Stanford HIVdb Research Articles

HIV-1 Drug Resistance in ART-Naïve Individuals in Myanmar.

BackgroundEstimating the prevalence and characterizing the transmission of HIV-1 drug resistance in treatment-naïve individuals are very important in the prevention and control of HIV/AIDS. As one of the areas most affected by HIV/AIDS, few data are currently available for HIV-1 drug resistance in antiretroviral therapy (ART)-naïve individuals in Myanmar, which borders Yunnan, China.MethodsHIV-1 pol sequences from ART-naïve HIV-1-infected individuals during 2008 and 2014 in Myanmar were retrieved from our previous studies. HIV-1 transmitted drug resistance (TDR) and susceptibility to antiretroviral drugs were predicted using the Stanford HIVdb program. HIV-1 transmission cluster (TC) was determined by Cluster Picker.ResultsA total of 169 partial pol sequences from ART-naïve HIV-1 positive Burmese were analyzed. The prevalence of TDR was 20.1%. CRF01_AE and BC recombinants appeared to have a higher prevalence of TDR than other subtypes. The V179D/T was found to be very common in the China–Myanmar border region and was involved in half of the transmission clusters formed by HIV-1 drug-resistance strains in this region. Comparison showed that drug-resistance mutation profile in Myanmar was very similar to that in Dehong prefecture of Yunnan. By further phylogenetic analysis with all available sequences from the China–Myanmar border region, four HIV-1 drug-resistance-related TCs were identified. Three of them were formed by Burmese long-distance truck drivers and the Burmese staying in Yunnan, and another was formed by Burmese injection drug users staying in Myanmar and Yunnan. These results suggest a potential transmission link of HIV-1 drug resistance between Myanmar and Yunnan.ConclusionGiven the high prevalence of TDR in Myanmar, and the potential risk of cross-border transmission of HIV-1 drug-resistant strains between Myanmar and Yunnan, China, ongoing monitoring of HIV-1 drug resistance in ART-naïve individuals will provide a guideline for clinical antiretroviral treatment and benefit the prevention and control of HIV/AIDS in this border region.

Impact of genotypic diversity on selection of subtype-specific drug resistance profiles during raltegravir-based therapy in individuals infected with B and BF recombinant HIV-1 strains.

Current knowledge on HIV-1 resistance to integrase inhibitors (INIs) is based mostly on subtype B strains. This contrasts with the increasing use of INIs in low- and middle-income countries, where non-B subtypes predominate. HIV-1 drug resistance genotyping was performed in 30 HIV-1-infected individuals undergoing virological failure to raltegravir. Drug resistance mutations (DRMs) and HIV-1 subtype were characterized using Stanford HIVdb and phylogenetic analyses. Of the 30 integrase (IN) sequences, 14 were characterized as subtype F (47%), 8 as subtype B (27%), 7 as BF recombinants (23%) and 1 as a putative CRF05_DF (3%). In 25 cases (83%), protease and reverse transcriptase (PR-RT) sequences from the same individuals confirmed the presence of different BF recombinants. Stanford HIVdb genotyping was concordant with phylogenetic inference in 70% of IN and 60% of PR-RT sequences. INI DRMs differed between B and F IN subtypes, with Q148K/R/H, G140S and E138K/A being more prevalent in subtype B (63% versus 0%, P = 0.0021; 50% versus 0%, P = 0.0096; and 50% versus 0%, P = 0.0096, respectively). These differences were independent of the time on raltegravir therapy or viral load at the time of genotyping. INI DRMs in subtype F IN genomes predicted a lower level of resistance to raltegravir and no cross-resistance to second-generation INIs. Alternative resistance pathways to raltegravir develop in subtypes B and F IN genomes, with implications for clinical practice. Evaluating the role of HIV-1 subtype in development and persistence of mutations that confer resistance to INIs will be important to improve algorithms for resistance testing and optimize the use of INIs.

Natural polymorphisms in HIV-1 CRF01_AE strain and profile of acquired drug resistance mutations in a long-term combination treatment cohort in northeastern China

