OBJECTIVETo explore the induction of L-forms of Mycobacterium abscess using isoniazid.

METHODSMycobacterium abscess were cultured in aqueous culture media in the presence or absence of 128 µg/ml isoniazid. The culture media containing isoniazid were filtered with 0.45 µm membrane, and the filtrate was subcultured in nutrient agar media for reversion. The isoniazid-free and isoniazid-containing media and the reversion bacteria were observed for cell wall integrity by cell wall staining and transmission electron microscopy, and the microstructures of the cell surfaces were observed by scanning electron microscopy. The isoniazid-containing culture was subcultured in L-form agar media, and the isoniazid-free culture and the reversed bacteria in nutrient agar media to observe the colony morphology. The reversed and non-induced bacteria were identified for 16S rDNA.

RESULTSThe bacteria induced with 128 µg/ml isoniazid showed cell wall defect, presenting with a spherical cell morphology and typical fried egg-like colonies in L-form agar media, while in isoniazid-free cultures, the cells showed intact cell walls with rod-like shapes and round colony morphologies on nutrient agar. The reversed bacteria, showing also intact cell walls with rod-like shapes and round colonies on nutrient agar, had identical 16S rDNA with the non-induced bacteria.

CONCLUSIONIsoniazid can induce the L-forms of mycobacterium abscess for use in studies of multidrug resistance and treatment of the bacteria.

Cell Wall ; drug effects ; Culture Media ; Isoniazid ; pharmacology ; L Forms ; drug effects ; Mycobacterium tuberculosis ; cytology ; drug effects

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*

With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.
Anti-Bacterial Agents ; pharmacology ; Antibiotics, Antitubercular ; pharmacology ; Antitubercular Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Drug Resistance, Bacterial ; Electroporation ; Ethambutol ; pharmacology ; Isoniazid ; pharmacology ; Microbial Sensitivity Tests ; Mycobacterium smegmatis ; drug effects ; genetics ; metabolism ; Oxidoreductases ; genetics ; metabolism ; Plasmids ; Rifampin ; pharmacology ; Streptomycin ; pharmacology

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

OBJECTIVETo compare the performance of MTBDRplus V2 and Xpert MTB/RIF for detecting smear negative pulmonary tuberculosis (PTB).

METHODSClinical PTB suspects were enrolled consecutively in Anhui Chest Hospital and Xi'an Chest Hospital from January to December in 2014. The sputum samples of smear negative PTB suspects were collected and decontaminated. The sediment was used to conduct MTBDRplus V2, Xpert MTB/RIF and drug susceptibility test (DST). All the samples with discrepant drug susceptibility result between molecular methods and phenotypic method were confirmed by DNA sequencing.

RESULTSA total of 1973 cases were enrolled in this study. The detection rates of Mycobacterium tuberculosis complex (MTBC) by MTBDRplus V2 and Xpert MTB/RIF were 27.67% and 27.98%, respectively. When setting MGIT culture result as a gold standard, the sensitivity and specificity of MTBDRplus V2 were 86.74% and 93.84%, and the sensitivity and specificity of Xpert MTB/RIF were 86.55% and 93.43%, respectively. For the detection of the resistance to rifampin, the sensitivity and specificity of MTBDRplus V2 were 94.34% and 96.62%, and the sensitivity and specificity of Xpert MTB/RIF were 88.68% and 95.96%, respectively. For the detection of the resistance to isoniazid, the sensitivity and specificity of MTBDRplus V2 were 77.38% and 98.02%, respectively.

CONCLUSIONMTBDRplus V2 and Xpert MTB/RIF can be used to detect MTBC in smear negative samples with satisfactory performance.

