An NGS amplicon tiling protocol for HIV-1 drug resistance detection using Illumina® COVIDSeq™ Assay Kit. v1

Introduction. Over the past 10 years, there has been an increase in HIV-1 resistance to antiretroviral drugs worldwide. HIV infection patients with low adherence to antiretroviral therapy (ART) are most often at risk of developing acquired HIV-1 resistance. The identification of key factors affecting the degree of adherence will allow timely detection of patients with a high risk of poor adherence to ART and treatment defaulting, and develop individual management tactics for such patients to reduce the risk of resistance development. Aim. To study the factors influencing adherence to ART in patients with virological failure and the effect of adherence on the risk of developing acquired HIV-1 resistance to first-line antiretroviral drugs. Materials and methods. Two hundred and seventy one patients with an confirmed diagnosis of HIV infection, with different stages of the disease, aged 21 to 59 years, who were followed-up from March 2018 to August 2022 at the Center for the Prevention and Control of AIDS (City Infectious Clinical Hospital No. 1, Novosibirsk) and received ART, were enrolled in a study. The criterion to include patients in the study was a virological breakthrough during previously effective ART with first-line drugs (when an undetectable level of HIV RNA load was recorded for 3–6 months). A retrospective analysis of the degree of adherence to ART of the studied patients with HIV infection and virological breakthrough was performed, considering various factors. The HIV-1 drug resistance testing was carried out based on the State Research Center of Virology and Biotechnology VECTOR. Results. Men predominated among the patients – 56.4% (n = 153). The average age was 35.4 ± 2.85 years. Heterosexual transmission of HIV was dominant – 64.8%. Parenteral transmission of HIV (intravenous drug abuse) accounted for 31.5% of cases. According to the results of the retrospective analysis of the degree of adherence to ART, it was found that 48.3% (131 out of 271) of patients had missed medications, including low adherence in 31.7% (86 out of 271) patients; average – in 16.6% of patients (45 out of 271). Most often, poor adherence was reported in people with parenteral HIV transmission– 75.3% compared with sexual transmission – 36% (p < 0.001), and in rural residents – 62.5% compared with urban residents – 34.1% (p < 0.001). Among men, missed medication was detected more often than in women – 57.5 and 36.4%, respectively (p < 0.001). Also, a significant factor was the lack of permanent employment – poor adherence was in 69.5% of patients in this group compared with patients having permanent employment – 26.3% (p < 0.05). On the contrary, high adherence was more often observed in female patients – 63.6% compared with male ones – 42.5%; in the presence of permanent employment – 73.7% compared with 30.5% in it’s absence; in the case of sexual transmission – 64.0% compared with parenteral transmission – 24.7%; in urban residents – 65.9% versus 37.5% in rural residents (p < 0.05). According to the totality of all mutations revealed, HIV drug-resistant variants were found in 54.6% of the patients with virological ART failure (148 out of 271). Of the 148 HIV-1 drug-resistant variants, a combination of drug resistance mutations from the group of nucleoside reverse transcriptase inhibitors (NRTIs) was most often detected and non-nucleoside reverse transcriptase inhibitors (NNRTIs) – in 60.1% (89 cases). Isolated mutations to NRTIs were found in 15.5% (23) cases, for NNRTIs – in 12.2% (18). A combination of mutations of resistance to NRTIs + integrase inhibitors (IIs) was found in 4.1% (6) patients. The proportion of patients with resistance to NRTIs and PIs (protease inhibitors) was 2% (3); to NRTIs + IIs – 1.4% (2). A combination of mutations to three classes of drugs at once was detected in 6 patients: NRTIs + NNRTIs + IIs – 2.7% (4) and NRTIs + NNRTIs + PIs – 1.4% (2). An isolated mutation to IIs was in one patient (0.6%). Conclusion. The analysis showed that non-adherence was reported in a significant part of patients – 48.3% (low – 31.7%, moderate – 16.6%). Factors contributing to poor adherence to ART were: parenteral transmission of HIV (in 75.3% of people with poor adherence); living in rural areas (in 62.5%); lack of permanent employment (in 69.5%); male sex (in 57.5%). Among patients with detected HIV drug resistance (n = 148), individuals with poor adherence prevailed (70.9%) compared with patients who did not have HIV drug resistance (23.6%) (p < 0.001). Taking into account the data obtained, when choosing the initial ART regimen, it is necessary to analyze the degree of risk of non-adherence depending on social and other factors. The high frequency of registration of HIV drug-resistant mutations found in the Novosibirsk Region necessitates timely testing for drug-resistant HIV patients with virological ART failure and the introduction of screening for primary drug resistance in patients at risk of poor adherence.

