Prevalence and patterns of HIV drug resistance in patients with suspected virological failure in North-Western Tanzania.

BACKGROUND: More than 15 million people in sub-Saharan Africa receive ART. Treatment failure is common, but the role of HIV drug resistance in treatment failure is largely unknown because drug resistance testing is not routinely done. This study determined the prevalence and patterns of HIV drug resistance in patients with suspected virological failure. MATERIALS AND METHODS: A single high viral load of >1000 viral RNA copies/mL of plasma at any point during ART was considered as suspected virological failure. HIV-1 RNA was extracted from plasma samples of these patients using the QIAamp Viral RNA kit. The protease and part of the RT regions of the HIV pol gene were characterized. RESULTS: Viral load was determined in 317 patients; 64 (20.2%) had suspected virological failure. We successfully genotyped 56 samples; 48 (85.7%) had at least one major resistance-associated mutation (RAM). Common mutations in RT were M184V (75%), T215Y (41.1%), K103N (39.3%), M41L (32.1%), D67DN (30.3%), G190A (28.6%) and A98G (26.8%). No RAMs were detected in ART regimens based on a ritonavir-boosted PI. CONCLUSIONS: The Tanzanian national guidelines define 'virological failure' as two consecutive viral load measurement results, at 3 month intervals, above the WHO threshold (1000 copies/mL). Here, we show that a single viral load above the WHO threshold is associated with high rates of RAMs. This suggests that a single high viral load measurement could be used to predict virological failure and avoid delays in switching patients from first-line to higher genetic barrier second-line regimens.

Interaction analysis of statistically enriched mutations identified in Cameroon recombinant subtype CRF02_AG that can influence the development of Dolutegravir drug resistance mutations.

BACKGROUND: The Integrase (IN) strand transfer inhibitor (INSTI), Dolutegravir (DTG), has been given the green light to form part of first-line combination antiretroviral therapy (cART) by the World Health Organization (WHO). DTG containing regimens have shown a high genetic barrier against HIV-1 isolates carrying specific resistance mutations when compared with other class of regimens. METHODS: We evaluated the HIV-1 CRF02_AG IN gene sequences from Cameroon for the presence of resistance-associated mutations (RAMs) against INSTIs and naturally occurring polymorphisms (NOPs), using study sequences (n = 20) and (n = 287) sequences data derived from HIV Los Alamos National Laboratory database. The possible impact of NOPs on protein structure caused by HIV-1 CRF02_AG variations was addressed within the context of a 3D model of the HIV-1 IN complex and interaction analysis was performed using PyMol to validate DTG binding to the Wild type and seven mutant structures. RESULTS: We observed 12.8% (37/287) sequences to contain RAMs, with only 1.0% (3/287) of the sequences having major INSTI RAMs: T66A, Q148H, R263K and N155H. Of these,11.8% (34/287) of the sequences contained five different IN accessory mutations; namely Q95K, T97A, G149A, E157Q and D232N. NOPs occurred at a frequency of 66% on the central core domain (CCD) position, 44% on the C-terminal domain (CTD) position and 35% of the N-terminal domain (NTD) position. The interaction analysis revealed that DTG bound to DNA, 2MG ions and DDE motif residues for T66A, T97A, Q148H, N155H and R263K comparable to the WT structure. Except for accessory mutant structure E157Q, only one MG contact was made with DTG, while DTG had no MG ion contacts and no DDE motif residue contacts for structure D232N. CONCLUSIONS: Our analysis indicated that all RAM's that resulted in a change in the number of interactions with encompassing residues does not affect DTG binding, while accessory mutations E157Q and D232N could affect DTG binding leading to possible DTG resistance. However, further experimental validation is required to validate the in silico findings of our study.

Short Communication: HIV-1 Drug Resistance Mutation Analyses of Cameroon-Derived Integrase Sequences.

HIV-1 integrase (IN) is a primary target for combination antiretroviral therapy. Only a limited number of studies report on the emergence of resistance-associated mutations (RAMs) in Cameroon. We observed that 1.4% of sequence from treatment-naive patients had IN strand transfer inhibitor (INSTI) RAMs. These mutations confer resistance to raltegravir and elvitegravir. We also observed that 10.1% of the sequences have INSTI accessory RAMs. HIV-1 CRF02_AG was the predominant subtype (44.7%) in this study analyses. The occurrence of INSTI RAMs among the sequences at baseline needs to be monitored carefully.

