Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

BACKGROUND: Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. METHODS: Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. RESULTS: We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08-6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39-7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. CONCLUSIONS: Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally

Gag-Protease Sequence Evolution Following Protease Inhibitor Monotherapy Treatment Failure in HIV-1 Viruses Circulating in East Africa.

Around 2.5 million HIV-infected individuals failing first-line therapy qualify for boosted protease inhibitor (bPI)-based second-line therapy globally. Major resistance mutations are rarely present at treatment failure in patients receiving bPI and the determinants of failure in these patients remain unknown. There is evidence that Gag can impact PI susceptibility. Here, we have sequenced Gag-Protease before and following failure in 23 patients in the SARA trial infected with subtypes A, C, and D viruses. Before bPI, significant variation in Protease and Gag was observed at positions previously associated with PI exposure and resistance including Gag mutations L449P, S451N, and L453P and Protease K20I and L63P. Following PI failure, previously described mutations in Protease and Gag were observed, including those at the cleavage sites such as R361K and P453L. However, the emergence of clear genetic determinants of therapy failure across patients was not observed. Larger Gag sequence datasets will be required to comprehensively identify mutational correlates of bPI failure across subtypes

Lack of effectiveness of adherence counselling in reversing virological failure among patients on long-term antiretroviral therapy in rural Uganda.

OBJECTIVES: The current World Health Organization and Uganda Ministry of Health HIV treatment guidelines recommend that asymptomatic patients who have a viral load (VL) ≥ 1000 HIV-1 RNA copies/mL should receive adherence counselling and repeat VL testing before switching to second-line therapy. We evaluated the effectiveness of this strategy in a large HIV treatment programme of The AIDS Support Organisation Jinja in Jinja, Uganda. METHODS: We measured the HIV VL at enrolment, and for participants with VL ≥ 1000 copies/mL we informed them of their result, offered enhanced adherence counselling and repeated the VL measurement after 3 months. All blood samples with VL ≥ 1000 copies/mL were sequenced in the polymerase (pol) region, a 1257-bp fragment spanning the protease and reverse transcriptase genes. RESULTS: One thousand and ninety-one participants were enrolled in the study; 74.7% were female and the median age was 44 years [interquartile range (IQR) 39-50 years]. The median time on antiretroviral therapy (ART) at enrolment was 6.75 years (IQR 5.3-7.6 years) and the median CD4 cell count was 494 cells/μL (IQR 351-691 cells/μL). A total of 113 participants (10.4%) had VLs ≥ 1000 copies/mL and were informed of the VL result and its implications and given adherence counselling. Of these 113 participants, 102 completed 3 months of follow-up and 93 (91%) still had VLs ≥ 1000 copies/mL. We successfully genotyped HIV for 105 patients (93%) and found that 103 (98%) had at least one mutation: eight (7.6%) had only one mutation, 94 (89.5%) had two mutations and one sample (1%) had three mutations. CONCLUSIONS: In this study, enhanced adherence counselling was not effective in reversing virologically defined treatment failure for patients on long-term ART who had not previously had a VL test

Sub-Saharan Africa preparedness and response to the COVID-19 pandemic : A perspective of early career African scientists

Emerging highly transmissible viral infections such as SARS-CoV-2 pose a significant global threat to human health and the economy. Since its first appearance in December 2019 in the city of Wuhan, Hubei province, China, SARS-CoV-2 infection has quickly spread across the globe, with the first case reported on the African continent, in Egypt on February 14 th, 2020. Although the global number of COVID-19 infections has increased exponentially since the beginning of the pandemic, the number of new infections and deaths recorded in African countries have been relatively modest, suggesting slower transmission dynamics of the virus on the continent, a lower case fatality rate, or simply a lack of testing or reliable data. Notably, there is no significant increase in unexplained pneumonias or deaths on the continent which could possibly indicate the effectiveness of interventions introduced by several African governments. However, there has not yet been a comprehensive assessment of sub-Saharan Africa's (SSA) preparedness and response to the COVID-19 pandemic that may have contributed to prevent an uncontrolled outbreak so far. As a group of early career scientists and the next generation of African scientific leaders with experience of working in medical and diverse health research fields in both SSA and resource-rich countries, we present a unique perspective on the current public health interventions to fight COVID-19 in Africa. Our perspective is based on extensive review of the available scientific publications, official technical reports and announcements released by governmental and non-governmental health organizations as well as from our personal experiences as workers on the COVID-19 battlefield in SSA. We documented public health interventions implemented in seven SSA countries including Uganda, Kenya, Rwanda, Cameroon, Zambia, South Africa and Botswana, the existing gaps and the important components of disease control that may strengthen SSA response to future outbreaks

Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally

Phenotypic and genotypic analyses to guide selection of reverse transcriptase inhibitors in second-line HIV therapy following extended virological failure in Uganda

Objectives We investigated phenotypic and genotypic resistance after 2 years of first-line therapy with two HIV treatment regimens in the absence of virological monitoring. Methods NORA [Nevirapine OR Abacavir study, a sub-study of the Development of AntiRetroviral Therapy in Africa (DART) trial] randomized 600 symptomatic HIV-infected Ugandan adults (CD4 cell count <200 cells/mm3) to receive zidovudine/lamivudine plus abacavir (cABC arm) or nevirapine (cNVP arm). All virological tests were performed retrospectively, including resistance tests on week 96 plasma samples with HIV RNA levels ≥1000 copies/mL. Phenotypic resistance was expressed as fold-change in IC50 (FC) relative to wild-type virus. Results HIV-1 RNA viral load ≥1000 copies/mL at week 96 was seen in 58/204 (28.4%) cABC participants and 21/159 (13.2%) cNVP participants. Resistance results were available in 35 cABC and 17 cNVP participants; 31 (89%) cABC and 16 (94%) cNVP isolates had a week 96 FC below the biological cut-off for tenofovir (2.2). In the cNVP arm, 16/17 participants had resistance mutations synonymous with high-level resistance to nevirapine and efavirenz; FC values for etravirine were above the biological cut-off in 9 (53%) isolates. In multivariate regression models, K65R, Y115F and the presence of thymidine analogue-associated mutations were associated with increased susceptibility to etravirine in the cABC arm. Conclusions Our data support the use of tenofovir following failure of a first-line zidovudine-containing regimen and shed further light on non-nucleoside reverse transcriptase inhibitor hypersusceptibility

Correction: Evidence for reduced drug susceptibility without emergence of major protease mutations following protease inhibitor monotherapy failure in the SARA Trial

[This corrects the article DOI: 10.1371/journal.pone.0137834.]

PI susceptibility and single round infectivity of patient Gag-Protease derived pre-PI therapy and at the time of treatment failure.

<p>Full-length Gag-Protease from pre-PI therapy and failure time-points was amplified or synthesised, and cloned into our Gag-Pol expression vector p8.9NSX+. PI susceptibility of VSV-g pseudotyped viruses from patients experiencing virological failure in the absence of major resistance mutations was measured in a cell-based, single-round, phenotypic assay to the PIs A) Lopinavir (LPV) and B) Darunavir (DRV). The patient numbers are shown for each data point and data are means of two independent repeats. Our data demonstrate no significant difference in susceptibility between pre-PI therapy and failure viruses to the PIs LPV (t test, p = 0.91) and DRV (t test, p = 0.78), in patients failing in the absence of major resistance mutations. (C) Single-round infectivity of viruses was measured in the absence of drug in HEK 293T cells and compared in patients failing without major resistance mutations. No significant decrease in single-round infectivity was present (t test, p = 0.07).</p