Stanford HIV Drug Resistance Database Research Articles

Spectrum of Non-Nucleoside Reverse Transcriptase Inhibitor-Associated Drug Resistance Mutations in Persons Living with HIV-1 Receiving Rilpivirine

Introduction: Few data are currently available on the nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) resistance mutations selected in persons living with HIV-1 (PLWH) who develop virological failure while receiving rilpivirine (RPV). Methods: We analyzed pooled HIV-1 RT genotypic data from 280 PLWH in the multicenter EuResist database and 115 PLWH in the Stanford HIV Drug Resistance Database (HIVDB) who received RPV as their only NNRTI. Results: Among the 395 PLWH receiving RPV, 180 (45.6%) had one or more NNRTI-associated DRMs. Overall, 44 NNRTI-associated DRMs were identified, including 26 that occurred in two or more PLWHs. Seven mutations had a prevalence ≥10% among the 180 PLWH with one or more NNRTI-associated DRM: E138K (32.2%), V90I (25.0%), K101E (17.8%), Y181C (17.2%), E138A (13.9%), H221Y (12.2%), and K103N (10.6%). Y181C was significantly more likely to co-occur with K101E, V179F, H221Y, and M230L. Ten novel non-polymorphic mutations at known NNRTI-associated mutation positions were also identified, usually in just one PLWH: L100F, V108A, T139I, P225S, M230V, Y232C, and T240A/I/M/S. Conclusions: Our analysis extends the spectrum of mutations emerging in PLWH receiving RPV. Additional phenotypic characterization of RPV-selected mutations is necessary to better understand their biological and possible clinical significance.

Emerging integrase resistance in an international perinatal virtual clinic.

The aim of this study was to identify the prevalence of emergent integrase drug resistance mutations (INSTI-DRMs) in international referrals to a perinatal virtual clinic (PVC). A retrospective cohort study. Monthly multidisciplinary PVC reviewing complex case management for children and adolescents with perinatally acquired HIV (CAWHIV). One hundred fourteen cases referred for virological failure between October 2018 and January 2024. Data collected included age, sex, weight, country of residence, antiretroviral therapy (ART) history, HIV viral load, CD4+ cell count, and comorbidities. Resistance mutations were interpreted using the Stanford HIV Drug Resistance database with emergent major INSTI-DRMs described. Of 114 referrals, 103 (90%) had resistance sequences available. Prior INSTI exposure was documented in 61/103 (59%) with 19/61 (31%) having INSTI-DRMs. For these 19, median (IQR) age was 11 years (6-14), weight 25 kg (17-50), CD4+ cell count 485 cells/μl (153-805), and viral load 84 000 copies/ml (2380-137 000). Twelve of 19 (65%) were from low/middle-income countries (LMIC), 6/19 (32%) had current AIDS diagnoses with 14/19 (74%) referred from 2022 onwards. There were a median three prior regimens with 13/19 (68%) having at least 3 class resistance. Two developed INSTI-DRMs on first-line dolutegravir (DTG)-based ART, 17 on second+ line therapy. PVC recommendations were for tenofovir+ lamivudine/emtricitabine (six split adult tablets) with boosted darunavir [19; six twice daily (b.i.d.)], with b.i.d. DTG (6), plus fostemsavir (1) and ibalizumab (1). Although uncommon, INSTI resistance is emerging, mainly in highly treatment experienced CAWHIV from LMIC, highlighting the global need for access to boosted protease inhibitors and novel classes, including formulations for children less than 35 kg.

Frequency of Major Transmitted Integrase Resistance in Poland Remains Low Despite Change in Subtype Variability.

With the widespread use of integrase inhibitors and the expanding use of long-acting cabotegravir in both pre-exposure prophylaxis and antiretroviral treatment, molecular surveillance on the transmission of integrase resistance has regained clinical significance. This study aimed to determine the frequency of INSTI-transmitted drug resistance mutations (DRMs) among treatment-naïve individuals in Poland from 2016 to 2023. INSTI resistance was analyzed in 882 antiretroviral treatment-naïve individuals using Sanger sequencing. Integrase DRMs were defined based on the Stanford HIV drug resistance database scores. Phylogeny was used to investigate subtyping and clustering. For the analysis of time-trends, logistic regression was used. Major (E138K and R263K) integrase mutations were detected in 0.45% of cases with minor resistance observed in 14.85%, most commonly (13.95%) E157Q. Overall, no major clusters of transmitted drug resistance were identified, and the transmission of E157Q showed a decreasing trend (p < 0.001). While the frequency of sub-subtype A6 increased, it was predominantly found among migrants and associated with L74 mutations. The frequency of major integrase-transmitted DRMs remains low, despite the changes in subtype variability. Surveillance of changing HIV molecular variation patterns is vital from the perspective of the optimal use of integrase inhibitors, especially due to expanding long-acting cabotegravir implementation.

