Drug Resistance Mutations Research Articles

Tilting the Scales toward EGFR Mutant Selectivity: Expanding the Scope of Bivalent "Type V" Kinase Inhibitors.

Binding multiple sites within proteins with bivalent compounds is a strategy for developing uniquely active agents. A new class of dual-site inhibitors has emerged targeting the epidermal growth factor receptor (EGFR) anchored to both the orthosteric (ATP) and allosteric sites. Despite proof-of-concept successes, enabling selectivity against oncogenic activating mutations has not been achieved and classifying these inhibitors among kinase inhibitors remains underexplored. This study investigates the structure-activity relationships, binding modes, and biological activity of ATP-allosteric bivalent inhibitors (AABIs). We find that AABIs selectively inhibit drug-resistant EGFR mutants (L858R/T790M and L858R/T790M/C797S) by anchoring a methyl isoindolinone moiety along the αC-helix channel of the allosteric site. In contrast, related Type I1/2 inhibitors target wild-type EGFR but are less effective against resistant mutants. This shift in selectivity demonstrates that mutant-selective AABIs classify as "Type V" bivalent inhibitors.

Assessment of Favipiravir and Remdesivir in Combination for SARS-CoV-2 Infection in Syrian Golden Hamsters

Favipiravir (FVP) and remdesivir (RDV) have demonstrable antiviral activity against SARS-CoV-2. Here, the efficacy of FVP, RDV, and FVP with RDV (FVP + RDV) in combination was assessed in Syrian golden hamsters challenged with SARS-CoV- 2 (B.1.1.7) following intraperitoneal administration. At day 4 post infection, viral RNA and viral antigen expression were significantly lower in lungs for all three treatment groups compared to the sham treatment. Similarly, viral titres in the lungs were lower in all treatment groups compared to the sham treatment. The FVP + RDV combination was the only treatment group where viral RNA in nasal turbinate and lung, virus titres in lung, and viral antigen expression (lung) were all lower than those for the sham treatment group. Moreover, lower viral titre values were observed in the FVP + RDV group compared to other treatment groups, albeit only significantly lower in comparison to those in the RDV-only-treated group. Further assessment of the potential utility of FVP in combination with RDV may be warranted. Future studies should also consider whether the combination of these two drugs may reduce the speed at which drug resistance mutations are selected.

Booster-free anti-retroviral therapy for persons living with HIV and multidrug resistance (B-Free): protocol for a multicentre, multistage, randomised, controlled, non-inferiority trial

IntroductionAnti-retroviral therapy (ART) simplification strategies are needed for treatment-experienced people with HIV (PWH) and multidrug-resistant viruses. These individuals are commonly treated with boosted ART regimens and are thereby at risk...

Outcomes after a virological failure to first-line second-generation INSTI-based therapy in a real-life setting.

Virological failures of first-line second-generation (SG) INSTI-based regimens are rare, usually characterized by low viremia and absence of drug resistance mutations. To explore the efficacy of rescue regimens introduced after virological failure (VF) to a first-line SG-INSTI therapy. This was a retrospective study on people living with HIV (PWH) failing a first-line SG-INSTI regimen [DTG/3TC, BIC/FTC/TAF, DTG-based three-drug regimen (DTG-3DR)] between 24 March 2016 and 31 December 2021. Follow-up accrued from the second viral load (VL) ≥ 50 copies/mL under SG-INSTI regimen (baseline) until virological success (VS, achievement of at least one VL < 50 copies/mL after baseline) or last visit. Cumulative probabilities of VS were estimated by Kaplan-Meier curves and compared using a log-rank test. Overall, of 521 naïve PWH who started a first-line SG-INSTI regimen, 45 (8.6%) had VF after a median of 14.9 (IQR = 6.9-25.9) months: 33/395 (8.4%) individuals failed a DTG-3DR, 11/102 (10.8%) a BIC/FTC/TAF and 1/24 (4.2%) failed a DTG/3TC.At baseline, 12/45 (27%) PWH changed antiretroviral therapy [median baseline VL 134 (IQR = 81-233) copies/mL], while 33 (73%) maintained their failing regimen [median baseline VL 75 (IQR = 60-145) copies/mL].During a median follow-up of 5.13 (IQR = 3.8-7.1) months, 34 (75.6%) PWH achieved VS: 25/33 (75.8%) maintaining their failing regimen, 9/12 (75%) switched regimen; the estimated 6- and 12-months probabilities of VS were 59% and 84%, respectively.There was no difference in VS curves between PWH who maintained their failing regimen and those who switched therapy. Most individuals remained on their failing regimen, achieving spontaneous virological suppression in most cases. These data help to understand a real-life VF scenario in the context of the current SG-INSTI era.

Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics.

It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations.

Genetic Diversity and Drug Resistance Mutations in HIV-1 pol Gene Sequences in the Philippines: A Retrospective Genomic Analysis

The Philippines has experienced a significant increase in HIV-1 infections in recent years, with a growing epidemic driven by the CRF01_AE strain. Understanding the genetic diversity of HIV-1 in the Philippines is crucial for the development of effective treatment strategies and the prevention of drug resistance. This study analyzed comprehensive data on common resistance mutation patterns from 2009 to 2017, revealing an increasing trend of mutations observed in NRTI and NNRTI resistance among the predominant CRF01_AE strains. The most common NRTI mutations observed were M184V, K65R, and S68G, whereas the most common NNRTI mutations were K103N, Y181C, and G190A. The study also found a high prevalence of M184V minority variants (0.5-20%) in treatment-naive patients, which could increase the risk of virological failure in 3TC-containing regimens. The findings of this study highlight the importance of comprehensive drug resistance surveillance and access to resistance testing to guide optimal first-line antiretroviral treatment selection and to manage the growing HIV-1 epidemic in the Philippines. The development of effective strategies to prevent and manage drug resistance is crucial to ensuring the long-term success of HIV treatment programs in the country.

Evaluation of genotype characteristics and drug resistance mutations in patients with chronic hepatitis B

Hepatitis B is one of the public health priorities worldwide, especially in the Southwest China. Our study aimed to investigate the relationship between genotypes and drug resistance mutations among HBV patients in Southwest China, with the objective of providing guidance for clinical antiviral treatment. A total of 4266 chronic hepatitis B (CHB) patients treated in the Qianjiang Hospital of Chongqing University were included in our study from 2014 to 2020. Both genotypes and drug-resistant mutations of CHB patients were determined by polymerase chain reaction (PCR). Genotype B and genotype C were the main HBV genotypes in our study. We found 54 mutation patterns, including 9 single-site mutations and 45 multiple-site mutations, accounting for 57.64% and 42.36%, respectively. rtM204I/V/S (485/1936) was the most common single-site mutation type, and rtL180M + rtM204I/V (482/1936) was the most common multiple-site mutation type. 1372 CHB patients were resistant to LAM + LDT, and 342 CHB patients were resistant to ADV. There was only 1 CHB patient who exhibited resistance to LAM + LDT + ADV + ETV, with a specific mutation pattern of rtA181T + rtT184L + rtM204V. Our study demonstrated trends in genetic mutations and drug resistance in CHB patients to enable timely adjustment of antiviral treatment strategies.

Leveraging large-scale Mycobacterium tuberculosis whole genome sequence data to characterise drug-resistant mutations using machine learning and statistical approaches

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a major global public health problem, resulting in > 1 million deaths each year. Drug resistance (DR), including the multi-drug form (MDR-TB), is challenging control of the disease. Whilst many DR mutations in the Mtb genome are known, analysis of large datasets generated using whole genome sequencing (WGS) platforms can reveal new variants through the assessment of genotype-phenotype associations. Here, we apply tree-based ensemble methods to a dataset comprised of 35,777 Mtb WGS and phenotypic drug-susceptibility test data across first- and second-line drugs. We compare model performance across models trained using mutations in drug-specific regions and genome-wide variants, and find high predictive ability for both first-line (area under ROC curve (AUC); range 88.3–96.5) and second-line (AUC range 84.1–95.4) drugs. To aggregate information from low-frequency variants, we pool mutations by functional impact and observe large improvements in predictive accuracy (e.g., sensitivity: pyrazinamide + 25%; ethionamide + 10%). We further characterise loss-of-function mutations observed in resistant phenotypes, uncovering putative markers of resistance (e.g., ndh 293dupG, Rv3861 78delC). Finally, we profile the distribution of known DR-associated single nucleotide polymorphisms across discretised minimum inhibitory concentration (MIC) data generated from phenotypic testing (n = 12,066), and identify mutations associated with highly resistant phenotypes (e.g., inhA − 779G > T and 62T > C). Overall, our work demonstrates that applying machine learning to large-scale WGS data is useful for providing insights into predicting Mtb binary drug resistance and MIC phenotypes, thereby potentially assisting diagnosis and treatment decision-making for infection control.

