Mycobacterium Tuberculosis Isolates Research Articles

Genetic Characterization and Population Structure of Drug-Resistant Mycobacterium tuberculosis Isolated from Brazilian Patients Using Whole-Genome Sequencing

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug-resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensive resistance (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides the analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definitions. Sub-lineage 4.3 (52%) was the most frequent genotype, and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected twenty novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. A cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug resistance prediction, presenting the best accuracy values for five of the nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) or genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil and the genetic and clinical data correlations and demonstrated its utility for surveillance efforts regarding the spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Evaluation of genetic correlation with fluoroquinolones resistance in rifampicin-resistant Mycobacterium tuberculosis isolates

ObjectiveTo detect levofloxacin (LFX) and moxifloxacin (MFX) resistance among rifampicin-resistant tuberculosis (RR-TB) isolates, and predict the resistance level based on specific mutations in gyrA and gyrB genes. MethodsA total of 686 RR-TB isolates were collected from Chinese Drug Resistance Surveillance Program from 2013 to 2020. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole genome sequencing (WGS) analysis. ResultsAmong the 686 RR isolates, the most prevalent resistance was to isoniazid (80.5 %) and ethambutol (28.4 %), followed by LFX (26.1 %) and MFX (21.9 %). The resistance rate of LFX (26.1%–99.4 %) was higher than that of MFX (21.9%–83.3 %) across various drug resistance patterns. Of the 180 fluoroquinolones (FQs) resistant isolates, 168 (93.3 %) had mutations in quinolone-resistant determining regions (QRDRs) with 21 mutation types, and Asp94Gly (32.7 %, 55/168) was the predominant mutation. Isolates with mutations in Asp94Asn and Asp94Gly were associated with high levels of resistance to LFX and MFX. Using broth microdilution method as gold standard, the sensitivities of WGS for LFX and MFX were 93.3 % and 98.0 %, and the specificities were 98.6 % and 95.0 %, respectively. ConclusionThe resistance rate of LFX was higher than that of MFX among various drug resistance patterns in RR-TB isolates. The gyrA Asp94Gly was the predominant mutation type underlying FQs resistance. However, no significant difference was observed between mutation patterns in gyrA gene and resistance level of FQs.

Identification of novel single nucleotide variants in the drug resistance mechanism of Mycobacterium tuberculosis isolates by whole-genome analysis

BackgroundTuberculosis (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.MethodsIn 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.ResultsWe 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.ConclusionThis comprehensive study elucidates novel resistance mechanisms in MTB against antibiotics, paving the way for future design and development of anti-tuberculosis drugs.

Discordant results between Xpert MTB/RIF assay and Bactec MGIT 960 culture system regarding the detection of rifampin-resistant Mycobacterium tuberculosis isolates in Wenzhou, China.

This study aimed to assess the possible causes of discordant results between Xpert MTB/RIF (Xpert) and Bactec MGIT 960 Culture System (MGIT960) regarding rifampicin (RIF) susceptibility in Mycobacterium tuberculosis. Patients with previous RIF-resistant tuberculosis who were admitted to Wenzhou Central Hospital from January 2020 to December 2022 were enrolled. The isolates obtained from these patients were subjected to RIF susceptibility tests using Xpert and MGIT960, and the minimum inhibitory concentration (MIC) of RIF was determined by the MYCOTB MIC plate test. Additionally, molecular docking and molecular dynamics (MD) simulations were performed to evaluate the binding efficacy of rpoB and RIF based on rpoB mutations detected in the isolates with discordant RIF susceptibility results. A total of 28 isolates with discordant RIF susceptibility test results were detected, 15 of them were RIF susceptible with MICs ≤ 0.5 µg/mL. Twelve out of 15 isolates contained borderline RIF resistance-associated mutations [L430P (n = 6), H445N (n = 6)], 1 isolate had D435Y and Q429H double mutation, and the remaining 2 isolates had a silent (Q432Q) mutation. Compared with the affinity of RIF toward the wild type (WT) (-45.83 kcal/mol) by MD, its affinity toward L452P (-55.52 kcal/mol), D435Y (-47.39 kcal/mol), L430P (approximately -69.72 kcal/mol), H445N (-49.53 kcal/mol), and Q429H (-55.67 kcal/mol) increased. Borderline RIF resistance-associated mutations were the main cause for the discordant RIF susceptibility results between Xpert and MGIT960, and the mechanisms of the resistance need further investigated.IMPORTANCEThis study is aimed at assessing discordant results between Xpert MTB/RIF (Xpert) assay and Bactec MGIT 960 Culture System (MGIT960) regarding the detection of rifampicin (RIF)-resistant Mycobacterium tuberculosis isolates in Wenzhou, China. The discordant results of RIF between these two assays were mainly caused by borderline RIF resistance-associated mutations, subsequently by silent mutations of rpoB. Borderline RIF resistance- associated mutations detected in our study were demonstrated to not be affected by the affinity of rpoB and RIF by molecular dynamics, and the mechanism of resistance was needed to be clarified. For the discordant results of RIF by Xpert and MGIT960 that occurred, rpoB DNA sequencing was recommended to investigate its association with resistance to RIF.