Lack of Resistance Mutations to the Novel HIV-1 Capsid Inhibitor Lenacapavir Among People Living with HIV in Guangdong, China.

The capsid inhibitor (CAI) lenacapavir (LEN) was approved for use in 2022, yet there are few reports about its drug resistance mutations (DRMs) and sensitivity. To delineate the prevalence of CAI DRMs and drug susceptibility among HIV-1 infected individuals living in Guangdong, China. A total of 1035 individuals with HIV-1 infection, including 660highly Active Anti-Retroviral Therapy (HAART) naive individuals and 375hAART experienced individuals whose protease (PR)/ reverse transcriptase (RT) fragments were amplified successfully during drug resistance surveillance between October 2021 and December 2023, were randomly included in this study. The entire HIV-1 gag gene was amplified from plasma in LEN-naive individuals with or without antiretroviral therapy. The epidemiological and demographic information of the enrolled individuals were collected. The Stanford HIV Drug Resistance Database HIVdb program for Capsid was used to interpret the CAI DRMs and the LEN susceptibility. Among 1035 samples, 805 gag sequences were amplified, sequenced and assembled successfully from 518hAART drugs naive individuals and 287hAART drugs experienced individuals. Among them, 0.50% (4/805) carried at least one CAI DRM, of which 0.19% (1/518) from HAART naive individuals and 1.05% (3/287) from HAART experienced individuals. Among the individuals with CAI DRMs, two patients carried CAI major mutations (Q67H) conferring intermediate resistance to LEN and two patients carried CAI accessory mutation (T107A) conferring low level resistance to LEN. Extremely low prevalence of CAI DRMs was detected among people living with HIV (PLWH) in Guangdong, China. Our observations indicate that LEN application may be promising when used in clinical practice in China. Before the administration of LEN, there is no need to consider detecting CAI mutations in PLWH through DRM examination for the time being.

Analysis of HIV-1 Subtypes and Transmitted Drug Resistance in Hospitalized Treatment-Native Patients With AIDS

To investigate the distribution characteristics of HIV-1 subtypes, the status of transmitted drug resistance (TDR), and the influencing factors of TDR in treatment-naive patients with AIDS who are hospitalized. Treatment-naive patients with AIDS who were admitted to the Infectious Disease Department, Public Health Clinical Center of Chengdu between January 2020 and December 2022 were enrolled in the study. The diagnosis and confirmation diagnosis of all the subjects were made at the same hospital. Blood samples were collected from the subjects before antiretroviral therapy (ART). The in-house method was used for HIV gene amplification and sequencing. A phylogenetic tree was constructed to analyze the HIV-1 subtypes. The Stanford HIV Drug Resistance Database was used to conduct an online comparative analysis of the drug resistance mutation sites and to determine the types and levels of drug resistance. The distribution characteristics of HIV-1 subtypes, the occurrence of TDR, and the influencing factors of TDR were analyzed. A total of 213 patients were included in the study and their blood samples were collected. HIV-1 subtypes were successfully amplified in 83.10% (177/213) of the subjects. Ten HIV subtypes were identified, with CRF07_BC being the most common subtypes, accounting for 43.50% (77/177), which was followed by CRF01_AE at 37.85%. Unique recombinant forms (URFs) were relatively uncommon, accounting for 8.47%. The other subtypes accounted for 10.17%. These 4 categories of HIV-1 subtypes were distributed with statistically significant differences in different age groups (P=0.024). Further analysis revealed significant differences in the distribution of the HIV-1 subtypes of CRF01_AE and URFs between the groups of patients aged 30-50 years and those over 50. In addition, URFs accounted for a higher proportion in patients aged 30 to 50 years (P=0.008). The incidences of TDR were 6.49%, 8.96%, 13.33%, and 5.56% for CRF07_BC, CRF01_AE, URFs, and other subtypes, respectively, showing no significant difference (P>0.05). The overall TDR was 6.57%. The TDR for non-nucleoside reverse transcriptase inhibitors (NNRTIs) was 5.16%, and the main mutation sites were V179D/E, E138A/G, V106M/I, and Y181C. The TDR for nucleoside reverse transcriptase inhibitors (NRTIs) was 1.88%, and the main mutation site was M184V. One patient was found to be resistant to both NNRTIs and NRTIs. The highly resistant rate was 4.23%, moderate resistance was 0.47%, and low resistance was 1.88%. No significant effects of the specific years, demographic characteristics, transmission route, baseline condition, and opportunistic infections on TDR were found in this study (P>0.05). The HIV-1 subtypes are diverse and complex in treatment-naive patients with AIDS who were hospitalized. The overall prevalence of TDR is relatively high. It is necessary to strengthen HIV drug resistance testing to optimize ART treatment and reduce the risk of drug resistance transmission.

