Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

BACKGROUND: Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). METHODS: EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. RESULTS: We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. CONCLUSION: From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Acquired drug resistance during the turnaround time for drug susceptibility testing impacts outcome of tuberculosis.

BACKGROUND: The impacts of acquired resistance to first-line drugs other than rifampicin during turnaround time (TAT) for drug susceptibility testing (DST) on tuberculosis (TB) treatment are unclear. METHOD: We performed a prospective cohort study to test acquired resistance to isoniazid, ethambutol and pyrazinamide during TAT for DST as risk factors for prolonged time to sputum culture conversion (SCC) and treatment failure in China. Participants included had a baseline DST result for a Mycobacterium tuberculosis (Mtb) isolate collected at TB diagnosis and a follow-up DST result for a Mtb isolate collected upon baseline DST results availability. Acquired drug resistance was identified by comparing baseline and follow-up DST results. RESULTS: This study included 65 patients with acquired resistance Mtb isolates and 130 patients with consistent drug susceptibility profiles. Cox proportional hazard regression analysis demonstrated acquired isoniazid resistance (aHR 0.50, 95%CI: 0.29-0.85) and acquired pyrazinamide resistance (aHR 0.54, 95%CI: 0.36-0.81) were associated with prolonged time to SCC. Moreover, acquired isoniazid resistance (aOR 7.64, 95%CI: 2.39-16.08) and acquired pyrazinamide resistance (aOR 5.71, 95%CI: 2.31-14.12) were independently associated with treatment failure. CONCLUSION: Acquired resistance to isoniazid and/or pyrazinamide during TAT for DST was associated with prolonged time to SCC as well as treatment failure.

Pyrazinamide-resistant Tuberculosis Obscured From Common Targeted Molecular Diagnostics.

Here, we describe a clinical case of pyrazinamide-resistant (PZA-R) tuberculosis (TB) reported as PZA-susceptible (PZA-S) by common molecular diagnostics. Phenotypic susceptibility testing (pDST) indicated PZA-R TB. Targeted Sanger sequencing reported wild-type PncA, indicating PZA-S TB. Whole Genome Sequencing (WGS) by PacBio and IonTorrent both detected deletion of a large portion of pncA, indicating PZA-R. Importantly, both WGS methods showed deletion of part of the primer region targeted by Sanger sequencing. Repeating Sanger sequencing from a culture in presence of PZA returned no result, revealing that 1) two minority susceptible subpopulations had vanished, 2) the PZA-R majority subpopulation harboring the pncA deletion could not be amplified by Sanger primers, and was thus obscured by amplification process. This case demonstrates how a small susceptible subpopulation can entirely obscure majority resistant populations from targeted molecular diagnostics and falsely imply homogenous susceptibility, leading to incorrect diagnosis. To our knowledge, this is the first report of a minority susceptible subpopulation masking a majority resistant population, causing targeted molecular diagnostics to call false susceptibility. The consequence of such genomic events is not limited to PZA. This phenomenon can impact molecular diagnostics' sensitivity whenever the resistance-conferring mutation is not fully within primer-targeted regions. This can be caused by structural changes of genomic context with phenotypic consequence as we report here, or by uncommon mechanisms of resistance. Such false susceptibility calls promote suboptimal treatment and spread of strains that challenge targeted molecular diagnostics. This motivates development of molecular diagnostics unreliant on primer conservation, and impels frequent WGS surveillance for variants that evade prevailing molecular diagnostics.

Novel and reported compensatory mutations in rpoABC genes found in drug resistant tuberculosis outbreaks.

