Resistance-conferring Mutations Research Articles

Applying whole genome sequencing to predict phenotypic drug resistance in Mycobacterium tuberculosis: leveraging 20 years of nationwide data from Denmark.

Infection with Mycobacterium tuberculosis remains one of the biggest causes of death from a single microorganism worldwide, and the continuous emergence of drug resistance aggravates our ability to cure the disease. New improved resistance detection methods are needed to provide adequate treatment, such as whole genome sequencing (WGS), which has been used increasingly to identify resistance-conferring mutations over the last decade. The steadily increasing knowledge of resistance-conferring mutations increases our ability to predict resistance based on genomic data alone. This study evaluates the performance of WGS to predict M. tuberculosis complex resistance. It compares WGS predictions with the phenotypic (culture-based) drug susceptibility results based on 20 years of nationwide Danish data. Analyzing 6,230 WGS-sequenced samples, the sensitivities for isoniazid, rifampicin, ethambutol, and pyrazinamide were 82.5% [78.0%-86.5%, 95% confidence interval (CI)], 97.3% (90.6%-99.7%, 95% CI), 58.0% (43.2%-71.8%, 95% CI), and 60.5% (49.0%-71.2%, 95% CI), respectively, and specificities were 99.8% (99.7%-99.9%, 95% CI), 99.8% (99.7%-99.9%, 95% CI), 99.4% (99.2%-99.6%, 95% CI), and 99.9% (99.7%-99.9%, 95% CI), respectively. A broader range of both sensitivities and specificities was observed for second-line drugs. The results conform with previously reported values and indicate that WGS is reliable for routine resistance detection in resource-rich tuberculosis low-incidence and low-resistance settings such as Denmark.

Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata).

The limited number of available antifungal drugs and the increasing number of fungal isolates that show drug or multidrug resistance pose a serious medical threat. Several yeast pathogens, such as Nakaseomyces glabratus (Candida glabrata), show a remarkable ability to develop drug resistance during treatment through the acquisition of genetic mutations. However, how stable this resistance and the underlying mutations are in non-selective conditions remains poorly characterized. The stability of acquired drug resistance has fundamental implications for our understanding of the appearance and spread of drug-resistant outbreaks and for defining efficient strategies to combat them. Here, we used an in vitro evolution approach to assess the stability under optimal growth conditions of resistance phenotypes and resistance-associated mutations that were previously acquired under exposure to antifungals. Our results reveal a remarkable stability of the resistant phenotype and the underlying mutations in a significant number of evolved populations, which conserved their phenotype for at least two months in the absence of drug-selective pressure. We observed a higher stability of anidulafungin resistance over fluconazole resistance, and of resistance-conferring point mutations as compared with aneuploidies. In addition, we detected accumulation of novel mutations in previously altered resistance-associated genes in non-selective conditions, which suggest a possible compensatory role. We conclude that acquired resistance, particularly to anidulafungin, is a long-lasting phenotype, which has important implications for the persistence and propagation of drug-resistant clinical outbreaks.

Diagnostic accuracy of the Xpert® MTB/XDR assay for detection of Isoniazid and second-line antituberculosis drugs resistance at central TB reference laboratory in Tanzania