Enhancing Precision in HIV Treatment: Validation of a Robust Next-Generation Sequencing System for Drug Resistance Mutation Analysis.

Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy. This study aimed to validate the Vela Diagnostics NGS platform for HIV drug resistance mutation analysis, rigorously assessed with clinical samples and CAP proficiency testing controls previously analyzed by Sanger sequencing. The experimental approach involved the following: RNA extraction from clinical specimens, reverse transcription polymerase chain reaction (RT-PCR) utilizing the Sentosa SX 101 platform, library preparation with the Sentosa SQ HIV Genotyping Assay, template preparation, sequencing using the Sentosa SQ301 instrument, and subsequent data analysis employing the Sentosa SQ Suite and SQ Reporter software. Drug resistance profiles were interpreted using the Stanford HIV Drug Resistance Database (HIVdb) with the HXB2 reference sequence. The Vela NGS system successfully identified a comprehensive array of drug resistance mutations across the tested samples: 28 nucleoside reverse transcriptase inhibitors (NRTI), 25 non-nucleoside reverse transcriptase inhibitors (NNRTI), 25 protease inhibitors (PI), and 10 integrase gene-specific variants. Dilution experiments further validated the system's sensitivity, detecting drug resistance mutations even at viral loads lower than the recommended threshold (1000 copies/mL) set by Vela Diagnostics. This study underscores the validation and clinical applicability of the Vela NGS system, and its implementation may offer clinicians enhanced precision in therapeutic decision-making for individuals living with HIV.

Exploring disparities in HIV-1 pretreatment and acquired drug resistance in China from 2003 to 2022.

To investigate the epidemic patterns of pretreatment drug resistance (PDR) and acquired drug resistance (ADR) in HIV-1 sequences from China. HIV-1 pol sequences and associated epidemiological data were collected from the Los Alamos HIV Sequence Database, NCBI, HIV Gene Sequence Database and PubMed. Genotypic resistance and subtypes were identified using the Stanford HIV Drug Resistance Database. A total of 36 263 sequences from ART-naïve individuals and 1548 sequences from ART-experienced individuals with virological failure were evaluated. PDR prevalence was 6.64%, initially decreasing and then increasing to 7.84% (2018-22) due to NNRTI. Pooled ADR prevalence (44.96%) increased, with NNRTI and NRTI aligning with the overall trend. The percentage of multidrug resistance was more than that of single-drug resistance in PDR and especially ADR annually. PDR was most prevalent in Central China followed by Southwest and North. ADR prevalence was highest in North China followed by Northwest and Southwest. In ADR sequences, high-level resistance was more common, especially in NRTI. PDR sequences exhibited low-level or intermediate resistance, especially PI. Drug resistance mutations revealed distinct patterns in PDR and ADR. CRF01_AE, the predominant subtype in China, exhibited the highest proportions among most ART drugs and drug resistance mutations, with a few exceptions where CRF07_BC (prominent in the Northwest), CRF55_01B and CRF08_BC (prominent in the Southwest) showed the highest proportions. HIV-1 PDR and ADR prevalence in China exhibited diverse epidemiological characteristics, underscoring the importance of ongoing national monitoring of PDR, ADR and subtype; patient education on adherence; and personalized regimens.

Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana

BackgroundThe successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. MethodFrom a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. ResultsSequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). ConclusionThe study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

Performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase.

