predictions that could affect choice of subsequent regimen. All CAPRISA 103 study sequences were HIV-1C as confirmed by phylogenetic analysis. Of the 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method were 100% concordant with EQA panel results. Overall genotyping cost per sample was estimated at ~ US$43-$US49, with a processing time of ~ 2 working days. CONCLUSIONS: We successfully designed an in-house HIVDR method that is suitable for genotyping HIV-1 PR, RT and IN genes, at an affordable cost and shorter turnaround time. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS.

Antiretroviral therapy adherence patterns, virological suppression, and emergence of drug resistance: A nested case-control study from Uganda and South Africa.

BACKGROUND: Relationships between distinct antiretroviral therapy (ART) adherence patterns and risk of drug resistance are not well understood. METHODS: We conducted a nested case-control analysis within a longitudinal cohort study of individuals initiating efavirenz-based ART. Primary outcomes of interest, measured at 6 and 12 months after treatment initiation, were: 1) virologic suppression, 2) virologic failure with resistance, and 3) virologic failure without resistance. Our primary exposure of interest was ART adherence, measured over the 6 months before each visit with electronic pill monitors, and categorized in three ways: 1) 6 months average adherence; 2) running adherence, defined as the proportion of days with average adherence over 9 days of less than or equal to 10%, 20%, and 30%; and 3) number of 3-, 7-, and 28-day treatment gaps in the prior 6 months. RESULTS: We analyzed data from 166 individuals (107 had virologic failure during observation and 59 had virologic suppression at 6 and 12 months). Average adherence was higher among those with virologic suppression (median 83%, IQR 58-96%) versus those with virologic failure with resistance (median 35%, IQR 20-77%, pairwise P < 0.01) and those with virologic failure without resistance (median 21%, IQR 2-54%, pairwise P < 0.01). Although treatment gaps generally predicted virologic failure (P < 0.01), they did not differentiate failure with and without drug resistance (P > 0.6). CONCLUSIONS: Average adherence patterns, but not the assessed frequency of treatment gaps, differentiated failure with versus without drug resistance among individuals initiating efavirenz-based ART. Future work should explore adherence-resistance relationships for integrase inhibitor-based regimens.

Imaging and biopsy of HIV-infected individuals undergoing analytic treatment interruption.

Background: HIV persistence during antiretroviral therapy (ART) is the principal obstacle to cure. Lymphoid tissue is a compartment for HIV, but mechanisms of persistence during ART and viral rebound when ART is interrupted are inadequately understood. Metabolic activity in lymphoid tissue of patients on long-term ART is relatively low, and increases when ART is stopped. Increases in metabolic activity can be detected by 18F-fluorodeoxyglucose Positron Emission Tomography (FDG-PET) and may represent sites of HIV replication or immune activation in response to HIV replication. Methods: FDG-PET imaging will be used to identify areas of high and low metabolic uptake in lymphoid tissue of individuals undergoing long-term ART. Baseline tissue samples will be collected. Participants will then be randomized 1:1 to continue or interrupt ART via analytic treatment interruption (ATI). Image-guided biopsy will be repeated 10 days after ATI initiation. After ART restart criteria are met, image-guided biopsy will be repeated once viral suppression is re-achieved. Participants who continued ART will have a second FDG-PET and biopsies 12-16 weeks after the first. Genetic characteristics of HIV populations in areas of high and low FDG uptake will be assesed. Optional assessments of non-lymphoid anatomic compartments may be performed to evaluate HIV populations in distinct anatomic compartments. Anticipated results: We anticipate that PET standardized uptake values (SUV) will correlate with HIV viral RNA in biopsies of those regions and that lymph nodes with high SUV will have more viral RNA than those with low SUV within a patient. Individuals who undergo ATI are expected to have diverse viral populations upon viral rebound in lymphoid tissue. HIV populations in tissues may initially be phylogenetically diverse after ATI, with emergence of dominant viral species (clone) over time in plasma. Dominant viral species may represent the same HIV population seen before ATI. Discussion: This study will allow us to explore utility of PET for identification of HIV infected cells and determine whether high FDG uptake respresents areas of HIV replication, immune activation or both. We will also characterize HIV infected cell populations in different anatomic locations. The protocol will represent a platform to investigate persistence and agents that may target HIV populations. Study protocol registration: Identifier: NCT05419024.