Efflux pump gene single nucleotide variants associated with resistance in Mycobacterium tuberculosis isolates with discrepant drug genotypes

IntroductionSingle nucleotide variants (SNVs) in Mycobacterium tuberculosis (MTB) genomes can predict multi-drug resistance (MDR) but not all phenotype – genotype correlations can be explained. We investigated SNVs in efflux pumps (EP) in the context of MTB drug resistance. MethodsWe analysed 2221 MTB genomes from 1432 susceptible and 200 MDR, 172 pre-XDR (extensively drug resistant) and 417 XDR isolates. Analysis of 47 EP genes was conducted using MTB-VCF, an in-house bioinformatics pipeline. SNVs were categorized according to their SIFT/Polyphen scores. Resistance genotypes were also called using the TB-Profiler tool. ResultsGenome comparisons between susceptible and DR isolates identified 418 unique SNVs in EP of which; 53.5% were in MDR, 68.9% in pre-XDR and 61.3% in XDR isolates. Twenty EP had unique SNVs with a high SIFT/PolyPhen score, comprising 38 unique SNVs. Sixteen SNVs across 12 EP genes were significantly associated with drug resistance and enriched in preXDR and XDR strains. These were 12 previously reported SNVs (in Rv0191, Rv0507, Rv0676, Rv1217, Rv1218, Rv1273, Rv1458, Rv1819 and Rv2688) and four novel SNVs (in Rv1877 and Rv2333). We investigated the 16 SNVs in 52 MDR isolates with phenotype-genotype discrepancy to rifampicin (RIF), isoniazid (INH) and flouroquinolones. SNVs associated with RIF and INH (Rv1217_1218, Rv1819, Rv0450, Rv1458, Rv3827, Rv0507, Rv0676, Rv1273 and Rv2333), and with fluoroquinolone (Rv2688) resistance were present in these discrepant strains. ConclusionsConsidering SNVs in efflux pumps as part of MTB genome-based resistance interpretation may add value especially in evaluation of XDR resistance in strains with phenotype-genotype discrepancies.

Identification of novel resistance-associated mutations and discrimination within whole-genome sequences of fluoroquinolone-resistant Mycobacterium tuberculosis isolates.

This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant Mycobacterium tuberculosis (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant (P = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, P < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in Mycobacterium tuberculosis (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.

Molecular analysis of bedaquiline resistance and genetic variations in clinical isolates of multidrug and fluoroquinolones-resistant Mycobacterium tuberculosis

BackgroundMycobacterium tuberculosis developing resistance to anti-tubercular therapy leads to pioneering innovative treatment protocols featuring carefully curated combinations of potent medications. The aim of this study was to analyze the frequency and pattern of fluoroquinolone (FQ) resistant mutations among the first-line drug-resistant specimens. By employing an elective sampling strategy, we deliberately focused our investigation on a targeted subset of multidrug resistant (MDR) and FQ resistant (FQR) TB cases. MethodSputum samples from first-line drug-resistant patients were subjected to a GenoType MTBDRsl VER 2.0 assay to determine FQR. To investigate the effect of bedaquiline (BDQ) on these mutation-conferring genes (Rv0678, pepQ, and atpE), sanger sequencing was carried out on the MDR + FQR subset that was obtained. We have also conducted a computational analysis of the gene products of Rv0678 and pepQ with BDQ using molecular docking methods. ResultsEighty-three samples were observed to have FQR, and the majority of these specimens were associated with naïve and treatment failure cases. Among them, 64 isolates exhibited mutations in gyrA gene, while 19 isolates showed mutations in gyrB gene. The predominant occurrence of genetic variations, particularly in D94G and A90V, was observed within the specimens displaying resistance in gyrA gene, and the same has been confirmed by PCR-based detection. And the sequence analysis of BDQ genes among MDR + FQR subset indicated that the majority of the samples did not exhibit mutations in pepQ and atpE genes. However, only 2 samples were found to have the genetic variant G337A, which involves replacing the amino acid glutamic acid (E) at codon position 113 of Rv0678 gene with lysine (K). ConclusionsMost of the mutations were harbored in rpoB + katG + gyrA gene pattern. These findings pave the way for the development of databases and rapid diagnostic probes.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