HIV genotyping is used to assess HIV susceptibility to antiretroviral drugs. The Applied Biosystems HIV-1 Genotyping Kit with Integrase (AB kit, Thermo Fisher Scientific) detects resistance-associated mutations (RAMs) in HIV protease (PR), reverse transcriptase (RT), and integrase (IN). We compared results from the AB kit with results obtained previously with the ViroSeq HIV-1 Genotyping System. DNA amplicons from the AB kit were also analyzed using next-generation sequencing (NGS). HIV RNA was extracted using the MagNA Pure 24 instrument (Roche Diagnostics; 96 plasma samples, HIV subtype B, viral load range: 530-737,741 copies/mL). FASTA files were generated from AB kit data using Exatype (Hyrax Biosciences). DNA amplicons from the AB kit were also analyzed by NGS using the Nextera XT kit (Illumina). Drug resistance was predicted using the Stanford HIV Drug Resistance Database. The mean genetic distance for sequences from ViroSeq and the AB kit was 0.02% for PR/RT and 0.04% for IN; 103 major RAMs were detected by both methods. Four additional major RAMs were detected by the AB kit only. These four major RAMs were also detected by NGS (detected in 18.1%-38.2% of NGS reads). NGS detected 27 major RAMs that were not detected with either of the Sanger sequencing-based kits. All major RAMs detected with ViroSeq were detected with the AB kit; additional RAMs were detected with the AB kit only. DNA amplicons from the AB kit can be used for NGS for more sensitive detection of RAMs.

Molecular genetic characterization analysis of a novel HIV-1 circulating recombinant form (CRF156_0755) in Guangdong, China.

The characteristic of human immunodeficiency virus type 1 (HIV-1) is its susceptibility to erroneous replication and recombination, which plays a crucial role in the diverse and dynamic variation of HIV-1. The spread of different subtypes in the same population often leads to the emergence of circulating recombination forms (CRFs). At present, the main recombinant subtypes of HIV-1 in China are CRF07_BC, CRF01_AE, CRF08_BC and B' subtypes, while CRF55_01B has become the fifth major epidemic strain in China after rapid growth in recent years since it was first reported in 2013. In this study, we obtained five nearly full-length genomes (NFLGs) and one half-length genome from five different cities in Guangdong. Here, we focused on analyzing their characteristics, parental origin and drug resistance. Plasma samples were collected from six HIV-1 infected patients in Guangdong Province who had no epidemiological association with each other. The NFLGs of HIV-1 were amplified in two overlapping segments by the near-terminal dilution method. The positive products were sequenced directly to obtain genomic sequences. The recombinant patterns and breakpoints of the NFLGs were determined using the Simplot software and confirmed by the maximum likelihood trees for segments using the IQ-TREE and BEAST software. The genotypic resistance profiles of the protease reverse transcriptase and integrase were resolved by the Stanford HIV drug resistance database. The six genomes shared highly similar recombinant pattern, with the CRF55_01B backbone substituted by CRF07_BC segments, therefore assigned as CRF156_0755. The evolutionary analysis of the segments showed that CRF07_BC segments were not clustered with the Chinese MSM variants in the CRF07_BC lineage. All the five NFLGs were identified with the non-nucleoside reverse-transcription inhibitors (NNRTIs) resistance mutation V179E. With the accumulation and evolution of recombination between CRF55_01B and CRF 07_BC, the prevalence of more recombinant strains of CRF55_01B and CRF 07_BC may occur. Therefore, it is necessary to strengthen the identification and monitoring of the recombination of CRF55_01B and CRF 07_BC.

High prevalence of pre-treatment and acquired HIV-1 drug resistance mutations among non-citizens living with HIV in Botswana.

Approximately 30,000 non-citizens are living with HIV in Botswana, all of whom as of 2020 are eligible to receive free antiretroviral treatment (ART) within the country. We assessed the prevalence of HIV-1 mutational profiles [pre-treatment drug resistance (PDR) and acquired drug resistance (ADR)] among treatment-experienced (TE) and treatment-naïve (TN) non-citizens living with HIV in Botswana. A total of 152 non-citizens living with HIV were enrolled from a migrant HIV clinic at Independence Surgery, a private practice in Botswana from 2019-2021. Viral RNA isolated from plasma samples were genotyped for HIV drug resistance (HIVDR) using Sanger sequencing. Major known HIV drug resistance mutations (DRMs) in the pol region were determined using the Stanford HIV Drug Resistance Database. The proportions of HIV DRMs amongst TE and TN non-citizens were estimated with 95% confidence intervals (95% CI) and compared between the two groups. A total of 60/152 (39.5%) participants had a detectable viral load (VL) >40 copies/mL and these were included in the subsequent analyses. The median age at enrollment was 43 years (Q1, Q3: 38-48). Among individuals with VL > 40 copies/mL, 60% (36/60) were treatment-experienced with 53% (19/36) of them on Atripla. Genotyping had a 62% (37/60) success rate - 24 were TE, and 13 were TN. A total of 29 participants (78.4, 95% CI: 0.12-0.35) had major HIV DRMs, including at least one non-nucleoside reverse transcriptase inhibitor (NNRTI) associated DRM. In TE individuals, ADR to any antiretroviral drug was 83.3% (20/24), while for PDR was 69.2% (9/13). The most frequent DRMs were nucleoside reverse transcriptase inhibitors (NRTIs) M184V (62.1%, 18/29), NNRTIs V106M (41.4%, 12/29), and K103N (34.4%, 10/29). No integrase strand transfer inhibitor-associated DRMs were reported. We report high rates of PDR and ADR in ART-experienced and ART-naïve non-citizens, respectively, in Botswana. Given the uncertainty of time of HIV acquisition and treatment adherence levels in this population, routine HIV-1C VL monitoring coupled with HIVDR genotyping is crucial for long-term ART success.