Background: Rifampicin (RIF) is a key first-line drug used to treat tuberculosis, a primarily pulmonary disease caused by Mycobacterium tuberculosis. RIF resistance is caused by mutations in rpoB, at the cost of slower growth and reduced transcription efficiency. Antibiotic resistance to RIF is prevalent despite this fitness cost. Compensatory mutations in rpoABC genes have been shown to alleviate the fitness cost of rpoB:S450L, explaining how RIF resistant strains harbor this mutation can spread so rapidly. Unfortunately, the full set of RIF compensatory mutations is still unknown, particularly those compensating for rarer RIF resistance mutations. Objectives: We performed an association study on a globally representative set of 4,309 whole genome sequenced clinical M. tuberculosis isolates to identify novel putative compensatory mutations, determine the prevalence of known and previously reported putative compensatory mutations, and determine which RIF resistance markers associate with these compensatory mutations. Results and conclusions: Of the 1,079 RIF resistant isolates, 638 carried previously reported putative and high-probability compensatory mutations. Our strict criteria identified 46 additional mutations in rpoABC for which no strong prior evidence of their compensatory role exists. Of these, 35 have previously been reported. As such, our independent corroboration adds to the mounting evidence that these 35 also carry a compensatory role. The remaining 11 are novel putative compensatory markers, reported here for the first time. Six of these 11 novel putative compensatory mutations had two or more mutation events. Most compensatory mutations appear to be specifically compensating for the fitness loss due to rpoB:S450L. However, an outbreak of 22 closely related isolates each carried three rpoB mutations, the rare RIFR markers D435G and L452P and the putative compensatory mutation I1106T. This suggests compensation may require specific combinations of rpoABC mutations. Here, we report only mutations that met our very strict criteria. It is highly likely that many additional rpoABC mutations compensate for rare resistance-causing mutations and therefore did not carry the statistical power to be reported here. These findings aid in the identification of RIF resistant M. tuberculosis strains with restored fitness, which pose a greater risk of causing resistant outbreaks.

Effect of COVID-19 pandemic on incidence of mycobacterial diseases among suspected tuberculosis pulmonary patients in Tehran, Iran.

Background: Recent pandemic of coronavirus SARS-CoV-2 (COVID-19) caused limitations in the country's strategies to fight against mycobacterial infections. The aim of this study was to compare the suspected tuberculosis (TB) pulmonary patients before and during the COVID-19 pandemic (January 2018-December 2021) who were referred to the National Reference TB Laboratory (NRL TB), Tehran, Iran. The mycobacterial isolated strains were identified and compared with previous data. Methods: A total of 16,899 clinical samples collected from 7041 suspected pulmonary TB patients were received from 2018 to 2021. Primary isolation of Mycobacterium isolates was done on Löwenstein-Jensen medium. Then, the DNA was extracted from acid-fast bacillus culture-positive samples and identification was performed by IS6110, Hsp65, and 16S-23S rRNA genes using polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and nested PCR methods. Results: A total of 11679 specimens (69.1%) from 4866 suspected TB patients were collected in 2018-2019 and 5220 specimens (30.8%; from 2175 patients) in 2020-2021. Out of 11679 specimens, 2046 samples that belong to 852 patients were infected with Mycobacterium tuberculosis, and the remaining were non-TB Mycobacterium (NTM) species (n = 244) isolated from 102 patients. The cultures for 12894 specimens were either negative (76.3%) or contaminated (845/16899; 5%). A comparison of the total number of patients who were referred for diagnosis and treatment (954/666 patients, P > 0.05) showed a 30.1% reduction during the COVID-19 pandemic. Although, with these low number of patients, the significant increases of NTM species (P < 0.05) among suspected TB pulmonary patients were observed. Besides, new species of NTM, for example, Mycobacterium peregrinum and Mycobacterium montefiorense, were detected. For the past 20 years, these two species were not reported from pulmonary patients in Iran. Conclusions: During the pandemic of COVID-19, the TB diagnosis network became irregular, as a consequence, many patients could not reach the treatment center, and this could increase the circulation of mycobacterial diseases (TB and NTM). The study shows the emergence of new opportunistic NTM species also.

Distribution of Common and Rare Genetic Markers of Second-Line-Injectable-Drug Resistance in Mycobacterium tuberculosis Revealed by a Genome-Wide Association Study.

Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.

Dynamics of Extensive Drug Resistance Evolution of Mycobacterium tuberculosis in a Single Patient During 9 Years of Disease and Treatment.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Detection of Pyrazinamide Heteroresistance in Mycobacterium tuberculosis.