Programs for the prevention of mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV) have been scaled up in many low- and middle-income countries. However, HIV drug resistance (HIVDR) data among HIV-1-infected young children remain limited. Surveys of pretreatment HIVDR among children aged <18 months who were diagnosed with HIV through early infant diagnosis were conducted in 5 sub-Saharan African countries (Mozambique, Swaziland, South Africa, Uganda, and Zimbabwe) between 2011 and 2014 following World Health Organization (WHO) guidance. Deidentified demographic and clinical data were used to explore risk factors associated with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance. Among the 1450 genotypes analyzed, 1048 had accompanying demographic and clinical data. The median age of children was 4 months; 50.4% were female. HIV from 54.1% showed resistance to 1 or more antiretroviral (ARV) drugs, with 53.0% and 8.8% having resistance to 1 or more NNRTI or nucleoside reverse transcriptase inhibitors, respectively. NNRTI resistance was particularly high in children exposed to ARV drugs through PMTCT; adjusted odds ratios were 1.8 (95% confidence interval [CI], 1.3-2.6) for maternal exposure only and 2.4 (CI, 1.6-3.6) for neonatal exposure only. Protease inhibitor-based regimens in children aged <3 years are currently recommended by WHO, but the implementation of this recommendation is suboptimal. These results reinforce the urgent need to overcome barriers to scaling up pediatric protease inhibitor-based regimens in sub-Saharan Africa and underscore the need to accelerate the study and approval of integrase inhibitors for use in young children.

Children living with human immunodeficiency virus (HIV) (CLHIV) receiving antiretroviral therapy (ART) in resource-limited settings are susceptible to high rates of acquired HIV drug resistance (HIVDR), but few studies include children initiating age-appropriate World Health Organization (WHO)-recommended first-line regimens. We report data from a cohort of ART-naive South African children who initiated first-line ART. ART-eligible CLHIV aged 0-12 years were enrolled from 2012 to 2014 at 5 public South African facilities and were followed for up to 24 months. Enrolled CLHIV received standard-of-care WHO-recommended first-line ART. At the final study visit, a dried blood spot sample was obtained for viral load and genotypic resistance testing. Among 72 successfully genotyped CLHIV, 49 (68.1%) received ABC/3TC/LPV/r, and 23 (31.9%) received ABC/3TC/EFV. All but 2 children on ABC/3TC/LPV/r were <3 years, and all CLHIV on ABC/3TC/EFV were ≥3 years. Overall, 80.6% (58/72) had at least one drug resistance mutation (DRM). DRMs to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) were found among 65% and 51% of all CLHIV, respectively, with no statistical difference by ART regimen. More CLHIV on ABC/3TC/EFV, 47.8% (11/23), were found to have 0 or only 1 effective antiretroviral drug remaining in their current regimen compared to 8.2% (4/49) on ABC/3TC/LPV/r. High levels of NNRTI and NRTI DRMs among CLHIV receiving ABC/3TC/LPV/r suggests a lasting impact of failed mother-to-child transmission interventions on DRMs. However, drug susceptibility analysis reveals that CLHIV with detectable viremia on ABC/3TC/LPV/r are more likely to have maintained at least 2 effective agents on their current HIV regimen than those on ABC/3TC/EFV.