Persistent Hepatitis B Viraemia with Polymerase Mutations among HIV/HBV Co-Infected Patients on HBV-Active ART in KwaZulu-Natal, South Africa.

To understand the problem of persistent Hepatitis B virus (HBV) viraemia in HIV/HBV co-infected patients on HBV-active antiretroviral therapy (ART), we assessed the rate of HBV virological response in patients on HBV-active ART in KwaZulu-Natal, South Africa, and analysed factors associated with persistent HBV viraemia. One hundred and fifty eligible participants with a chronic HBV diagnosis, with or without HIV coinfection, were enrolled and followed up after 6 months. The HBV pol gene was sequenced by next-generation sequencing and mutations were determined using the Stanford HBVseq database. Logistic regression analysis was used to assess factors associated with HBV viraemia at 6-month follow-up. The mean duration of HBV-active ART was 24 months. Thirty-seven of one hundred and six (35%) participants receiving HBV-active ART for longer than 6 months had virological failure. Advanced immunosuppression with CD4+ cell counts <200 cells/µL was independently associated with persistent HBV viraemia, aOR 5.276 (95% CI 1.575−17.670) p = 0.007. A high proportion of patients on HBV-active ART are unsuppressed, which will ultimately have an impact on global elimination goals. Better monitoring should be implemented, especially in HIV-coinfected patients with low CD4+ cell counts and followed by early HBV drug-resistance testing.

Adherence Measured Using Electronic Dose Monitoring is Associated with Emergent Antiretroviral Resistance and Poor Outcomes in People with Human Immunodeficiency Virus/AIDS and Multidrug-Resistant Tuberculosis.

BACKGROUND: Medication adherence is known to challenge treatment of human immunodeficiency virus (HIV)/AIDS and multidrug-resistant tuberculosis (MDR-TB). We hypothesized that adherence using electronic dose monitoring (EDM) would identify an antiretroviral therapy (ART) adherence threshold for emergent ART resistance and predict treatment outcomes in patients with MDR-TB and HIV on ART and bedaquiline-containing TB regimens. METHODS: A prospective cohort of adults with MDR-TB and HIV on ART and initiating MDR-TB treatment with bedaquiline were enrolled at a public hospital in KwaZulu-Natal, South Africa (PRAXIS Study). Participants received separate EDM devices that measure adherence to bedaquiline and ART (nevirapine or lopinavir/ritonavir). Adherence was calculated cumulatively over 6 months. Participants were followed through completion of MDR-TB treatment. HIV genome sequencing was performed at baseline and 2 and 6 months on samples with HIV RNA ≥1000 copies/mL. RESULTS: From November 2016 through February 2018, 198 persons with MDR-TB and HIV were enrolled and followed (median, 17.2 months; interquartile range, 12.2-19.6). Eleven percent had baseline ART resistance mutations, and 7.5% developed emergent ART resistance at 6 months. ART adherence was independently associated with ART resistance and mortality. Modeling identified a significant (P < .001), linear association between ART adherence and emergent resistance, suggesting a strong association without a specific threshold. CONCLUSIONS: Our findings highlight the need for ART resistance testing, especially in patients with MDR-TB and HIV, which is currently not the standard of care in resource-limited settings. Despite short follow-up duration, reduced ART adherence was significantly associated with emergent resistance and increased mortality. CLINICAL TRIALS REGISTRATION: NCT03162107.

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.

Acquired HIV drug resistance and virologic monitoring in a HIV hyper-endemic setting in KwaZulu-Natal Province, South Africa.

