Investigation of the HIV diversity in the Cape Winelands, Overberg and West Coast districts of the Western Cape Province of South Africa

Mikasi, Sello Given (2017-03)

Thesis (MMedSc)--Stellenbosch University, 2018.

Thesis

ENGLISH SUMMARY: The Western Cape Province of South Africa has a well-established program that monitors active combination antiretroviral therapy (cART) against HIV-1. The HIV-1 prevalence rate in the Province has increased from 5.0% in 2011 to 18.0% in 2015. South Africa has the highest rate of infections worldwide (19.2%). In this study, we analyzed the Protease (PR), Reverse Transcriptase (RT) and Intergrase (IN) regions of HIV-1 for diversity and resistanceassociated mutations (RAMs) from samples obtained from the Cape Winelands, West Coast and Overberg districts of the Province, where no such study has ever been conducted. Samples were received from our diagnostic laboratory for HIV-1 viral load testing, through the National Health Laboratory Services (NHLS). Two hundred and five (205) patient samples with a viral load of 2000 copies/ml and above were included, based on Gall et al., (2012) who showed that a sensitivity of at least 2000 copies/ml is a limit of amplification for the SuperScsript ® III one-step RT with Platinum Taq DNA Polymerase kit, used in this study. We screened for HIV-1 diversity and RAMs using the pol PR, RT and IN regions with a laboratory-based PCR and sequencing protocol. Sequence-specific subtype analyses were executed with the REGA HIV subtyping tool 3.0, Recombinant Identification Program (RIP) 3.0 and subtype classification using evolutionary algorithms (SCUEAL) software. Sequences were screened for RAMs using the Stanford University HIV Drug Resistance Database (HIVdb) 8.1. We successfully PCR amplified 170 (82.9%) PR and 166 (80.9%) RT fragments. For the IN region, only 176 samples had sufficient plasma and RNA left after genotyping of the PR and RT regions. For IN we successfully amplified 143 (81.3%) of the patient samples. A total of 197 (96.1%) samples could be amplified for at least one of the pol regions. Of these, 62 (53.4%) PR, 103 (62.0%) RT and 93 (86.1%) IN sequences were obtained, respectively. We could successfully sequence 173 (84.4%) of the samples included. HIV-1 subtype C was predominant (n = 144; 93.7%), with 5.3% of other subtypes detected. This includes A1 (n = 2; 1.3%), B (n = 4; 2.6%), D (n = 1; 0.7%) and H (n =1; 0.7%). No major RAMs were detected against PI and IN inhibitors. Minor RAMs were detected in 4 PR (3.7%) and 15 IN (16.1%) sequences analysed. RAMs against RT inhibitors were detected in 63 (61.7%) of the sequences analyzed. This includes 39 NRTI mutations (36.1%) and 71 NNRTI mutations (63.5%) identified. As the national cART program continues to expand, HIV-1 diversity, viral load monitoring and drug resistance screening remains critical for the success of cART outcomes and reducing transmission rates. Our results reflect that subtype C is still the driving force of the epidemic in South Africa. However, we cannot ignore the potential impact of non-C subtypes. Sequence analyses confirm that the majority of patients receiving viral load testing have major RAMs against RT inhibitors used in first line therapy. Better surveillance systems for HIV diversity and drug resistance testing are required to ensure success of cART.

AFRIKAANSE OPSOMMING: Die Wes-Kaap Provinsie van Suid-Afrika het 'n goed gevestigde program wat aktief kombinasie antiretrovirale terapie (cART) teen MIV-1 monitor. Die MIV-1 voorkomssyfer in die Provinsie het toegeneem van 5,0% in 2011 tot 18,0% in 2015. Suid-Afrika het die hoogste koers van infeksies wêreldwyd (19,2%). In hierdie studie het ons die Protease (PR), Trutranskriptase (RT) en Intergrase (IN) streke van MIV-1 vir diversiteit en weerstand geassosieerde mutasies (RAMS) ontleed. Die monsters is verky vanuit die Kaapse Wynland, Weskus en Overberg distrikte van die Provinsie, waar geen sodanige studie ooit gedoen is nie. Monsters wat getoets was vir MIV-1 viruslading is van ons diagnostiese laboratorium seksie deur die NHLS verkry. 205 pasiënt monsters met 'n virale lading van 2000 kopieë / ml of meer is in ons studie ingesluit. Ons kriteria is gebaseer op Gall et al., (2012) wat getoon het dat 'n sensitiwiteit van ten minste 2000 kopieë / ml 'n beperking is vir amplifisering met die SuperScsript ® III een-stap RT met Platinum Taq DNA-polimerase kit, wat in hierdie studie gebruik is. Ons ondersoeke is gedoen met ’n laboratorium-gebaseerde PCR en DNS volgorde bepalings protokol. MIV-1 subtipe analises is uitgevoer met die volgende aanlyn sagteware: REGA 3.0; RIP 3.0 en SCUEAL. Die Stanford Universiteit se MIV weerstand aanlyn databasis (HIVdb) 8.1 is gebruik om die voorkomssyfer van RAMs te bepaal. Uit 'n versameling van 205 monsters het ons suksesvol 170 (82.9%) PR en 166 (80.9%) RT fragmente geamplifiseer. Vir die IN gebied was slegs 176 monsters oor met genoeg plasma en RNA vir verdere toetse. Vir die IN gebied kon ons 143 (81.3%) monsters suksesvol amplifiseer. Ons kon vir 197 (96.1%) van die monsters ten minste een van die pol streke amplifiseer. Hiervan is 62 (53.4%) PR, 103 (62.0%) RT en 93 IN (86.1%) DNS volgordes onderskeidelik verkry. Ons kon suksesvol die MIV-1 DNS volgorde bepaal van ten minste een pol gebied van 173 (84.4%) van ons totale monsters. MIV-1 subtipe C was oorheersend (n = 144; 93,7%), met 5.3% wat as ander subtipes geklasifiseer is. Dit sluit in A1 (n = 2; 1.3%), B (n = 4; 2.6%), D (n = 1; 0.7%) en H (N = 1; 0.7%). Daar was geen groot RAMs teen PR en IN middels nie. Kleiner RAMs is identifiseer in 4 (3.7%) PR en 15 (16.1%) volgordes wat ontleed is. RAMs teen RT-middels is opgespoor in 63 (61,7%) van die volgordes ontleed. Dit sluit in 39 NRTI mutasies (36,1%) en 71 NNRTI mutasies (63,5%) geïdentifiseer. Soos die nasionale cART program uitgebrei word, word MIV-1 diversiteit, viruslading en weerstandstoetse al hoe belangriker, veral om die sukses van cART te bepaal. Ons resultate toon dat subtipe C steeds die dryfkrag van die epidemie in Suid-Afrika is. Ons kan egter nie die potensiële impak van nie-C subtipes ignoreer nie. Volgorde analises bevestig dat die meerderheid van die pasiënte wat virale lading toetse ondergaan weerstand het teen RTmiddels wat in die eerste lyn behandeling gebruik word. Beter toesig stelsels is nodig om optimale sukses cART te verseker.

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