Evolution of hepatitis B virus polymerase and surface genes in patients receiving finite antiviral therapy.

Hepatitis B virus (HBV) reactivation could develop after withdrawal following a finite course of nucleoside analog (NA) therapy, leading to virological and clinical relapses. The genetic heterogeneity in the HBV surface and polymerase genes during finite NA therapy has not been carefully studied. Seven chronic HBV-infected patients experiencing relapses following entecavir (ETV; n=5; Patients 1 to 5) or tenofovir disoproxil fumarate (TDF; n=2; Patients 6 and 7) withdrawal were included. Sera obtained before treatment and at relapses were retrieved and submitted for DNA extraction and amplicon-specific deep sequencing. ETV-treated patients had a longer time-to-relapse than that of TDF-treated patients (P=0.0357). No drug-resistance related polymerase mutation was detected during relapses, except for a low percentage (1.4%) of rtM204I mutation in Patient 1. Two surface truncation mutations (sW216*; 40.9% and sW182*; 4.7%) detected before treatment in two TDF-treated patients (Patients 6 and 7, respectively) were overtaken by the wild types during subsequent drug-withdrawal-related relapses. The simultaneous presence of sG44E (T-cell epitope) and sE164G (B-cell epitope) mutations was associated with failure of HBV e antigen (HBeAg) seroclearance in ETV-treated patients. In conclusion, HBV genome continues to evolve during the courses of finite antiviral therapies. Pre-existing surface truncation mutations can be overtaken by the wild types after relapses. The sG44E/sE164G mutations are associated with failure of HBeAg seroclearance.

Analysis of genotype resistance and HIV-1 transmission risk in HIV-1-infected men who have sex with men in Guiyang, China.