BACKGROUND: Introduction of tenofovir (TDF) plus lamivudine (3TC) and dolutegravir (DTG) in first- and second-line HIV treatment regimens in South Africa warrants characterization of acquired HIV-1 drug resistance (ADR) mutations that could impact DTG-based antiretroviral therapy (ART). In this study, we sought to determine prevalence of ADR mutations and their potential impact on susceptibility to drugs used in combination with DTG among HIV-positive adults (≥ 18 years) accessing routine care at a selected ART facility in KwaZulu-Natal, South Africa. METHODS: We enrolled adult participants in a cross-sectional study between May and September 2019. Eligible participants had a most recent documented viral load (VL) ≥ 1000 copies/mL after at least 6 months on ART. We genotyped HIV-1 reverse transcriptase and protease genes by Sanger sequencing and assessed ADR. We characterized the effect of ADR mutations on the predicted susceptibility to drugs used in combination with DTG. RESULTS: From 143 participants enrolled, we obtained sequence data for 115 (80%), and 92.2% (95% CI 85.7-96.4) had ADR. The proportion with ADR was similar for participants on first-line ART (65/70, 92.9%, 95% CI 84.1-97.6) and those on second-line ART (40/44, 90.9%, 95% CI 78.3-97.5), and was present for the single participant on third-line ART. Approximately 89% (62/70) of those on first-line ART had dual class NRTI and NNRTI resistance and only six (13.6%) of those on second-line ART had major PI mutations. Most participants (82%) with first-line viraemia maintained susceptibility to Zidovudine (AZT), and the majority of them had lost susceptibility to TDF (71%) and 3TC (84%). Approximately two in every five TDF-treated individuals had thymidine analogue mutations (TAMs). CONCLUSIONS: Susceptibility to AZT among most participants with first-line viraemia suggests that a new second-line regimen of AZT + 3TC + DTG could be effective. However, atypical occurrence of TAMs in TDF-treated individuals suggests a less effective AZT + 3TC + DTG regimen in a subpopulation of patients. As most patients with first-line viraemia had at least low-level resistance to TDF and 3TC, identifying viraemia before switch to TDF + 3TC + DTG is important to avoid DTG functional monotherapy. These findings highlight a need for close monitoring of outcomes on new standardized treatment regimens.

HIV-1 Genetic Diversity and Natural Polymorphisms of the Integrase Gene in Integrase Inhibitor-Naive Patients in Harare, Zimbabwe.

Previously used as part of salvage therapy, integrase strand transfer inhibitors (INSTIs) have become part of the preferred antiretroviral therapy (ART) first-line regimen in most low- to middle-income countries. With the extensive use of dolutegravir in first-line ART, drug resistance mutations to INSTIs are inevitable. Therefore, active monitoring and surveillance of INSTI drug resistance is required. The aim of this study was to evaluate the genetic diversity of the integrase gene and determine pretreatment INSTI resistance in Harare, Zimbabwe. Forty-four HIV-1 Integrase sequences from 65 were obtained from treatment-naive individuals using a custom genotyping method. Drug resistance mutations were determined using the Stanford HIV Drug Resistance Interpretation program. Viral subtyping was done by phylogenetic analysis and the REGA HIV subtyping tool determined recombinants. Natural polymorphisms were evaluated relative to the global subtype B and C consensus sequences. One hundred ninety-two sequences from the region were accessed from GenBank to assess differences between the Zimbabwean sequences and those from neighboring countries. No major INSTI resistance mutations were detected; however, the L74I polymorphism was detected in three sequences of the 44 (6.8%). There was little genetic variability in the Integrase gene, with a mean genetic distance range of 0.053015. The subtype C consensus was identical to the global subtype C consensus and varied from the global subtype B consensus at five major positions: T124A, V201I, T218I, D278A, and S283G. This study has provided baseline sequence data on the presence of HIV-1 subtype C Integrase gene drug resistance mutations from Harare, Zimbabwe.

HIV-1 Drug Resistance Genotyping in Resource Limited Settings: Current and Future Perspectives in Sequencing Technologies.

Affordable, sensitive, and scalable technologies are needed for monitoring antiretroviral treatment (ART) success with the goal of eradicating HIV-1 infection. This review discusses use of Sanger sequencing and next generation sequencing (NGS) methods for HIV-1 drug resistance (HIVDR) genotyping, focusing on their use in resource limited settings (RLS). Sanger sequencing remains the gold-standard method for detecting HIVDR mutations of clinical relevance but is mainly limited by high sequencing costs and low-throughput. NGS is becoming a more common sequencing method, with the ability to detect low-abundance drug-resistant variants and reduce per sample costs through sample pooling and massive parallel sequencing. However, use of NGS in RLS is mainly limited by infrastructure costs. Given these shortcomings, our review discusses sequencing technologies for HIVDR genotyping, focusing on common in-house and commercial assays, challenges with Sanger sequencing in keeping up with changes in HIV-1 treatment programs, as well as challenges with NGS that limit its implementation in RLS and in clinical diagnostics. We further discuss knowledge gaps and offer recommendations on how to overcome existing barriers for implementing HIVDR genotyping in RLS, to make informed clinical decisions that improve quality of life for people living with HIV.

