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PloS One 2024To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency...
To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency testing performed DST, and the sensitivity, specificity, reproducibility, and accordance rate were calculated from data of 13 rounds proficiency testing results for DST from 2008 to 2021. A total of 30 and 20 strains of Mycobacterium tuberculosis with known susceptibility results were sent to each laboratory in 2008 to 2019, 2020 and 2021, respectively. The number of participating laboratories ranged from 30 in 2009 to 546 in 2021. L-J DST was the predominant method. The specificity presented relatively higher than sensitivity. Improvement of specificity were observed for all drugs through the years, while sensitivity did not show improvement for amikacin and capreomycin. Accordance rate of pyrazinamide and kanamycin and reproducibility of capreomycin and pyrazinamide were not significantly improved through the years. Most of the participating laboratories significantly improved the quality of their DST through the consecutive rounds of proficiency testing except for second-line injectable drugs and pyrazinamide. The results highlight the importance of developing novel and/or improving existing methods for phenotypic DST for certain drugs.
Topics: Mycobacterium tuberculosis; Microbial Sensitivity Tests; China; Antitubercular Agents; Humans; Laboratory Proficiency Testing; Reproducibility of Results; Phenotype; Amikacin; Pyrazinamide
PubMed: 38809914
DOI: 10.1371/journal.pone.0304265 -
Infection and Drug Resistance 2024We compared the MeltPro assay to whole-genome sequencing (WGS) to investigate the molecular characterization of second-line injectable drug (SLID) resistance in...
OBJECTIVE
We compared the MeltPro assay to whole-genome sequencing (WGS) to investigate the molecular characterization of second-line injectable drug (SLID) resistance in multidrug-resistant tuberculosis (MDR-TB) isolates in Chongqing, China.
METHODS
A total of 122 MDR-TB patient isolates were collected between March 2019 and June 2020 from Chongqing Municipality, China. Conventional drug-susceptibility testing was performed using the proportion method, followed to generate minimum inhibitory concentrations (MICs) of SLIDs determined by microplate alamarblue assay. All strains were subjected to both MeltPro and WGS assays.
RESULTS
Among 122 MDR-TB isolates, 30 (24.6%), 22 (18.0%), and 14 (11.5%) were resistant to kanamycin (KM), amikacin (AM), and capreomycin (CM), respectively. Of the 31 SLID-resistant isolates, 24 (77.4%, 24/31) isolates harbored mutations in the gene, with the most prevalent mutations in A1401G (22/24, 91.7%). Mutation in A1401G was associated with high levels of resistance to KM (MIC, ≥40 μg/mL) and AM (MIC, ≥64 μg/mL), but disparities in CM-resistance levels. Using phenotypic drug-susceptibility testing as gold standard, we found that the overall sensitivity of MeltPro and WGS was 87.1% and 90.32% and specificity 100% and 97.8%, respectively. Seven isolates had discordant results between phenotypic and genotypic resistance of SLIDs.
CONCLUSION
MeltPro is a promising diagnostic tool for accurate identification of SLID-resistant MTB isolates with mutations in the and genes. There was a disparity between MeltPro with WGS results in the proportion of heterogeneous drug-resistant bacteria with mutation and limited probes. Resistance mechanisms other than genetic mutations will affect the consistency of MeltPro and WGS with phenotypic drug-susceptibility results.
PubMed: 38807773
DOI: 10.2147/IDR.S459142 -
The Lancet. Infectious Diseases May 2024Targeted next-generation sequencing (NGS) can rapidly and simultaneously detect mutations associated with resistance to tuberculosis drugs across multiple gene targets....
BACKGROUND
Targeted next-generation sequencing (NGS) can rapidly and simultaneously detect mutations associated with resistance to tuberculosis drugs across multiple gene targets. The use of targeted NGS to diagnose drug-resistant tuberculosis, as described in publicly available data, has not been comprehensively reviewed. We aimed to identify targeted NGS assays that diagnose drug-resistant tuberculosis, determine how widely this technology has been used, and assess the diagnostic accuracy of these assays.