Genetic analysis of pyrazinamide resistance: mutations in the pncA gene among clinical isolates of multi-drug resistant Mycobacterium Tuberculosis

Pyrazinamide (PZA) resistance presents a significant challenge in tuberculosis (TB) management, particularly in multi-drug resistant (MDR) cases. Understanding the genetic basis of PZA resistance is crucial for effective treatment strategies. This review provides a comprehensive overview of recent advancements in molecular characterization of PZA resistance, focusing on mutations in the pncA, rpsA, and panD genes. We discuss the prevalence of PZA resistance, mechanisms of resistance, and the utility of genetic analysis in early diagnosis. Key findings from studies investigating PZA resistance in clinical isolates are summarized, highlighting the predominant role of pncA mutations. Additionally, we explore the significance of mutations in rpsA and panD genes, elucidating alternative pathways to PZA resistance. Insights from genetic analysis have implications for personalized treatment approaches and TB control programs. Future directions in research and the potential of molecular methods in enhancing PZA susceptibility testing are also discussed.

The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran

BackgroundTuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates.MethodsNineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP).ResultsAll 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ.ConclusionsOur results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

Discordance in Phenotypic and Genotypic Susceptibility Testing for Streptomycin Due to Nonsynonymous/Nonsense/Deletion Frame-Shift Mutations in gidB among Clinical Mycobacterium tuberculosis Isolates in Kuwait

Objective: Increasing reports of resistance to newer antituberculosis drugs have prompted the search for other alternative drugs. Streptomycin (STR) could be used for the treatment of drug-resistant tuberculosis if susceptibility of Mycobacterium tuberculosis isolate to STR could be accurately detected. We performed phenotypic and genotypic drug susceptibility testing (DST) of 118 M. tuberculosis isolates for STR. Materials and Methods: Fifty pansusceptible and 68 multidrug-resistant M. tuberculosis (MDR-TB) isolates were used. Phenotypic DST for STR, rifampicin, isoniazid, and ethambutol was performed by mycobacteria growth indicator tube 960 System. Genotypic DST was done by GenoTypeMTBDRplus assay for rifampicin and isoniazid and by PCR-sequencing of rpsL, rrs, and gidB genes for STR. MDR-TB isolates were genotyped by spoligotyping. Results: Phenotypic DST identified 50 isolates susceptible to all four drugs (pansusceptible). Sixty-one of 68 MDR-TB isolates were resistant to STR. Genotypic testing for rifampicin and isoniazid yielded expected results. Fifty pansusceptible and 7 STR-susceptible MDR-TB isolates contained no mutation in rpsL or rrs, while 47, 2, and 1 STR-resistant isolate contained rpsL, rrs, and rpsL + rrs mutations, respectively. Of the remaining 11 STR-resistant MDR-TB, 9 isolates contained deletion frame-shift/nonsynonymous mutations in gidB. Surprisingly, 13 pansusceptible isolates also contained deletion frame-shift/nonsense/nonsynonymous mutations in gidB. Also, 30 of 68 MDR-TB but only 2 of 50 pansusceptible isolates belonged to the Beijing genotype. Conclusions: Our data show that, like rifampicin, ethambutol, and pyrazinamide, STR also exhibits discordant phenotypic and genotypic DST results for some M. tuberculosis isolates. Hence, STR should be included in therapy regimens only if both phenotypic and genotypic resistance testing indicate susceptibility to avoid amplification of resistance and drug toxicity.

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Transcriptomic responses to antibiotic exposure in Mycobacterium tuberculosis.