BackgroundThe impacts of genetic polymorphisms on drug resistance mutations (DRMs) among various HIV-1 subtypes have long been debated. In this study, we aimed to analyze the natural polymorphisms and acquired DRM profile in HIV-1 CRF01_AE-infected patients in a large first-line antiretroviral therapy (ART) cohort in northeastern China.MethodsThe natural polymorphisms of CRF01_AE were analyzed in 2034 patients from a long-term ART cohort in northeastern China. The polymorphisms in 105 treatment failure (TF) patients were compared with those in 1148 treatment success (TS) patients. The acquired DRM profile of 42 patients who experienced TF with tenofovir/lamivudine/efavirenz (TDF/3TC/EFV) treatment was analyzed by comparing the mutations at TF time point to those at baseline. The Stanford HIVdb algorithm was used to interpret the DRMs. Binomial distribution, McNemar test, Wilcoxon test and CorMut package were used to analyze the mutation rates and co-variation. Deep sequencing was used to analyze the evolutionary dynamics of co-variation.ResultsBefore ART, there were significantly more natural polymorphisms of 31 sites on reverse transcriptase (RT) in CRF01_AE than subtype B HIV-1 (|Z value| ≥ 3), including five known drug resistance-associated sites (238, 118, 179, 103, and 40). However, only the polymorphism at site 75 was associated with TF (|Z value| ≥ 3). The mutation rate at 14 sites increased significantly at TF time point compared to baseline, with the most common DRMs comprising G190S/C, K65R, K101E/N/Q, M184 V/I, and V179D/I/A/T/E, ranging from 66.7 to 45.2%. Moreover, two unknown mutations (V75 L and L228R) increased by 19.0 and 11.9% respectively, and they were under positive selection (Ka/Ks > 1, log odds ratio [LOD] > 2) and were associated with several other DRMs (cKa/Ks > 1, LOD > 2). Deep sequencing of longitudinal plasma samples showed that L228R occurred simultaneously or followed the appearance of Y181C.ConclusionThe high levels of natural polymorphisms in CRF01_AE had little impact on treatment outcomes. The findings regarding potential new CRF01_AE-specific minor DRMs indicate the need for more studies on the drug resistance phenotype of CRF01_AE.

Trend of HIV-1 drug resistance in China: A systematic review and meta-analysis of data accumulated over 17 years (2001–2017)

BackgroundThe emergence and spread of HIV-1 drug resistance may compromise HIV control globally. In response to HIV/AIDS epidemic, China launched national HIV/AIDS treatment program in 2003, and started to accumulate drug resistance data since 2001. In this study we aimed to assess the level, trend and distribution of HIV-1 drug resistance during a period of 17 years from 2001 to 2017, and to characterize crucial drug resistance mutations. MethodsWe systematically reviewed 4737 studies published between January 1, 2001 and March 31, 2019 in PubMed, Embase, China National Knowledge Infrastructure (CNKI), WanFang Database, Web of Science, conference abstracts from the Chinese Medical Association and the Chinese AIDS Academic Conferences, and selected 170 studies that met our study criteria. To assess the prevalence of drug resistance in whole country or a local region, we performed pooled analyses of raw data. The transformed proportions were pooled using the inverse variance fixed effects methods or the DerSimonian-Laired random effects methods. The temporal trend of transmitted drug resistance (TDR) was determined using generalized additive model implemented in the Mgcv version 1.8 package. HIV-1 genotypic resistance was analyzed using the Stanford HIVdb algorithm. FindingsWe assembled 218 datasets from 170 selected studies (129 in Chinese and 41 in English), covering 21,451 ART-naïve and 30,475 ART-treated individuals with HIV-1 infection. The pooled prevalence of TDR was 3.0% (95%CI: 2.8–3.2), including 0.7% (95%CI: 0.4–1.0), 1.4% (95%CI: 1.3–1.6) and 0.5% (95%CI: 0.4–0.6) for nucleoside reverse transcriptase inhibitor (NRTI), non-NRTI (NNRTI) and protease inhibitor (PI) resistance, respectively. The acquired drug resistance (ADR) prevalence was 44.7% (95%CI: 39.3–50.2), including 31.4% (95%CI: 28.2–34.6), 39.5% (95%CI: 35.6–43.5) and 1.0% (95%CI: 0.8–1.2) for NRTI, NNRTI and PI resistance, respectively. TDR and ADR prevalence had characteristic regional patterns. The worst prevalence of drug resistance occurred in Central China, and higher ADR prevalence occurred in South China than North China. TDR in whole country has risen since 2012, and this rise was driven mainly by NNRTI resistance. One NRTI-associated (M184V/I) and three NNRTI-associated (K103N/S, Y181C/I and G190A/S) mutations had high percentages in ART-naïve and ART-treated individuals, and these mutations conferred high-level resistance to 3TC, EFV and/or NVP. InterpretationThese findings suggest that the current available first-line ART regimens containing 3TC and/or EFV or NVP need to be revised. In addition, scale-up of multiple viral load measurements per year and drug resistance testing prior to ART initiation are recommended. Furthermore, implementation of pre-treatment education and counseling to improve patient adherence to ART is encouraged. FundingThis work was supported by grants from the National Natural Science Foundation of China (81672033, U1302224, and 81271888) and Open Research Fund Program of the State Key Laboratory of Virology of China (2019IOV002).

Survey of Pretreatment HIV Drug Resistance and Genetic Transmission Network Analysis Among HIV Patients in a High Drug-Use Area of Southwest China.