IntroductionEarly diagnosis of tuberculosis (TB) and universal access to drug-susceptibility testing (DST) are critical elements of the WHO End TB Strategy. Current rapid tests (e.g., Xpert® MTB/RIF and Ultra-assays) can detect rifampicin resistance-conferring mutations, but cannot detect resistance to Isoniazid and second-line anti-TB agents. Although Line Probe Assay is capable of detecting resistance to second-line anti-TB agents, it requires sophisticated laboratory infrastructure and advanced skills which are often not readily available in settings replete with TB. A rapid test capable of detecting Isoniazid and second-line anti-TB drug resistance is highly needed.MethodsWe conducted a diagnostic accuracy study to evaluate a new automated Xpert MTB/XDR 10-colour assay for rapid detection of Isoniazid and second-line drugs, including ethionamide, fluoroquinolones, and injectable drugs (Amikacin, Kanamycin, and Capreomycin). Positive Xpert MTB/RIF respiratory specimens were prospectively collected through routine diagnosis and surveillance of drug resistance at the Central TB Reference Laboratory in Tanzania. Specimens were tested by both Xpert XDR assay and LPA against culture-based phenotypic DST as the reference standard.FindingsWe analysed specimens from 151 TB patients with a mean age (SD) of 36.2 (12.7) years. The majority (n = 109, 72.2%) were males. The sensitivity for Xpert MTB/XDR was 93.5% (95% CI, 87.4–96.7); for Isoniazid, 96.6 (95% CI, 92.1–98.6); for Fluoroquinolone, 98.7% (95% Cl 94.8–99.7); for Amikacin, 96.6%; and (95% CI 92.1–98.6) for Ethionamide. Ethionamide had the lowest specificity of 50% and the highest was 100% for Fluoroquinolone. The diagnostic performance was generally comparable to that of LPA with slight variations between the two assays. The non-determinate rate (i.e., invalid M. tuberculosis complex detection) of Xpert MTB/XDR was 2·96%.ConclusionThe Xpert MTB/XDR demonstrated high sensitivity and specificity for detecting resistance to Isoniazid, Fluoroquinolones, and injectable agents. This assay can be used in clinical settings to facilitate rapid diagnosis of mono-isoniazid and extensively drug-resistant TB.

Overcoming Secondary Mutations of Type II Kinase Inhibitors.

Type II kinase inhibitors bind in the "DFG-out" kinase conformation and are generally considered to be more potent and selective than type I inhibitors, which target a DFG-in conformation. Nine type II inhibitors are currently clinically approved, with more undergoing clinical development. Resistance-conferring secondary mutations emerged with the first series of type II inhibitors, most commonly at residues within the kinase activation loop and at the "gatekeeper" position. Recently, new inhibitors have been developed to overcome such mutations; however, mutations activating other pathways (and/or other targets) have subsequently emerged on occasion. Here, we systematically summarize the secondary mutations that confer resistance to type II inhibitors, the structural basis for resistance, newer inhibitors designed to overcome resistance, as well as the challenges and opportunities for the development of new inhibitors to overcome secondary kinase domain mutations.

Frequency-Dependent Ecological Interactions Increase the Prevalence, and Shape the Distribution, of Preexisting Drug Resistance

The evolution of resistance remains one of the primary challenges for modern medicine, from infectious diseases to cancers. Many of these resistance-conferring mutations often carry a substantial fitness cost in the absence of treatment. As a result, we would expect these mutants to undergo purifying selection and be rapidly driven to extinction. Nevertheless, preexisting resistance is frequently observed from drug-resistant malaria to targeted cancer therapies in non-small-cell lung cancer (NSCLC) and melanoma. Solutions to this apparent paradox have taken several forms, from spatial rescue to simple mutation supply arguments. Recently, in an evolved resistant NSCLC cell line, we found that frequency-dependent ecological interactions between ancestor and resistant mutant ameliorate the cost of resistance in the absence of treatment. Here, we hypothesize that frequency-dependent ecological interactions in general play a major role in the prevalence of preexisting resistance. We combine numerical simulations with robust analytical approximations to provide a rigorous mathematical framework for studying the effects of frequency-dependent ecological interactions on the evolutionary dynamics of preexisting resistance. First, we find that ecological interactions significantly expand the parameter regime under which we expect to observe preexisting resistance. Next, even when positive ecological interactions between mutants and ancestors are rare, these resistant clones provide the primary mode of evolved resistance because even weak positive interaction leads to significantly longer extinction times. We then find that even in the case where mutation supply alone is sufficient to predict preexisting resistance, frequency-dependent ecological forces still contribute a strong evolutionary pressure that selects for increasingly positive ecological effects (negative frequency-dependent selection). Finally, we genetically engineer several of the most common clinically observed resistance mechanisms to targeted therapies in NSCLC, a treatment notorious for preexisting resistance. We find that each engineered mutant displays a positive ecological interaction with their ancestor. As a whole, these results suggest that frequency-dependent ecological effects can play a crucial role in shaping the evolutionary dynamics of preexisting resistance. Published by the American Physical Society 2024

Antibiotic resistance in Mycobacterium tuberculosis alters tolerance to cell wall-targeting inhibitors.