As the social economy has developed and population mobility has increased, differences in the Human immunodeficiency virus type 1 (HIV-1) genotype distribution among men who have sex with men (MSM) have become apparent in the provinces and cities across China. The high variability and drug resistance characteristics of HIV-1 can lead to the widespread spread of resistant strains, which may also result in antiretroviral therapy failure and an increase in the mortality rate. The genotypic drug resistance characteristics and HIV-1 transmission risks among HIV-1-infected MSM in Guiyang, Guizhou Province were analyzed in the current study. The aim of the study was to provide a scientific basis for preventing the spread of HIV-1 strains among MSM and develop intervention measures. A cross-sectional study was conducted at the Guiyang Public Health Clinical Center. A total of 181 HIV-1-infected MSM who not received treatment at the center between 1 January 2020 and 31 December 2022 were selected. The HIV-1 pol region gene fragment, including the protease and reverse transcriptase regions, was amplified by nested PCR and RT-PCR. The maximum likelihood method was used to construct a phylogenetic tree for analyzing the HIV-1 genotypes in MSM. HIV-1 genotypic resistance was evaluated using the Stanford University HIV drug resistance database. A molecular transmission network of HIV was constructed and the risk of HIV-1 transmission was determined. We successfully amplified 173 pol gene sequences from blood samples obtained from 181 patients. The main subtype was CRF07_BC (60.69% [105/173]), followed by CRF01_AE (26.59% [46/173]), CRF08_BC (4.05% [7/173]), CRF55_01B (4.62% [8/173]), B (3.47% [6/173]), and C (0.58% [1/173]). The distribution of HIV-1 genotypes in MSM showed that there was a significant difference in the genotype composition of HIV-1-infected MSM according to registered residences and ages (p < .05). The CRF55_01B subtype accounted for the lowest proportion in Guiyang City and individuals >30 years of age. Multivariate logistic regression analysis of risk factors for drug resistance in HIV-1-infected MSM showed that the overall prevalence of pretreatment drug-resistant species was 12.72% (22/173), and age >30 years, CRF55_01B subtype, and CD4+ T lymphocyte count >350 cells/mm3 were risk factors for drug resistance in MSM HIV-1 strains. Among the pretreatment drug-resistant species, non-nucleoside reverse transcriptase inhibitors with ≥1 drug resistant-species accounted for 9.25% (16/173), followed by protease inhibitors at 4.05% (7/173) and nucleoside reverse transcriptase inhibitors at 1.73% (3/173). Non-nucleoside reverse transcriptase inhibitors resistant to the CRF07_BC and CRF01_AE genotypes were predominant. The CRF55_01B genotype was shown to be most likely to carry the V179E mutation. The molecular network included CRF07_BC and B genotypes. The results of multi-factor logistic regression analysis on the factors affecting the rate of joining the network showed that individuals >30 years of age were less likely to join the network compared to those individuals <30 years of age. The distribution of HIV-1 genotypes among MSM in Guiyang is diverse and complex. The main genotypes were shown to be CRF07_BC and CRF01_AE. The drug resistance mutation rate is high and pretreatment drug-resistant species is at a moderate level of prevalence, with NNRTIs being the most common site for drug resistance mutations. The CRF07_BC subtype and patients <30 years of age were identified as the key intervention targets in Guiyang based on the molecular transmission network. Patients should routinely undergo drug resistance testing before starting antiretroviral therapy to avoid virologic treatment failure and prevent the spread of HIV-1-resistant strains in MSM.

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

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). 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. 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. 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.

Archived HIV-1 Drug Resistance Mutations: Role of Proviral HIV-1 DNA Genotype for the Management of Virological Responder People Living with HIV.

Despite its effectiveness in controlling plasma viremia, antiretroviral therapy (ART) cannot target proviral DNA, which remains an obstacle to HIV-1 eradication. When treatment is interrupted, the reservoirs can act as a source of viral rebound, highlighting the value of proviral DNA as an additional source of information on an individual's overall resistance burden. In cases where the viral load is too low for successful HIV-1 RNA genotyping, HIV-1 DNA can help identify resistance mutations in treated individuals. The absence of treatment history, the need to adjust ART despite undetectable viremia, or the presence of LLV further support the use of genotypic resistance tests (GRTs) on HIV-1 DNA. Conventionally, GRTs have been achieved through Sanger sequencing, but the advances in NGS are leading to an increase in its use, allowing the detection of minority variants present in less than 20% of the viral population. The clinical significance of these mutations remains under debate, with interpretations varying based on context. Additionally, proviral DNA is subject to APOBEC3-induced hypermutation, which can lead to defective, nonviable viral genomes, a factor that must be considered when performing GRTs on HIV-1 DNA.

Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication.

Coronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. All CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, though the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug-resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating coronavirus infections.

HIV-1 transmission clusters and drug resistance in men who have sex with men in Portugal

Abstract Introduction In 2022, 45.8% of diagnoses in the EU/EEA with known route of transmission were in Men who have Sex with Men (MSM). We aim to characterise HIV transmission clusters and transmission of drug resistance (TDR), as well as its determinants, using integrated sociodemographic, behavioural, clinical, and viral genomic data of MSMs newly diagnosed in Portugal between 2014 and 2019. Methods This study included data from 340 MSM who were diagnosed with HIV-1 infection. These individuals were newly diagnosed at 17 hospitals in Portugal between September 2014 and December 2019. Phylogenies were constructed and transmission clusters were identified with branch support ≥ 90% and 1.5% genetic distance. Logistic regression models were computed to examine the factors associated with clusters of transmission and with the presence of TDR. Results We identified 38 transmission clusters with 104 MSM, which included 26.6% of the total 305 MSM from our database used for cluster analysis. The overall prevalence of TDR was 8.2%. Only subtype C was significantly associated with TDR. 10.5% of the clusters had at least 1 drug resistance mutation (K103N, L90M or N88S). There was no significant difference in the prevalence of TDR between MSM inside and outside clusters. 50% of the clusters were composed of portuguese native MSM only, whereas 16% had exclusively migrant MSMs. No significant difference was found in the proportion of portuguese and migrant MSM inside and outside clusters. Factors associated with HIV-1 transmission clusters were age at diagnosis, district of residence, unprotected anal or vaginal sex with a woman, HIV testing frequency, presenter status and subtype. 87% of men engaged in sexual activity only with other men, while 13% with both men and women. Conclusions Specific subgroups of MSM are contributing to HIV dissemination in Portugal. Direct and precise prevention measures based on molecular epidemiology should be developed. Key messages • Specific subgroups of MSM are contributing to HIV dissemination in Portugal. • Direct and precise prevention measures based on molecular epidemiology should be developed.