Transcriptional responses in bacteria following antibiotic exposure offer insights into antibiotic mechanism of action, bacterial responses, and characterization of antimicrobial resistance. We aimed to define the transcriptional antibiotic response (TAR) in Mycobacterium tuberculosis (Mtb) isolates for clinically relevant drugs by pooling and analyzing Mtb microarray and RNA-seq data sets. We generated 99 antibiotic transcription profiles across 17 antibiotics, with 76% of profiles generated using 3-24 hours of antibiotic exposure and 49% within one doubling of the WHO antibiotic critical concentration. TAR genes were time-dependent, and largely specific to the antibiotic mechanism of action. TAR signatures performed well at predicting antibiotic exposure, with the area under the receiver operating curve (AUC) ranging from 0.84-1.00 (TAR <6 hours of antibiotic exposure) and 0.76-1.00 (>6 hours of antibiotic exposure) for upregulated genes and 0.57-0.90 and 0.87-1.00, respectfully, for downregulated genes. This work desmonstrates that transcriptomics allows for the assessment of antibiotic activity in Mtb within 6 hours of exposure.

Spiritual Holy Water Sites in Ethiopia: Unrecognized High-Risk Settings for Transmission of Pulmonary Tuberculosis.

Ethiopia is a high-tuberculosis (TB) burden country with 157 new cases per 100,000 people, with 23,800 TB-related deaths in 2020. In Ethiopia, TB patients have different healthcare-seeking behaviors. They frequently visit spiritual places, such as holy water sites (HWSs), to seek treatment for their illness spiritually. This study examined the prevalence of pulmonary TB (PTB) and drug susceptibility profiles of Mycobacterium tuberculosis (MTB) isolates among spiritual HWS attendees in Northwest Ethiopia. A cross-sectional study was conducted from June 2019 to March 2020. Sputum samples were collected, processed, and cultured using Löwenstein-Jensen (LJ) culture medium. Second-generation line probe assays (LPAs), GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0, were used to detect anti-TB drug-resistant isolates. STATA 17 was utilized to perform descriptive statistics, bivariate, and multivariate regression analyses. Of 560 PTB-symptomatic participants, 21.8% ((95% confidence interval (95 CI): 18.4-25.2%)) were culture-positive, resulting in a point prevalence of 1,183/100,000 attendees. Amongst HWS attendees, culture-positive TB occurred most commonly in persons 18-33 years of age (28.5% (95 CI 23.4-34.3%)). Other participant characteristics significantly associated with culture-positive PTB were as follows: rural residents (adjusted odds ratio (aOR) 2.65; 95 CI 1.38-5.10), married participants (aOR 2.43; 95 CI 1.28-4.63), family members >5 per household (aOR 1.84; 95 CI 1.04-3.24), and sharing living space (aOR 10.57; 95 CI 3.60-31.13). Also, among 438 participants followed for 12 months after showing negative TB culture results while at the HWS, 6.8% (95 CI 4.4-9.4%) developed or contracted culture-positive TB post-residency at the HWSs. Of the 122 tested isolates, 20 (16.4%) were isoniazid (INH) and/or rifampicin (RIF) resistant. Multidrug-resistant (MDR) TB was detected in 15 cases (12.3%), five of which were fluoroquinolones (FLQs) resistant. The findings from this study should raise a concern about HWSs as potential high-risk settings for TB transmission. It is recommended that appropriate control measures be instituted that include compulsory TB testing and tightened infection control at HWSs, where an increased risk exists for transmission of TB.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

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.

Novel mutation in efflux pump Rv1258c (Tap) gene in drug resistant clinical isolates of Mycobacterium tuberculosis in Iran.

Tuberculosis (TB) remains a serious public health problem worldwide. Drug-resistant TB is considered a major and growing global threat. Despite the great variety of described mutations in Mycobacterium tuberculosis (MTB) resistance genes, the mechanisms of drug resistance are still controversial. Recently, a report on the role of efflux pump genes in drug resistance added to this complexity. Therefore, a thorough understanding of efflux pump genes in drug-resistant TB clinical isolates is needed. We performed molecular analysis of the efflux pump gene (Rv1258c) in 33 drug-resistant and 20 drug-sensitive clinical MTB isolates by sequencing the amplicons' targets in both the forward and reverse directions. A novel mutation of the Rv1258c gene was identified at G442A (Ala148Thr) in rifampicin mono-resistant clinical strain, as compared to the H37Rv reference strain. In addition, a cytosine nucleotide insertion was found between the positions 580 and 581 (denominated Tap580) in two drug-sensitive strains at identical gene positions. These results indicated the possibility of mutation in the efflux pump genes and the important role of Tap efflux pump genes in drug-resistant MTB isolates. However, further research is required to determine the direct association of these mutations with resistant MTB.