Delayed identification of treatment failure causes high levels of acquired drug resistance and less future drug options among HIV-1-infected South Indians

PurposeHIV-1 Drug Resistance Mutations (DRMs) among Immunological failure (IF) on NRTI based first-line regimens, Thymidine analogue (TA) - AZT & D4T and Non-Thymidine Analogue (NTA) -TDF; and predict viral drug susceptibility to gain vision about optimal treatment strategies for second-line. MethodsCross-sectionally, 300 HIV-1 infected patients, failing first-line HAART were included. HIV-1 pol gene spanning 20–240 codons of RT was genotyped and mutation pattern was examined, (IAS-USA 2014 and Stanford HIV drug resistance database v7.0). ResultsThe median age of the participants was 35 years (IQR 29–40), CD4 T cell count of TDF failures was low at 172 cells/μL (IQR 80–252), and treatment duration was low among TDF failures (24 months vs. 61 months) (p < 0.0001). Majority of the TDF failures were on EFV based first-line (89 % vs 45 %) (p < 0.0001). Level of resistance for TDF and AZT shows, that resistance to TDF was about one-third (37 %) of TDF participants and onefourth (23 %) of AZT participants; resistance to AZT was 17 % among TDF participants and 47 % among AZT participants; resistance to both AZT and TDF was significantly high among AZT participants [21 % vs. 8 %, OR 3.057 (95 % CI 1.4–6.8), p < 0.0001]. ConclusionAlthough delayed identification of treatment failure caused high levels of acquired drug resistance in our study. Thus, we must include measures to regularize virological monitoring with integrated resistance testing in LMIC (Low and Middle Income Countries) like in India; this will help to preserve the effectiveness of ARV and ensure the success of ending AIDS as public health by 2030.

High viral suppression and detection of dolutegravir-resistance associated mutations in treatment-experienced Tanzanian adults living with HIV-1 in Dar es Salaam

To curb HIV infection rate in Tanzania, antiretroviral therapy (ART) has been scaled up since 2006, and in 2019, the country shifted to regimen including dolutegravir as a default first line. We assessed the success of ART and the contribution of HIV drug resistance (HIVDR) to unsuppressed viral loads. Between February and May 2023 a cross-sectional survey with random sampling was conducted in the six clinics in an urban cohort in Dar es Salaam. Patients with unsuppresed viral loads (local criteria viral load (VL) ≥ 1000 copies/mL) were tested for HIVDR mutations using the WHO adapted protocol for plasma samples. Mutations were interpreted using the Stanford HIVDR database. In total 600 individuals participated in this survey, the majority were female (76.83%), mean age (pm standard deviation) was 44.0 (pm 11.6) years. The median duration on ART (interquartile range) was 6.5 (3.9–10.2) years. Approximately 99% were receiving tenofovir + lamivudine + dolutegravir as a fixed dose combination. VL testing was successful in 99.67% (598/600) of survey patients and only 33 had VL ≥ 1000 copies/mL, resulting in a viral suppression level of 94.48% (565/598, 95% CI 92.34–96.17%). For 23 samples, protease and reverse transcriptase (RT) genotyping were successful, with 13 sequences containing RT inhibitor surveillance drug resistance mutations (SDRMs) (56.5%). No SDRM against protease inhibitors were detected. Thirty samples were successfully genotyped for integrase with 3 sequences (10.08%) containing integrase strand transfer inhibitor (INSTI) SDRMs. In samples successfully genotyped in the three genetic regions, 68.18% (16/22) had a genotypic susceptibility score (GSS) ≥ 2.5 for the concurrent regimen, implying factors beyond drug resistance caused the unsuppressed viral load. For five patients, GSS indicated that HIVDR may have caused the unsuppressed viral load. All three patients with INSTI resistance mutations were highly resistant to dolutegravir and accumulated nucleoside and non-nucleoside RT inhibitor HIVDR mutations. Although in this cohort the last 95 UNAIDS target was almost achieved, HIVDR mutations, including INSTIs resistance mutations were detected in HIV-positive individuals taking ART for at least one year. We recommend the design and implementation of high-impact interventions to prevent the increase of HIVDR, failure of dolutegravir and address the non-resistance factors in the study area.