A limited ability to eliminate drug-resistant strains of Mycobacterium tuberculosis is a major contributor to the morbidity of TB. Complicating this problem, little is known about how drug resistance-conferring mutations alter the ability of M. tuberculosis to tolerate antibiotic killing. Here, we investigated if drug-resistant strains of M. tuberculosis have an altered ability to tolerate killing by cell wall-targeting inhibitors. Bacterial killing and MIC assays were used to test for antibiotic tolerance and synergy against a panel of drug-resistant M. tuberculosis strains. Our results demonstrate that vancomycin and thioacetazone exhibit increased killing of diverse drug-resistant strains. Mutations in mmaA4 and mmpL3 increased vancomycin killing, which was consistent with vancomycin synergizing with thioacetazone and MmpL3-targeting inhibitors. In contrast, mutations in the mce1 operon conferred tolerance to vancomycin. Overall, this work demonstrates how drug-resistant strains experience perturbations in cell-wall production that alters their tolerance to killing by cell wall-targeting inhibitors.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone

ObjectivesWe evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. MethodsFTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. ResultsOf the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DiscussionFTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.

A protective role for type I interferon signaling following infection with Mycobacterium tuberculosis carrying the rifampicin drug resistance-conferring RpoB mutation H445Y.

Interleukin-1 (IL-1) signaling is essential for controlling virulent Mycobacterium tuberculosis (Mtb) infection since antagonism of this pathway leads to exacerbated pathology and increased susceptibility. In contrast, the triggering of type I interferon (IFN) signaling is associated with the progression of tuberculosis (TB) disease and linked with negative regulation of IL-1 signaling. However, mice lacking IL-1 signaling can control Mtb infection if infected with an Mtb strain carrying the rifampin-resistance conferring mutation H445Y in its RNA polymerase β subunit (rpoB-H445Y Mtb). The mechanisms that govern protection in the absence of IL-1 signaling during rpoB-H445Y Mtb infection are unknown. In this study, we show that in the absence of IL-1 signaling, type I IFN signaling controls rpoB-H445Y Mtb replication, lung pathology, and excessive myeloid cell infiltration. Additionally, type I IFN is produced predominantly by monocytes and recruited macrophages and acts on LysM-expressing cells to drive protection through nitric oxide (NO) production to restrict intracellular rpoB-H445Y Mtb. These findings reveal an unexpected protective role for type I IFN signaling in compensating for deficiencies in IL-1 pathways during rpoB-H445Y Mtb infection.

Whole-genome sequencing of clinical isolates from tuberculosis patients in India: real-world data indicates a high proportion of pre-XDR cases.

The current study is based on real-world data on the TB drug-resistance profile by whole-genome sequencing of 600 clinical samples from patients with TB in India. This study indicates the clinicians' reasons for sending samples for WGS, which is for difficult-to-treat cases and/or relapse and treatment failure. The study reports a significant proportion of cases with pre-XDR-TB strains that warrant policy makers' attention. It reflects the current iterative nature of the diagnostic tests under programmatic conditions that leads to delays in appropriate diagnosis and empirical treatment. India had an estimated burden of 2.95 million TB cases in 2020 and 135,000 multidrug-resistant cases. However, WGS profiles of M.tb from India remains disproportionately poorly represented. This study adds a significant body of data on the mutation profiles seen in M.tb isolated from patients with TB in India, mutations outside the RRDR, disputed mutations, and resistance-conferring mutations to newer drugs such as bedaquiline and linezolid.

The recent rapid expansion of multidrug resistant Ural lineage Mycobacterium tuberculosis in Moldova

The projected trajectory of multidrug resistant tuberculosis (MDR-TB) epidemics depends on the reproductive fitness of circulating strains of MDR M. tuberculosis (Mtb). Previous efforts to characterize the fitness of MDR Mtb have found that Mtb strains of the Beijing sublineage (Lineage 2.2.1) may be more prone to develop resistance and retain fitness in the presence of resistance-conferring mutations than other lineages. Using Mtb genome sequences from all culture-positive cases collected over two years in Moldova, we estimate the fitness of Ural (Lineage 4.2) and Beijing strains, the two lineages in which MDR is concentrated in the country. We estimate that the fitness of MDR Ural strains substantially exceeds that of other susceptible and MDR strains, and we identify several mutations specific to these MDR Ural strains. Our findings suggest that MDR Ural Mtb has been transmitting efficiently in Moldova and poses a substantial risk of spreading further in the region.