METHODS
In this systematic review and meta-analysis, we searched MEDLINE, Embase, Cochrane Library, Web of Science Core Collection, Global Index Medicus, Google Scholar, ClinicalTrials.gov, and the WHO International Clinical Trials Registry Platform for published and unpublished reports on targeted NGS for drug-resistant tuberculosis from Jan 1, 2005, to Oct 14, 2022, with updates to our search in Embase and Google Scholar until Feb 13, 2024. Studies eligible for the systematic review described targeted NGS approaches to predict drug resistance in Mycobacterium tuberculosis infections using primary samples, reference strain collections, or cultured isolates from individuals with presumed or confirmed tuberculosis. Our search had no limitations on study type or language, although only reports in English, German, and French were screened for eligibility. For the meta-analysis, we included test accuracy studies that used any reference standard, and we assessed risk of bias using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The primary outcomes for the meta-analysis were sensitivity and specificity of targeted NGS to diagnose drug-resistant tuberculosis compared to phenotypic and genotypic drug susceptibility testing. We used a Bayesian bivariate model to generate summary receiver operating characteristic plots and diagnostic accuracy measures, overall and stratified by drug and sample type. This study is registered with PROSPERO, CRD42022368707.
FINDINGS
We identified and screened 2920 reports, of which 124 were eligible for our systematic review, including 37 review articles and 87 reports of studies collecting samples for targeted NGS. Sequencing was mainly done in the USA (14 [16%] of 87), western Europe (ten [11%]), India (ten [11%]), and China (nine [10%]). We included 24 test accuracy studies in the meta-analysis, in which 23 different tuberculosis drugs or drug groups were assessed, covering first-line drugs, injectable drugs, and fluoroquinolones and predominantly comparing targeted NGS with phenotypic drug susceptibility testing. The combined sensitivity of targeted NGS across all drugs was 94·1% (95% credible interval [CrI] 90·9-96·3) and specificity was 98·1% (97·0-98·9). Sensitivity for individual drugs ranged from 76·5% (52·5-92·3) for capreomycin to 99·1% (98·3-99·7) for rifampicin; specificity ranged from 93·1% (88·0-96·3) for ethambutol to 99·4% (98·3-99·8) for amikacin. Diagnostic accuracy was similar for primary clinical samples and culture isolates overall and for rifampicin, isoniazid, ethambutol, streptomycin, and fluoroquinolones, and similar after excluding studies at high risk of bias (overall sensitivity 95·2% [95% CrI 91·7-97·1] and specificity 98·6% [97·4-99·3]).
INTERPRETATION
Targeted NGS is highly sensitive and specific for detecting drug resistance across panels of tuberculosis drugs and can be performed directly on clinical samples. There is a paucity of data on performance for some currently recommended drugs. The barriers preventing the use of targeted NGS to diagnose drug-resistant tuberculosis in high-burden countries need to be addressed.
FUNDING
National Institutes of Allergy and Infectious Diseases and Swiss National Science Foundation.
PubMed: 38795712
DOI: 10.1016/S1473-3099(24)00263-9 -
BMC Genomics May 2024Tuberculosis (TB) represents a major global health challenge. Drug resistance in Mycobacterium tuberculosis (MTB) poses a substantial obstacle to effective TB treatment....
BACKGROUND
Tuberculosis (TB) represents a major global health challenge. Drug resistance in Mycobacterium tuberculosis (MTB) poses a substantial obstacle to effective TB treatment. Identifying genomic mutations in MTB isolates holds promise for unraveling the underlying mechanisms of drug resistance in this bacterium.
METHODS
In this study, we investigated the roles of single nucleotide variants (SNVs) in MTB isolates resistant to four antibiotics (moxifloxacin, ofloxacin, amikacin, and capreomycin) through whole-genome analysis. We identified the drug-resistance-associated SNVs by comparing the genomes of MTB isolates with reference genomes using the MuMmer4 tool.
RESULTS
We observed a strikingly high proportion (94.2%) of MTB isolates resistant to ofloxacin, underscoring the current prevalence of drug resistance in MTB. An average of 3529 SNVs were detected in a single ofloxacin-resistant isolate, indicating a mutation rate of approximately 0.08% under the selective pressure of ofloxacin exposure. We identified a set of 60 SNVs associated with extensively drug-resistant tuberculosis (XDR-TB), among which 42 SNVs were non-synonymous mutations located in the coding regions of nine key genes (ctpI, desA3, mce1R, moeB1, ndhA, PE_PGRS4, PPE18, rpsA, secF). Protein structure modeling revealed that SNVs of three genes (PE_PGRS4, desA3, secF) are close to the critical catalytic active sites in the three-dimensional structure of the coding proteins.