Heteroresistance is defined as the coexistence of both susceptible and resistant bacteria in a bacterial population. Previously published data show that it may occur in 9 to 57% of Mycobacterium tuberculosis isolates for various drugs. Pyrazinamide (PZA) is an important first-line drug used for treatment of both drug-susceptible and PZA-susceptible multidrug-resistant TB. Clinical PZA resistance is defined as a proportion of resistant bacteria in the isolate exceeding 10%, when the drug is no longer considered clinically effective. The ability of traditional drug susceptibility testing techniques to detect PZA heteroresistance has not yet been evaluated. The aim of this study was to compare the capacity of Bactec MGIT 960, Wayne's test, and whole-genome sequencing (WGS) to detect PZA-resistant subpopulations in bacterial suspensions prepared with different proportions of mutant strains. Both Bactec MGIT 960 and WGS were able to detect the critical level of 10% PZA heteroresistance, whereas Wayne's test failed to do so, with the latter falsely reporting highly resistant samples as PZA susceptible. Failure to detect drug-resistant subpopulations may lead to inadvertently weak treatment regimens if ineffective drugs are included, with the risk of treatment failure with the selective growth of resistant subpopulations. We need clinical awareness of heteroresistance as well as evaluation of new diagnostic tools for their capacity to detect heteroresistance in TB.

The 7H11 Agar Medium Supplemented with Calf Bovine Serum for Susceptibility Testing of Mycobacterium tuberculosis Isolates Against Pyrazinamide.

Despite its importance, pyrazinamide (PZA) is a blind spot in drug susceptibility testing in tuberculosis laboratories. The aim of this study was to set up a reliable agar-based proportion method for detection of PZA-resistant phenotypes using Middlebrook 7H11 agar supplemented with calf bovine serum (CBS) compared with albumin/dextrose/catalase (ADC) enrichment and pncA/rpsA sequencing results. The 7H11 agar medium supplemented with 10% ADC or 10% CBS (pH 6.2) and 100 µg/mL PZA was used to detect PZA resistance among 64 Mycobacterium tuberculosis isolates. Sanger sequencing and whole-genome sequencing were performed to track mutations in the pncA, rpsA, and their upstream regions. A total of 43 rifampicin/multidrug-resistant, 20 drug-susceptible, and 1 isoniazid mono-resistant M. tuberculosis isolates were investigated. The 7H11+ADC and 7H11+CBS could detect 22 and 23 PZA-resistant strains, respectively. With the same specificity, the sensitivity and accuracy of 7H11+CBS was found to be a little greater than 7H11+ADC in PZA resistance detection compared with sequencing results. Twenty-four mutant strains were found to have different mutations in pncA-upstream, pncA and rpsA genes, in which Gly97Asp was the most dominant mutation. The results obtained from 7H11+CBS were comparable to the results of 7H11+ADC. Therefore, the 7H11 agar proportion method would be a less-expensive test using CBS and produces reliable results.

Additional drug resistance for Mycobacterium tuberculosis during turnaround time for drug-susceptibility testing in China: A multicenter observational cohort study.

BACKGROUND: Although phenotypic drug susceptibility testing (DST) of Mycobacterium tuberculosis (Mtb) takes up to 6-8 weeks, little is known about how drug susceptibility is affected during this period. METHODS: We performed a prospective cohort study to investigate the development of drug resistance (DR) during turnaround time (TAT), including 359 pulmonary tuberculosis (PTB) patients with a baseline DST result of an Mtb isolate collected at TB diagnosis and a follow-up DST result of an Mtb isolate collected when baseline DST result was available between 2013 and 2018. Whole-genome sequencing (WGS) was used to differentiate between acquired drug resistance, exogenous reinfection, and mixed infection. RESULTS: Among the studied patients, during TAT for DST, 116 (32.3%) developed DR to four first-line drugs (rifampicin, isoniazid, pyrazinamide, ethambutol). Among 116 pairs of isolates included for WGS, 21 pairs were classified as acquired drug resistance with single nucleotide polymorphisms (SNPs) differences less than 12. Four pairs with an intermediate SNPs differences displayed minor differences in related genotypes and were assessed as mixed infection. The remaining 91 pairs had high SNPs differences consistent with exogenous reinfection. CONCLUSIONS: The exogenous reinfection of drug-resistant strains played a vital role in the development of DR of Mtb isolates during TAT for DST, highlighting the need for both rapid DST methods and improved infection control.

Emergence of additional drug resistance during treatment of multidrug-resistant tuberculosis in China: a prospective cohort study.

