Kinetic coevolutionary models predict the temporal emergence of HIV-1 resistance mutations under drug selection pressure.

Drug resistance in HIV type 1 (HIV-1) is a pervasive problem that affects the lives of millions of people worldwide. Although records of drug-resistant mutations (DRMs) have been extensively tabulated within public repositories, our understanding of the evolutionary kinetics of DRMs and how they evolve together remains limited. Epistasis, the interaction between a DRM and other residues in HIV-1 protein sequences, is key to the temporal evolution of drug resistance. We use a Potts sequence-covariation statistical-energy model of HIV-1 protein fitness under drug selection pressure, which captures epistatic interactions between all positions, combined with kinetic Monte-Carlo simulations of sequence evolutionary trajectories, to explore the acquisition of DRMs as they arise in an ensemble of drug-naive patient protein sequences. We follow the time course of 52 DRMs in the enzymes protease, RT, and integrase, the primary targets of antiretroviral therapy. The rates at which DRMs emerge are highly correlated with their observed acquisition rates reported in the literature when drug pressure is applied. This result highlights the central role of epistasis in determining the kinetics governing DRM emergence. Whereas rapidly acquired DRMs begin to accumulate as soon as drug pressure is applied, slowly acquired DRMs are contingent on accessory mutations that appear only after prolonged drug pressure. We provide a foundation for using computational methods to determine the temporal evolution of drug resistance using Potts statistical potentials, which can be used to gain mechanistic insights into drug resistance pathways in HIV-1 and other infectious agents.

Bioinformatics toolbox for exploring target mutation-induced drug resistance.

Drug resistance is increasingly among the main issues affecting human health and threatening agriculture and food security. In particular, developing approaches to overcome target mutation-induced drug resistance has long been an essential part of biological research. During the past decade, many bioinformatics tools have been developed to explore this type of drug resistance, and they have become popular for elucidating drug resistance mechanisms in a low cost, fast and effective way. However, these resources are scattered and underutilized, and their strengths and limitations have not been systematically analyzed and compared. Here, we systematically surveyed 59 freely available bioinformatics tools for exploring target mutation-induced drug resistance. We analyzed and summarized these resources based on their functionality, data volume, data source, operating principle, performance, etc. And we concisely discussed the strengths, limitations and application examples of these tools. Specifically, we tested some predictive tools and offered some thoughts from the clinician's perspective. Hopefully, this work will provide a useful toolbox for researchers working in the biomedical, pesticide, bioinformatics and pharmaceutical engineering fields, and a good platform for non-specialists to quickly understand drug resistance prediction.

Low clinical impact of HIV drug resistance mutations in oral pre-exposure prophylaxis: a systematic review and meta-analysis.

INTRODUCTION: Despite the widespread use of pre-exposure prophylaxis (PrEP) in preventing human immunodeficiency virus (HIV) transmission, scant information on HIV drug resistance mutations (DRMs) has been gathered over the past decade. This review aimed to estimate the pooled prevalence of pre-exposure prophylaxis and its two-way impact on DRM. METHODS: We systematically reviewed studies on DRM in pre-exposure prophylaxis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis 2020 guidelines. PubMed, Cochrane, and SAGE databases were searched for English-language primary studies published between January 2001 and December 2023. The initial search was conducted on 9 August 2021 and was updated through 31 December 2023 to ensure the inclusion of the most recent findings. The registration number for this protocol review was CRD42022356061. RESULTS: A total of 26,367 participants and 562 seroconversion cases across 12 studies were included in this review. The pooled prevalence estimate for all mutations was 6.47% (95% Confidence Interval-CI 3.65-9.93), while Tenofovir Disoproxil Fumarate/Emtricitabine-associated drug resistance mutation prevalence was 1.52% (95% CI 0.23-3.60) in the pre-exposure prophylaxis arm after enrolment. A subgroup analysis, based on the study population, showed the prevalence in the heterosexual and men who have sex with men (MSM) groups was 5.53% (95% CI 2.55-9.40) and 7.47% (95% CI 3.80-12.11), respectively. Notably, there was no significant difference in the incidence of DRM between the pre-exposure prophylaxis and placebo groups (log-OR = 0.99, 95% CI -0.20 to 2.18, I2 = 0%; p = 0.10). DISCUSSION: Given the constrained prevalence of DRM, the World Health Organization (WHO) advocates the extensive adoption of pre-exposure prophylaxis. Our study demonstrated no increased risk of DRM with pre-exposure prophylaxis (p > 0.05), which is consistent with these settings. These findings align with the previous meta-analysis, which reported a 3.14-fold higher risk in the pre-exposure prophylaxis group than the placebo group, although the observed difference did not reach statistical significance (p = 0.21). CONCLUSIONS: Despite the low prevalence of DRM, pre-exposure prophylaxis did not significantly increase the risk of DRM compared to placebo. However, long-term observation is required to determine further disadvantages of extensive pre-exposure prophylaxis use. PROSPERO Number: CRD42022356061.

