Medical Virology

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Browsing Medical Virology by browse.metadata.advisor "Ikomey, George Mondinde"

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    HIV-1 molecular diversity and drug resistance mutations amongst immuno-competent, therapy naïve infants/children and adults in Yaounde, Cameroon
    (Stellenbosch : Stellenbosch University, 2017-12) Gichana, Josiah Otwoma; Jacobs, Graeme Brendon; Ikomey, George Mondinde; Stellenbosch University. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
    Background: In Cameroon, HIV infections range between 550, 000 to 690, 000 for adults aged 15 to 49 years and a prevalence rate at 4.5%. In children of 0 – 14 years, HIV infections range between 34, 000 to 44, 000. The country harbors both HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV groups found in Cameroon include M, N, O, P variants. Group M subtypes are the most prevalent, with CRF02_AG accounting for approximately 40% of all HIV infections. This is unlike other regions globally where other group M subtypes like C are the predominant ones. The high genomic diversity of HIV-1 and the emergence of drug resistant associated mutations (RAMs) continue to be a major challenge in designing standardized laboratory protocols for HIV testing, vaccine development and providing successful lifelong therapy to HIV infected patients. In Cameroon, drug resistance rates for therapy naïve individuals are currently at 3.8% in adults and 3.6% in children. This study aimed at identifying HIV-1 diversity and evaluate drug resistant mutations (DRMs) in two different cohorts of therapy-naïve infants/children and adults in Cameroon. Methods: A total of 180 plasma samples were collected from therapy naïve HIV positive patients that included: (1) 55 plasma samples from proxy-consented infants/children aged 9-72 months old with unknown prevention of mother-to-child transmission (PMTCT) exposure and (2) 125 plasma samples from adults of 15 to 50 years old. The CD4+ T-cell count was performed using standard methods following manufacturer’s instructions. To study the HIV-1 diversity and resistance in the two cohorts, partial pol Protease (PR), Reverse Transcriptase (RT) and Integrase (IN) regions of the HIV-1 genome were targeted for conventional PCR amplification and Sanger DNA sequencing. Phylogenetic inference using Neighbor-Joining (NJ) trees were used to cluster and infer subtypes. Results: In the infants/children cohort, the CD4+ T-cell count ranged between 500-2000 cells/m3 (a median of 33.0%) and the HIV-1 viral load between 3000-6000 copies/ml (a median of 4.96 RNA copies/ml). A total of 37/55 (67.3%) paediatric cohort samples were amplified for at least one of the HIV-1 pol fragments. These include 29/55 (52.0%) for the PR, 27/55 (49.0%) for the RT and 28/55 (51.0%) for IN. The most predominant HIV-1 strain was G/CRF02_AG at 62.5% (n = 15). Other subtypes detected include subtype A (20.8%; n = 5), C (8.3%; n = 2) and F2 (8.3%; n = 2). Three sequences (11.1%) could not be assigned to any subtype with confidence. Levels of DRMs to Protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTI were 27.6% (only minor DRMS were observed for PR), 3.7% and 40.7%, respectively. The NRTI mutations observed showed high-level resistance to Zidovudine (AZT), Tenofovir (TDF), Didanosine (DDI) and Stavudine (D4T), and low to intermediate-level resistance to Lamivudine (3TC), Abacavir (ABC), and Emtricitabine (FTC). The NNRTI mutations observed showed high level resistance to Nevirapine (NVP) and Efavirenz (EFV) with reduced susceptibility to Etravirine (ETR) and Rilpivirine (RPV). In the adult cohort, the RT fragment (n = 55) was used for phylogenetic analysis with majority of the sample sequences clustered with HIV-1 subtype G/CRF02_AG which accounted for 40% (n = 22), CRF22_01A1 (10.9%; n = 6), C (1.8%; n = 1), B (1.8%; n = 1), other complex forms – 37_cpx/11_cpx (3.6%; n = 2). Twenty three samples (41.8%) could not be assigned to any subtype with confidence. The levels of drug resistance for adults was 5.4% for both NRT and NNRT inhibitors - 4.0% had low level resistance to EFV, ETR, NVP and RPV while 1.4% had intermediate to high level resistance to ABC, FTC, TDF, EFV and NVP. Conclusion: Cameroon continues to harbor many HIV-1 subtypes and circulating recombinant forms (CRFs) as observed in both cohorts. Furthermore, rare group O and other group M subtypes like C were noticed within the study cohorts suggesting an improvement in sensitivity of detection methodologies currently used. Drug resistance is a major challenge to current antiretroviral drug regimens as illustrated by the detection of RAMS in both cohorts of this study. Continuous surveillance of the HIV diversity and drug resistance is therefore necessary to better manage the HIV-1 pandemic.