Background: Pretreatment drug resistance (PDR) poses an increasing threat to the success of antiretroviral treatment (ART) programs in China. We aimed to conduct a survey of PDR among HIV patients in an area in Southwest China with extensive drug trafficking.Methods: Consecutive cross-sectional surveys were conducted in Liangshan Prefecture of Sichuan Province from 2009 to 2018 based on the WHO-recommended method. PDR was identified by testing pol region sequences with the Stanford HIVdb algorithm (version 7.0). PDR prevalence and related factors were assessed by multivariable logistic regression. The transmission of HIV drug resistance was analyzed using a genetic transmission network.Results: HIV-1 pol genes from 1889 patients were successfully amplified. The distribution of HIV-1 genotypes was as follows: CRF07_BC (94.0%), CRF08_BC (2.3%), CRF01_AE (2.0%) and others (1.4%). Of the participants, 6.9% (95% CI: 4.1-8.1%) had pretreatment resistance to 12 antiretroviral drugs recommended by the WHO, and nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitors (PI) resistance were identified among 1.4% (95% CI: 0.7-3.4%), 5.8% (95% CI: 1.2-8.7%) and 0.4% (95% CI: 0.1-3.0%) of the patients, respectively. In the multivariate logistic model, the prevalence of PDR was 1.52-fold higher among intravenous drug users (IDUs) than among patients infected by heterosexual transmission (95% CI: 1.07-2.38; P=0.049), and the prevalence of PDR among patients diagnosed from 2017-2018 was 2.03-fold higher than that among patients diagnosed from 2009-2016 (95% CI: 1.18-5.76; P=0.018). A total of 26 clusters containing PDR and a rapidly growing drug resistance-related cluster containing the E138Q and V179D mutations were identified by genetic transmission network analysis.Conclusion: The results show a moderate overall level of PDR prevalence and rapidly growing drug resistance over time. Preventive intervention should be focused on controlling the HIV epidemic among drug users, and surveillance is urgently needed to monitor the trend of PDR.

High levels of pretreatment and acquired HIV drug resistance in Nicaragua: results from the first nationally representative survey, 2016.

IntroductionA nationally representative HIV drug resistance (HIVDR) survey in Nicaragua was conducted to estimate the prevalence of pretreatment HIVDR (PDR) among antiretroviral therapy (ART) initiators and acquired HIVDR among people living with HIV (PLHIV) who had received ART for 12 ± 3 months (ADR12) and ≥48 months (ADR48).MethodsA nationwide cross‐sectional survey with a two‐stage cluster sampling was conducted from March to November 2016. Nineteen of 45 total ART clinics representing >90% of the national cohort of adults on ART were included. ART initiators were defined as PLHIV initiating or reinitiating first‐line ART. HIVDR was assessed for protease, reverse transcriptase and integrase Sanger sequences using the Stanford HIVdb algorithm. Viral load (VL) suppression was defined as <1000 copies/mL. Results were weighted according to the survey design.Results and discussionA total of 638 participants were enrolled (PDR: 171; ADR12: 114; ADR48: 353). The proportion of ART initiators with prior exposure to antiretrovirals (ARVs) was 12.3% (95% CI: 5.8% to 24.3%). PDR prevalence to any drug was 23.4% (95% CI: 14.4% to 35.6%), and 19.3% (95% CI: 12.2% to 29.1%) to non‐nucleoside reverse transcriptase inhibitors (NNRTI). NNRTI PDR was higher in ART initiators with previous ARV exposure compared with those with no exposure (76.2% vs. 11.0%, p < 0.001). Protease inhibitors (PI) and integrase strand transfer inhibitors PDR was not observed. VL suppression rate was 77.8% (95% CI: 67.1% to 85.8%) in ADR12 and 70.3% (95% CI: 66.7% to 73.8%) in ADR48. ADR12 prevalence to any drug among PLHIV without VL suppression was 85.1% (95% CI: 66.1% to 94.4%), 82.4% to NNRTI and 70.2% to nucleoside reverse transcriptase inhibitors (NRTI). ADR48 prevalence to any drug among PLHIV without VL suppression was 75.5% (95% CI: 63.5% to 84.5 %), 70.7% to NNRTI, 59.4% to NRTI and 4.6% to PI.ConclusionsDespite implementation challenges yielding low‐precision HIVDR estimates, high rates of NNRTI PDR were observed in Nicaragua, suggesting consideration of non‐NNRTI‐based first‐line regimens for ART initiators. Strengthened HIVDR monitoring, systematic VL testing, and improved ART adherence support are also warranted.

Validation of publicly-available software used in analyzing NGS data for HIV-1 drug resistance mutations and transmission networks in a Washington, DC, Cohort.