Rate of response to initial antiretroviral therapy according to level of pre-existing HIV-1 drug resistance detected by next-generation sequencing in the strategic timing of antiretroviral treatment (START) study.

The main objective of this analysis was to evaluate the impact of pre-existing drug resistance by next-generation sequencing (NGS) on the risk of treatment failure (TF) of first-line regimens in participants enrolled in the START study. Stored plasma from participants with entry HIV RNA >1000 copies/mL were analysed using NGS (llumina MiSeq). Pre-existing drug resistance was defined using the mutations considered by the Stanford HIV Drug Resistance Database (HIVDB v8.6) to calculate the genotypic susceptibility score (GSS, estimating the number of active drugs) for the first-line regimen at the detection threshold windows of >20%, >5%, and >2% of the viral population. Survival analysis was conducted to evaluate the association between the GSS and risk of TF (viral load >200 copies/mL plus treatment change). Baseline NGS data were available for 1380 antiretroviral therapy (ART)-naïve participants enrolled over 2009-2013. First-line ART included a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 976 (71%), a boosted protease inhibitor in 297 (22%), or an integrase strand transfer inhibitor in 107 (8%). The proportions of participants with GSS <3 were 7% for >20%, 10% for >5%, and 17% for the >2% thresholds, respectively. The adjusted hazard ratio of TF associated with a GSS of 0-2.75 versus 3 in the subset of participants with mutations detected at the >2% threshold was 1.66 (95% confidence interval 1.01-2.74; p = 0.05) and 2.32 (95% confidence interval 1.32-4.09; p = 0.003) after restricting the analysis to participants who started an NNRTI-based regimen. Up to 17% of participants initiated ART with a GSS <3 on the basis of NGS data. Minority variants were predictive of TF, especially for participants starting NNRTI-based regimens.

Archived rilpivirine-associated resistance mutations among ART-naive and virologically suppressed people living with HIV-1 subtype C in Botswana: implications for cabotegravir/rilpivirine use.

Pre-existing rilpivirine resistance-associated mutations (RVP-RAMs) have been found to predict HIV-1 virological failure in those switching to long-acting injectable cabotegravir/rilpivirine. We here evaluated the prevalence of archived RPV-RAMs in a cohort of people living with HIV (PWH). We analysed near full-length HIV-1 pol sequences from proviral DNA for the presence of RPV-RAMs, which were defined according to the 2022 IAS-USA drug resistance mutation list and Stanford HIV drug resistance database. RPV-RAMs were identified in 757/5805 sequences, giving a prevalence of 13.0% (95% CI 12%-13.9%). Amongst the ART-naive group, 137/1281 (10.7%, 95% CI 9.1%-12.5%) had at least one RPV-RAM. Of the 4524 PWH with viral suppression on ART (VL <400 copies/mL), 620 (13.7%, 95% CI 12.7%-14.7%) had at least one RPV-RAM. E138A was the most prevalent RPV-RAM in the ART-naive group (7.9%) and the ART-suppressed group (9.3%). The rest of the mutations observed (L100I, K101E, E138G, E138K, E138Q, Y181C, H221Y, M230L, A98G, V179D, G190A, G190E and M230I) were below a prevalence of 1%. RPV-RAMs were present in 10.7% of ART-naive and 13.7% of ART-suppressed PWH in Botswana. The most common RPV-RAM in both groups was E138A. Since individuals with the E138A mutation may be more likely to fail cabotegravir/rilpivirine, monitoring RPV-RAMs will be crucial for effective cabotegravir/rilpivirine implementation in this setting.