Structural Comparison of Diverse HIV-1 Subtypes using Molecular Modelling and Docking Analyses of Integrase Inhibitors.

The process of viral integration into the host genome is an essential step of the HIV-1 life cycle. The viral integrase (IN) enzyme catalyzes integration. IN is an ideal therapeutic enzyme targeted by several drugs; raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), and bictegravir (BIC) having been approved by the USA Food and Drug Administration (FDA). Due to high HIV-1 diversity, it is not well understood how specific naturally occurring polymorphisms (NOPs) in IN may affect the structure/function and binding affinity of integrase strand transfer inhibitors (INSTIs). We applied computational methods of molecular modelling and docking to analyze the effect of NOPs on the full-length IN structure and INSTI binding. We identified 13 NOPs within the Cameroonian-derived CRF02_AG IN sequences and further identified 17 NOPs within HIV-1C South African sequences. The NOPs in the IN structures did not show any differences in INSTI binding affinity. However, linear regression analysis revealed a positive correlation between the Ki and EC50 values for DTG and BIC as strong inhibitors of HIV-1 IN subtypes. All INSTIs are clinically effective against diverse HIV-1 strains from INSTI treatment-naïve populations. This study supports the use of second-generation INSTIs such as DTG and BIC as part of first-line combination antiretroviral therapy (cART) regimens, due to a stronger genetic barrier to the emergence of drug resistance.

Correction: Molecular dynamic simulations to investigate the structural impact of known drug resistance mutations on HIV-1C Integrase-Dolutegravir binding.

[This corrects the article DOI: 10.1371/journal.pone.0223464.].

Molecular dynamic simulations to investigate the structural impact of known drug resistance mutations on HIV-1C Integrase-Dolutegravir binding.

Resistance associated mutations (RAMs) threaten the long-term success of combination antiretroviral therapy (cART) outcomes for HIV-1 treatment. HIV-1 Integrase (IN) strand transfer inhibitors (INSTIs) have proven to be a viable option for highly specific HIV-1 therapy. The INSTI, Dolutegravir is recommended by the World Health Organization for use as first-line cART. This study aims to understand how RAMs affect the stability of IN, as well as the binding of the drug Dolutegravir to the catalytic pocket of the protein. A homology model of HIV-1 subtype C IN was successfully constructed and validated. The site directed mutator webserver was used to predict destabilizing and/or stabilizing effects of known RAMs while FoldX confirmed any changes in protein energy upon introduction of mutation. Also, interaction analysis was performed between neighbouring residues. Three mutations known to be associated with Raltegravir, Elvitegravir and Dolutegravir resistance were selected; E92Q, G140S and Y143R, for molecular dynamics simulations. The structural quality assessment indicated high reliability of the HIV-1C IN tetrameric structure, with more than 90% confidence in modelled regions. Change in free energy for the three mutants indicated different effects, while simulation analysis showed G140S to have the largest affect on protein stability and flexibility. This was further supported by weaker non-bonded pairwise interaction energy and binding free energy values between the drug DTG and E92Q, Y143R and G140S mutants suggesting reduced binding affinity, as indicated by interaction analysis in comparison to the WT. Our findings suggest the G140S mutant has the strongest effect on the HIV-1C IN protein structure and Dolutegravir binding. To the best of our knowledge, this is the first study that uses the consensus wild type HIV-1C IN sequence to build an accurate 3D model to understand the effect of three known mutations on DTG drug binding in a South Africa context.

Drug Resistance Mutations Against Protease, Reverse Transcriptase and Integrase Inhibitors in People Living With HIV-1 Receiving Boosted Protease Inhibitors in South Africa.

