Humans ; Isoniazid/pharmacology* ; Mycobacterium tuberculosis/genetics* ; Rifampin/pharmacology* ; Antitubercular Agents/pharmacology* ; Mutation ; Microbial Sensitivity Tests ; Tuberculosis, Multidrug-Resistant/microbiology* ; Drug Resistance, Multiple, Bacterial/genetics* ; Bacterial Proteins/genetics*

Objective: To evaluate the clinical value of the MeltPro MTB assays in the diagnosis of drug-resistant tuberculosis. Methods: A cross-sectional study design was used to retrospectively collect all 4 551 patients with confirmed tuberculosis between January 2018 and December 2019 at Beijing Chest Hospital, Capital Medical University. Phenotypic drug sensitivity test and GeneXpert MTB/RIF (hereafter referred to as "Xpert") assay were used as gold standards to analyze the accuracy of the probe melting curve method. The clinical value of this technique was also evaluated as a complementary method to conventional assays of drug resistance to increase the detective rate of drug-resistant tuberculosis. Results: By taking the phenotypic drug susceptibility test as the gold standard, the sensitivity of the MeltPro MTB assays to detect resistance to rifampicin, isoniazid, ethambutol and fluoroquinolone was 14/15, 95.7%(22/23), 2/4 and 8/9,respectively; and the specificity was 92.0%(115/125), 93.2%(109/117), 90.4%(123/136) and 93.9%(123/131),respectively; the overall concordance rate was 92.1%(95%CI:89.6%-94.1%),and the Kappa value of the consistency test was 0.63(95%CI:0.55-0.72).By taking the Xpert test results as the reference, the sensitivity of this technology to the detection of rifampicin resistance was 93.6%(44/47), the specificity was100%(310/310), the concordance rate was 99.2%(95%CI:97.6%-99.7%), and the Kappa value of the consistency test was 0.96(95%CI:0.93-0.99). The MeltPro MTB assays had been used in 4 551 confirmed patients; the proportion of patients who obtained effective drug resistance results increased from 83.3% to 87.8%(P<0.01); and detection rate of rifampicin, isoniazid, ethambutol, fluoroquinolone resistance, multidrug and pre-extensive drug resistance cases were increased by 3.2%, 14.7%, 22.2%, 13.7%, 11.2% and 12.5%, respectively. Conclusion: The MeltPro MTB assays show satisfactory accuracy in the diagnosis of drug-resistant tuberculosis. This molecular pathological test is an effective complementary method in improving test positivity of drug-resistant tuberculosis.
Humans ; Rifampin/therapeutic use* ; Antibiotics, Antitubercular/therapeutic use* ; Mycobacterium tuberculosis ; Ethambutol/pharmacology* ; Isoniazid/pharmacology* ; Paraffin Embedding ; Retrospective Studies ; Cross-Sectional Studies ; Drug Resistance, Bacterial ; Sensitivity and Specificity ; Tuberculosis, Multidrug-Resistant/drug therapy*

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.
Bacterial Proteins/genetics* ; DNA-Directed RNA Polymerases/genetics* ; Drug Resistance, Bacterial/genetics* ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis/genetics* ; Reproducibility of Results ; Rifampin/pharmacology* ; Technology