Antitubercular Agents ; pharmacology ; Bacteriological Techniques ; methods ; Drug Resistance, Bacterial ; Humans ; Isoniazid ; pharmacology ; Mycobacterium tuberculosis ; drug effects ; isolation & purification ; Sensitivity and Specificity ; Tuberculosis, Pulmonary ; diagnosis ; microbiology

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*

BACKGROUND: The purpose of this study was to compare the turnaround time for liquid culturing and primary anti-tuberculous drug susceptibility testing (DST) performed using the mycobacteria growth indicator tube (MGIT) 960 system (Becton Dickinson, USA) with that for conventional culturing and DST (by the absolute concentration method) performed using solid culture medium and to determine the concordance rates of DST results obtained using these 2 methods. METHODS: In this retrospective study, we compared the turnaround times from receiving the request for mycobacterial culture to reporting the DST results before and after the introduction of the MGIT 960 system. Further, we determined the concordance between DST results for isoniazid and rifampin for Mycobacterium tuberculosis isolates obtained using the MGIT 960 system and the absolute concentration method, which was conducted at the Korean Institute of Tuberculosis. RESULTS: The overall turnaround time for mycobacterial culturing and DST was 27 days for liquid culturing and DST using the MGIT 960 system versus approximately 70 days for culturing on solid medium and DST with the absolute concentration method (P<0.001). There was a good concordance between findings of DST obtained with the 2 methods (97.2%, kappa coefficient=0.855 for rifampin; and 95.6%, kappa coefficient=0.864 for isoniazid), for 1,083 clinical isolates. CONCLUSIONS: The automated MGIT 960 system for culturing and DST of M. tuberculosis was successfully introduced in a hospital laboratory setting in Korea with significant shortening of the turnaround time.
Antitubercular Agents/*pharmacology ; Automation ; *Drug Resistance, Multiple, Bacterial/drug effects ; Humans ; Isoniazid/pharmacology ; *Microbial Sensitivity Tests/instrumentation/methods ; Mycobacterium tuberculosis/*drug effects/growth & development/isolation & purification ; Retrospective Studies ; Rifampin/pharmacology ; Time Factors ; Tuberculosis/*diagnosis

BACKGROUND: In Korea, tuberculosis is resistant to isoniazid (INH) and/or rifampicin (RIF) in more than 10% of cases. To prevent the spread of resistant Mycobacterium tuberculosis strains, it is crucial to develop more rapid resistance detection methods. METHODS: To determine the feasibility of using direct sequencing for detecting INH- and RIF-resistant strains, the katG gene, the regulatory region of the inhA gene, and the 81-bp hot-spot region of the rpoB gene from 95 culture isolates and 46 respiratory specimens were sequenced. Total 141 culture isolates were classified by conventional drug susceptibility testing (DST) as INH(R)/RIF(R) (N=30), INH(R)/RIF(S) (N=23), INH(S)/RIF(R) (N=15), and INH(S)/RIF(S) (N=73). RESULTS: Compared with phenotypic DST, the overall sensitivity and specificity of sequencing were 83.0% (44/53) and 96.6% (85/88), respectively, for INH resistance, and 93.3% (42/45) and 100% (96/96), respectively, for RIF resistance. The rates were similar between culture isolates and respiratory specimens. Interestingly, three specimens with inhA -15C>T mutation were susceptible to INH by conventional DST. CONCLUSIONS: Detection of mutations in the katG codon 315, the inhA regulatory region, and the hot-spot region of rpoB would be useful for rapid detection of INH and RIF resistance in Korea.
Antitubercular Agents/*pharmacology ; Bacterial Proteins/*genetics ; Catalase/*genetics ; Drug Resistance, Multiple, Bacterial ; Genotype ; Humans ; Isoniazid/*pharmacology ; Mutation ; Mycobacterium tuberculosis/*genetics/isolation & purification ; Oxidoreductases/*genetics ; Republic of Korea ; Rifampin/*pharmacology ; Sensitivity and Specificity ; Sequence Analysis, DNA/*methods

BACKGROUNDChina is one of the high burden countries of Mycobacterium tuberculosis (TB) infection globally, with high incidence and mortality. We studied the molecular characteristics of rifampin (RIF) and isoniazid (INH) resistant Mycobacterium tuberculosis strains from Beijing, China, in order to find out the genetic marker for rapid detection of specific drug resistance.