On the onset and dispersal of a major MDR TB clone among HIV-negative patients, Tunisia

BackgroundTo carry out a whole genome sequencing (WGS)-based investigation on the emergence and spread of the largest multidrug-resistant tuberculosis (MDR TB) outbreak that has been thriving among HIV-negative patients, Tunisia, since the early 2000s.MethodsWe performed phylogeographic analyses and molecular dating based on a WGS dataset representing 68 unique Mycobacterium tuberculosis isolates, covering almost the entire MDR TB outbreak for the time period 2001–2016.ResultsThe data indicate that the ancestor of the MDR TB outbreak emerged in the region of Bizerte, as early as 1974 (95% CI 1951–1985), from where it spread to other regions by 1992 (95% CI 1980–1996). Analysis of a minimum spanning tree based on core genome Multilocus Sequence Typing (cgMLST) uncovered the early spill-over of the fitness-compensated MDR TB strain from the prison into the general population. Indeed, cases with history of incarceration were found to be directly or indirectly linked to up to 22 new outbreak cases (32.35%) among the non-imprisoned population. By around 2008, the MDR TB outbreak strain had acquired additional resistance, leading to an XDR phenotype.ConclusionsWGS allowed refining our understanding of the emergence and evolution of the largest MDR TB outbreak in Tunisia, whose causative strain has been circulating silently for almost 26 years before. Our study lends further support to the critical role of prisons-related cases in the early spread of the outbreak among the general population. The shift to an XDR phenotype of such an epidemic clone prompts an urgent need to undertake drastic control measures.

Transmission of drug-resistant Mycobacterium tuberculosis isolates between Finnish- and foreign-born cases, 2014–2021: A molecular epidemiological study

BackgroundData on the molecular epidemiology and transmission of drug-resistant Mycobacterium tuberculosis (MTB) in low-incidence settings with immigration from high-incidence settings is limited. MethodWe included 115 drug-resistant (DR) MTB isolates with whole-genome sequencing data isolated in Finland between 2014 and 2021. Potential transmission clusters were identified using a threshold of 12 single-nucleotide polymorphisms (SNPs). Highly related clusters were identified using a threshold of 5 SNPs. ResultOf the 115 DR MTB isolates, 31 (27.0%) isolates were from Finnish-born cases and 84 (73.0%) were from foreign-born cases. The proportion of multidrug-resistant (MDR) MTB isolates (30/84, 35.7%) from foreign-born cases was higher than that of MDR MTB isolates from Finnish-born cases (8/31, 25.8%). Lineage 2 (40/115, 34.8%) and lineage 4 (40/115, 34.8%) were the most prevalent lineages. A total of 25 (21.7%) isolates were classified into eight potential transmission clusters (≤12 SNPs). Furthermore, five highly related clusters (≤5 SNPs) were identified, including three DR MTB isolates from Finnish-born cases and 14 DR isolates from foreign-born cases. ConclusionThe risk of DR MTB transmission between Finnish- and foreign-born persons is not negligible. Further research on clustering analysis in drug-susceptible MTB is worth to inform tuberculosis management and control in low-incidence settings with increasing immigration.

Prevalence, Transmission and Genetic Diversity of Pyrazinamide Resistance Among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Hunan, China.

China is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB). However, published data on pyrazinamide (PZA) resistance are still limited in Hunan province, China. This study investigated the prevalence, transmission, and genetic diversity of PZA resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Hunan province. Drug susceptibility testing (DST) with the Bactec MGIT 960 PZA kit and pyrazinamidase (PZase) testing were conducted on all 298 MDR clinical isolates. Moreover, 24-locus MIRU-VNTR and DNA sequencing of pncA, rpsA, and panD genes were conducted on 180 PZA-resistant (PZA-R) isolates. The prevalence of PZA resistance among MDR-TB strains reached 60.4%. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA, rpsA, and panD mutations showed that transmission of PZA-R isolates played a significant role in the formation of PZA-R TB. Ninety-eight mutation patterns were observed in the pncA among 180 PZA-R isolates, and seventy-one (72.4%) were point mutations. Twenty-four of these mutations are new, including 2 base substitutions (V93G and T153S) and 22 nucleotide deletions or insertions. The W119C was found in PZA-S isolates, on the other hand, F94L and V155A mutations were found in both PZA resistant and susceptible isolates with positive PZase activity, indicating that they were not associated with PZA resistance. This is not entirely in line with the WHO catalogue. Ten novel rpsA mutations were found in 10 PZA-R isolates, which all combined with mutations in pncA. Thus, it is unpredictable whether these mutations in rpsA can impact PZA resistance. No panD mutation was found in all PZA-R isolates. DNA sequencing of pncA and PZase activity testing have great potential in predicting PZA resistance.