Use of Next-Generation Sequencing in a State-Wide Strategy of HIV-1 Surveillance: Impact of the SARS-COV-2 Pandemic on HIV-1 Diagnosis and Transmission.

BACKGROUND: The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic posed an unpreceded threat to the management of other pandemics such as human immunodeficiency virus-1 (HIV-1) in the United States. The full impact of the SARS-CoV-2 pandemic on the HIV-1 pandemic needs to be evaluated. METHODS: All individuals with newly reported HIV-1 diagnoses from NC State Laboratory of Public Health were enrolled in this prospective observational study, 2018-2021. We used a sequencing-based recency assay to identify recent HIV-1 infections and to determine the days postinfection (DPI) for each person at the time of diagnosis. RESULTS: Sequencing used diagnostic serum samples from 814 individuals with new HIV-1 diagnoses spanning this 4-year period. Characteristics of individuals diagnosed in 2020 differed from those in other years. People of color diagnosed in 2021 were on average 6 months delayed in their diagnosis compared to those diagnosed in 2020. There was a trend that genetic networks were more known for individuals diagnosed in 2021. We observed no major integrase resistance mutations over the course of the study. CONCLUSIONS: SARS-CoV-2 pandemic may contribute to the spread of HIV-1. Public health resources need to focus on restoring HIV-1 testing and interrupting active, ongoing, transmission.

Absence of Proviral Human Immunodeficiency Virus (HIV) Type 1 Evolution in Early-Treated Individuals With HIV Switching to Dolutegravir Monotherapy During 48 Weeks.

Human immunodeficiency virus type 1 (HIV-1) infection is treated with antiretroviral therapy (ART), usually consisting of 2-3 different drugs, referred to as combination ART (cART). Our recent randomized clinical trial comparing a switch to dolutegravir monotherapy with continuation of cART in early-treated individuals demonstrated sustained virological suppression over 48 weeks. Here, we characterize the longitudinal landscape of the HIV-1 reservoir in these participants, with particular attention to potential differences between treatment groups regarding evidence of evolution as a proxy for low-level replication. Near full-length HIV-1 proviral polymerase chain reaction and next-generation sequencing was applied to longitudinal peripheral blood mononuclear cell samples to assess proviral evolution and the potential emergence of drug resistance mutations (DRMs). Neither an increase in genetic distance nor diversity over time was detected in participants of both treatment groups. Single proviral analysis showed high proportions of defective proviruses and low DRM numbers. No evidence for evolution during dolutegravir monotherapy was found in these early-treated individuals.

"I feel drug resistance testing allowed us to make an informed decision": qualitative insights on the role of HIV drug resistance mutation testing among children and pregnant women living with HIV in western Kenya.