Antiretroviral drug resistance limits treatment options for people with human immunodeficiency virus (HIV) 1 and may reduce the effectiveness of preexposure prophylaxis. Novel treatment options with enhanced efficacy and more convenient formulations have become available from 2016 to 2021. Large-scale studies of trends in the prevalences of plasma RNA drug resistance mutations (DRMs) since 2018 are lacking, and there have been no systematic studies of trends in proviral DNA DRMs. We retrospectively analyzed deidentified HIV-1 plasma RNA and proviral DNA sequences from specimens submitted to a reference laboratory between January 2018 and May 2024. We analyzed the annual prevalence of DRMs with a Stanford HIV Drug Resistance Database score of ≥30 for nucleoside and nonnucleoside reverse-transcriptase inhibitors (NRTIs and NNRTIs), protease inhibitors, and integrase strand transfer inhibitors (INSTIs). The prevalence of resistance declined for both RNA and DNA sequences. Single-class and dual-class NRTI + NNRTI resistance declined but was higher for DNA (NRTI + NNRTI, declined from 6.1% to 3.5% for RNA and from 12.1% to 7.8% for DNA). Rilpivirine DRMs remained low, with prevalences of 6.3% (RNA) and 10.2% (DNA) in 2024. The doravirine DRM prevalences in 2024 were 2% (RNA) and 2.9% (DNA). INSTI and dual-class NRTI + INSTI resistance also declined, but the prevalence of integrase DRM R263K increased. Prevalence of NRTI and NNRTI resistance has declined, consistent with increased use of regimens with higher resistance barriers, improved tolerability, and more convenient dosing. Proviral DNA resistance trends were correlated with those for RNA. Continued advances in antiretroviral therapy efficacy, durability, and tolerability may lead to increased rates of virologic suppression and further reduce the incidence of archived resistance mutations in proviral DNA.

This study sought to examine the human immunodeficiency virus type 1 (HIV-1) genetic diversity on drug resistance among men who have sex with men (MSM) with virologic failure in antiretroviral therapy (ART), and investigate linking-associated factors for genetic transmission networks.Seven hundred and thirty-four HIV-positive MSM with virologic failure in ART were recruited into our study from 2011 to 2017. HIV-1 pol gene sequences were used for phylogenetic and genotypic drug resistance analyses. The drug resistance mutations were determined using the Stanford University HIV Drug Resistance Database. The genetic transmission networks were analyzed for CRF01_AE and CRF07_BC sequences by the genetic distance-based method.Of 734 subjects, 372 (50.68%) showed drug resistance, in which CRF01_AE and CRF07_BC were the predominating subtypes. Drug resistance more frequently occurred in non-nucleoside reverse transcriptase inhibitors (NNRTIs) treatment (48.64%), and followed by nucleoside reverse transcriptase inhibitors (NRTIs) (36.51%) and PIs (4.03%). The most common drug resistance-associated mutations in protease inhibitors (PIs), NRTIs and NNRTIs were K20I/R, M184V/I and K103N/KN, respectively. For 283CRF01_AE sequences, 64 (22.61%) fell into clusters at a genetic distance of 0.011, resulting in 17 clusters ranging in size from 2 to 16 individuals. For 230 CRF07_BC sequences, 66 (28.69%) were connected to at least one other sequence with 0.005 genetic distances, resulting in 8 clusters ranging in size from 2 to 52 individuals. Individuals who showed drug resistance to ART were less likely to fall into clusters than those who did not. The genetic linkage was robust by the exclusion of sites associated with drug resistance.CRF01_AE and CRF07_BC were the main strains among MSM with virologic failure in ART, and the drug resistance more frequently occurred in NNRTIs, followed by NRTIs and PIs. Genetic transmission networks revealed a complexity of transmission pattern, suggesting early-diagnosis and in-time intervention among MSM.

Introduction of HIV drug-resistance testing in clinical practice.