Genetic characterization of HIV-1 viruses among cases with antiretroviral therapy failure in Suzhou City, China

BackgroundThis retrospective study aimed to characterize the distribution of HIV-1 genotypes and the prevalence of drug resistance mutations in people with antiretroviral treatment (ART) failure in Suzhou City, China.MethodsPol gene of HIV-1 viruses in blood samples of EDTA anticoagulants from 398 patients with failed antiviral treatment was successfully amplified by using an in-house assay. Drug resistance mutations were analyzed by using the Stanford HIV Drug Resistance Database system (https://hivdb.stanford.edu/hivdb/by-mutations/). HIV-1 genotypes were determined by the REGA HIV subtyping tool (version 3.46, https://www.genomedetective.com/app/typingtool/hiv). Near full-length genomes (NFLG) of HIV-1 viruses were obtained by next generation sequencing method.ResultsSequences analysis of the pol gene revealed that CRF 01_AE (57.29%, 228/398) was the dominant subtype circulating in Suzhou City, followed by CRF 07_BC (17.34%, 69/398), subtype B (7.54%, 30/398), CRF 08_BC (6.53%, 26/398), CRF 67_01B (3.02%, 12/398) and CRF55_01B (2.51%, 10/398). The overall prevalence of drug-resistant mutations in cases with ART failure was 64.57% (257/398), including 45.48% (181/398) for nucleotide reverse transcriptase inhibitors (NRTIs) mutations, 63.32% (252/398) for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations, and 3.02% (12/398) for protease inhibitors (PIs) mutations. Ten near full-length genomes (NFLG) of HIV-1 viruses were identified, including six recombinants of CRF 01_AE and subtype B, two recombinants of CRF 01_AE, subtype B and subtype C sequences, one recombinant of CRF 01_AE and subtype C and one recombinant of CRF 01_AE, subtype A1 and subtype C.ConclusionsThe high prevalence of drug-resistant HIV-1 viruses was a serious challenge for HIV prevention and treatment of people with HIV infection. Treatment regimens for ART failure patients should be adjusted over time based on the outcome of drug resistance tests. NFLG sequencing facilitates the identification of new recombinants of HIV-1.

Low level of HIV-1C integrase strand transfer inhibitor resistance mutations among recently diagnosed ART-naive Ethiopians

With the widespread use of Integrase strand transfer inhibitors (INSTIs), surveillance of HIV-1 pretreatment drug resistance is critical in optimizing antiretroviral treatment efficacy. However, despite the introduction of these drugs, data concerning their resistance mutations (RMs) is still limited in Ethiopia. Thus, this study aimed to assess INSTI RMs and polymorphisms at the gene locus coding for Integrase (IN) among viral isolates from ART-naive HIV-1 infected Ethiopian population. This was a cross-sectional study involving isolation of HIV-1 from plasma of 49 newly diagnosed drug-naive HIV-1 infected individuals in Addis-Ababa during the period between June to December 2018. The IN region covering the first 263 codons of blood samples was amplified and sequenced using an in-house assay. INSTIs RMs were examined using calibrated population resistance tool version 8.0 from Stanford HIV drug resistance database while both REGA version 3 online HIV-1 subtyping tool and the jumping profile Hidden Markov Model from GOBICS were used to examine HIV-1 genetic diversity. Among the 49 study participants, 1 (1/49; 2%) harbored a major INSTIs RM (R263K). In addition, blood specimens from 14 (14/49; 28.5%) patients had accessory mutations. Among these, the M50I accessory mutation was observed in a highest frequency (13/49; 28.3%) followed by L74I (1/49; 2%), S119R (1/49; 2%), and S230N (1/49; 2%). Concerning HIV-1 subtype distribution, all the entire study subjects were detected to harbor HIV-1C strain as per the IN gene analysis. This study showed that the level of primary HIV-1 drug resistance to INSTIs is still low in Ethiopia reflecting the cumulative natural occurrence of these mutations in the absence of selective drug pressure and supports the use of INSTIs in the country. However, continues monitoring of drug resistance should be enhanced since the virus potentially develop resistance to this drug classes as time goes by.

Increasing trend of transmitted integrase inhibitor resistance in a cohort of antiretroviral therapy-naive people living with HIV.

Drug regimens containing integrase strand transfer inhibitors (INSTIs) are widely recommended by international guidelines for ART-naive people living with HIV (PLWH).1,2 However, INSTI resistance mutations are uncommon and therefore their surveillance is not suggested before starting ART. The aim of the study was to evaluate the prevalence trend of transmitted INSTI drug resistance (DR) and to assess factors associated with transmitted INSTI resistance among ART-naive PLWH over the past decade. We conducted a time-trend, single-centre study over the period 2009–19 on adult naive PLWH with a genotypic resistance test (GRT) performed before the ART initiation. GRT was determined by Sanger sequencing using an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA); we included in the analysis the immediate pre-ART GRT available in people newly diagnosed with HIV infection. The degree of resistance to each INSTI drug was calculated using the Genotypic Resistance Interpretation Algorithm of the Stanford HIV Drug Resistance Database Program (version 9.1.1) (https://hivdb.stanford.edu/hivdb/by-patterns/). The obtained scores were used to classify the presence of low-level, intermediate or high-level resistance to each INSTI drug and to the INSTI drug class. DR within the INSTI class was defined by the presence of at least low-level resistance to ≥1 drug of the INSTI class.

