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.
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.
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.
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 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.
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 for detecting armazide resistance in mycobacterium tuberculosis. Methods We searched PUBMED, EMBASE, CBM, CSJD and CJFD. QUADAS items were used to evaluate the quality of included studies. Meta-disc software was used to handle data from included studies. Results Twelve studies were included. Meta-analyses showed that the summary sensitivity and summary specificity of nitrate reductase assay were 92% and 99%, and those of BACTEC MGIT 960 system were 93% and 96%, respectively. The SROC of nitrate reductase assay and BACTEC MGIT 960 system were 0.9836 and 0.9862, respectively. Conclusion We recommend that proportion method can be replaced by nitrate reductase assay as a screening test for detecting armazide resistance in mycobacterium tuberculosis, and BACTEC 460 can be replaced by BACTEC MGIT 960 system as a final diagnostic test for detecting armazide resistance in mycobacterium tuberculosis.