The South African national combination antiretroviral therapy (cART) roll-out program started in 2006, with over 4.4 million people accessing treatment since it was first introduced. HIV-1 drug resistance can hamper the success of cART. This study determined the patterns of HIV-1 drug-resistance associated mutations (RAMs) in People Living with HIV-1 (PLHIV-1). Receiving first (for children below 3 years of age) and second-line (for adults) cART regimens in South Africa. During 2017 and 2018, 110 patients plasma samples were selected, 96 samples including those of 17 children and infants were successfully analyzed. All patients were receiving a boosted protease inhibitor (bPI) as part of their cART regimen. The viral sequences were analyzed for RAMs through genotypic resistance testing. We performed genotypic resistance testing (GRT) for Protease inhibitors (PIs), Reverse transcriptase inhibitors (RTIs) and Integrase strand transfer inhibitors (InSTIs). Viral sequences were subtyped using REGAv3 and COMET. Based on the PR/RT sequences, HIV-1 subtypes were classified as 95 (99%) HIV-1 subtype C (HIV-1C) while one sample as 02_AG. Integrase sequencing was successful for 89 sequences, and all the sequences were classified as HIV-1C (99%, 88/89) except one sequence classified CRF02_AG, as observed in PR/RT. Of the 96 PR/RT sequences analyzed, M184V/I (52/96; 54%) had the most frequent RAM nucleoside reverse transcriptase inhibitor (NRTI). The most frequent non-nucleoside reverse transcriptase inhibitor (NNRTI) RAM was K103N/S (40/96, 42%). Protease inhibitor (PI) RAMs M46I and V82A were present in 12 (13%) of the sequences analyzed. Among the InSTI major RAM two (2.2%) sequences have Y143R and T97A mutations while one sample had T66I. The accessory RAM E157Q was identified in two (2.2%). The data indicates that the majority of the patients failed on bPIs didn't have any mutation; therefore adherence could be major issue in these groups of individuals. We propose continued viral load monitoring for better management of infected PLHIV.

HIV-1 Integrase Diversity and Resistance-Associated Mutations and Polymorphisms Among Integrase Strand Transfer Inhibitor-Naive HIV-1 Patients from Cameroon.

The World Health Organization (WHO) has put forth recommendations for the use of integrase (IN) strand transfer inhibitors (INSTIs) to be part of the first-line combination antiretroviral therapy regimen to treat HIV infections. The knowledge of pretreatment drug resistance against INSTIs is still scarce in resource-limited settings (RLS). We characterized the integrase gene to identify resistance-associated mutations (RAMs) in 56 INSTI-naive patient viral sequences from Cameroon. Study analysis used 37 sequences with fragment size ≥500 bp or of good quality .The majority of the sequences were identified as CRF02_AG 54.% (n = 20/37) and 45.9% (n = 17/37), other subtype viral sequences include (A, CRF36_cpx, F,G, and C). A total of 18.9% (n = 7/37) of the sequences had RAMs, with only 5.4% (n = 2/37) having major RAMs (Y143R/C/D/G and P145S), against INSTIs. Accessory RAMs were present in 8.1% (n = 3/37) of the sequences, of which one sequence contained solely E157Q, and another Q95K. One patient sequence had three accessory RAMs (G140E, E157Q, and G163R). We identified major RAMs to INSTIs, which might have a potential clinical impact to dolutegravir rollout in RLS, including Cameroon. This is the first study to describe RAMs among INSTI-naive people living with HIV-1 (PLHIV-1) infected with CRF02_AG and other subtypes in Cameroon.

Observed HIV drug resistance associated mutations amongst naïve immunocompetent children in Yaoundé, Cameroon.

INTRODUCTION: The emergence of drug resistance mutations (DRMs) has been a major threat for successful lifelong combination antiretroviral therapy (cART), especially for HIV-vertically infected children within the context of the prevention of mother-to-child transmission (PMTCT). This study aimed to evaluate DRMs amongst immune competent treatment-naïve children in Cameroon. METHODS: A cross-sectional study was conducted between 2015 and 2016 amongst 55 proxy consented HIV-1 positive children, aged 9 months to 6 years. They were all immune competent, cART naïve and with unknown history of PMTCT. CD4 cell counts and genotypic drug resistance testing were performed using standard methods. RESULTS: Levels of DRMs to protease (PR) inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs were 27.6%, 3.7% and 40.7%, respectively. Only minor DRMs were observed for PR. The observed mutations for NRTI were K65R, T215I and K219E (33.0% each) and for NNRTI: V106M, Y181C and Y188H (6.0% each). Only minor accessory mutations were found in the integrase (IN) region. CONCLUSION: Despite widely available cART we still observe naïve HIV children, especially from the rural communities. We observe that a proportion of study participants had HIV-1 drug resistance associated mutations (RAMs). Data generated could help strengthen the current PMTCT programmes within the country. There is a need to upscale approaches for drug resistance testing for children in Cameroon and many other resource-limited settings.