Prediction of HIV-1 protease resistance using genotypic, phenotypic, and molecular information with artificial neural networks.

Drug resistance is a primary barrier to effective treatments of HIV/AIDS. Calculating quantitative relations between genotype and phenotype observations for each inhibitor with cell-based assays requires time and money-consuming experiments. Machine learning models are good options for tackling these problems by generalizing the available data with suitable linear or nonlinear mappings. The main aim of this study is to construct drug isolate fold (DIF) change-based artificial neural network (ANN) models for estimating the resistance potential of molecules inhibiting the HIV-1 protease (PR) enzyme. Throughout the study, seven of eight protease inhibitors (PIs) have been included in the training set and the remaining ones in the test set. We have obtained 11,803 genotype-phenotype data points for eight PIs from Stanford HIV drug resistance database. Using the leave-one-out (LVO) procedure, eight ANN models have been produced to measure the learning capacity of models from the descriptors of the inhibitors. Mean R2 value of eight ANN models for unseen inhibitors is 0.716, and the 95% confidence interval (CI) is [0.592-0.840]. Predicting the fold change resistance for hundreds of isolates allowed a robust comparison of drug pairs. These eight models have predicted the drug resistance tendencies of each inhibitor pair with the mean 2D correlation coefficient of 0.933 and 95% CI [0.930-0.938]. A classification problem has been created to predict the ordered relationship of the PIs, and the mean accuracy, sensitivity, specificity, and Matthews correlation coefficient (MCC) values are calculated as 0.954, 0.791, 0.791, and 0.688, respectively. Furthermore, we have created an external test dataset consisting of 51 unique known HIV-1 PR inhibitors and 87 genotype-phenotype relations. Our developed ANN model has accuracy and area under the curve (AUC) values of 0.749 and 0.818 to predict the ordered relationships of molecules on the same strain for the external dataset. The currently derived ANN models can accurately predict the drug resistance tendencies of PI pairs. This observation could help test new inhibitors with various isolates.

Affordable drug resistance genotyping of HIV-1 reverse transcriptase, protease and integrase genes, for resource limited settings

BackgroundAs use of dolutegravir (DTG) becomes more common in resource limited settings (RLS), the demand for integrase resistance testing is increasing. Affordable methods for genotyping all relevant HIV-1 pol genes (i.e., protease (PR), reverse transcriptase (RT) and integrase (IN)) are required to guide choice of future antiretroviral therapy (ART). We designed an in-house HIV-1 drug resistance (HIVDR) genotyping method that is affordable and suitable for use in RLS.MethodsWe obtained remnant plasma samples from CAPRISA 103 study and amplified HIV-1 PR, RT and IN genes, using an innovative PCR assay. We validated the assay using remnant plasma samples from an external quality assessment (EQA) programme. We genotyped samples by Sanger sequencing and assessed HIVDR mutations using the Stanford HIV drug resistance database. We compared drug resistance mutations with previous genotypes and calculated method cost-estimates.ResultsFrom 96 samples processed, we obtained sequence data for 78 (81%), of which 75 (96%) had a least one HIVDR mutation, with no major-IN mutations observed. Only one sample had an E157Q INSTI-accessory mutation. When compared to previous genotypes, 18/78 (23%) had at least one discordant mutation, but only 2/78 (3%) resulted in different phenotypic predictions that could affect choice of subsequent regimen. All CAPRISA 103 study sequences were HIV-1C as confirmed by phylogenetic analysis. Of the 7 EQA samples, 4 were HIV-1C, 2 were HIV-1D, and 1 was HIV-1A. Genotypic resistance data generated using the IDR method were 100% concordant with EQA panel results. Overall genotyping cost per sample was estimated at ~ US$43–$US49, with a processing time of ~ 2 working days.ConclusionsWe successfully designed an in-house HIVDR method that is suitable for genotyping HIV-1 PR, RT and IN genes, at an affordable cost and shorter turnaround time. This HIVDR genotyping method accommodates changes in ART regimens and will help to guide HIV-1 treatment decisions in RLS.