Mycobacterium tuberculosis is the causative agent of human tuberculosis. Through the genotyping of Mycobacterium tuberculosis, we can find the epidemic situation and characteristics of tuberculosis in time, analyze the transmission chain between patients in different jurisdictions, and formulate effective intervention measures in time, to provide a strong basis for clinical diagnosis and treatment. At present, several genotyping techniques for Mycobacterium tuberculosis have their advantages and disadvantages in application. This article reviews the genotyping technology, population genetics and genotyping naming rules of Mycobacterium tuberculosis.
ObjectiveTo explore distribution characteristics of drug-resistant mutations and analyze drug-resistant genotypes in Mycobacterium tuberculosis in Deyang district, Sichuan. MethodsA total of 257 patients infected with Mycobacterium tuberculosis and positive for mycobacterium tuberculosis DNA who were detected from February 2010 to March 2013 were included in our research. Drug-resistance mutations were detected and analyzed using gene chip technology combining by polymerase chain reaction (PCR) and reverse dot hybridization (RDB). ResultsIn these 257 pulmonary tuberculosis patients, drug-resistance mutations were detected in 49 with pulmonary tuberculosis. Drug-resistance mutation rate at katG 315, rpsL 43, embB 306 and rpoB 531 (S531L) was 11.67% (30/257), 7.00% (18/257), 4.28% (11/257) and 3.89% (10/257), respectively. In 234 initially treated pulmonary tuberculosis patients, the rate of isoniazid-resistant genotype, rifampicin-resistant genotype, ethambutol-resistant genotype, streptomycin-resistant genotype and multi-drug resistant genotype was 9.83%, 4.27%, 3.42%, 5.13% and 2.99%, respectively. In 23 retreated pulmonary tuberculosis patients, these rates was 52.17%, 26.09%, 13.04%, 43.48% and 13.04%, respectively. ConclusionIn Deyang district, Sichuan, drug-resistant genotypes for isoniazid, rifampicin, ethambutol and streptomycin are detected in Mycobacterium tuberculosis. Most of the drug-resistant mutations occur at katG 315, rpsL 43, embB 306 and rpoB 531. The rates of drug-resistant genotypes and multi-drug resistance in initially treated pulmonary tuberculosis patients are lower than those in retreated patients. Multi-drug resistant rate is relatively low in our research.
ObjectiveTo investigate the clinical characteristics of non-tuberculous mycobacterium (NTM) pulmonary disease and pulmonary tuberculosis, as well as the bacterial distribution of NTM pulmonary disease. Methods The bacterial distribution and clinical characteristics of 104 patients with NTM lung disease hospitalized in Jiangxi Provincial People’s Hospital from May 2017 to May 2020 were retrospectively analyzed, as well as the clinicplal characteristics of 155 patients with tuberculosis hospitalized during the same period. Results The age of NTM lung disease group [(60±15) years] was higher than that of tuberculosis group [(55±19) years]. There were statistically significant differences in basic diseases (such as malignant tumor, type 2 diabetes, old tuberculosis, bronchiectasis), laboratory examination (such as blood routine examination, albumin) and chest imaging characteristics between the two groups (P<0.05). There was no significant difference in clinical symptoms (such as cough, sputum or fever) (P>0.05). The common underlying diseases of NTM lung disease were malignant tumor (29%), bronchiectasis (21%), chronic obstructive pulmonary disease (19%), etc. The common clinical symptoms of NTM lung disease included cough, sputum, fever, hemoptysis, chest tightness and shortness of breath, and other non-specific respiratory symptoms. The common manifestations of NTM lung disease on chest high-resolution CT (HRCT) included patchy images (82%), mediastinal lymph node enalargement (35%), pleural thickening (31%), pleural effusion (26%) and other signs. The isolates of NTM included Mycobacterium avium (50%), Mycobacterium intracellulare (21%), Mycobacterium chelonae/abscessus (14%), Mycobacterium fortuitum (5%), Mycobacterium gordonae (4%), Mycobacterium gilvum (3%), and Mycobacterium smegmatis (3%). Multivariate Logistic regression analysis showed that advanced age (OR=1.027) was a risk factor for NTM lung disease. Conclusions The clinical manifestations of NTM lung disease and tuberculosis are similar and difficult to distinguish. For male patients over 60 years old with malignant tumor, old tuberculosis, bronchiectasis and other basic diseases, and the chest HRCT findings are mainly bronchiectasis, NTM lung disease should be actively excluded. There is little difference in clinical manifestations between different strains of NTM lung disease, and the treatment cycle of NTM lung disease is long and easy to be interrupted, requiring enhanced follow-up.