Transmission dynamics of SARS-CoV-2 within-host diversity in two major hospital outbreaks in South Africa.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in humans and a wide range of animal species. Its rapid global spread has resulted in a major public health emergency, necessitating commensurately rapid research to improve control strategies. In particular, the ability to effectively retrace transmission chains in outbreaks remains a major challenge, partly due to our limited understanding of the virus' underlying evolutionary dynamics within and between hosts. We used high-throughput sequencing whole-genome data coupled with bottleneck analysis to retrace the pathways of viral transmission in two nosocomial outbreaks that were previously characterised by epidemiological and phylogenetic methods. Additionally, we assessed the mutational landscape, selection pressures, and diversity at the within-host level for both outbreaks. Our findings show evidence of within-host selection and transmission of variants between samples. Both bottleneck and diversity analyses highlight within-host and consensus-level variants shared by putative source-recipient pairs in both outbreaks, suggesting that certain within-host variants in these outbreaks may have been transmitted upon infection rather than arising de novo independently within multiple hosts. Overall, our findings demonstrate the utility of combining within-host diversity and bottleneck estimations for elucidating transmission events in SARS-CoV-2 outbreaks, provide insight into the maintenance of viral genetic diversity, provide a list of candidate targets of positive selection for further investigation, and demonstrate that within-host variants can be transferred between patients. Together these results will help in developing strategies to understand the nature of transmission events and curtail the spread of SARS-CoV-2.

HIV-1 integrase strand transfer inhibitors: a review of current drugs, recent advances and drug resistance.

Antiretroviral therapy has been imperative in controlling the human immunodeficiency virus (HIV) epidemic. Most low- and middle-income countries have used nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors extensively in the treatment of HIV. However, integrase strand transfer inhibitors (INSTIs) are becoming more common. Since their identification as a promising therapeutic drug, significant progress has been made that has led to the approval of five INSTIs by the US Food and Drug Administration (FDA), i.e. dolutegravir (DTG), raltegravir (RAL), elvitegravir (EVG), bictegravir (BIC) and cabotegravir (CAB). INSTIs have been shown to effectively halt HIV-1 replication and are commended for having a higher genetic barrier to resistance compared with NRTIs and NNRTIs. More interestingly, DTG has shown a higher genetic barrier to resistance compared with RAL and EVG, and CAB is being used as the first long-acting agent in HIV-1 treatment. Considering the increasing interest in INSTIs for HIV-1 treatment, we focus our review on the retroviral integrase, development of INSTIs and their mode of action. We also discuss each of the INSTI drugs, including potential drug resistance and known side effects.

Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report.

OBJECTIVES: The Network for Genomic Surveillance in South Africa (NGS-SA) was formed to investigate the introduction and understand the early transmission dynamics of the SARS-CoV-2 epidemic in South-Africa. DESIGN: This paper presents the first results from this group, which is a molecular epidemiological study of the first 21 SARS-CoV-2 whole genomes sampled in the first port of entry - KwaZulu-Natal (KZN) - during the first month of the epidemic. By combining this with calculations of the effective reproduction number (R), it aimed to shed light on the patterns of infections in South Africa. RESULTS: Two of the largest provinces - Gauteng and KZN - had a slow growth rate for the number of detected cases, while the epidemic spread faster in the Western Cape and Eastern Cape. The estimates of transmission potential suggested a decrease towards R = 1 since the first cases and deaths, but a subsequent estimated R average of 1.39 between 6-18 May 2020. It was also demonstrated that early transmission in KZN was associated with multiple international introductions and dominated by lineages B1 and B. Evidence for locally acquired infections in a hospital in Durban within the first month of the epidemic was also provided. CONCLUSION: The COVID-19 pandemic in South Africa was very heterogeneous in its spatial dimension, with many distinct introductions of SARS-CoV2 in KZN and evidence of nosocomial transmission, which inflated early mortality in KZN. The epidemic at the local level was still developing and NGS-SA aimed to clarify the dynamics in South Africa and devise the most effective measures as the outbreak evolved.

Reduced efficacy of HIV-1 integrase inhibitors in patients with drug resistance mutations in reverse transcriptase.