Patterns and profiles of drug resistance-conferring mutations in Mycobacterium tuberculosis genotypes isolated from tuberculosis-suspected attendees of spiritual holy water sites in Northwest Ethiopia.

This study examined the patterns and frequency of genetic changes responsible for resistance to first-line (rifampicin and isoniazid), fluoroquinolones, and second-line injectable drugs in drug-resistant Mycobacterium tuberculosis (MTB) isolated from culture-positive pulmonary tuberculosis (PTB) symptomatic attendees of spiritual holy water sites (HWSs) in the Amhara region. From June 2019 to March 2020, a cross-sectional study was carried out. A total of 122 culture-positive MTB isolates from PTB-suspected attendees of HWSs in the Amhara region were evaluated for their drug resistance profiles, and characterized gene mutations conferring resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs), and second-line injectable drugs (SLIDs) using GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0. Drug-resistant MTB isolates were Spoligotyped following the manufacturer's protocol. Genetic changes (mutations) responsible for resistance to RIF, INH, and FLQs were identified in 15/122 (12.3%), 20/122 (16.4%), and 5/20 (25%) of MTB isolates, respectively. In RIF-resistant, rpoB/Ser531Lue (n = 12, 80%) was most frequent followed by His526Tyr (6.7%). Amongst INH-resistant isolates, katG/Ser315Thr1 (n = 19, 95%) was the most frequent. Of 15 MDR-TB, the majority (n = 12, 80%) isolates had mutations at both rpoB/Ser531Leu and katG/Ser315Thr1. All 20 INH and/or RIF-resistant isolates were tested with the MTBDRsl VER 2.0, yielding 5 FLQs-resistant isolates with gene mutations at rpoB/Ser531Lue, katG/Ser315Thr1, and gyrA/Asp94Ala genes. Of 20 Spoligotyped drug-resistant MTB isolates, the majority (n = 11, 55%) and 6 (30%) were SIT149/T3-ETH and SIT21/CAS1-Kili sublineages, respectively; and they were any INH-resistant (mono-hetero/multi-). Of 15 RIF-resistant (RR/MDR-TB) isolates, 7 were SIT149/T3-ETH, while 6 were SIT21/CAS1-Kili sublineages. FLQ resistance was detected in four SIT21/CAS1-Kili lineages. In the current study, the most common gene mutations responsible for resistance to INH, RIF, and FLQs were identified. SIT149/T3-ETH and SIT21/CAS1-Kili constitute the majority of drug-resistant TB (DR-TB) isolates. To further understand the complete spectrum of genetic changes/mutations and related genotypes, a sequencing technology is warranted.

An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations.

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance.

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.

Co-resistance to isoniazid and second-line anti-tuberculosis drugs in isoniazid-resistant tuberculosis at a tertiary care hospital in Thailand.

The prevalence of isoniazid-resistant tuberculosis (Hr-TB) is the highest among other types of drug-resistant tuberculosis. Currently, the World Health Organization (WHO) guidelines recommend the treatment of Hr-TB with rifampicin, ethambutol, pyrazinamide, and levofloxacin for 6 months. The susceptibility profiles of Hr-TB clinical isolates, especially when they are co-resistant to second-line drugs, are critical in the selection of the appropriate treatment regimen to prevent treatment failure. This study highlights the susceptibility profiles of the WHO-recommended treatment regimen in Hr-TB clinical isolates from a tertiary care hospital in Thailand and the concordance and importance of using the phenotypic drug susceptibility testing or genotypic drug susceptibility testing for accurate and comprehensive interpretation of results.

Compensatory mutations are associated with increased in vitro growth in resistant clinical samples of Mycobacterium tuberculosis.