The DC Cohort is an ongoing longitudinal observational study of persons living with HIV. To better understand HIV-1 drug resistance and potential transmission clusters among these participants, we performed targeted, paired-end next-generation sequencing (NGS) of protease, reverse transcriptase and integrase amplicons. We elected to use free, publicly-available software (HyDRA Web, Stanford HIVdb and HIV-TRACE) for data analyses so that laboratory personnel without extensive bioinformatics expertise could use it; making the approach accessible and affordable for labs worldwide. With more laboratories transitioning away from Sanger-based chemistries to NGS platforms, lower frequency drug resistance mutations (DRMs) can be detected, yet their clinical relevance is uncertain. We looked at the impact choice in cutoff percentage had on number of DRMs detected and found an inverse correlation between the two. Longitudinal studies will be needed to determine whether low frequency DRMs are an early indicator of emerging resistance. We successfully validated this pipeline against a commercial pipeline, and another free, publicly-available pipeline. RT DRM results from HyDRA Web were compared to both SmartGene and PASeq Web; using the Mantel test, R2 values were 0.9332 (p<0.0001) and 0.9097 (p<0.0001), respectively. PR and IN DRM results from HyDRA Web were then compared with PASeq Web only; using the Mantel test, R2 values were 0.9993 (p<0.0001) and 0.9765 (p<0.0001), respectively. Drug resistance was highest for the NRTI drug class and lowest for the PI drug class in this cohort. RT DRM interpretation reports from this pipeline were also highly correlative compared to SmartGene pipeline; using the Spearman’s Correlation, rs value was 0.97757 (p<0.0001). HIV-TRACE was used to identify potential transmission clusters to better understand potential linkages among an urban cohort of persons living with HIV; more individuals were male, of black race, with an HIV risk factor of either MSM or High-risk Heterosexual. Common DRMs existed among individuals within a cluster. In summary, we validated a comprehensive, easy-to-use and affordable NGS approach for tracking HIV-1 drug resistance and identifying potential transmission clusters within the community.

First-line HIV treatment failures in non-B subtypes and recombinants: a cross-sectional analysis of multiple populations in Uganda

BackgroundOur understanding of HIV-1 and antiretroviral treatment (ART) is strongly biased towards subtype B, the predominant subtype in North America and western Europe. Efforts to characterize the response to first-line treatments in other HIV-1 subtypes have been hindered by the availability of large study cohorts in resource-limited settings. To maximize our statistical power, we combined HIV-1 sequence and clinical data from every available study population associated with the Joint Clinical Research Centre (JCRC) in Uganda. These records were combined with contemporaneous ART-naive records from Uganda in the Stanford HIVdb database.MethodsTreatment failures were defined by the presence of HIV genotype records with sample collection dates after the ART start dates in the JCRC database. Drug resistances were predicted by the Stanford HIVdb algorithm, and HIV subtype classification and recombination detection was performed with SCUEAL. We used Bayesian network analysis to evaluate associations between drug exposures and subtypes, and binomial regression for associations with recombination.ResultsThis is the largest database of first-line treatment failures (n=1724) in Uganda to date, with a predicted statistical power of 80% to detect subtype associations at an odds ratio of ge 1.2. In the subset where drug regimen data were available, we observed that use of 3TC was associated with a higher rate of first line treatment failure, whereas regimens containing AZT and TDF were associated with reduced rates of failure. In the complete database, we found limited evidence of associations between HIV-1 subtypes and treatment failure, with the exception of a significantly lower frequency of failures among A/D recombinants that comprised about 7% of the population. First-line treatment failure was significantly associated with reduced numbers of recombination breakpoints across subtypes.ConclusionsExpanding access to first-line ART should confer the anticipated public health benefits in Uganda, despite known differences in the pathogenesis of HIV-1 subtypes. Furthermore, the impact of ART may actually be enhanced by frequent inter-subtype recombination in this region.

Prevalence and persistence of transmitted drug resistance mutations in the German HIV-1 Seroconverter Study Cohort.

The prevalence of transmitted drug resistance (TDR) in antiretroviral therapy (ART)-naïve individuals remains stable in most developed countries despite a decrease in the prevalence of acquired drug resistance. This suggests that persistence and further transmission of HIV-1 that encodes transmitted drug resistance mutations (TDRMs) is occurring in ART-naïve individuals. In this study, we analysed the prevalence and persistence of TDRMs in the protease and reverse transcriptase-sequences of ART-naïve patients within the German HIV-1 Seroconverter Study Cohort who were infected between 1996 and 2017. The prevalence of TDRMs and baseline susceptibility to antiretroviral drugs were assessed using the Stanford HIVdb list and algorithm. Mean survival times of TDRMs were calculated by Kaplan-Meier analysis. The overall prevalence of TDR was 17.2% (95% CI 15.7–18.6, N = 466/2715). Transmitted NNRTI resistance was observed most frequently with 7.8% (95% CI 6.8–8.8), followed by NRTI resistance (5.0%, 95% CI 4.2–5.9) and PI resistance (2.8%, 95% CI 2.2–3.4). Total TDR (OR = 0.89, p = 0.034) and transmitted NRTI resistance (OR = 0.65, p = 0.000) decreased between 1996 and 2017 but has remained stable during the last decade. Viral susceptibility to NNRTIs (6.5%-6.9% for individual drugs) was mainly reduced, while <3% of the recommended NRTIs and PIs were affected. The longest mean survival times were calculated for the NNRTI mutations K103N (5.3 years, 95% CI 4.2–5.6) and E138A/G/K (8.0 years, 95% CI 5.8–10.2 / 7.9 years, 95% CI 5.4–10.3 / 6.7 years, 95% CI 6.7–6.7) and for the NRTI mutation M41L (6.4 years, 95% CI 6.0–6.7).The long persistence of single TDRMs indicates that onward transmission from ART-naïve individuals is the main cause for TDR in Germany. Transmitted NNRTI resistance was the most frequent TDR, showing simultaneously the highest impact on baseline ART susceptibility and on TDRMs with prolonged persistence. These results give cause for concern regarding the use of NNRTI in first-line regimens.

