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.
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 analyze the clinical characteristics of Mycobacterium tuberculosis (Mtb) infection of the sternum after cardiac surgery. Methods A computerized search was conducted in PubMed, Web of Science, Wanfang, and CNKI databases for published case reports of post-cardiac surgery sternal Mtb infections up to July 18, 2023. Two researchers independently screened the included cases, extracted relevant data, and analyzed clinical features. Results A total of 15 articles involving 26 patients were included, comprising 16 males and 10 females, with a mean age of (61.9±15.6) years (range 5-80). Previous surgical histories included coronary artery bypass grafting (14 patients, 53.8%), valve surgery (8 patients, 30.8%), congenital heart disease surgery (3 patients, 11.5%), and aortic surgery (2 patients, 7.7%). The main symptoms and signs were wound abscesses and poor healing, often accompanied by pain, with or without redness and swelling, while systemic symptoms such as low-grade fever, night sweats, and weight loss were typically absent. The median time for infection to be discovered in 13 patients after surgery was 10.0 (3.0, 13.0) months, and the median interval between infection discovery and diagnosis in 9 patients was 3.0 (2.0, 6.0) months. The primary diagnostic methods included Mtb culture, histopathology, and acid-fast staining of debrided bone tissue or pus. One patient died of cardiac arrest before diagnosis. Among the 25 diagnosed patients, 13 received only antitubercular therapy (11 of whom had undergone surgical treatment before diagnosis), while 12 received combined antitubercular and surgical treatment. All 25 patients were successfully cured, with antitubercular therapy lasting 6-12 months. Conclusion Post-cardiac surgery sternal Mtb infection has a prolonged latency period and poses significant diagnostic challenges, leading to high rates of missed and misdiagnosed cases. Timely diagnosis and treatment can markedly improve prognosis.
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.
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.
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.
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.
ObjectiveTo understand the drug resistance of Mycobacterium tuberculosis complex in West China Hospital, Sichuan University, analyze its drug resistance characteristics, and provide reference for the monitoring of drug-resistant tuberculosis.MethodsFrom January 2016 to March 2018, Mycobacterium tuberculosis drug susceptibility testing kit was used to detect the drug susceptibility of Mycobacterium tuberculosis culture-positive strains in Department of Laboratory Medicine, West China Hospital, Sichuan University. The tested drugs included four of the first-line anti-tuberculosis drugs: rifampicin, isoniazid, ethambutol, and streptomycin, and ten of the second-line anti-tuberculosis drugs: capreomycin, ofloxacin, ethionamide, p-aminosalicylic acid, levofloxacin, moxifloxacin, rifabutin, amikacin, kanamycin, and chlorine phenazine.ResultsA total of 130 patients (130 strains) were enrolled, including 82 newly diagnosed patients (82 strains) and 48 re-treated patients (42 strains). The drug resistance rate of the 130 patients was 37.69%. The drug resistance rate of the newly diagnosed patients (28.05%) was significantly lower than that of the re-treated patients (54.17%), and there was a statistical difference (χ2=8.794, P=0.003). The multi-drug resistance rate of the newly diagnosed patients (6.10%) was significantly lower than that of the re-treated patients (25.00%), and the difference was statistically significant (χ2=9.517, P=0.002). The resistance rate of isoniazid, rifampicin, and streptomycin in newly diagnosed patients (23.17%, 8.54%, and 7.32%, respectively) were significantly lower than those in the re-treated patients (45.83%, 41.67%, and 29.17%, respectively), and the differences were statistically significant (P<0.05). The resistance rate of ofloxacin, moxifloxacin, rifabutin and ethionamide in the newly diagnosed patients (9.76%, 8.54%, 7.31%, and 4.88%, respectively) were significantly lower than those in the re-treated patients (39.58%, 27.08%, 25.00%, and 22.92%, respectively), and the differences were statistically significant (P<0.05).ConclusionIt is necessary to strengthen the standardized treatment of patients with newly diagnosed tuberculosis, increase the treatment and management of re-treated tuberculosis patients, and prevent the generation and spread of drug-resistant patients, especially multidrug-resistant patients.
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.