CONCLUSION
This comprehensive study elucidates novel resistance mechanisms in MTB against antibiotics, paving the way for future design and development of anti-tuberculosis drugs.
Topics: Mycobacterium tuberculosis; Polymorphism, Single Nucleotide; Whole Genome Sequencing; Genome, Bacterial; Humans; Drug Resistance, Bacterial; Microbial Sensitivity Tests; Mutation; Antitubercular Agents; Bacterial Proteins
PubMed: 38745294
DOI: 10.1186/s12864-024-10390-3 -
BMC Genomics Apr 2024Drug-resistant tuberculosis (TB) is a major threat to global public health. Whole-genome sequencing (WGS) is a useful tool for species identification and drug resistance...
BACKGROUND
Drug-resistant tuberculosis (TB) is a major threat to global public health. Whole-genome sequencing (WGS) is a useful tool for species identification and drug resistance prediction, and many clinical laboratories are transitioning to WGS as a routine diagnostic tool. However, user-friendly and high-confidence automated bioinformatics tools are needed to rapidly identify M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), detect drug resistance, and further guide treatment options.
RESULTS
We developed GenoMycAnalyzer, a web-based software that integrates functions for identifying MTBC and NTM species, lineage and spoligotype prediction, variant calling, annotation, drug-resistance determination, and data visualization. The accuracy of GenoMycAnalyzer for genotypic drug susceptibility testing (gDST) was evaluated using 5,473 MTBC isolates that underwent phenotypic DST (pDST). The GenoMycAnalyzer database was built to predict the gDST for 15 antituberculosis drugs using the World Health Organization mutational catalogue. Compared to pDST, the sensitivity of drug susceptibilities by the GenoMycAnalyzer for first-line drugs ranged from 95.9% for rifampicin (95% CI 94.8-96.7%) to 79.6% for pyrazinamide (95% CI 76.9-82.2%), whereas those for second-line drugs ranged from 98.2% for levofloxacin (95% CI 90.1-100.0%) to 74.9% for capreomycin (95% CI 69.3-80.0%). Notably, the integration of large deletions of the four resistance-conferring genes increased gDST sensitivity. The specificity of drug susceptibilities by the GenoMycAnalyzer ranged from 98.7% for amikacin (95% CI 97.8-99.3%) to 79.5% for ethionamide (95% CI 76.4-82.3%). The incorporated Kraken2 software identified 1,284 mycobacterial species with an accuracy of 98.8%. GenoMycAnalyzer also perfectly predicted lineages for 1,935 MTBC and spoligotypes for 54 MTBC.
CONCLUSIONS
GenoMycAnalyzer offers both web-based and graphical user interfaces, which can help biologists with limited access to high-performance computing systems or limited bioinformatics skills. By streamlining the interpretation of WGS data, the GenoMycAnalyzer has the potential to significantly impact TB management and contribute to global efforts to combat this infectious disease. GenoMycAnalyzer is available at http://www.mycochase.org .
Topics: Humans; Antitubercular Agents; Mycobacterium tuberculosis; Microbial Sensitivity Tests; Tuberculosis, Multidrug-Resistant; Nontuberculous Mycobacteria; Drug Resistance; Internet
PubMed: 38643090
DOI: 10.1186/s12864-024-10320-3 -
Communications Biology Dec 2023Metagenomic-based studies have predicted an extraordinary number of potential antibiotic-resistance genes (ARGs). These ARGs are hidden in various environmental bacteria...