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.

Prevalence of major INSTI and HIV-1 drug resistance mutations in pre- and antiretroviral-treated patients in Indonesia

Indonesia has one of the highest HIV infection rates in Southeast Asia. The use of dolutegravir, an integrase strand transfer inhibitor (INSTI), as a first-line treatment underscores the need for detailed data on INSTI drug resistance mutations (DRMs). Currently, there is a lack of comprehensive data on DRMs INSTI and other HIV drug resistance in Indonesian patients, both pre- and post-treatment. The aim of this study was to identify the subtypes and drug resistance mutations of the protease, reverse transcriptase, and integrase genes in both treatment-naive and ARV-treated patients in Bandung, West Java, Indonesia. A cross-sectional study was conducted involving HIV-positive patients at Hasan Sadikin Hospital, Bandung, Indonesia, from September 2022 to January 2023. The patients were categorized into two groups: ARV-treated and pre-treatment patients. Peripheral blood mononuclear cells (PBMCs) were processed for DNA extraction, followed by amplification and sequencing of the pol gene to detect mutations and subtypes. The study found that the predominant subtype was CRF01_AE, accounting for 85.4% and 69% of pre-treatment and treated patients, respectively, followed by recombinant forms such as A1/CRF01_AE, CRF01_AE/CRF02_AG, subtype B, and other subtypes. Among ARV-treated/INSTI-naive patients, major INSTI DRMs R263K and Y143H were identified, while pre-treatment patients exhibited accessory integrase DRMs. The most common DRMs detected were non-nucleoside reverse transcriptase inhibitor (NNRTI) DRMs, with prevalences of 14.6% and 7% in pre-treatment and ARV-treated patients, respectively. In conclusion, CRF01_AE emerged as the predominant subtype in both pre-treatment and ARV-treated patients in Bandung, underscoring the necessity for ongoing surveillance of integrase DRMs, particularly given the presence of major INSTI DRMs in patients undergoing INSTI treatment.

Epidemiological dynamics and molecular characterization of HIV drug resistance in eastern China from 2020 to 2023.

HIV drug resistance (HIVDR) has become a threat to the elimination of the AIDS epidemic due to the global scale-up of antiretroviral therapy (ART) for HIV-infected individuals. This study aims to investigate the epidemiological dynamics and molecular characterization of HIV pretreatment drug resistance (PDR) and acquired drug resistance (ADR) in Hangzhou, a developed region in China. An epidemiological survey combined with a molecular transmission network and Bayesian analysis was conducted. A total of 3,596 individuals with newly confirmed HIV infections (from 2020 to 2023) and 164 individuals with ART failure (from 2021 to 2023) were included. The molecular transmission network was used to identify key drug-resistant transmission clusters, while the Bayesian analysis was utilized to trace the origins and spread of these clusters. The overall prevalence of PDR was found to be 8.4% (303/3596). Among these cases, PDR to non-nucleoside reverse transcriptase inhibitors (NNRTIs) accounted for 4.7% (170/3596), significantly higher than the resistance observed for protease inhibitors (PIs; 2.8%, p < 0.001) and nucleoside reverse transcriptase inhibitors (NRTIs; 1.4%, p < 0.001). Multivariate logistic regression analysis revealed a significantly higher PDR value among individuals infected with the CRF07_BC subtype compared to those with the CRF08_BC subtype (aOR = 0.56, 95% CI = 0.359-0.859, p = 0.008). The molecular transmission network analysis identified the transmission of the drug resistance mutation (DRM) Q58E within the clusters of the CRF07_BC subtype. The Bayesian analysis suggested that these clusters were introduced into Hangzhou from Shenzhen between 2005 and 2012. Furthermore, the study highlighted 50.6% (83/164) prevalence of ADR among individuals experiencing ART failure. The combined molecular network analysis of virological failure and newly confirmed HIV infections indicated the transmission of the K103N mutation between these groups. In conclusion, targeted interventions may be necessary for specific subtypes and transmission clusters to control the spread of drug-resistant HIV. Continuous monitoring of resistance patterns is critical to inform treatment strategies and optimize ART regimens.