METHODSForty pansusceptible and 81 resistant strains of Mycobacterium tuberculosis isolated from Beijing, China during 2002-2005 were analyzed. The modified rifampin oligonucleotide (RIFO) assay based on reverse line blot hybridization was used to detect mutations in the 81 bp hot-spot region of rpoB gene, which is associated with RIF resistance. The INH resistance associated genes, regulatory region mab-inhA (-15C/T) and structural gene katG S315T were detected by reverse line blot hybridization and PCR-restriction fragment length polymorphism (RFLP) method respectively. All the strains were typed by spoligotying and the Beijing genotype was further subdivided by NTF locus analysis. The distribution of drug resistance associated mutations in the above genes was compared in these groups.

RESULTSSixty-five (91.5%) of 71 RIF resistant and 52 (92.9%) of 56 multidrug-resistant (MDR, i.e. resistant to at least RIF and INH) strains were found to harbor mutations in the rpoB hot-spot region. No mutation was detected in RIF sensitive strains. The specificity and sensitivity of the modified RIFO assay were 100% and 91.5%, respectively. katG315 AGC>ACC and inhA-15C>T mutations were found in 40 (60.6%) and 10 (15.2%) of 66 INH resistant strains, respectively; 7.6% of INH-resistant strains had mutations in both of these genes. Therefore, a combined use of both katG315 and inhA-15 identified 68.2% of INH-resistant strains. The Beijing genotype accounted for 91.7% of total strains and was further subdivided into "modern" (76.6%) and "ancestral" (23.4%) group. There is no significant difference between "ancestral" and "modern" group in prevalance of drug resistance-associated gene mutations.

CONCLUSIONSThe hot-spot region of rpoB gene can be used as genetic marker for detection of RIF resistant strains; a combined use of both katG315 and inhA-15 can improve the detection rate of INH resistant strains; the Beijing genotype is prevalent in Beijing, China; the modified RIFO assay can be a practical tool for rapid detection of RIF resistant and MDR isolates in the routine diagnostic work.

Antitubercular Agents ; pharmacology ; Bacterial Proteins ; genetics ; Catalase ; genetics ; DNA Transposable Elements ; DNA, Bacterial ; analysis ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; Isoniazid ; pharmacology ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Rifampin ; pharmacology

OBJECTIVETo explore the molecular and epidemic characteristics of rifampin (RFP) and isoniazid (INH) resistance of mycobacterium tuberculosis (MTB) in Sichuan.

METHODSGenoType reg; MTBDRplus Assay GTplus was used to examine 68 clinical isolates of MTB and 105 clinical specimens for mutations in rpoB, katG and inhA genes related to RFP and INH resistance.

RESULTSOf the 151 valid tests obtained, 44 (29.14%) and 26 (17.22%) showed drug resistance and multidrug resistance, respectively. Resistance to RFP and INH was found in 21.85% (33/151) and 24.50% (37/151) of the samples, respectively. The most prevalent mutations were rpoB S531L, katG S315T1 and inhA C-15T. The multidrug resistance rate in the sputum specimens was significantly higher than that in the non-respiratory samples (19.35% vs 7.41%).

CONCLUSIONDrug-resistant, especially multidrug-resistant tuberculosis is highly prevalent in Sichuan. The multidrug-resistant bacteria most frequently show rpoB S531L combined with katG S315T1 mutations, suggesting the necessity of developing rapid clinical identification methods for drug-resistant MTB to control the spread of the resistant strains.

DNA, Bacterial ; analysis ; Drug Resistance, Multiple, Bacterial ; Genotype ; Humans ; Isoniazid ; pharmacology ; Mycobacterium tuberculosis ; drug effects ; isolation & purification ; Reagent Kits, Diagnostic ; Rifampin ; pharmacology ; Sputum ; microbiology ; Tuberculosis, Multidrug-Resistant ; diagnosis ; microbiology