Epidemiological surveillance of HIV-1 transmitted drug resistance among newly diagnosed individuals in Shijiazhuang, northern China, 2014-2015.

BackgroundThe widespread use of antiretroviral therapy (ART) has led to considerable concerns about the prevalence of transmitted drug resistance (TDR). Sexual contact, particularly men who have sex with men (MSM) was the most prevalent form of HIV transmission in Shijiazhuang. Hence, we conducted an epidemiological surveillance study on TDR among newly diagnosed individuals who infected-HIV through sexual contact in from 2014–2015.MethodsGenotypic resistance mutations were defined using the WHO-2009 surveillance list. Potential impact on antiretroviral drug was predicted according to the Stanford HIV db program version 7.0. The role of transmission clusters in drug resistant strains was evaluated by phylogenetic and network analyses.ResultsIn this study, 589 individuals were recruited and 542 samples were amplified and sequenced successfully. The over prevalence of TDR was 6.1%: 1.8% to nucleoside reverse transcriptase inhibitors (NRTIs), 2.0% to non- NRTIs (NNRTIs) and 2.4% to protease inhibitors (PIs), respectively. We did not find significant differences in the TDR prevalence by demographic and clinical characteristics (p > 0.05). Using network and phylogenetic analysis, almost 60.0% sequences were clustered together. Of these clusters, 2 included at least two individuals carrying the same resistance mutation, accounting for 21.2% (7/33) individuals with TDR. No significant difference was observed in the clustering rate between the individuals with and without TDR.ConclusionsWe obtained a moderate level TDR rate in studied region. These findings enhance our understanding of HIV-1 drug resistance prevalence in Shijiazhuang, and may be helpful for the comprehensive prevention and control of HIV-1.

The global burden of HIV-1 drug resistance in the past 20 years.

Genotypic drug resistance testing has been an integral part of the clinical management of HIV patients for almost 20 years, not only assisting treatment choices but also informing drug development. Accurate estimations on the worldwide circulation of drug resistance are difficult to obtain, particularly in low/middle-income countries. In this work, we queried two of the largest public HIV sequence repositories in the world—Los Alamos and Stanford HIVdb—to derive global prevalence, time trends and geodemographic predictors of HIV drug resistance. Different genotypic interpretation systems were used to ascertain resistance to reverse transcriptase and protease inhibitors. Continental, subtype-specific (including circulating recombinant forms) stratification as well as analysis on drug-naïve isolates were performed. Geographic information system analysis correlated country-specific drug resistance to sociodemographic and health indicators obtained from the World Bank. By looking at over 33,000 sequences worldwide between 1996 and 2016, increasing drug resistance trends with non-B subtypes and recombinants were found; transmitted drug resistance appeared to remain stable in the last decade. While an increase in drug resistance is expected with antiretroviral therapy rollout in resource-constrained areas, the plateau effect in areas covered by the most modern drug regimens warns against the downgrading of the resistance issue.

Primary resistance to integrase strand transfer inhibitors in patients infected with diverse HIV-1 subtypes in sub-Saharan Africa

To investigate the prevalence and patterns of major and accessory resistance mutations associated with integrase strand transfer inhibitors (INSTIs), across diverse HIV-1 subtypes in sub-Saharan Africa. pol gene sequences were obtained using Illumina next-generation sequencing from 425 INSTI-naive HIV-infected adults from Kenya (21.2%), Nigeria (7.3%), South Africa (22.8%), Uganda (25.2%) and Zambia (23.5%). Drug resistance interpretation was based on the IAS 2017 mutation list and accessory mutations from Stanford HIVdb with resistance penalty scores of ≥10 to at least 1 INSTI. Resistance was further classified based on sensitivity thresholds of ≥20% (Sanger sequencing) and 1%-20% for low-frequency variants (next-generation sequencing). Of 425 genotypes, 48.7% were subtype C, 28.5% A, 10.1% D, 2.8% G and 9.9% were recombinants. Major INSTI resistance mutations were detected only at <20% threshold, at a prevalence of 2.4% (2.5% in subtype A, 2.4% C, 0% D, 8.3% G and 2.4% in recombinants) and included T66A/I (0.7%), E92G (0.5%), Y143C/S (0.7%), S147G (0.2%) and Q148R (0.5%). Accessory mutations occurred at a prevalence of 15.1% at the ≥20% threshold (23.1% in subtype A, 8.7% C, 11.6% D, 25% G and 23.8% in recombinants), and included L74I/M (10.4%), Q95K (0.5%), T97A (4%), E157Q (0.7%) and G163R/K (0.7%). Major INSTI resistance mutations were rare and only occurred at low-level resistance detection thresholds. INSTI-based regimens are expected to be effective across the different major HIV-1 subtypes in the region.