Replication capacity and susceptibility of nirmatrelvir-resistant mutants to next-generation Mpro inhibitors in a SARS-CoV-2 replicon system

There is an ongoing need to expand the anti-SARS-CoV-2 armamentarium to include agents capable of suppressing replication of drug-resistant mutants emerging during monotherapy with approved direct-acting antivirals. Using a subgenomic SARS-CoV-2 replicon system, we studied the RNA replication capacity of nirmatrelvir (NTV)-resistant mutants and their susceptibility to next-generation Mpro inhibitors, including ibuzatrelvir (ITV), ensitrelvir (ETV), and ML2006a4. Our findings revealed that E166V Mpro mutants reduced viral RNA replication, whereas other Mpro mutations retained or increased the replication capacity, suggesting the potential of the latter to dominate under NTV selective pressure. Except for having an advantage against E166A mutants, ITV largely showed the same mutational sensitivity as NTV. ETV was more effective than NTV against E166V mutants but less effective against S144A, E166A, and L167F mutants. ML2006a4 demonstrated the most effective suppression across most mutants (S144A, E166V, S144A + L50F, E166 A/V + L50F, L167F + L50F, and E166A + L167F + L50F). Thus, ML2006a4 represents an attractive investigational candidate against clinically relevant NTV-resistant SARS-CoV-2 mutants.

Genetic analysis of drug resistance mechanisms and phylogenetic clustering in Candida auris isolates from Western India

Objectives: Candida auris is an emerging multidrug-resistant fungal pathogen that poses a significant threat to global health. Limited information is available from the Indian subcontinent regarding mutations associated with drug resistance and genetic variability among the isolates. In this study, we employed whole-genome sequencing (WGS) to investigate the genetic variations and drug resistance mechanisms within C. auris isolates from the western region of India. Materials and Methods: A total of twenty archived isolates were subjected to WGS on the Illumina NextSeq 2000 platform. A set of 18 genes was analyzed to check for the presence of drug-resistant mutations. Phylogenetic analysis was done using MEGA v6.06 software to identify the C. auris subgroup or clade and to check genetic relatedness among species. Statistical Analysis: The data related to drug resistance were presented in numbers and percentages. Results: Through manual analysis, drug-resistant mutations were detected in ERG11, CDR1, and TAC1b genes, which are known to be associated with reduced susceptibility to antifungal agents. Phylogenetic analysis revealed that all the isolates clustered within Clade I, indicating a high degree of genetic similarity among isolates. The absence of comprehensive antifungal mutation databases and automated tools for drug resistance detection necessitated the utilization of specialized computational skills of bioinformaticians for data analysis. Conclusions: The study provides valuable insights into the genetic diversity and drug resistance mechanisms of C. auris isolates in the western region of India and emphasizes the need for continued research and surveillance to combat this emerging pathogen. Our findings underscore the need for the development of user-friendly automated tools and comprehensive databases to facilitate rapid and accurate identification of drug resistance in C. auris.

The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir.

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents that potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into phase 2a clinical trials. Previous cell culture-based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. Although both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect the direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor-mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR-induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared with the WT virus. By rationally modifying PIR, we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, the evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds that work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild-type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here, we have elucidated the structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cells that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir-resistant virus.