OBJECTIVE: To systematically review the diagnostic accuracy of Xpert^® Mycobacterium tuberculosis/rifampicin (Xpert^® MTB/RIF) for the detection of active tuberculosis (TB) and rifampicin-resistance TB in Chinese patients. METHODS: Four Chinese databases (SinoMed, CNKI, WanFang database, and VIP) and three English databases (PubMed, Embase, and The Cochrane Library) were searched from January 1, 2000 to September 15, 2017, to identify diagnostic tests about the accuracy of Xpert^® MTB/RIF in Chinese patients. Two investigators screened the articles and extracted the information independently, and then the quality of each included study was evaluated by Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2. Bivariate random-effects meta-analysis was conducted to pool the sensitivity and specificity. In addition, subgroup analyses were performed based on patient type (TB patient and TB suspected patient), sample type (sputum, bronchoalveolar lavage fluid and others). All statistical analyses were conducted with Stata version 13.0. RESULTS: A total of 47 articles were included in this systematic review. Most of them (38 articles) were in Chinese and only 9 articles were in English. All the articles were published during 2014 to 2017, and the sample size ranged from 31 to 3 151. Forty articles including 42 comparisons about TB were finally included with the pooled sensitivity of 0.94 (95%CI: 0.92, 0.95) and the pooled specificity of 0.87 (95%CI: 0.84, 0.91). Subgroup analysis showed that different patient and specimen types had no significant differences on sensitivity, but the specificity of sputum group was higher than that of bronchoalveolar lavage fluid. As for the detection of rifampicin-resistant TB, 33 articles (38 comparisons) were analyzed, the pooled sensitivity and specificity were 0.92 (95%CI: 0.89, 0.94) and 0.98 (95%CI: 0.97, 0.99) respectively. There were no significant differences between the patient and specimen in the subgroup analyses. The Deeks funnel plot showed a possible publication bias for detecting active tuberculosis (P=0.08) and no publication bias for rifampicin-resistant TB (P=0.24). The likelihood ratio scatter gram showed that in clinical applications, Xpert^® MTB/RIF had a good diagnostic ability for detecting active tuberculosis, and it had good clinical diagnostic value in detecting rifampicin-resistant TB. CONCLUSION Xpert^® MTB/RIF has good sensitivity and specificity in detecting TB and rifampicin-resistant TB in Chinese people. In particular, it has good clinical value in diagnosing rifampicin-resistance TB.
Antibiotics, Antitubercular/therapeutic use* ; China ; Diagnostic Tests, Routine ; Drug Resistance, Bacterial ; Humans ; Rifampin/pharmacology* ; Sensitivity and Specificity ; Tuberculosis/drug therapy* ; Tuberculosis, Multidrug-Resistant/drug therapy* ; Tuberculosis, Pulmonary/drug therapy*

Objective: To evaluate the diagnostic accuracy of line probe assays for drug- resistant tuberculosis (TB) in China. Methods: Chinese databases (CNKI, Wanfang, SinoMed, VIP Information) and English databases (PubMed, Embase, Cochrane Library) were used to retrieve the literatures regarding the accuracy of line probe assays in the diagnosis of drug-resistant tuberculosis in China between January 1, 2000 and September 1, 2017. Quality Assessment of Diagnostic Accuracy Studies-2 was used to evaluate the quality of the included studies. Sensitivity and specificity in different studies (using drug sensitivity test or gene sequencing as gold standard) were combined by Meta-analysis using bivariate or univariate model. In addition, subgroup analysis (GenoType MTBDRplus, GenoType MTBDRsl and Reverse dot blot hybridization) and sensitivity analysis were also carried out. Results: A total of 24 literatures involving 82 studies were included in the final analysis. The sensitivity and specificity of line probe assays for rifampicin resistant TB were 0.91(0.88-0.94) and 0.98 (0.97-0.99), respectively. The sensitivity and specificity of line probe assays for isoniazid resistant TB were 0.80 (0.77-0.83) and 0.98 (0.96-0.99), respectively. The sensitivity and specificity of line probe assays for multidrug-resistant TB were 0.81 (0.76-0.85) and 0.99 (0.99-1.00), respectively. The sensitivity and specificity of line probe assays for quinolone resistant TB were 0.92(0.88-0.95) and 0.94 (0.91-0.97), respectively. The sensitivity and specificity of line probe assays for second-line injectable drug resistant TB (including kanamycin, Capreomycin, amikacin) were 0.79(0.58-0.91) and 0.98 (0.90-1.00), respectively. The sensitivity and specificity of line probe assays for extensively drug-resistant TB were 0.46 (0.19-0.75) and 1.00 (0.98-1.00), respectively. Subgroup analysis showed that the overall diagnostic accuracy of GenoType MTBDRplus and GenoType MTBDRsl was higher than that of Reverse dot blot hybridization. According to the results of sensitivity analysis, the results of this study were robust. Conclusion: The diagnostic accuracy of line probe assays for drug-resistant TB is high.
Antitubercular Agents/therapeutic use* ; Biological Assay/methods* ; China ; Humans ; Isoniazid/pharmacology* ; Microbial Sensitivity Tests/methods* ; Mycobacterium tuberculosis/isolation & purification* ; Rifampin/pharmacology* ; Sensitivity and Specificity ; Tuberculosis, Multidrug-Resistant/drug therapy*