OBJECTIVES: Little is known about how additional second-line drug resistance emerges during multidrug-resistant tuberculosis (MDR-TB) treatment. The present study aimed to investigate the influence of microevolution, exogenous reinfection and mixed infection on second-line drug resistance during the recommended 2-year MDR-TB treatment. METHODS: Individuals with MDR-TB were enrolled between 2013 and 2016 in a multicentre prospective observational cohort study and were followed up for 2 years until treatment completion. Whole-genome sequencing (WGS) was applied for serial Mycobacterium tuberculosis isolates from study participants throughout the treatment, to study the role of microevolution, exogenous reinfection and mixed infection in the development of second-line drug resistance. RESULTS: Of the 286 enrolled patients with MDR-TB, 63 (22.0%) M. tuberculosis isolates developed additional drug resistance during the MDR-TB treatment, including 5 that fulfilled the criteria of extensively drug-resistant TB. By comparing WGS data of serial isolates retrieved from the patients throughout treatment, 41 (65.1%) of the cases of additional second-line drug resistance were the result of exogenous reinfection, 18 (28.6%) were caused by acquired drug resistance, i.e. microevolution, while the remaining 4 (6.3%) were caused by mixed infections with drug-resistant and drug-susceptible strains. In multivariate analysis, previous TB treatment (adjusted hazard ratio (aHR) 2.51, 95% CI 1.51-4.18), extensive disease on chest X-ray (aHR 3.39, 95% CI 2.03-5.66) and type 2 diabetes mellitus (aHR 4.00, 95% CI 2.22-7.21) were independent risk factors associated with the development of additional second-line drug resistance. CONCLUSIONS: A large proportion of additional second-line drug resistance emerging during MDR-TB treatment was attributed to exogenous reinfection, indicating the urgency of infection control in health facilities as well as the need for repeated drug susceptibility testing throughout MDR-TB treatment.

Atypical Genetic Basis of Pyrazinamide Resistance in Monoresistant Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in Mycobacterium tuberculosis strains with existing resistance to isoniazid and rifampin (i.e., multidrug resistance [MDR]) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotyped pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of 19 phenotypically PZA-monoresistant isolates collected in Sweden and compared their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and the N terminus of ClpC1 (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro, and the N-terminal region of ClpC1 has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-Oceanic sublineages. Indo-Oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (odds ratio [OR] = 22.2, P < 0.00001) among PZA-monoresistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-Oceanic isolates' overrepresentation in PZA-monoresistant tuberculosis (TB) remains undetermined, but substantial circumstantial evidence suggests that ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

Pyrazinamide Susceptibility Is Driven by Activation of the SigE-Dependent Cell Envelope Stress Response in Mycobacterium tuberculosis.

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We found that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, exposure to peptidoglycan synthesis inhibitors, such as beta-lactams and d-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response. IMPORTANCE For decades, pyrazinamide has served as a cornerstone of tuberculosis therapy. Unlike any other antitubercular drug, pyrazinamide requires an acidic environment to exert its action. Despite its importance, the driver of this conditional susceptibility has remained unknown. In this study, a genome-wide approach revealed that pyrazinamide action is governed by the cell envelope stress response. This observation was validated by orthologous approaches that demonstrate that a central player of this response, SigE, is both necessary and sufficient for potentiation of pyrazinamide action. Moreover, constitutive activation of this response through deletion of the anti-sigma factor gene rseA or exposure of bacilli to drugs that target the cell wall was found to potently drive pyrazinamide susceptibility independent of environmental pH. These findings force a paradigm shift in our understanding of pyrazinamide action and open new avenues for improving diagnostic and therapeutic tools for tuberculosis.

Drug Exposure and Minimum Inhibitory Concentration Predict Pulmonary Tuberculosis Treatment Response.

BACKGROUND: Prospective studies correlating pharmacokinetic/pharmacodynamic (PK/PD) indices to clinical responses are urgently needed. This study aimed to find clinically relevant PK/PD thresholds that can be used for treatment optimization. METHODS: Pharmacokinetic sampling and minimum inhibitory concentration (MIC) measurements were performed for patients with culture-confirmed tuberculosis (TB). Classification and regression tree (CART) analysis was applied to obtain PK and/or PD thresholds for first-line drugs predictive of 2-week/month culture conversion, treatment outcome determined at 6-8 months, acute kidney injury (AKI), and drug-induced liver injury (DILI). Least absolute shrinkage and selection operator (LASSO) logistic regression was used for model development and validation. RESULTS: Finally, 168 and 52 patients with TB were included in development and validation cohorts for analysis, respectively. Area under the concentration-time curve (AUC)/MIC below CART-derived thresholds for pyrazinamide of 8.42, pyrazinamide of 2.79, or rifampicin of 435.45 were the predominant predictors of 2-week culture conversion, 2-month culture conversion, or treatment success, respectively. Isoniazid AUC >21.78 mg · h/L or rifampicin AUC >82.01 mg · h/L were predictive of DILI or AKI during TB treatment. The predictive performance of trained LASSO models in the validation cohort was evaluated by receiver operating characteristic curves and ranged from 0.625 to 0.978. CONCLUSIONS: PK/PD indices and drug exposure of TB drugs were associated with clinical outcome and adverse events. The effect of CART-derived thresholds for individualized dosing on treatment outcome should be studied in a randomized controlled trial.