Little is known about the impact of pretreatment drug resistance (PDR) on the efficacy of second generation integrase inhibitors. We sequenced pretreatment plasma specimens from the ADVANCE trial (NCT03122262). Our primary outcome was 96-week virologic success, defined as a sustained viral load <1000 copies/mL from 12 weeks onwards, <200 copies/mL from 24 weeks onwards, and <50 copies/mL after 48 weeks. Here we report how this outcome was impacted by PDR, defined by the World Health Organization (WHO) mutation list. Of 1053 trial participants, 874 (83%) have successful sequencing, including 289 (33%) randomized to EFV-based therapy and 585 (67%) randomized to DTG-based therapy. Fourteen percent (122/874) have ≥1 WHO-defined mutation, of which 98% (120/122) are NNRTI mutations. Rates of virologic suppression are lower in the total cohort among those with PDR 65% (73/112) compared to those without PDR (85% [605/713], P < 0.001), and for those on EFV-based treatment (60% [12/20] vs 86% [214/248], P = 0.002) and for those on DTG-based treatment (61/92 [66%] vs 84% [391/465] P < 0.001, P for interaction by regimen 0.49). Results are similar in multivariable models adjusted for clinical characteristics and adherence. NNRTI resistance prior to treatment is associated with long-term failure of integrase inhibitor-containing first-line regimens, and portends high rates of first-line failure in sub Saharan Africa.

Next Generation Sequencing and Bioinformatics Analysis of Family Genetic Inheritance.

Mendelian and complex genetic trait diseases continue to burden and affect society both socially and economically. The lack of effective tests has hampered diagnosis thus, the affected lack proper prognosis. Mendelian diseases are caused by genetic mutations in a singular gene while complex trait diseases are caused by the accumulation of mutations in either linked or unlinked genomic regions. Significant advances have been made in identifying novel diseases associated mutations especially with the introduction of next generation and third generation sequencing. Regardless, some diseases are still without diagnosis as most tests rely on SNP genotyping panels developed from population based genetic analyses. Analysis of family genetic inheritance using whole genomes, whole exomes or a panel of genes has been shown to be effective in identifying disease-causing mutations. In this review, we discuss next generation and third generation sequencing platforms, bioinformatic tools and genetic resources commonly used to analyze family based genomic data with a focus on identifying inherited or novel disease-causing mutations. Additionally, we also highlight the analytical, ethical and regulatory challenges associated with analyzing personal genomes which constitute the data used for family genetic inheritance.

High Resolution analysis of Transmission Dynamics of Sars-Cov-2 in Two Major Hospital Outbreaks in South Africa Leveraging Intrahost Diversity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes acute, highly transmissible respiratory infection in both humans and wide range of animal species. Its rapid spread globally and devasting effects have resulted into a major public health emergency prompting the need for methodological interventions to understand and control its spread. In particular, The ability to effectively retrace its transmission pathways in outbreaks remains a major challenge. This is further exacerbated by our limited understanding of its underlying evolutionary mechanism. Using NGS whole-genome data, we determined whether inter- and intra-host diversity coupled with bottleneck analysis can retrace the pathway of viral transmission in two epidemiologically well characterised nosocomial outbreaks in healthcare settings supported by phylogenetic analysis. Additionally, we assessed the mutational landscape, selection pressure and diversity of the identified variants. Our findings showed evidence of intrahost variant transmission and evolution of SARS-CoV-2 after infection These observations were consistent with the results from the bottleneck analysis suggesting that certain intrahost variants in this study could have been transmitted to recipients. In both outbreaks, we observed iSNVs and SNVs shared by putative source-recipients pairs. Majority of the observed iSNVs were positioned in the S and ORF1ab region. AG, CT and TC nucleotide changes were enriched across SARS-COV-2 genome. Moreover, SARS-COV-2 genome had limited diversity in some loci while being highly conserved in others. Overall, Our findings show that the synergistic effect of combining withinhost diversity and bottleneck estimations greatly enhances resolution of transmission events in Sars-Cov-2 outbreaks. They also provide insight into the genome diversity suggesting purifying selection may be involved in the transmission. Together these results will help in developing strategies to elucidate transmission events and curtail the spread of Sars-Cov-2.

Impact of pretreatment low-abundance HIV-1 drug-resistant variants on virological failure among HIV-1/TB-co-infected individuals.