Next generation sequencing of HIV-1 protease in the PIVOT trial of protease inhibitor monotherapy

BackgroundThe PIVOT trial examined whether patients with suppressed viral load on combination antiretroviral therapy could be safely switched long-term to ritonavir-boosted protease inhibitor (PI) monotherapy. The main trial publication reported that only one of 296 patients allocated to PI monotherapy experienced a loss of drug options due to protease mutations (identified by local Sanger sequencing resistance tests) likely selected by study drug. ObjectivesTo assess if we had missed low frequency mutations, using a more sensitive methodology. Study designWe performed next generation sequencing (NGS) on all available frozen plasma samples with VL >1000 copies/ml from patients who were randomised to PI monotherapy. Assays were performed at Public Health England laboratories using a previously described method.Median coverage depth was 76,000 and the threshold for detection of minority variants was 2%. Drug susceptibility was predicted using the Stanford HIVdb algorithm. Results17 of 26 potential samples, all from different patients, were identified and successfully tested. The median viral load was 6780 copies/ml and the median time since randomisation was 43 weeks. NGS revealed previously unidentified minority variant protease mutations (G73D, I54T, L89V) in three samples, at frequencies ranging between 2% and 10%. None of these mutations predicted intermediate or high level resistance, the trial primary outcome. DiscussionThis report adds to the body of evidence that ritonavir-boosted PI monotherapy, when used as a switch strategy with prompt detection of viral load rebound and early re-introduction of combination therapy, rarely leads to the development of clinically important protease resistance mutations.

HIV-1 Drug Resistance and Third-Line Therapy Outcomes in Patients Failing Second-Line Therapy in Zimbabwe.

ObjectivesTo analyze the patterns and risk factors of HIV drug resistance mutations among patients failing second-line treatment and to describe early treatment responses to recommended third-line antiretroviral therapy (ART) in a national referral HIV clinic in Zimbabwe.MethodsPatients on boosted protease inhibitor (PI) regimens for more than 6 months with treatment failure confirmed by 2 viral load (VL) tests >1000 copies/mL were genotyped, and susceptibility to available antiretroviral drugs was estimated by the Stanford HIVdb program. Risk factors for major PI resistance were assessed by logistic regression. Third-line treatment was provided as Darunavir/r, Raltegravir, or Dolutegravir and Zidovudine, Abacavir Lamivudine, or Tenofovir.ResultsGenotypes were performed on 86 patients who had good adherence to treatment. The median duration of first- and second-line ART was 3.8 years (interquartile range [IQR], 2.3–5.1) and 2.6 years (IQR, 1.6–4.9), respectively. The median HIV viral load and CD4 cell count were 65 210 copies/mL (IQR, 8728–208 920 copies/mL) and 201 cells/mm3 (IQR, 49–333 cells/mm3). Major PI resistance-associated mutations (RAMs) were demonstrated in 44 (51%) non-nucleoside reverse transcriptase inhibitor RAMs in 72 patients (83%) and nucleoside reverse transcriptase inhibitors RAMs in 62 patients (72%). PI resistance was associated with age >24 years (P = .003) and CD4 cell count <200 cells/mm3 (P = .007). In multivariable analysis, only age >24 years was significantly associated (adjusted odds ratio, 4.75; 95% confidence interval, 1.69–13.38; P = .003) with major PI mutations. Third-line DRV/r- and InSTI-based therapy achieved virologic suppression in 29/36 patients (81%) after 6 months.ConclusionsThe prevelance of PI mutations was high. Adolescents and young adults had a lower risk of acquiring major PI resistance mutations, possibly due to poor adherence to ART. Third-line treatment with a regimen of Darunavir/r, Raltegravir/Dolutegravir, and optimized nucleoside reverse transcriptase inhibitors was effective.

ACQUIRED DRUG RESISTANCE TO NRTI CLASS IN TREATMENT-EXPERIENCED HIV INFECTED PATIENTS FROM THE CONSTANTA COUNTY: THERAPEUTIC IMPLICATIONS

Objective. To determine the prevalence of acquired drug resistance (ADR) and of resistance patterns in treatment-experienced HIV infected patients from Constanta in order to establish the best therapeutic options in NRTI class. Material and methods. A retrospective study which included 144 treatment-experienced HIV patients with confirmed viral failure. The strains isolated from these patients were analysed in the Molecular Genetic Laboratory of „Matei Bals“ National Institute of Infectious Diseases, Bucharest and the resulting sequences were saved in FASTA format. The HIV-1 subtyping was based on „REGA HIV01&amp;2 Automated subtyping tool version 2.0“ algorithm. „Stanford HIVdb Program version 8.4“ was used in order to determine the therapeutic options. For statistical calculations, the R-Project software was used. Graphic representations were performed using GNUPLOT program. Results. The prevalence of the acquired drug resistance was 92.36%. The most frequent mutation occurred at the level of the codon 184. The TAM-2 path was more frequently selected compared to TAM-1. Association between TAM1 and TAM 2 were also found, mutation K65R being rarely met. Conclusions. The prevalence of the acquired drug resistance in our study was high, The most valuable therapeutic option in the INRT class remains tenofovir, due to the mutational profile, which was selected on account of the extensive use of thymidine analogues.