BACKGROUND: Pregnant women and children living with HIV in Kenya achieve viral suppression (VS) at lower rates than other adults. While many factors contribute to these low rates, the acquisition and development of HIV drug resistance mutations (DRMs) are a contributing factor. Recognizing the significance of DRMs in treatment decisions, resource-limited settings are scaling up national DRM testing programs. From provider and patient perspectives, however, optimal ways to operationalize and scale-up DRM testing in such settings remain unclear. METHODS: Our mixed methods study evaluates the attitudes towards, facilitators to, and barriers to DRM testing approaches among children and pregnant women on antiretroviral therapy (ART) in five HIV treatment facilities in Kenya. We conducted 68 key informant interviews (KIIs) from December 2019 to December 2020 with adolescents, caregivers, pregnant women newly initiating ART or with a high viral load, and providers, laboratory/facility leadership, and policy makers. Our KII guides covered the following domains: (1) DRM testing experiences in routine care and through our intervention and (2) barriers and facilitators to routine and point-of-care DRM testing scale-up. We used inductive coding and thematic analysis to identify dominant themes with convergent and divergent subthemes. RESULTS: The following themes emerged from our analysis: (1) DRM testing and counseling were valuable to clinical decision-making and reassuring to patients, with timely results allowing providers to change patient ART regimens faster; (2) providers and policymakers desired an amended and potentially decentralized DRM testing process that incorporates quicker sample-to-results turn-around-time, less burdensome procedures, and greater patient and provider "empowerment" to increase comfort with testing protocols; (3) facility-level delays, deriving from overworked facilities and sample tracking difficulties, were highlighted as areas for improvement. CONCLUSIONS: DRM testing has the potential to considerably improve patient health outcomes. Key informants recognized several obstacles to implementation and desired a more simplified, time-efficient, and potentially decentralized DRM testing process that builds provider comfort and confidence with DRM testing protocols. Further investigating the implementation, endurance, and effectiveness of DRM testing training is critical to addressing the barriers and areas of improvement highlighted in our study. TRIAL REGISTRATION: NCT03820323.

Characterization of mutants with single and combined Qi and Qo site mutations in Saccharomyces cerevisiae reveals interactions between the picolinamide fungicide CAS-649 and azoxystrobin.

Picolinamide and strobilurin fungicides bind to the Qi and Qo sites on cytochrome b, respectively, and target many of the same plant pathogens. Using Saccharomyces cerevisiae as a model system, we explore effects of amino acid changes at each site on sensitivity to a fungicide acting at the opposite site and examine the relationship between altered sensitivity and growth penalty. In addition, double mutants containing the G143A or F129L mutations responsible for strobilurin resistance in combination with Qi site mutations that confer resistance to picolinamides are characterized in terms of their sensitivity to QiI and QoI fungicides and growth rate. Mutants containing amino acid changes at the Qo site varied in their growth rate and sensitivity to the picolinamide CAS-649, and increased sensitivity was associated with a greater growth penalty. Conversely, changes at the Qi site affected sensitivity to azoxystrobin and also showed a correlation between increased sensitivity and reduced growth. There was no overall correlation between resistance to azoxystrobin and CAS-649 among mutants, however negative cross-resistance occurred in the case of mutations which conferred resistance to either compound and also carried a growth penalty. These results suggest the use of QoI fungicides to delay the emergence of pathogen resistance to QiIs, and vice versa. Double mutants containing G143A or F129L in combination with Qi site changes N31K, G37C/V or L198F that cause resistance to picolinamides generally exhibited lower resistance factors for both azoxystrobin and CAS-649 than corresponding resistant strains with a single mutation. Reduced growth was observed for all F129L-containing double mutants, whereas the growth rate of double mutants containing G143A was significantly reduced only by the Qi site mutations N31K and G37V that confer a larger growth penalty. Our results suggest that resistance to picolinamides in pathogens could emerge more readily in a strobilurin-sensitive genetic background than in a strobilurin-resistant one.

Inference of selection pressures that drive insecticide resistance in Anopheles and Culex mosquitoes in Korea.

Pyrethroids are primarily used for mosquito control in Korea. However, high frequencies of mutations conferring resistance to not only pyrethroids but also to other insecticides have been found in mosquito populations. This study aimed to examine the hypothesis that insecticides used outside of public health may play a role in selection. Briefly, the resistance mutation frequencies to three insecticide groups (pyrethroids, organophosphates, and cyclodienes) were estimated in two representative groups of mosquito species (Anopheles Hyrcanus Group and Culex pipiens complex). The relationship between these frequencies and the land-use status of the collection sites was investigated through multiple regression analysis. In the Anopheles Hyrcanus Group, the frequencies of both ace1 (organophosphate resistance) and rdl (cyclodiene resistance) mutations were positively correlated with 'proximity to golf course', possibly be due to the insecticides used for turf maintenance. They also showed positive correlations with field area and rice paddy area, respectively, suggesting the role of agricultural insecticides in the selection of these resistance traits. For the Cx. pipiens complex, the kdr (pyrethroid resistance), ace1, and rdl mutations were positively correlated with the residential area, field, and rice paddy, respectively. Therefore, pyrethroids used for public health could serve as a direct source of resistance selection pressure against kdr, whereas non-public health insecticides may pose primary selection pressure against the ace1 and rdl traits. The current findings suggest that the insecticides used in agriculture and the golf industry play a significant role in mosquito selection, despite variations in the extent of indirect selection pressure according to the mosquito groups and insecticide classes.

