(P29) Protease inhibitor drug resistance mutations in minor viral quasispecies of HIV-1 infected patients failing second line therapy in South Africa
Soham Gupta , Adetayo Emmanuel Obasa [1,2], Anoop T Ambikan , Graeme Brendon Jacobs , Ujjwal Neogi 
 Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden.  Division of Medical Virology, Department of Pathology, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
Background: HIV-1 subtype C (HIV-1C) is the major HIV-1 subtype in South Africa, responsible for more than 90% of infections. The recommended second-line cART consists of the nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine (AZT) or tenofovir (TDF) and lamivudine (3TC) and a ritonavir-boosted (/r) protease inhibitor (PI), usually lopinavir (LPV/r). HIV-1C have been shown to have a greater risk of virological failure and reduced susceptibility towards boosted-protease inhibitors (bPIs) and data on acquired minority mutations on treatment failure patients from South Africa are limited. This study aimed at evaluating the HIV-1 drug resistance mutations at the minor viral populations, by high throughput sequencing genotypic resistance testing (HTS-GRT), in patients suspected of failing on South African national second-line cART regimen with bPIs.
Materials and methods: Plasma samples were obtained from 56 patients suspected of failing bPIs. The complete pol gene was amplified and sequenced with Illumina HiSeq2500, followed by bioinformatics analysis to quantify the resistance associated mutations (RAMs) according to the Stanford HIV drug resistance database.
Results: Among the 56 patients 5.3% (n=3) were on boosted ATV, while only one patient was on DRV/r and the rest 93% (n=52) were receiving LPV/r. The median (range) viral load was 71814 (937-5500000) copies/mL. HIV-1 subtyping identified 55 samples as HIV-1 subtype C and one as CRF02_AG. The PI, NRTI, NNRTI and INI RAMs were observed at 27% (15/56), 57% (32/56), 50% (28/56) and 7% (4/56) respectively. Statistically significant (p<0.001) higher PI RAMs were observed in minor viral quasispecies (25%; 14/56), compared to nucleoside reverse transcriptase inhibitors (NRTIs) (11%; 6/56), non-nucleoside reverse transcriptase inhibitors NNRTIs (9%; 5/56) and integrase inhibitors (INIs) RAM (4%; 2/56). The majority of the DRMs in the minor viral quasispecies were observed in the V82A mutation (n=13) in protease, K65R (n=5), K103N (n=7) and M184V (n=5) in reverse transcriptase.
Conclusion: HTS-GRT improved identification of PI and RTI RAMs in second-line cART failing patients from South Africa, compared to the conventional GRT. Several RTI RAMs like K65R, M184V or K103N and PI RAM V82A were identified in <20% population. Deep sequencing could be of greater value in detecting the acquired resistance mutations early.