HIV pretreatment drug resistance trends in three geographic areas of Mexico.

Pretreatment drug resistance (PDR) levels to NNRTI approaching 10% have recently been reported in Mexico. However, subnational differences may exist in PDR prevalence and transmission dynamics. We longitudinally assessed HIV PDR in three geographic areas of Mexico. HIV-infected, antiretroviral-naive individuals were recruited from 2008 to 2016, from the Central Metropolitan Zone (CMZ), Cancun and Tijuana (1194, 773 and 668 respectively). PDR was estimated using the Stanford HIVdb tool from plasma HIV pol sequences. A higher proportion of females, lower education and lower employment rate were observed in Tijuana, while a higher proportion of MSM was observed in the CMZ (P < 0.0001, all cases). For 2012-16, PDR was 13.4%, 8.9% and 11.2% in the CMZ, Tijuana and Cancun respectively. NNRTI PDR was highest in the three regions (8.7%, 4.8% and 8.1% respectively, P < 0.05); nevertheless, NNRTI PDR in Tijuana was lower than in the CMZ (P = 0.01). For 2008-16, we observed increasing efavirenz resistance trends in all regions (P < 0.05, all cases), reaching 11.8%, 6.1% and 8.3% respectively in 2016. Increasing efavirenz resistance was mostly associated with increasing K103N frequency (P = 0.007 CMZ, P = 0.03 Tijuana, not significant for Cancun). Our study suggests different NNRTI PDR prevalence and transmission dynamics in three geographical areas of Mexico. Even when increasing trends in efavirenz resistance were observed in the three areas, our observations support that, in a large country such as Mexico, subnational surveillance and locally tailored interventions to address drug resistance may be a reasonable option.

Impact of lopinavir-ritonavir exposure in HIV-1 infected children and adolescents in Madrid, Spain during 2000-2014.

BackgroundThe most-used protease-inhibitor in children is Lopinavir-ritonavir (LPV/r), which provides durable suppression of viral load and increases CD4+T-counts. This study describes the virological outcome of the HIV-1-infected paediatric population exposed to LPV/r during 15 years in Spain.MethodologyPatients from the Madrid Cohort of HIV-1-infected-children and adolescents exposed to LPV/r as different line therapy during 2000–2014 were selected. The baseline epidemiological-clinical features, viral suppression, changes in CD4+T-CD8+T cell counts and drug susceptibility were recorded before and during LPV/r exposure. Drug resistance mutations (DRM) were identified in viruses from samples collected until 2011. We predicted drug susceptibility to 19 antiretrovirals among those carrying DRM using the Stanford′s HIVdb Algorithm.ResultsA total of 199 (37.3%) of the 534 patients from the cohort were exposed to LPV/r during 2000–2014 in first (group 1), second (group 2) or more line-therapies (group 3). Patients were mainly Spaniards (81.9%), perinatally infected (96.5%) with subtype-B (65.3%) and HIV-diagnosed before year 2000 (67.8%). The mean age at first LPV/r exposure was 9.7 years. After protease-inhibitor exposure, viral suppression was higher in groups 1 and 2 than in group 3. Viral suppression occurred in 87.5%, 68.6% and 64.8% patients from groups 1, 2 and 3, respectively. Among the 64 patients with available resistance data during LPV/r treatment, 27(42.3%) carried DRM to protease-inhibitor, 28 (58.3%) to reverse-transcriptase-inhibitors and 21 (43.7%) to non-reverse-transcriptase-inhibitors. Darunavir/ritonavir, atazanavir-ritonavir and tipranavir/ritonavir presented the highest susceptibility and nelfinavir the lowest.ConclusionsA better lymphocyte recovering occurred when protease-inhibitor was taken as part of a first-line regimen and a higher number of patients reached viral suppression. The least compromised antiretrovirals for rescue antiretroviral regimens, according to DRM in the LPV/r-exposed-paediatric cohort, were mainly the new protease inhibitors.

Surveillance of HIV-1 pol transmitted drug resistance in acutely and recently infected antiretroviral drug-naïve persons in rural western Kenya.