HIV drug resistance (HIVDR) presents a significant challenge to antiretroviral therapy (ART) success, particularly in resource-limited settings like Ethiopia. This cross-sectional study investigated viral suppression rates and resistance patterns among patients on second-line ART across 28 Ethiopian health facilities. Blood samples collected from 586 participants were analyzed to measure CD4 count and viral load and assess HIVDR in patients experiencing virological failure (VF) (viral load ≥ 1000 copies/mL). Demographic and clinical data were analyzed using logistic regression to identify factors associated with VF. Results showed that 13.82% of participants experienced VF, with 67.57% of genotyped samples exhibiting at least one drug resistance mutation. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) was detected in 48.64%, 64.86%, and 18.92% of cases, respectively. Dual-class resistance was identified in 48.64% of patients, while triple-class resistance was detected in 18.92%. VF was more likely among students and those with CD4 counts below 200 cells/mm³, but less likely in patients on second-line treatment for 12 months or more. Our findings highlight a substantial HIVDR burden among patients on second-line ART with VF, emphasizing the need for comprehensive HIV care, including adherence support, regular viral load monitoring, and HIVDR testing.

In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007. HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences. Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF. Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

BackgroundThe World Health Organization (WHO) recommends that HIV treatment scale-up is accompanied by a robust assessment of drug resistance emergence and transmission. The WHO HIV Drug Resistance (HIVDR) monitoring and surveillance strategy includes HIVDR testing in adults both initiating and receiving antiretroviral therapy (ART). Due to limited information about HIVDR in Mozambique, we conducted two nationally representative surveys of adults initiating and receiving first-line ART regimes to better inform the HIV program.MethodsWe carried out a cross-sectional study between March 2017 and December 2019. Adults (older than 15 years) living with HIV (PLHIV) initiating ART or receiving first-line ART for between 9-15 months at 25 health facilities across all eleven provinces in Mozambique were included. Genotypic HIVDR was assessed on dried blood spots (DBS) when viral loads were ≥ 1000 copies/ml. Genotypic resistance for non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), and protease inhibitors (PIs) was determined using the Stanford HIV database algorithm 9.5 and calibrated population resistance tool 8.1.ResultsOf 828 participants -enrolled, viral load (VL) testing was performed on 408 initiators and 409 ART experienced. Unsuppressed VL was found in 68.1% 419 initiators and 18.8% (77/409) of the ART experienced. Of the 278 initiators and 70 ART experienced who underwent sequencing, 51.7% (144/278) and 75.7% (53/70) were sequenced successfully. Among the new initiators, pretreatment drug resistance (PDR) for NNRTI and PI was found in 16.0% (23/144) and 1.4% (2/144) of the participants, respectively. Acquired drug resistance (ADR) was found in 56.5% (30/53) of the ART-experienced participants of whom 24.5% (13/53) were resistant to both NRTI and NNRTI.ConclusionHigh rates of PDR and ADR for NNRTI and ADR for NRTI were observed in our study. These findings support the replacement of NNRTIs with dolutegravir (DTG) but high levels of NRTI resistance in highly treatment-experienced individuals still require attention when transitioning to new regimens. Moreover, the study underlines the need for routine VL testing and HIVDR surveillance to improve treatment management strategies.

The use of highly active antiretroviral therapy against human immunodeficiency virus (HIV) can lead to rare instances of treatment failure and the emergence of drug resistance. HIV drug-resistant strains are archived in cellular reservoirs, and this can exclude the future efficacy of drugs or drug classes against which resistance has emerged. In addition, drug-resistant viruses can be transmitted between individuals. HIV drug resistance has been countered through the constant development of new antiretroviral drugs. Integrase strand transfer inhibitors, that actively block the integration of the HIV genome into the host DNA, represent the most recent antiretroviral drugs. Of these, raltegravir, elvitegravir, and dolutegravir are the only integrase strand transfer inhibitors that have been approved for human therapy by the US Food and Drug Administration. Dolutegravir is unique in its ability to seemingly evade HIV drug resistance in treatment-naïve individuals. Here, we review the use of integrase strand transfer inhibitors in the management of HIV, focusing on HIV resistance.