Epistatic interactions promote persistence of NS3-Q80K in HCV infection by compensating for protein folding instability.

The Q80K polymorphism in the NS3-4A protease of the hepatitis C virus is associated with treatment failure of direct-acting antiviral agents. This polymorphism is highly prevalent in genotype 1a infections and stably transmitted between hosts. Here, we investigated the underlying molecular mechanisms of evolutionarily conserved coevolving amino acids in NS3-Q80K and revealed potential implications of epistatic interactions in immune escape and variants persistence. Using purified protein, we characterized the impact of epistatic amino acid substitutions on the physicochemical properties and peptide cleavage kinetics of the NS3-Q80K protease. We found that Q80K destabilized the protease protein fold (p < 0.0001). Although NS3-Q80K showed reduced peptide substrate turnover (p < 0.0002), replicative fitness in an H77S.3 cell culture model of infection was not significantly inferior to the WT virus. Epistatic substitutions at residues 91 and 174 in NS3-Q80K stabilized the protein fold (p < 0.0001) and leveraged the WT protease stability. However, changes in protease stability inversely correlated with enzymatic activity. In infectious cell culture, these secondary substitutions were not associated with a gain of replicative fitness in NS3-Q80K variants. Using molecular dynamics, we observed that the total number of residue contacts in NS3-Q80K mutants correlated with protein folding stability. Changes in the number of contacts reflected the compensatory effect on protein folding instability by epistatic substitutions. In summary, epistatic substitutions in NS3-Q80K contribute to viral fitness by mechanisms not directly related to RNA replication. By compensating for protein-folding instability, epistatic interactions likely protect NS3-Q80K variants from immune cell recognition.

Impact of Integrase Sequences from HIV-1 Subtypes A6/A1 on the In Vitro Potency of Cabotegravir or Rilpivirine.

The FLAIR study demonstrated noninferiority of monthly long-acting cabotegravir + rilpivirine versus daily oral dolutegravir/abacavir/lamivudine for maintaining virologic suppression. Three participants who received long-acting therapy had confirmed virologic failure (CVF) at Week 48, and all had HIV-1 that was originally classified as subtype A1 and contained the baseline integrase polymorphism L74I; updated classification algorithms reclassified all 3 as HIV-1 subtype A6. Retrospectively, the impact of L74I on in vitro sensitivity and durability of response to cabotegravir in HIV-1 subtype B and A6 backgrounds was studied. Site-directed L74I and mutations observed in participants with CVF were generated in HIV-1 subtype B and a consensus integrase derived from 3 subtype A6 CVF baseline sequences. Rilpivirine susceptibility was assessed in HIV-1 subtype B and A1 containing reverse transcriptase mutations observed in participants with CVF. HIV-1 subtype B L74I and L74I/G140R mutants and HIV-1 subtype A6 I74L and I74/G140R mutants remained susceptible to cabotegravir; L74I/Q148R double mutants exhibited reduced susceptibility in HIV-1 subtypes B and A6 (half maximal effective capacity fold change, 4.4 and 4.1, respectively). Reduced rilpivirine susceptibility was observed across HIV-1 subtypes B and A1 with resistance-associated mutations K101E or E138K (half maximal effective capacity fold change, 2.21 to 3.09). In cabotegravir breakthrough experiments, time to breakthrough was similar between L74 and I74 viruses across HIV-1 subtypes B and A6; Q148R was selected at low cabotegravir concentrations. Therefore, the L74I integrase polymorphism did not differentially impact in vitro sensitivity to cabotegravir across HIV-1 subtype B and A6 integrase genes (ClinicalTrials.gov identifier: NCT02938520).

Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production.

Antibiotic-resistant bacteria in the genus Enterococcus are a major cause of nosocomial infections and are an emergent public health concern. Similar to a number of bacterial species, resistance to the antibiotic rifampicin (RifR) in enterococci is associated with mutations in the gene encoding the ß subunit of RNA polymerase (rpoB). In Mycobacterium tuberculosis, RifRrpoB mutations alter mycobacterial surface lipid expression and are associated with an altered IL-1 cytokine response in macrophages upon infection. However, it is not clear if RifR mutations modulate host cytokine responses by other bacteria. To address this question, we utilized Enterococcus faecalis (E. faecalis). Here, we treated human monocyte-derived macrophages with heat-inactivated wild type or RifRrpoB mutants of E. faecalis and found that RifR mutations reduced IL-1ß cytokine production. However, RifR mutations elicited other potent pro- and anti-inflammatory responses, indicating that they can impact other immune pathways beyond IL-1R1 signaling. Our findings suggest that immunomodulation by mutations in rpoB may be conserved across diverse bacterial species and that subversion of IL-1R1 pathway is shared by RifR bacteria.

Ripretinib in advanced gastrointestinal stromal tumors: an overview of current evidence and drug approval.

Over the past 20 years, the management of gastrointestinal stromal tumors has acted as an important model in the advancement of molecularly targeted therapies for solid tumors. The success of imatinib has established it as a lasting therapy in the management of early-stage and advanced disease in the first-line setting. Imatinib resistance inevitably develops, resulting in the need for further lines of therapy. Ripretinib is an orally administered switch-control tyrosine kinase inhibitor, specifically developed to target both primary and secondary KIT and PDGFRα resistance mutations. Herein, the authors discuss the molecular rationale, the preclinical evidence and the clinical use of ripretinib in the treatment of gastrointestinal stromal tumors in the advanced stages of disease.

A novel naturally Phe206Tyr mutation confers tolerance to ALS-inhibiting herbicides in Alopecurus myosuroides.

Herbicide-resistant weeds pose a serious threat to world food production. The rapid and widespread development of target-site based resistance limits the application of herbicides. Alopecurus myosuroides Huds. (blackgrass) has spread rapidly in winter wheat regions in China, and the field recommended dose of ALS herbicides no longer controls blackgrass populations in recent years. A highly resistant population TW18(R) was collected in 2018 from Shandong Province. Dose-response assays showed that the TW18 was resistant to mesosulfuron-methyl, flucarbazone-sodium, and imazethapyr, with resistance index values of 5.96, 6.1, and 4.09, respectively. DNA sequencing of the TW18 population revealed a Phe206Tyr (F206Y) mutation in the ALS, which was not yet reported. Blackgrass ALS gene with the F206Y mutation (R gene) was expressed in Arabidopsis and rice. Transgenic studies have shown that both Arabidopsis and rice expressing this R gene have resistance to imazethapyr. However, it did not confer resistance to tribenuron methyl and florasulam in transgenic Arabidopsis. This study showed that the F206Y substitution caused herbicide resistance in blackgrass. To our knowledge, this is the first-reported F206Y mutation of a weed species in the natural environment. Transgenic plants showed this functional site could be utilized to generate imazethapyr-resistant rice to control herbicide-resistant weed damage.

Rapid detection of kdr mutation F1534C in Aedes aegypti using recombinase polymerase amplification and lateral flow dipsticks.

Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes. We trialled the rapid F1534C pyrethroid resistance test using insecticide resistant Ae. aegypti mosquito bodies and compared results to a conventional, allele-specific quantitative PCR (AS-qPCR) coupled with melt curve genotyping in corresponding mosquito heads. From a strain of Ae. aegypti established from an insecticide resistant population in Merida, Mexico (n = 27), all the mosquito bodies (n = 27) tested positive with the rapid F1534C test regardless of whether they were homozygous or heterozygous. To assess diagnostic test specificity, we confirmed that F1534 was not detected in laboratory-reared, fully susceptible Ae. aegypti mosquito bodies (n = 28) using the rapid F1534C test or the conventional AS-qPCR melt curve analysis. All corresponding mosquito heads (n = 28) were homozygous wild-type FF1534. The rapid F1534C test thus demonstrated 100% diagnostic sensitivity (95% CI: 87.23% to 100%) and 100% diagnostic specificity (95% CI: 87.66% to 100.00%) for detection of the F1534C pyrethroid resistant single nucleotide polymorphism (SNP) in both heterozygous and homozygous Ae. aegypti. In the collection of mutant mosquitoes from Mexico, CC1534 homozygous mutants occurred at a frequency of 74.1% (n = 20) and FC heterozygous mutants at a frequency of 25.9% (n = 7). The rapid F1534C test significantly reduced the sample processing and testing time from approximately 6 h for the AS-qPCR melt curve analysis to only 25 min. These results demonstrate significant potential for our approach to resistance testing as a field-based, low-resource, rapid alternative to time-consuming and expensive laboratory-based detection.

Predicting rifampicin resistance mutations in bacterial RNA polymerase subunit beta based on majority consensus.

BACKGROUND: Mutations in an enzyme target are one of the most common mechanisms whereby antibiotic resistance arises. Identification of the resistance mutations in bacteria is essential for understanding the structural basis of antibiotic resistance and design of new drugs. However, the traditionally used experimental approaches to identify resistance mutations were usually labor-intensive and costly. RESULTS: We present a machine learning (ML)-based classifier for predicting rifampicin (Rif) resistance mutations in bacterial RNA Polymerase subunit ß (RpoB). A total of 186 mutations were gathered from the literature for developing the classifier, using 80% of the data as the training set and the rest as the test set. The features of the mutated RpoB and their binding energies with Rif were calculated through computational methods, and used as the mutation attributes for modeling. Classifiers based on five ML algorithms, i.e. decision tree, k nearest neighbors, naïve Bayes, probabilistic neural network and support vector machine, were first built, and a majority consensus (MC) approach was then used to obtain a new classifier based on the classifications of the five individual ML algorithms. The MC classifier comprehensively improved the predictive performance, with accuracy, F-measure and AUC of 0.78, 0.83 and 0.81for training set whilst 0.84, 0.87 and 0.83 for test set, respectively. CONCLUSION: The MC classifier provides an alternative methodology for rapid identification of resistance mutations in bacteria, which may help with early detection of antibiotic resistance and new drug discovery.

Extended interaction networks with HCV protease NS3-4A substrates explain the lack of adaptive capability against protease inhibitors.

Inhibitors against the NS3-4A protease of hepatitis C virus (HCV) have proven to be useful drugs in the treatment of HCV infection. Although variants have been identified with mutations that confer resistance to these inhibitors, the mutations do not restore replicative fitness and no secondary mutations that rescue fitness have been found. To gain insight into the molecular mechanisms underlying the lack of fitness compensation, we screened known resistance mutations in infectious HCV cell culture with different genomic backgrounds. We observed that the Q41R mutation of NS3-4A efficiently rescues the replicative fitness in cell culture for virus variants containing mutations at NS3-Asp168 To understand how the Q41R mutation rescues activity, we performed protease activity assays complemented by molecular dynamics simulations, which showed that protease-peptide interactions far outside the targeted peptide cleavage sites mediate substrate recognition by NS3-4A and support protease cleavage kinetics. These interactions shed new light on the mechanisms by which NS3-4A cleaves its substrates, viral polyproteins and a prime cellular antiviral adaptor protein, the mitochondrial antiviral signaling protein MAVS. Peptide binding is mediated by an extended hydrogen-bond network in NS3-4A that was effectively optimized for protease-MAVS binding in Asp168 variants with rescued replicative fitness from NS3-Q41R. In the protease harboring NS3-Q41R, the N-terminal cleavage products of MAVS retained high affinity to the active site, rendering the protease susceptible for potential product inhibition. Our findings reveal delicately balanced protease-peptide interactions in viral replication and immune escape that likely restrict the protease adaptive capability and narrow the virus evolutionary space.

Triazole-Resistant Aspergillus fumigatus in an Israeli Patient with Chronic Cavitary Pulmonary Aspergillosis Due to a Novel E306K Substitution in Hmg1.