Metagenomic-based studies have predicted an extraordinary number of potential antibiotic-resistance genes (ARGs). These ARGs are hidden in various environmental bacteria and may become a latent crisis for antibiotic therapy via horizontal gene transfer. In this study, we focus on a resistance gene cph, which encodes a phosphotransferase (Cph) that confers resistance to the antituberculosis drug capreomycin (CMN). Sequence Similarity Network (SSN) analysis classified 353 Cph homologues into five major clusters, where the proteins in cluster I were found in a broad range of actinobacteria. We examine the function and antibiotics targeted by three putative resistance proteins in cluster I via biochemical and protein structural analysis. Our findings reveal that these three proteins in cluster I confer resistance to CMN, highlighting an important aspect of CMN resistance within this gene family. This study contributes towards understanding the sequence-structure-function relationships of the phosphorylation resistance genes that confer resistance to CMN.
Topics: Capreomycin; Anti-Bacterial Agents; Bacteria; Genes, Bacterial; Immunity, Innate
PubMed: 38114770
DOI: 10.1038/s42003-023-05681-6 -
Infection and Drug Resistance 2023New antituberculosis drugs have recently been approved for the treatment of multidrug-resistant tuberculosis TB (MDR-TB). We aimed to describe the distributions of...
BACKGROUND
New antituberculosis drugs have recently been approved for the treatment of multidrug-resistant tuberculosis TB (MDR-TB). We aimed to describe the distributions of bedaquiline, delamanid, linezolid, clofazimine, and capreomycin MIC values for .
METHODS
clinical isolates were originally isolated from 2020 to 2021 from 1452 different pulmonary tuberculosis patients of the Shanghai Pulmonary Hospital in China. The drug susceptibility testing was performed using the Sensititre custom plates (SHTBMY) (TREK Diagnostic Systems, Thermo Fisher Scientific In., USA) consisting of a 96-well microtitre plate containing 4 (bedaquiline, delamanid, clofazimine, capreomycin) antimicrobial agents. MICs were determined for linezolid using a microdilution method.
RESULTS
Based on the latest definitions, 156 (10.74%) were MDR-TB, 93 (6.40%) were pre-XDR-TB, and 27 (1.86%) were XDR-TB. The rate of BDQ resistance in cases of MDR-TB was 7.69%, while it was observed to be 10.75% in cases of pre-XDR-TB, and significantly higher at 37.04% in cases of XDR-TB. The lowest rate of drug resistance against was DLM (0.14%). For LZD, 11 (0.76%) clinical isolates were resistant, based on the CLSI breakpoint of 1μg/mL. The five strains with a MIC value of >32 for LZD resistance were XDR-TB isolates. Among all MDR, pre-XDR, and XDR isolates tested, LZD' MIC increased from 0.25 and 0.5 to 1μg/mL. The MIC value of LZD against XDR-TB isolates was 32μg/mL. For CFZ, six isolates with elevated MICs of ≥2μg/mL. CFZ's MIC and MIC values in all isolates were 0.12μg/mL and 0.25μg/mL, respectively.
CONCLUSION
The study findings indicate that BDQ, DLM, CFZ, and LZD may exhibited excellent in vitro activity against MDR-TB isolates. Detection of resistance to BDQ and LZD was alarming for XDR-TB isolates. It is necessary to perform universal drug sensitivity testing for , especially MDR-TB and XDR-TB patients.
PubMed: 38107433
DOI: 10.2147/IDR.S440711 -
New Microbes and New Infections Oct 2023Tuberculosis (TB), caused by (MTB), is one of the top infectious killer diseases in the world. The emergence of drug-resistant MTB strains has thrown challenges in...
BACKGROUND
Tuberculosis (TB), caused by (MTB), is one of the top infectious killer diseases in the world. The emergence of drug-resistant MTB strains has thrown challenges in controlling TB worldwide. This study investigated the prevalence of drug-resistant tuberculosis in the states of Nigeria and the risk factors that can increase the incidence of tuberculosis.
METHODS
The study is a cross-sectional epidemiological research carried out in the six senatorial districts of Ekiti and Ondo states, Nigeria, between February 2019 and January 2020. A structured questionnaire was administered to 1203 respondents for socio-demographic information, and sputum samples were collected from them for TB investigation. GeneXpert technique was used to diagnose TB from the sputum samples, followed by bacterial isolation using Löweinstein-Jensen medium and antibiotic susceptibility testing.