Mutations in Mycobacterium tuberculosis associated with resistance to antibiotics often come with a fitness cost for the bacteria. Resistance to the first-line drug rifampicin leads to lower competitive fitness of M. tuberculosis populations when compared to susceptible populations. This fitness cost, introduced by resistance mutations in the RNA polymerase, can be alleviated by compensatory mutations (CMs) in other regions of the affected protein. CMs are of particular interest clinically since they could lock in resistance mutations, encouraging the spread of resistant strains worldwide. Here, we report the statistical inference of a comprehensive set of CMs in the RNA polymerase of M. tuberculosis, using over 70 000 M. tuberculosis genomes that were collated as part of the CRyPTIC project. The unprecedented size of this data set gave the statistical tests more power to investigate the association of putative CMs with resistance-conferring mutations. Overall, we propose 51 high-confidence CMs by means of statistical association testing and suggest hypotheses for how they exert their compensatory mechanism by mapping them onto the protein structure. In addition, we were able to show an association of CMs with higher in vitro growth densities, and hence presumably with higher fitness, in resistant samples in the more virulent M. tuberculosis lineage 2. Our results suggest the association of CM presence with significantly higher in vitro growth than for wild-type samples, although this association is confounded with lineage and sub-lineage affiliation. Our findings emphasize the integral role of CMs and lineage affiliation in resistance spread and increases the urgency of antibiotic stewardship, which implies accurate, cheap and widely accessible diagnostics for M. tuberculosis infections to not only improve patient outcomes but also prevent the spread of resistant strains.

Modeling stress-induced responses: plasticity in continuous state space and gradual clonal evolution.

Mathematical models of cancer and bacterial evolution have generally stemmed from a gene-centric framework, assuming clonal evolution via acquisition of resistance-conferring mutations and selection of their corresponding subpopulations. More recently, the role of phenotypic plasticity has been recognized and models accounting for phenotypic switching between discrete cell states (e.g., epithelial and mesenchymal) have been developed. However, seldom do models incorporate both plasticity and mutationally driven resistance, particularly when the state space is continuous and resistance evolves in a continuous fashion. In this paper, we develop a framework to model plastic and mutational mechanisms of acquiring resistance in a continuous gradual fashion. We use this framework to examine ways in which cancer and bacterial populations can respond to stress and consider implications for therapeutic strategies. Although we primarily discuss our framework in the context of cancer and bacteria, it applies broadly to any system capable of evolving via plasticity and genetic evolution.

Pancreatic cancer acquires resistance to MAPK pathway inhibition by clonal expansion and adaptive DNA hypermethylation

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data.ResultsWe found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells.ConclusionOverall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.

Transmission of Fluoroquinolones Resistance among Multidrug-Resistant Tuberculosis in Shanghai, China: A Retrospective population-based Genomic Epidemiology Study

ABSTRACT Fluoroquinolones (FQ) are essential for the treatment of multidrug-resistant tuberculosis (MDR-TB). The FQ resistance (FQ-R) rate in MDR-TB in China and its risk factors remain poorly understood. We conducted a retrospective, population-based genomic epidemiology study of MDR-TB patients in Shanghai, China, from 2009 to 2018. A genomic cluster was defined as strains with genetic distances ≤ 12 single nucleotide polymorphisms. The transmitted FQ-R was defined as the same FQ resistance-conferring mutations shared by ≥ 2 strains in a genomic cluster. We used multivariable logistic regression analysis to identify the risk factors for drug resistance. Among the total 850 MDR-TB patients included in the study, 72.8% (619/850) were male, the median age was 39 (interquartile range 28, 55) years, 52.7% (448/850) were migrants, and 34.5% (293/850) were previously treated patients. Most of the MDR-TB strains belong to the Beijing lineage (91.7%, 779/850). Overall, the genotypic resistance rate of FQ was 34.7% (295/850), and 47.1% (139/295) FQ-R patients were in genomic clusters, of which 98 (33.2%, 98/295) were presumed as transmitted FQ-R. Patients with treatment-naïve (aOR = 1.84; 95% CI: 1.09, 3.16), diagnosed in a district-level hospital (aOR = 2.69; 95% CI: 1.56, 4.75), and streptomycin resistance (aOR = 3.69; 95% CI: 1.65, 9.42) were significantly associated with the transmission of FQ-R. In summary, the prevalence of FQ-R among MDR-TB patients was high in Shanghai, and at least one-third were transmitted. Enforced interventions including surveillance of FQ drug susceptibility testing and screening among MDR-TB before initiation of treatment were urgently needed.