Triazole resistance in the pathogenic mold Aspergillus fumigatus has increased worldwide, posing a growing therapeutic challenge. Recently, mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase gene (hmg1) have been associated with triazole resistance. Here, we describe a novel E306K triazole resistance-conferring mutation in the HMG-CoA reductase gene from an Israeli patient with chronic cavitary pulmonary aspergillosis (CCPA).

Drug Resistance Mutation Frequency of Single-Genome Amplification-Derived HIV-1 Polymerase Genomes in the Cerebrospinal Fluid and Plasma of HIV-1-Infected Individuals under Nonsuppressive Therapy.

HIV-1 evolution in the cerebrospinal fluid (CSF) and plasma may result in discordant drug resistance mutations (DRMs) in the compartments. Single-genome amplification (SGA) was used to generate partial HIV-1 polymerase genomes in paired CSF and plasma samples from 12 HIV-1-positive participants in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) study who were classified as neurocognitively unimpaired or with various degrees of HIV-associated neurocognitive disorders (HAND). Subjects were viremic on combination antiretroviral therapy (cART). HIV-1 DRMs and phylogenetic characteristics were determined using the Stanford HIVdb program and phylogenetic analyses. Individual DRMs were identified more frequently in plasma than in paired CSF (P = 0.0078). Significant differences in the ratios of DRMs in CSF and plasma were found in 3 individuals with HAND (3/7 = 43%). Two HAND subjects (2/7 = 29%) demonstrated one DRM in CSF not identified in paired plasma. Longitudinal analyses (n = 4) revealed significant temporal differences in the ratios of DRMs in the compartments. Statistically significant differences in the frequency of DRMs in the CSF and plasma are readily found in those on nonsuppressive cART. While compartment-based DRM discordance was largely consistent with increased drug-selective pressures in the plasma, overrepresentation of DRMs in the central nervous system (CNS) can occur. Underlying mechanisms of HAND are complex and multifactorial. The clinical impact of DRM discordance on viral persistence and HAND pathogenesis remains unclear and warrants further investigation in larger, longitudinal cohorts.IMPORTANCE Several antiretroviral agents do not efficiently enter the CNS, and independent evolution of HIV-1 viral variants in the CNS and plasma can occur. We used single-genome amplification (SGA) in cross-sectional and longitudinal analyses to uniquely define both the identity and relative proportions of drug resistance mutations (DRMs) on individual HIV-1 polymerase genomes in the cerebrospinal fluid (CSF) and plasma in individuals with incomplete viral suppression and known neurocognitive status. Statistically significant differences in the ratio of DRMs in the CSF and plasma were readily found in those on nonsuppressive cART, and overrepresentation of DRMs in the CNS can occur. Although questions about the clinical significance of DRM discordance remain, in the quest for viral eradication, it is important to recognize that a significant, dynamic, compartment-based DRM ratio imbalance can exist, as it has the potential to go unnoticed in the setting of standard clinical drug resistance testing.

Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance.

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

Molecular basis of cross-resistance to acetohydroxy acid synthase-inhibiting herbicides in Sagittaria trifolia L.

Acetohydroxy acid synthase (AHAS)-inhibiting herbicides are one of the most commonly used herbicides for controlling the growth of Sagittaria trifolia L. in paddy fields in Northeastern China. In this study, we collected five suspected resistant populations of S. trifolia (R1-R5) from three different provinces of Northeastern China. The results of whole-plant bioassays revealed that those populations showed high level of resistance to bensulfuron-methyl with resistance index (GR50 R/S) ranging from 39.90 to 88.50. The results of AHAS-activity assays were consistent with the results of the whole-plant bioassays. The AHAS gene analysis showed that R2 and R3 populations contained Pro-197-Leu mutations that were highly resistant to penoxsulam; R1 and R4 populations contained Pro-197-Ser mutations that were highly resistant to bispyribac­sodium; R5 population contained Trp-574-Leu mutation that showed high resistance to IMI, PT, PTB and SU herbicides. The AHAS with resistance mutations showed less sensitivity to feedback inhibition by BCAAs and R genotypes had increased free BCAAs.