We evaluate the performance of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis (EPTB) in China. The performance of Xpert was evaluated compared to the composite reference standard (CRS), drug susceptibility testing (DST), and imaging examination. The overall sensitivity and specificity of Xpert were 64.1% (195/304) and 100% (24/24), respectively, using CRS as the gold standard. The sensitivity was significantly higher than that of culture for pus (P<0.05). The proportion of EPTB-positive cases diagnosed by imaging was two times more than that diagnosed using Xpert; however, 6 out of 19 cases may have been overdiagnosed by imaging. Compared to phenotypic DST, the sensitivity and specificity of Xpert were 80% (12/15) and 100% (75/75), respectively. Considering its high sensitivity and specificity, Xpert MTB/RIF may be used as a rapid initial test for EPTB diagnosis, and may also support a quicker decision on the treatment regimen. The combination of imaging and Xpert testing could provide high efficiency and accurate diagnosis of suspected EPTB.
Bacterial Proteins ; genetics ; metabolism ; China ; DNA-Directed RNA Polymerases ; genetics ; metabolism ; Diagnostic Tests, Routine ; instrumentation ; methods ; Drug Resistance, Bacterial ; Humans ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; metabolism ; Retrospective Studies ; Rifampin ; pharmacology ; Sensitivity and Specificity ; Sputum ; Tuberculosis ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

In order to evaluate the performance of a molecular Hain line probe assay (Hain LPA) for rapid detection of rifampicin and isoniazid resistance of Mycobacterium tuberculosis in China, 1612 smear positive patients were consecutively enrolled in this study. Smear positive sputum specimens were collected for Hain LPA and conventional drug susceptibility testing (DST). The sensitivity and specificity of Hain LPA were analyzed by using conventional DST as golden reference. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for rifampicin resistance detection were 88.33%, 97.66%, 81.54%, and 98.62%, respectively. The sensitivity, specificity, PPV and NPV for isoniazid resistance detection were 80.25%, 98.07%, 87.25%, and 96.78%, respectively. These findings suggested that Hain LPA can be an effective method worthy of broader use in China.
China ; Genotyping Techniques ; methods ; Humans ; Isoniazid ; pharmacology ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Rifampin ; pharmacology ; Tuberculosis, Multidrug-Resistant ; diagnosis ; microbiology

OBJECTIVEA PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis).

METHODS12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay.

RESULTSThe sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively.

CONCLUSIONOur findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

Antitubercular Agents ; pharmacology ; Drug Resistance, Bacterial ; Genotype ; Immunoblotting ; methods ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; Polymerase Chain Reaction ; methods ; Rifampin ; pharmacology ; Sensitivity and Specificity ; Time Factors

We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 microg/mL), azithromycin (80 microg/mL), doxycycline (20 microg/mL), or chloramphenicol (100 microg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.
Antibiotics, Antitubercular/*pharmacology ; Cell Line, Tumor ; Drug Resistance, Bacterial ; Fluorescent Antibody Technique ; Humans ; Orientia tsutsugamushi/*drug effects/growth & development/metabolism ; Rifampin/*pharmacology

Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
ATP-Dependent Proteases ; metabolism ; Drug Resistance, Bacterial ; Endopeptidase Clp ; metabolism ; Mycobacterium smegmatis ; drug effects ; metabolism ; Proteolysis ; Proteomics ; Rifampin ; pharmacology