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.
To screen new tuberculosis diagnostic antigens and vaccine candidates, we predicted the epitopes of Mycobacterium tuberculosis latent infection-associated protein Rv2004c by means of bioinformatics. The homology between Rv2004c protein and human protein sequences was analyzed with BLAST method. The second structures, hydrophilicity, antigenicity, flexibility and surface probability of the protein were analyzed to predict B cell epitopes and T cell epitopes by Protean software of DNAStar software package. The Th epitopes were predicted by RANKPEP and SYFPEITHI supermotif method, the CTL epitopes were predicted by means of combination analyses of SYFPEITHI supermotif method, BIMAS quantitative motif method and NetCTL prediction method. The peptide sequences with higher scores were chosen as the candidate epitopes. Blast analysis showed that Rv2004c protein had low homology with human protein. This protein had abundant secondary structures through analysis of DNAStar software, the peptide segments with high index of hydrophilicity, antigenicity, surface probability and flexibility were widely distributed and were consistent with segments having beta turn or irregular coil. Ten candidates of B cell epitopes were predicted. The Th epitopes of Rv2004c protein were located after the 200th amino acid. Of 37 Th cell epitopes predicted, there were more epitopes of HLA-DRB1*0401 and HLA-DRB1*0701 phenotypes, and the MHC restrictive types of some Th cell epitopes exist cross overlap. Of 10 CTL epitopes predicted, there were more number and higher score of HLA-A2 restricted epitopes. Therefore Mycobacterium tuberculosis Rv2004c protein is a protein antigen with T cell and B cell epitopes, and is expected to be a new target protein candidate for tuberculosis diagnosis and vaccine.
Objective To evaluate the diagnostic value of all diagnostic tests detecting the ethambutol resistance in Mycobacterium tuberculosis. Methods PubMed, EMbase, Chinese Biomedical Database (CBM), Chinese Scientific Journals Full-Text Database (CSJD), and Chinese Journal Full-text Database (CJFD) were searched, and QUADAS items were used to evaluate the quality of included studies. Meta-disc software was used to handle data from included studies. Such index as sensitivity, specificity, and SROC were applied to assess the diagnostic value of individual diagnostic test. Results Nine studies were included. The results of meta-analyses showed that compared with proportion method, the summary sensitivity, summary specificity, positive likelihood ratio, negative likelihood ratio, and SROC area under curve of a nitrate reductase assay were 92%, 99%, 30.50, 0.13, and 0.975 2, respectively, while compared with BACTEC 460 TB, the above mentioned indexes of BACTEC MGIT 960 System were 92%, 99%, 6.27, 0.11, and 0.9, respectively. Bacteriophage biological amplification method revealed relative good analysis effectiveness on MB/BacT. Conclusion According to the results, it is recommended that nitrate reductase assay can replace proportion method as screening test of ethambutol resistance in Mycobacterium tuberculosis, and BACTEC MGIT 960 System can replace BACTEC 460 as final diagnostic test of ethambutol resistance in Mycobacterium tuberculosis.
Objective To investigate the clinical application value of GeneXpert Mycobacterium tuberculosis (MTB)/ rifampin (RIF) in urine samples for tuberculosis diagnosis. Methods The patients with clinically highly suspected tuberculosis admitted to West China Hospital of Sichuan University between January 1, 2018 and June 1, 2023 were selected retrospectively. The diagnostic efficacy of urine GeneXpert MTB/RIF detection, such as sensitivity, specificity, positive predictive value, and negative predictive value, were retrospectively analyzed to evaluate its clinical value in the diagnosis of tuberculosis. Correlation analysis was further conducted to explore the correlation between positive levels of GeneXpert MTB/RIF in urine samples and laboratory test indicators. Results A total of 400 patients were included. Among them, 163 cases were in the clinical tuberculosis group and 237 cases were in the clinical non tuberculosis group. In the clinical tuberculosis group, 112 cases were urogenital tuberculosis patients and 51 cases were non-urogenital tuberculosis patients. The sensitivity, specificity, positive predictive value, and negative predictive value of urine GeneXpert MTB/RIF in the diagnosis of tuberculosis were 55.2%, 97.5%, 93.8% and 76.0%, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of urine GeneXpert MTB/RIF in the diagnosis of urogenital tuberculosis were 65.2%, 92.0%, 76.0% and 87.2%, respectively, and the diagnostic sensitivity was further improved. Correlation analysis showed that the positive degree of urine GeneXpert MTB/RIF was correlated with the levels of hemoglobin, serum total protein, blood serum albumin, and other indicators. Conclusions Urine GeneXpert MTB/RIF detection offers high sensitivity and specificity in the diagnosis of tuberculosis, especially in urogenital tuberculosis, which is helpful for the early and rapid diagnosis of tuberculosis patients. The positive degree reported by the GeneXpert MTB/RIF in urine may indicate disease severity.