Molecular basis of human immunodeficiency virus drug resistance: An update

BackgroundAlthough the introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in considerable decrease in HIV related mortality; it has also induced the development of multiple drug-resistant HIV-1 variants.The few studies on HIV-1 mutagenesis in HIV infected children have not evaluated the impact of HIV-1 mutations on the clinical, virological and immunological presentation of HIV disease that is fundamental to optimizing the treatment regimens for these patients.ResultsA cross sectional study was conducted to evaluate the impact of treatment regimens and resistance mutation patterns on the clinical, virological, and immunological presentation of HIV disease in 41 children (25 male and 16 female) at the Robert Wood Johnson Pediatric AIDS Program in New Brunswick, New Jersey. The study participants were symptomatic and had preceding treatment history with combined ARV regimens including protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Fifteen (36.6%) children were treated with NRTI+NNRTI+ PI, 6 (14.6%) with NRTI+NNRTIs, 13 (31.7%) with NRTI+PIs, and the remaining 7 (17.1%) received NRTIs only.Combined ARV regimens did not significantly influence the incidence of NRTI and NNRTI associated mutations. The duration of ARV therapy and the child's age had no significant impact on the ARV related mutations. The clinico-immunological presentation of the HIV disease was not associated with ARV treatment regimens or number of resistance mutations. However, primary mutations in the protease (PR) gene increased the likelihood of plasma viral load (PVL) ≥ 10,000 copies/mL irrespective of the child's age, duration of ARV therapy, presence of NRTI and NNRTI mutation. Viremia ≥ 10,000 copies/mL was recorded in almost all the children with primary mutations in the PR region (n = 12/13, 92.3%) as compared with only 50.0% (n = 14/28) of HIV infected children without (PR-), P < 0.008. However, CD-4 T cells were not affected by the mutations in the PR gene of the HIV-1 isolates.ConclusionPrimary PR resistance mutations significantly increase the likelihood for high viral replication in pediatric patients with moderate/severe HIV-1 infection, which may affect the long-term clinical prognosis of the HIV infected children.

Are HIV-infected patients candidates for liver transplantation?

Application of high-throughput sequencing technology in HIV drug resistance detection

There are 28 approved antiretroviral drugs for the treatment of HIV infection; combinations of these drugs clearly change the natural history of HIV, dramatically decreasing HIV-related morbidity and mortality and promoting and prolonging healthy survival. Despite the benefits of HIV treatment, some antiretroviral regimens may be inconvenient, toxic, and/or have suboptimal antiretroviral activity, particularly against drug-resistant viral strains. Thus, newer compounds are needed that continue to improve convenience and tolerability, reduce toxicity, and improve antiretroviral activity, particularly against drug-resistant viruses. Additionally, newer drugs may better penetrate tissue reservoirs (e.g. genital tract, central nervous system) and/or exploit new viral or cellular targets with new mechanisms of action. In addition to the direct antiretroviral action, some investigational agents may target inflammation and/or immune responses. Finally, newer formulations of antiretroviral agents may allow additional options for one-pill, once-daily dosing or allow less frequent dosing than once-daily, perhaps as infrequently as once-monthly or once-quarterly parenteral dosing. There are a number of investigational antiretroviral agents currently in development. These include newer antiretroviral agents in existing classes, including new nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, integrase inhibitors, and entry inhibitors. Of those in the pipeline, a few compounds are in advanced stages of development: Tenofovir alafenamide fumarate (TAF) is a nucleoside analogue reverse transcriptase inhibitor currently in phase 3 that is potent and potentially less toxic than the current TDF formulation. Doravirine (formerly MK-1439) is a non-nucleoside analogue reverse transcriptase inhibitor currently in phase 2 that demonstrates activity against NNRTI-resistant viral strains. GSK ‘744 is an integrase inhibitor currently in phase 2, being developed in a long-acting preparation. In addition there are investigational drugs with new mechanisms of action in development: BMS-663,068, is a small molecule CD4 attachment inhibitor that completed phase 1 testing. Cenicriviroc is a novel CCR5/CCR2 antagonist currently in phase 3 that offers both antiretroviral activity and the potential for an anti-inflammatory effect (through CCR2 antagonism). A number of other compounds are in earlier stages of pre-clinical and clinical development. The clinical use of these newer agents and formulations will depend on the results of clinical trials, and the timeline for development and availability. Continued progress in HIV drug development will allow improvements in the clinical care of patients living with HIV infection.