Fungicide-Resistant Botrytis in Forest Nurseries May Impact Disease Control in Norway Spruce.

Gray mold, caused by Botrytis spp., is a serious problem in Norway spruce seedling production in forest nurseries. From 2013 to 2019, 125 isolates of Botrytis were obtained from eight forest nurseries in Norway: 53 from Norway spruce seedlings, 16 from indoor air, 52 from indoor surfaces, and four from weeds growing close to seedlings. The majority of isolates were identified as B. cinerea, and over 60% of these were characterized as Botrytis group S. B. pseudocinerea isolates were obtained along with isolates with DNA sequence similarities to B. prunorum. Fungicide resistance was assessed with a mycelial growth assay, and resistance was found for the following: boscalid (8.8%), fenhexamid (33.6%), fludioxonil (17.6%), pyraclostrobin (36.0%), pyrimethanil (13.6%), and thiophanate-methyl (50.4%). Many isolates (38.4%) were resistant to two to six different fungicides. A selection of isolates was analyzed for the presence of known resistance-conferring mutations in the cytb, erg27, mrr1, sdhB, and tubA genes, and mutations leading to G143A, F412S, ΔL497, H272R, and E198A/F200Y were detected, respectively. Detection of fungicide resistance in Botrytis from Norway spruce and forest nursery facilities reinforces the necessity of employing resistance management strategies to improve control and delay development of fungicide resistance in the gray mold pathogens.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Meta-analysis on Plasmodium falciparum sulfadoxine-pyrimethamine resistance-conferring mutations in India identifies hot spots for genetic surveillance

BackgroundIndia is on track to eliminate malaria by 2030 but emerging resistance to first-line antimalarials is a recognised threat. Two instances of rapid development, spread, and natural selection of drug-resistant mutant parasites in India (chloroquine across the country and artesunate + sulfadoxine-pyrimethamine [AS+SP] in the northeastern states) translated into drug policy changes for Plasmodium falciparum malaria in 2010 and 2013, respectively. Considering these rapid changes in the SP drug resistance-conferring mutation profile of P. falciparum, there is a need to systematically monitor the validated mutations in Pfdhfr and Pfdhps genes across India alongside AS+SP therapeutic efficacy studies. There has been no robust, systematic countrywide surveillance reported for these parameters in India, hence the current study was undertaken. MethodsStudies that reported data on WHO-validated SP resistance markers in P. falciparum across India from 2008 to January 2023 were included. Five major databases, PubMedⓇ, Web of ScienceTM, ScopusⓇ, EmbaseⓇ, and Google Scholar, were exhaustively searched. Individual and pooled prevalence estimates of mutations were obtained through random- and fixed-effect models. Data were depicted using forest plots created with a 95% confidence interval. The study is registered with PROSPERO (CRD42021236012). ResultsA total of 37 publications, and 533 Pfdhfr and 134 Pfdhps National Centre of Biotechnology Information (NCBI) DNA sequences were included from >4000 samples. The study included information from 80 districts, 21 states and 3 union territories (UTs) from India. The two PfDHFR mutations, C59R (62%) and S108N (74%), were the most prevalent mutations (pooled estimates 61% and 71%, respectively) and appeared to be stabilised/fixed. Although rarest overall, the prevalence of I164L was observed to be as high as 32%. The PfDHFR double mutants were the most prevalent overall (51%; pooled 42%). The prevalence of triple and quadruple mutations was 6% and 5%, respectively, and is an immediate concern for some states. The most prevalent PfDHPS mutation was A437G (39%), followed by K540E (25%) and A581G (12%). There was a low overall prevalence of PfDHFR/PfDHPS quintuple and sextuple mutations but surveillance for these mutations is critical for some areas. ConclusionThe analyses span the two critical policy changes, highlight the areas of concern, and guide policymakers in strategising and refining the anti-malaria drug policy for malaria elimination. The results of the analyses also highlight the SP-resistance hot spots, critical gaps and challenges, and indicate that focal and local malaria genetic surveillance (including drug-resistance markers) is needed until malaria is successfully eliminated.