Drivers and sites of diversity in the DNA adenine methylomes of 93 Mycobacterium tuberculosis complex clinical isolates.

This study assembles DNA adenine methylomes for 93 Mycobacterium tuberculosis complex (MTBC) isolates from seven lineages paired with fully-annotated, finished, de novo assembled genomes. Integrative analysis yielded four key results. First, methyltransferase allele-methylome mapping corrected methyltransferase variant effects previously obscured by reference-based variant calling. Second, heterogeneity analysis of partially active methyltransferase alleles revealed that intracellular stochastic methylation generates a mosaic of methylomes within isogenic cultures, which we formalize as 'intercellular mosaic methylation' (IMM). Mutation-driven IMM was nearly ubiquitous in the globally prominent Beijing sublineage. Third, promoter methylation is widespread and associated with differential expression in the ΔhsdM transcriptome, suggesting promoter HsdM-methylation directly influences transcription. Finally, comparative and functional analyses identified 351 sites hypervariable across isolates and numerous putative regulatory interactions. This multi-omic integration revealed features of methylomic variability in clinical isolates and provides a rational basis for hypothesizing the functions of DNA adenine methylation in MTBC physiology and adaptive evolution.

Treatment quality and outcome for multidrug-resistant tuberculosis patients in four regions of China: a cohort study.

BACKGROUND: China incurs an extremely low treatment coverage of multidrug-resistant tuberculosis (MDR-TB). This study aimed to understand the experience of MDR-TB patients on quality of health care, and the clinical impact through an up to six-year follow-up. METHODS: Cohorts of MDR-TB patients were built in TB/MDR-TB designated hospitals in four regions of China from 2014 to 2015. Patients were followed up during treatment course, and yearly confirmation afterward until 2019. Delay in MDR-TB diagnosis and treatment was calculated upon bacteriological confirmation and treatment initiation. Risk factors for unfavourable outcomes were identified by multivariate logistic regression. RESULTS: Among 1168 bacteriological-positive TB patients identified from a 12-million population, 58 (5.0%) MDR-TB cases were detected. The median delay for MDR-TB diagnosis was 90.0 days, with 13.8% having a delay above 180.0 days. MDR-TB treatment was only recommended to 19 (32.8%) participants, while the rest continued with regimen for drug-susceptible TB. In MDR-TB treatment group, 36.8% achieved treatment success, while the others had incomplete treatment (21.1%), loss to follow-up (36.8%) and TB relapse (5.3%). For non-MDR-TB treatment group, 33.3% succeeded, 25.6% relapsed, 2.6% failed, 23.1% died, and 15.4% were lost to follow-up. Overall, only 35.7% (20/56) of detected MDR-TB patients had favourable outcomes and higher education level was positively associated with it (adjusted odds ratio [aOR]: 3.60, 95% confidence interval [CI]: 1.04-12.5). CONCLUSIONS: A large proportion of patients did not receive MDR-TB treatment and had unfavourable outcomes. Delayed MDR-TB diagnosis resulted in poor quality of MDR-TB care. Rapid diagnosis, regulated patient management and high-quality MDR-TB treatment should be enhanced in China.

Genotypic and phenotypic characterization of Mycobacterium tuberculosis resistance against fluoroquinolones in the northeast of Iran.