In this study, loop-mediated isothermal amplification (LAMP) assay in conjunction with calcein for visualized detection of Mycobacterium tuberculosis (MTB) was established. Firstly, four LAMP primers were designed according to the region of 16S rDNA sequences of MTB. Secondly, clinical sputum samples were collected, decontaminated and their DNA was extracted. Thirdly, standard MTB strains were used to evaluate the specificity and sensitivity of LAMP. At the same time, electrophoresis was used for products detection and calcein was used for visualized verification. At last, Chi-squared test function in SPSS 17.0 software was used for consistency evaluation of LAMP assay as compared with the gold standard (culture method). Results showed that there was no nonspecific amplification appeared in the specificity assay and the detection limit was 10 copies/tube in the sensitivity assay. In addition, visualized method by calcein had a comparable sensitivity with that of electrophoresis method. After evaluation of clinical practicability, the sensitivity of LAMP was calculated as 94.74% and the specificity was 90%, respectively. And Chi-squared test showed that LAMP and culture method had no statistic difference, and the two methods were in good consistency (P>0.05). In conclusion, LAMP assay introduced in our study has the characteristics of high efficiency and visualized detection so that this technique has great application prospects in the resource-limited environment, such as work field and primary care hospitals.
ObjectiveTo evaluate the accuracy and practicability of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) in clinical isolates of mycobacteria.MethodsWe collected all tested strains, which were positive for Mycobacterium tuberculosis culture and positive for acid-fast staining, from West China Hospital of Sichuan University from 2014 to 2017, eliminating duplicate strains sent by the same patient at the same time. The traditional method was used with the P-nitrobenzoic acid (PNB)/ 2-Thiophenecarboxylic acid hydrazide (TCH) indicator to initially identify acid-resistant positive strains. Mycobacteria was identified by MALDI-TOF MS; the specificity and sensitivity of the MALDI-TOF MS was analyzed by duplex primer-polymerase chain reaction (Duplex-PCR) method and DNA sequencing method as the "gold standard" for the identification.ResultsA total of 237 anti-acid positive strains were collected; Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacteria (NTM) were identified by mycobacterium double primer PCR, and NTM was identified by 16S rRNA gene sequencing. There were 218 cases of MTC and 19 cases of NTM. The results of preliminary identification using the traditional identification method of PNB/TCH indicator showed that there were 209 cases of MTC (with the sensitivity of 95.9%, specificity of 100.0%, positive predictive value of 100.0%, and negative predictive value of 67.9%) and 28 cases of NTM (with the sensitivity of 100.0%, specificity of 95.9%, positive predictive value of 67.9%, and negative predictive value of 100.0%). The results of MALDI-TOF MS method indicated that there were 199 cases of MTC (with the sensitivity of 91.3%, specificity of 100.0%, positive predictive value of 100.0%, and negative predictive value of 50.0%), 32 cases of NTM (with the sensitivity of 68.4%. specificity of 94.0%, positive predictive value of 40.6%, and negative predictive value of 97.1%), and 6 cases of others. There were 168 strains (84.4%) with the identification score>1.9 obtained by MALDI-TOF MS method.ConclusionsMALDI-TOF MS is a better method for identifying mycobacteria, which has the same identification results as the traditional methods, and has low cost and is suitable for routine use in clinical microbiology laboratories.