HIV-1 transmitted drug resistance (TDR) is of increasing public health concern in sub-Saharan Africa with the rollout of antiretroviral (ARV) therapy. Such data are, however, limited in Kenya, where HIV-1 drug resistance testing is not routinely performed. From a population-based household survey conducted between September and November 2012 in rural western Kenya, we retrospectively assessed HIV-1 TDR baseline rates, its determinants, and genetic diversity among drug-naïve persons aged 15–59 years with acute HIV-1 infections (AHI) and recent HIV-1 infections (RHI) as determined by nucleic acid amplification test and both Limiting Antigen and BioRad avidity immunoassays, respectively. HIV-1 pol sequences were scored for drug resistance mutations using Stanford HIVdb and WHO 2009 mutation guidelines. HIV-1 subtyping was computed in MEGA6. Eighty seven (93.5%) of the eligible samples were successfully sequenced. Of these, 8 had at least one TDR mutation, resulting in a TDR prevalence of 9.2% (95% CI 4.7–17.1). No TDR was observed among persons with AHI (n = 7). TDR prevalence was 4.6% (95% CI 1.8–11.2) for nucleoside reverse transcriptase inhibitors (NRTIs), 6.9% (95% CI 3.2–14.2) for non- nucleoside reverse transcriptase inhibitors (NNRTIs), and 1.2% (95% CI 0.2–6.2) for protease inhibitors. Three (3.4% 95% CI 0.8–10.1) persons had dual-class NRTI/NNRTI resistance. Predominant TDR mutations in the reverse transcriptase included K103N/S (4.6%) and M184V (2.3%); only M46I/L (1.1%) occurred in the protease. All the eight persons were predicted to have different grades of resistance to the ARV regimens, ranging from potential low-level to high-level resistance. HIV-1 subtype distribution was heterogeneous: A (57.5%), C (6.9%), D (21.8%), G (2.3%), and circulating recombinant forms (11.5%). Only low CD4 count was associated with TDR (p = 0.0145). Our findings warrant the need for enhanced HIV-1 TDR monitoring in order to inform on population-based therapeutic guidelines and public health interventions.

English

HIV-1 genetic diversity in sub-Saharan Africa is broad and the AIDS epidemic is driven predominantly by recombinants in Central and West Africa. The classification of HIV-1 strains is therefore necessary to understand diagnostic efficiency, individual treatment responses as well as options for designing vaccines and antiretroviral (ARV) treatment guidelines. More so, accurate subtyping of a partial or full genome would represent the population dynamics of HIV and provide evidence for designing surveillance strategies within a geographic region. Evaluating the performance of rapid subtyping tools with options that incorporate phylogeny could be fast, more user-friendly and of high performance. A total of 570 HIV-1 partial sequences from Cameroon, Angola, Democratic Republic of Congo, Gabon and Senegal were obtained from the Los Alamos National Library (LANL) HIV Sequence Database. Phylogeny was performed using MEGA v6 and the results were used to evaluate the performance of eleven different rapid HIV-1 subtyping tools: REGA v2, REGA v3, NCBI, Stanford HIVDB, SUDI, Geno2Pheno, Euresist, STAR, jpHMM, COMET and SCUEAL. The performance of these subtyping tools differed among HIV-1 clades and across different viral genes. NCBI and SUDI showed the highest performance in subtyping. The discordance observed between the rapid subtyping tools and phylogeny implies that phylogenetic analysis is still the more suitable method for HIV-1 classification. However, the need to update the reference datasets of the subtyping tools, and validate algorithms for rapid subtyping and quality control is imperative as this information is relevant for clinical use and policy-making to the AIDS response. &nbsp; Key words: HIV, phylogeny, performance, subtyping tools, algorithm.

Prevalence of Integrase Strand Transfer Inhibitors (INSTI) Resistance Mutations in Taiwan.

Antiretroviral therapy containing an integrase strand transfer inhibitor (INSTI) plus two NRTIs has become the recommended treatment for antiretroviral-naive HIV-1-infected patients in the updated guidelines. We aimed to determine the prevalence of INSTI-related mutations in Taiwan. Genotypic resistance assays were performed on plasma from ARV-naïve patients (N = 948), ARV-experienced but INSTI-naive patients (N = 359), and raltegravir-experienced patients (N = 63) from 2006 to 2015. Major INSTI mutations were defined according to the IAS-USA list and other substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were defined as minor mutations. Of 1307 HIV-1 samples from patients never exposed to INSTIs, the overall prevalence of major resistance mutations to INSTIs was 0.9% (n = 12), with an increase to 1.2% in 2013. Of these 12 sequences, 11 harboured Q148H/K/R, one Y143R, and none N155H. Of 30 sequences (47.6%) with INSTI-resistant mutations from raltegravir-experienced patients, 17 harboured Q148H/K/R, 8 N155H, and 6 Y143C/R. Other than these major mutations, the prevalence of minor mutations were 5.3% and 38.1%, respectively, in ARV-naive and raltegravir-experienced patients. The overall prevalence of INSTI mutations remains low in Taiwan. Surveillance of INSTI resistance is warranted due to circulation of polymorphisms contributing to INSTI resistance and expected increasing use of INSTIs.