Use of amplification refractory mutation system PCR assay as a simple and effective tool to detect HIV-1 drug resistance mutations

Access to genotyping assays to determine successful antiretroviral treatment (ART) is limited in resource-constrained settings by high cost, suggesting the need for a cost-effective and simplified method to identify HIV-1 drug resistance (HIVDR) mutations. In this study, an amplification refractory mutation system (ARMS)-PCR assay was developed and used to investigate the most frequent HIVDR mutations affecting first-line ART in settings where WHO ART guidelines are applied. Seventy-five HIV-positive (HIV(+)) samples from Cameroon were used to assess the performance of this assay. Sequencing of HIV-1 reverse transcriptase was simultaneously performed for comparison, and discordant samples were tested with a Trugene HIV-1 genotyping kit. The ARMS-PCR assay was able to detect M184V, T215Y/F, K103N, and Y181C mutations with sensitivities of 96.8%, 85.7%, 91.3%, and 70%, respectively, and specificities of 90.6%, 95%, 100%, 96.9%, respectively, compared with data on sequencing. The results indicated the highest positive predictive value for K103N (100%) and the highest negative predictive value for M184V (97.5%). ARMS-PCR's limits of detection for mutations M184V, T215Y/F, K103N, and Y181C were <75 copies/ml, 143 copies/ml, 143 copies/ml, and 836 copies/ml, respectively. ARMS-PCR efficiently identified mutations in individuals harboring different HIV-1 clades (CRF02_AG and non-CRF02_AG). In addition, this approach was more cost-effective than other genotyping assays. The high throughput, the cost-effectiveness, and the simplicity of the ARMS-PCR assay make it a suitable tool to monitor HIVDR patterns in resource-constrained settings with broad HIV-1 genetic diversity

HIV-1 drug resistance testing is essential for heavily-treated patients switching from first- to second-line regimens in resource-limited settings: Evidence from routine clinical practice in Cameroon

BackgroundWith the phase-out of stavudine (d4T), change to first-line regimens with zidovudine (AZT) or tenofovir (TDF) in resource-limited settings (RLS) might increase risks of cross-resistance to nucleos(t) ide reverse transcriptase inhibitors (NRTI). This would restrict the scope of switching to the World Health Organisation (WHO)-recommended standard second-line combinations (SLC) without HIV drug resistance (HIVDR)-testing in routine clinical practice.MethodsAn observational study was conducted among 101 Cameroonian patients (55.4% male, median [IQR] age 34 [10-41] years) failing first-line antiretroviral therapy (ART) in 2016, and stratified into three groups according to NRTIs exposure: exposure to both thymidine analogues AZT and D4T (group-A, n=55); exposure to both TDF and AZT or D4T (group-B, n=22); exposure solely to D4T (group-C, n=24). Protease-reverse transcriptase HIVDR was interpreted using the HIVdb penalty scores (60: high-resistance; 20-59: intermediate-resistance; &lt;20: susceptible). The acceptable threshold for potential-efficacy was set at 80%.ResultsThe median [IQR] CD4, viral RNA, and time on ART, were respectively 129 [29-466] cells/l, 71,630 [19,041-368,000] copies/ml, and 4 [2-5] years. Overall HIVDR-level was 89.11% (90/101), with 83.2% harbouring M184V (high-level 3TC/FTC-resistance) and only 1.98% (2/101) major HIVDR-mutations to ritonavir-boosted protease-inhibitors (PI/r). Thymidine-analogue mutations (TAMs)-1 [T215FY (46.53%), M41L (22.77%), L210W (8.91%)], with cross-resistance to AZT and TDF, were higher compared to TAMs-2 [D67N (21.78%), K70R (19.80%), K219QE (18.81%)]. As expected, K65R was related with TDF-exposure: 0% (0/55) in group-A, 22.72% (5/22) group-B, 4.17% (1/24) group-C (p=0.0013). The potential-efficacy of AZT vs. TDF was respectively 43.64% (24/55) vs. 70.91% (39/55) in group-A (p=0.0038); 63.64% (14/22) vs. 68.28% (15/22) in group-B (p=1.0000); and 37.50% (9/24) vs. 83.33% (20/24) in group-C (p=0.0032). CRF02_AG was the prevailing subtype (63.40%), followed by CRF11.cpx (8.91%), A(1) (7.92%), G (5.94%); without any significant effect of the subtype-distribution on HIVDR (92.2% in CRF02_AG vs. 83.8% in non-AG; p=0.204).ConclusionFirst-line ART-failure exhibits high-level NRTI-resistance, with potential lower-efficacy of AZT compared to TDF. Significantly, using our 80% efficacy-threshold, only patients without NRTI-substitution on first-line could effectively switch to SLC following the WHO-approach. Patients with multiple NRTI-substitutions (exposed to both thymidine-analogues and TDF) on first-line ART would require HIVDR-testing to select active NRTIs for SLC

Next-generation sequencing provides an added value in determining drug resistance and viral tropism in Cameroonian HIV-1 vertically infected children

With limited and low-genetic barrier drugs used for the prevention of mother-to-child transmission (PMTCT) of HIV in sub-Saharan Africa, vertically transmitted HIV-1 drug-resistance (HIVDR) is concerning and might prompt optimal pediatric strategies.The aim of this study was to ascertain HIVDR and viral-tropism in majority and minority populations among Cameroonian vertically infected children.A comparative analysis among 18 HIV-infected children (7 from PMTCT-exposed mothers and 11 from mothers without PMTCT-exposure) was performed. HIVDR and HIV-1 co-receptor usage was evaluated by analyzing sequences obtained by both Sanger sequencing and ultra-deep 454-pyrosequencing (UDPS), set at 1% threshold.Overall, median (interquartile range) age, viremia, and CD4 count were 6 (4-10) years, 5.5 (4.9-6.0) log10 copies/mL, and 526 (282-645) cells/mm, respectively. All children had wild-type viruses through both Sanger sequencing and UDPS, except for 1 PMTCT-exposed infant harboring minority K103N (8.31%), born to a mother exposed to AZT+3TC+NVP. X4-tropic viruses were found in 5 of 15 (33.3%) children (including 2 cases detected only by UDPS). Rate of X4-tropic viruses was 0% (0/6) below 5 years (also as minority species), and became relatively high above 5 years (55.6% [5/9], P = .040. X4-tropic viruses were higher with CD4 ≤15% (4/9 [44.4%]) versus CD4 >15% (1/6 [16.7%], P = .580); similarly for CD4 ≤200 (3/4 [75%]) versus CD4 >200 (2/11 [18.2%] cells/mm, P = .077.NGS has the ability of excluding NRTI- and NNRTI-mutations as minority species in all but 1 children, thus supporting the safe use of these drug-classes in those without such mutations, henceforth sparing ritonavir-boosted protease inhibitors or integrase inhibitors for the few remaining cases. In children under five years, X4-tropic variants would be rare, suggesting vertical-transmission with CCR5-tropic viruses and possible maraviroc usage at younger ages

HIV-1 Drug Susceptibility to Potential Second- and Third-Line Antiretroviral Regimens among Cameroonian Patients: Evidence from a Cross-sectional Design

Scale-up of antiretroviral therapy (ART) and the growing number of long-term treated patients may favor multi-HIV drug resistance (HIVDR) in resource-limited settings. Understanding the burden of HIVDR with ART-exposure may provide new insights for an effective long-term management of infected patients