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.
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.
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 evaluate the clinical values of phage amplified biologically assay (PhaB) for diagnosis of tuberculosis by comparatively analyzing the diagnostic performances of PhaB, acid fast stain and culture. MethodsThe samples of random sputum and morning sputum from 157 tuberculosis patients diagnosed between January and December 2014 were detected by mycobacteria culture, PhaB, acid fast stain and culture method. The differences of diagnostic performances were analyzed by chi-square test. ResultsThe diagnostic sensitivity was 89.8% (mycobacteria culture), 68.2% (PhaB) and 52.2% (acid fast stain); according to the gold standard of culture method, the positive coincident rate was 74.5% and 57.4%, respectively in PhaB and acid fast stain (P<0.05), and the general coincident rate was 75.8% and 60.5% (P<0.05); of those patients with two negative sputum smears, the positive rate was 33.3% (25/75) in PhaB; the detection time was 1 hour (acid fast stain), 46 hours (PhaB) and 9.5 days (mycobacteria culture), respectively. ConclusionBecause of its high sensitivity, high specificity and short turn around time, simple operation, distinguishing dead isolates and live isolates and drug resistance detection, PhaB is a new method for screening test of tuberculosis or as an effective complementary testing for traditional assays.
Objective To evaluate the diagnostic accuracy of LiPA and phage-based assays in detecting rifampicin resistance in Mycobacterium tuberculosis. Methods A fully recursive literature search was conducted in PUBMED, EMBASE, CBMWeb, CSJD and CJFD. QUADAS items were used to evaluate the quality of the included studies. Meta-disc software was used to handle data from the included studies. SEN, SPE and SROC were used to assess the diagnostic accuracy of every individual diagnostic test. Results A total of 42 studies were included finally. (1) LiPA for detection of rifampicin resistant Mycobacterium tuberculosis: 7 studies took BACTEC 460 assay as the reference test, and meta-analysis showed that the summary SEN = 0.98, summary SPE = 0.98, SROC (AUC) = 0.9924; 6 studies chose proportion assay as the reference test, and meta-analysis showed that the summary SEN = 0.97, summary SPE = 1.00, SROC (AUC) = 0.9961; and 3 studies took both BACTEC 460 assay and proportion assay as the reference tests, and meta-analysis showed that the summary SEN = 0.92, summary SPE = 0.98, SROC (AUC) = 0.9842. (2)Seven studies detected the rifampicin resistant Mycobacterium tuberculosis using Phage amplification assays (Commercial), taking BACTEC 460 assay and proportion assay as the reference tests. Meta-analysis showed that the summary SEN = 0.95, summary SPE = 0.95, SROC (AUC) = 0.9842. (3) Seven studies detected the rifampicin resistant Mycobacterium tuberculosis using Phage amplification assays (in-house), taking BACTEC 460 assay, proportion assay and absolute concentration as the reference tests. Meta-analysis showed that the summary SEN = 0.98, summary SPE = 0.98, SROC (AUC) = 0.9949. (4)Seven studies detected the rifampicin resistant Mycobacterium tuberculosis using Luciferase reporter phage assays (In-house), taking BACTEC 460 assay, proportion assay and absolute concentration as the reference tests. Meta-analysis showed that the summary SEN = 0.98, summary SPE = 0.98, SROC (AUC) = 0.9788. Conclusion Current research confirms that Phage assay is a highly sensitive and specific test for the detection of rifampicin resistance in culture isolates and has a potential in improving the diagnostic accuracy of all diagnostic tests in detecting the rifampicin resistant Mycobacterium tuberculosis. LiPA is also a highly sensitive and specific test for the detection of rifampicin resistance, but the sensitivity appears to relatively decrease when it was used directly on clinical specimens. The results mentioned above need to be further confirmed by more high-quality studies.
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.