RESULTS
Prevalence of TB in the two states combined was 15 %; with 13.8 % for Ekiti state and 16.1 % for Ondo State. The distribution of TB in the senatorial districts was such that: Ondo South > Ekiti Central > Ekiti South > Ondo North > Ekiti North > Ondo Central. The risk factors identified for TB prevalence in two states were gender, male > female (OR = 0.548, p = 0.004); overcrowding (OR = 0.733, p = 0.026); room size (OR = 0.580, p = 0.002); smoking (OR = 0.682, p = 0.019) and dry and dusty season (OR = 0.468, p = 0.005). The prevalence of MDR-TB in Ekiti and Ondo States were 1.2 % and 1.3 % respectively. The identified risk factors for MDR were education (OR = 0.739, p = 0.017), age (OR = 0.846, p = 0.048), religion (OR = 1.95, p = 0.0003), family income (OR = 1.76, p = 0.008), previous TB treatment (OR = 3.64, p = 0.004), smoking (OR = 1.33, p = 0.035) and HIV status (OR = 1.85, p = 0.006). Rifampicin monoresistant was reported in 6.7 % of the rifampicin-resistant strains, while 93.3 % were rifampicin polyresistant strains. Two (13.3 %) of the MDR-TB strains were resistant to all the 3 first-line antimycobacterial agents. All the Rifampicin-resistant TB strains were susceptible to the aminoglycosides (Amikacin, Capreomycin and Kanamycin), also with high susceptibility to the fluoroquinilones: Moxifloxacin (100 %) and Levofloxacin (86.7 %). Sixteen (94.1 %) of the 17 Rifampicin-susceptible strains were susceptible to all the eight antibiotics tested, while one (5.9 %) was susceptible to Rifampicin and Isoniazid but resistant to the rest antibiotics. Conclusion: The study showed that there is high prevalence of TB and MDR-TB in Ekiti and Ondo States Nigeria, hence, to meet the SDG Target 3.3 of ending TB epidemic by 2030, culturing and antibiotic susceptibility testing should be carried out on every TB-positive sputum and the patients treated accordingly.
PubMed: 38046896
DOI: 10.1016/j.nmni.2023.101192 -
Genomics & Informatics Sep 2023Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and...
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Toll-like receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.
PubMed: 37813638
DOI: 10.5808/gi.23021 -
Journal of Nanobiotechnology Sep 2023In recent years, the development of BMSCs-derived exosomes (EXO) for the treatment of osteosarcoma (OS) is a safe and promising modality for OS treatment, which can...
BACKGROUND
In recent years, the development of BMSCs-derived exosomes (EXO) for the treatment of osteosarcoma (OS) is a safe and promising modality for OS treatment, which can effectively deliver drugs to tumor cells in vivo. However, the differences in the drugs carried, and the binding of EXOs to other organs limit their therapeutic efficacy. Therefore, improving the OS-targeting ability of BMSCs EXOs and developing new drugs is crucial for the clinical application of targeted therapy for OS.
RESULTS
In this study, we constructed a potential therapeutic nano platform by modifying BMSCs EXOs using the bone-targeting peptide SDSSD and encapsulated capreomycin (CAP) within a shell. These constructed nanoparticles (NPs) showed the ability of homologous targeting and bone-targeting exosomes (BT-EXO) significantly promotes cellular endocytosis in vitro and tumor accumulation in vivo. Furthermore, our results revealed that the constructed NPs induced ferroptosis in OS cells by prompting excessive accumulation of reactive oxygen species (ROS), Fe aggregation, and lipid peroxidation and further identified the potential anticancer molecular mechanism of ferroptosis as transduced by the Keap1/Nrf2/GPX4 signaling pathway. Also, these constructed NP-directed ferroptosis showed significant inhibition of tumor growth in vivo with no significant side effects.
CONCLUSION
These results suggest that these constructed NPs have superior anticancer activity in mouse models of OS in vitro and in vivo, providing a new and promising strategy for combining ferroptosis-based chemotherapy with targeted therapy for OS.
Topics: Animals; Mice; Exosomes; NF-E2-Related Factor 2; Ferroptosis; Kelch-Like ECH-Associated Protein 1; Signal Transduction; Osteosarcoma; Nanoparticles; Bone Neoplasms
PubMed: 37775799
DOI: 10.1186/s12951-023-02129-1