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    Investigating the structural impact of hiv-1 integrase natural occurring polymorphisms and novel mutations identified among group m subtypes circulating in sub-Saharan Africa
    (Stellenbosch : Stellenbosch University, 2020-12) Mikasi, Sello Given; Jacobs, Graeme Brendon; Cloete, Ruben; Van Zyl, Gert Uves; Engelbrecht, Susan; Ikomey, George Mondinde; Kasang, Christa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
    Introduction HIV/AIDS remains a major health concern worldwide, with sub-Saharan Africa (SSA) carrying the largest burden.HIV is characterised by extremely high genetic diversity, with all the major groups and subtypes circulating in SSA. Combination antiretroviral therapy (cART) have substantially reduced HIV related deaths, but this is counteracted by the development of HIVdrug resistance, caused by certain drug resistance-associated mutations (RAMS). Integrase (IN) strand transferase inhibitors (INSTIs), the newest class of antiretroviral drugs,has a high genetic barrier and can be used in individuals that previously exhibited resistance to other classes of drugs. The World Health Organisation (WHO) approved the use of Dolutegravir (DTG) as part of first-line cART. Methods This is a descriptive experimental design study, which aimed to identify IN natural occurring polymorphisms (NOP) among different HIV-1 group M subtypes and Drug resistance mutations within the HIV-1 pol gene fragment of INSTI naïve patients from South Africa (SA) and Cameroon (CR), using the Stanford University genotypic resistance interpretation algorithm. Structural computational methods that included; homology modelling, molecular docking, molecular dynamics simulations and interaction analysis was performed to understand the structural impact of mutations from diverse HIV-1 subtypes on DTG drug binding. ResultsWe observed low-level RAMs against INSTIs in SA (2.2%) and CR sequences (5.4%). Through Fisher’sexact test we noted that the two NOPs occurred: VI72I and R269K, with p-values ≤0. 05, were statistically enriched. The impact of having these mutations are yet to be fully understood. Through molecular modelling and stability predictions, we observed a destabilizing effect of the known G140S mutant on the HIV-1C IN protein structure and simulation analysis showed that it affected structural stability and flexibility of the protein structure. Interactions analysis of different drug binding conformations to different HIV-1 IN subtypes reported differences in the number of binding interactions to different HIV-1 IN subtypes, but we did not observe any significant differences in binding affinity for each INSTIs. This implies no significant alteration to the binding site in the wild type IN, which may not prevent INSTIs drug binding. In addition, all accessory mutations that resulted in a change in the number of interactions encompassing residues were found within the stable alpha-helix secondary structure element and not in close proximity to the drug active site.ConclusionThe study data indicate that RAMS against INSTIs remain low both in SA and in CR.Subtype C in SA and CRF02_AG in CR continues to be the driving force ofthe epidemic. We further reported on the impact of various NOPs on drug susceptibility. The analyses suggested that NOPs does not have an impact on IN protein structure and stability,and does not affect drug binding in the WT IN, but the known mutation G140S affect DTG binding. The study support recommendations made by the WHO to use DTG as part of salvage therapy in patients with RAM’s and accessory mutations. Data obtained from thisstudy can help to tailor effective treatment strategies in the African population, where diverse HIV subtypes circulate.