Objective To analyze the drug resistance of Mycobacterium tuberculosis complex (MTBC) in West China Hospital of Sichuan University in recent years to provide reference for drug resistance monitoring and prevention strategies of tuberculosis in general hospitals. Methods The clinical strains of MTBC that performed drug susceptibility tests in West China Hospital of Sichuan University between January 2019 and December 2022 were collected. The drug susceptibility information of 13 anti-tuberculosis drugs, namely rifampicin, isoniazid, ethambutol, streptomycin, rifabutin, amikacin, kanamycin, ofloxacin, levofloxacin, moxifloxacin, para-aminosalicylic acid, ethionamide, and capreomycin, was collected and retrospectively analyzed. Results A total of 502 clinical strains of MTBC were included, and 366 of them were isolated from newly-treated patients while 136 form re-treated patients. The resistance rates of MTBC strains to the first-line anti-tuberculosis drugs in descending order were 28.69% (isoniazid), 19.72% (ethambutol), and 14.94% (rifampicin). Among the second-line drugs, the resistance rates to ofloxacin, levofloxacin, and moxifloxacin were 13.55%, 12.15%, and 11.95%, respectively. The resistance rates to amikacin, kanamycin, para-aminosalicylic acid, and ethionamide were all less than 10%. The resistance rates to streptomycin, capreomycin, and rifabutin were 17.53%, 13.55%, and 12.15%, respectively. The resistance rates to the remaining 12 anti-tuberculosis drugs except capreomycin of MTBC strains isolated from re-treated patients were higher than those of MTBC strains isolated from newly-treated patients, and the differences were statistically significant (P<0.05). The isolation rates of monodrug-resistant, polydrug-resistant, multidrug-resistant (MDR) and pre-extensively drug-resistant (pre-XDR) strains were 9.36%, 7.37%, 7.17%, and 7.77%, respectively. The isolation rates of strains with the four drug-resistant phenotypes generally showed a downward trend during the four years, and the changing trends were statistically significant (P<0.05). The isolation rates of MDR and pre-XDR strains from re-treated patients were higher than those from newly-treated patients, and the differences were statistically significant (P<0.001). Conclusion Tuberculosis drug resistance in West China Hospital of Sichuan University, which is a comprehensive tuberculosis-designated hospital, remained severe during the four years from 2019 to 2022, and the prevention of tuberculosis and the monitoring of drug resistance should be further strengthened.
Objective To investigate the initial drug resistance of Mycobacterium tuberculosis ( M.tuberculosis) in patients with culture positive pulmonary tuberculosis. Methods 1184 patients who hospitalized in Shandong Provincial Chest Hospital with culture positive pulmonary tuberculosis were enrolled. The absolute density method was used to assess the drug resistance of M. tuberculosis. Results M.tuberculosis were sensitive to all anti-tuberculosis drugs in 834 cases( 70. 44% ) , and resistant in 350 cases( 29. 56% ) , in which initial resistance and secondary resistance accounted for 44. 86% ( 157/350) and 55. 14% ( 193 /350) respectively. In 157 cases with initial resistance, 53 cases ( 33. 8% ) were mono-drug resistant tuberculosis( MonoDR-TB) , of which 38 cases were resistance to Streptomycin( 24. 2% ) ; 72 cases( 45. 9% ) were polydrug-resistant tuberculosis ( PDR-TB) ; 20 cases ( 12. 7% ) were multidrug-resistant tuberculosis ( MDR-TB) ; 12 cases ( 7. 6% ) were extensively drug resistant tuberculosis ( XDR-TB) . There was no totally drug-resistant tuberculosis ( TDR-TB) . Conclusions The initial drug resistance of M.tuberculosis in patients with pulmonary tuberculosis is still serious. Unified management of TB control programs and full supervision of chemotherapy are very imperative.
ObjectiveTo explore application value of next-generation sequencing (NGS) technology in diagnosis of pathogenic microorganism infection through two cases report and literature review.MethodsThe NGS technology was used to make clear diagnosis of two cases of suspected pulmonary tuberculosis and pulmonary nontuberculous mycobacterial diseases. Bronchoalveolar lavage fluid of these two patients was collected for gene detection of pathogens using the NGS technology. A systematic literature review was performed for similar published cases in WanFang and CNKI database, using the keywords (next-generation sequencing) OR (NGS) AND (microorganism OR infection) from January 2000 to January 2018, using the PubMed database to retrieve the English literature before January 2018 with the " NGS, infectious diseases, China” as keywords.ResultsOne case of Mycobacterium tuberculosis and one case of non-tuberculous Mycobacteria were detected respectively. A total of 221 Chinese literatures and 3 English literatures were retrieved, excluding dissertations, conferences and newspapers. Finally, 10 articles were published in the infectious diseases and respiratory diseases subjects. The role of NGS technology in the diagnosis and study of related pathogens is proposed.ConclusionThe NGS method is expected to achieve precision medical purposes, such as early diagnosis of infectious diseases, transmission control, accurate treatment, good prognosis and so on.