OBJECTIVES: To determine the impact of pretreatment low-abundance HIV-1 drug-resistant variants (LA-DRVs) on virological failure (VF) among HIV-1/TB-co-infected individuals treated with NNRTI first-line ART. METHODS: We conducted a case-control study of 170 adults with HIV-1/TB co-infection. Cases had at least one viral load (VL) ≥1000 RNA copies/mL after ≥6 months on NNRTI-based ART, and controls had sustained VLs <1000 copies/mL. We sequenced plasma viruses by Sanger and MiSeq next-generation sequencing (NGS). We assessed drug resistance mutations (DRMs) using the Stanford drug resistance database, and analysed NGS data for DRMs at ≥20%, 10%, 5% and 2% thresholds. We assessed the effect of pretreatment drug resistance (PDR) on VF. RESULTS: We analysed sequences from 45 cases and 125 controls. Overall prevalence of PDR detected at a ≥20% threshold was 4.7% (8/170) and was higher in cases than in controls (8.9% versus 3.2%), P = 0.210. Participants with PDR at ≥20% had almost 4-fold higher odds of VF (adjusted OR 3.7, 95% CI 0.8-18.3) compared with those without, P = 0.104. PDR prevalence increased to 18.2% (31/170) when LA-DRVs at ≥2% were included. Participants with pretreatment LA-DRVs only had 1.6-fold higher odds of VF (adjusted OR 1.6, 95% CI 0.6-4.3) compared with those without, P = 0.398. CONCLUSIONS: Pretreatment DRMs and LA-DRVs increased the odds of developing VF on NNRTI-based ART, although without statistical significance. NGS increased detection of DRMs but provided no additional benefit in identifying participants at risk of VF at lower thresholds. More studies assessing mutation thresholds predictive of VF are required to inform use of NGS in treatment decisions.

Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic.

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium.

Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report.

Background: The emergence of a novel coronavirus, SARS-CoV-2, in December 2019, progressed to become a world pandemic in a few months and reached South Africa at the beginning of March. To investigate introduction and understand the early transmission dynamics of the virus, we formed the South African Network for Genomics Surveillance of COVID (SANGS_COVID), a network of ten government and university laboratories. Here, we present the first results of this effort, which is a molecular epidemiological study of the first twenty-one SARS-CoV-2 whole genomes sampled in the first port of entry, KwaZulu-Natal (KZN), during the first month of the epidemic. By combining this with calculations of the effective reproduction number (R), we aim to shed light on the patterns of infections that define the epidemic in South Africa. Methods: R was calculated using positive cases and deaths from reports provided by the four major provinces. Molecular epidemiology investigation involved sequencing viral genomes from patients in KZN using ARCTIC protocols and assembling whole genomes using meticulous alignment methods. Phylogenetic analysis was performed using maximum likelihood (ML) and Bayesian trees, lineage classification and molecular clock calculations. Findings: The epidemic in South Africa has been very heterogeneous. Two of the largest provinces, Gauteng, home of the two large metropolis Johannesburg and Pretoria, and KwaZulu-Natal, home of the third largest city in the country Durban, had a slow growth rate on the number of detected cases. Whereas, Western Cape, home of Cape Town, and the Eastern Cape provinces the epidemic is spreading fast. Our estimates of transmission potential for South Africa suggest a decreasing transmission potential towards R=1 since the first cases and deaths have been reported. However, between 06 May and 18 May 2020, we estimate that R was on average 1.39 (1.04 - 2.15, 95% CI). We also demonstrate that early transmission in KZN, and most probably in all main regions of SA, was associated with multiple international introductions and dominated by lineages B1 and B. The study also provides evidence for locally acquired infections in a hospital in Durban within the first month of the epidemic, which inflated early mortality in KZN. Interpretation: This first report of SANGS_COVID consortium focuses on understanding the epidemic heterogeneity and introduction of SARS-CoV-2 strains in the first month of the epidemic in South Africa. The early introduction of SARS-CoV-2 in KZN included caused a localized outbreak in a hospital, provides potential explanations for the initially high death rates in the province. The current high rate of transmission of COVID-19 in the Western Cape and Eastern Cape highlights the crucial need to strength local genomic surveillance in South Africa. Funding: UKZN Flagship Program entitled: Afrocentric Precision Approach to Control Health Epidemic, by a research Flagship grant from the South African Medical Research Council (MRC-RFA-UFSP-01-2013/UKZN HIVEPI, by the the Technology Innovation Agency and the the Department of Science and Innovation and by National Human Genome Re- search Institute of the National Institutes of Health under Award Number U24HG006941. H3ABioNet is an initiative of the Human Health and Heredity in Africa Consortium (H3Africa).