BACKGROUND: Fluoroquinolones are broad-spectrum antibiotics that are recommended, and increasingly important, for the treatment of multidrug-resistant tuberculosis (MDR-TB). Resistance to fluoroquinolones is caused by mutations in the Quinolone Resistance Determining Region (QRDR) of gyrA and gyrB genes of Mycobacterium tuberculosis. In this study, we characterized the phenotypic and genotypic resistance to fluoroquinolones for the first time in northeast Iran. METHODS: A total of 123 Mycobacterium tuberculosis isolates, including 111 clinical and 12 collected multidrug-resistant isolates were studied. Also, 19 WHO quality control strains were included in the study. The phenotypic susceptibility was determined by the proportion method on Löwenstein-Jensen medium. The molecular cause of resistance to the fluoroquinolone drugs ofloxacin and levofloxacin was investigated by sequencing of the QRDR region of the gyrA and gyrB genes. RESULTS: Among 123 isolates, six (4.8%) were fluoroquinolone-resistant according to phenotypic methods, and genotypically three of them had a mutation at codon 94 of the gyrA gene (Asp→ Gly) which was earlier reported to cause resistance. All three remaining phenotypically resistant isolates had a nucleotide change in codon 95. No mutations were found in the gyrB gene. Five of the 19 WHO quality control strains, were phenotypically fluoroquinolone-resistant, four of them were genotypically resistant with mutations at codon 90, 91 of the gyrA gene and one resistant strain had no detected mutation. CONCLUSIONS: Mutation at codon 94 of the gyrA gene, was the main cause of fluoroquinolone resistance among M. tuberculosis isolates in our region. In 3/6 fluoroquinolone-resistant isolates, no mutations were found in either gyrA or gyrB. Therefore, it can be concluded that various other factors may lead to fluoroquinolone resistance, such as active efflux pumps, decreased cell wall permeability, and drug inactivation.

Rising challenge of multidrug-resistant tuberculosis in China: a predictive study using Markov modeling.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) is on the rise in China. This study used a dynamic Markov model to predict the longitudinal trends of MDR-TB in China by 2050 and to assess the effects of alternative control measures. METHODS: Eight states of tuberculosis transmission were set up in the Markov model using a hypothetical cohort of 100 000 people. The prevalence of MDR-TB and bacteriologically confirmed drug-susceptible tuberculosis (DS-TB+) were simulated and MDR-TB was stratified into whether the disease was treated with the recommended regimen or not. RESULTS: Without any intervention changes to current conditions, the prevalence of DS-TB+ was projected to decline 67.7% by 2050, decreasing to 20 per 100 000 people, whereas that of MDR-TB was expected to triple to 58/100 000. Furthermore, 86.2% of the MDR-TB cases would be left untreated by the year of 2050. In the case where MDR-TB detection rate reaches 50% or 70% at 5% per year, the decline in prevalence of MDR-TB would be 25.9 and 36.2% respectively. In the case where treatment coverage was improved to 70% or 100% at 5% per year, MDR-TB prevalence in 2050 would decrease by 13.8 and 24.1%, respectively. If both detection rate and treatment coverage reach 70%, the prevalence of MDR-TB by 2050 would be reduced to 28/100 000 by a 51.7% reduction. CONCLUSIONS: MDR-TB, especially untreated MDR-TB, would rise rapidly under China's current MDR-TB control strategies. Interventions designed to promote effective detection and treatment of MDR-TB are imperative in the fights against MDR-TB epidemics.

Improved treatment outcome of multidrug-resistant tuberculosis with the use of a rapid molecular test to detect drug resistance in China.

OBJECTIVES: Numerous studies investigate the advantages of rapid molecular drug susceptibility testing (DST) in comparison to phenotypic DST, but the clinical impact on treating multi/extensively drug resistant TB(M/XDR-TB) is less studied. Therefore, we examined how molecular DST testing may improve MDR-TB treatment management and outcome in Chinese settings. METHODS: We performed a comparative study of patient cohorts before and after the implementation of molecular DST diagnosis with Genotype MTBDRsl/MTBDRplus assay in two Chinese hospitals. We collected clinical information including time to sputum culture conversion and final treatment outcome. RESULTS: In total, 242 MDR-TB patients were studied including 114 before (pre-implementation group) and 128 after the implementation (post-implementation group) of molecular DST. Time to MDR-TB diagnosis was significantly reduced for patients in the post-implementation group, as compared to the pre-implementation group (median,16 vs 62 days; P < 0.001). Patients with early available molecular DST results had a more rapid culture conversion (aHR1.94 95% CI: 1.37-2.73; median,12 vs 24 months, respectively; P < 0.001) and higher rate of treatment success (68% vs 47%, P < 0.01). CONCLUSIONS: The use of molecular DST in routine care for MDR-TB diagnosis as compared to phenotypic DST was associated with a decreased time to culture conversion and improved treatment outcome, highlighting its important clinical value.