Objective:To explore the genetic diversity of Mycobacterium tuberculosis (MTB) in different types of tuberculosis and its association with clinical features, providing evidence for precise diagnosis and treatment of tuberculosis. Methods:This cross-sectional study included 103 cases of tuberculosis (38 with simple pulmonary tuberculosis, 43 with tuberculous pleurisy, and 22 with pulmonary combined with extrapulmonary tuberculosis) from Shenzhen Third People′s Hospital from 2015 to 2018. Paired bacterial strains from lung and pleural effusion/extrapulmonary sites were collected. Whole-genome sequencing (WGS) was used for drug resistance prediction, and genetic diversity (π value) was calculated as well as differential genes screening. Statistical analysis included paired t-tests and χ2 tests to compare clinical, bacteriological and genetic diversity features among groups.Results:The simple pulmonary tuberculosis group exhibited significantly higher rates of retreatment (71.7%, 27/38), cavitation (70.4%, 19/27), and multidrug-resistant or rifampicin-resistant (MDR/RR) (60.5%, 23/38) compared to the tuberculous pleurisy group (retreatment 11.9%, 5/42; cavitation 11.9%, 5/42; MDR/RR 16.3%, 7/43) and extrapulmonary tuberculosis group (retreatment 9.1%, 2/22; cavitation 18.2%, 4/22; MDR/RR 13.6%, 3/22) ( P<0.05). The overall π values of the MTB strain genomes in lung [(5.94±3.93)×10 ?5], pleural effusion[(6.22±3.51)×10 ?5], and extrapulmonary tissues [(5.83±3.54)×10 ?5] showed no significant differences ( H=0.10, P=0.94). Differential gene diversity analysis revealed that π value alternating genes related to respiration and intermediate metabolism were prominently high [tuberculous pleurisy 32.4% (11/34) and extrapulmonary tuberculosis groups 31.4% (32/102)], while cell wall-associated genes dominated in the simple pulmonary tuberculosis group (42.9%, 6/14). Drug resistance profiles and mutation spectra were identical across isolates from different sites within the same patient. Conclusion:WGS revealed the MTB diversity among different types of tuberculosis. Difference between pulmonary and extrapulmonary environments may impel the adaptive alternations of the bacterial strains to maintain survival with higher overall genome stability. Drug resistance testing of lung-derived isolates may provide references on extrapulmonary tuberculosis treatment.

Objective:To explore the genetic diversity of Mycobacterium tuberculosis (MTB) in different types of tuberculosis and its association with clinical features, providing evidence for precise diagnosis and treatment of tuberculosis. Methods:This cross-sectional study included 103 cases of tuberculosis (38 with simple pulmonary tuberculosis, 43 with tuberculous pleurisy, and 22 with pulmonary combined with extrapulmonary tuberculosis) from Shenzhen Third People′s Hospital from 2015 to 2018. Paired bacterial strains from lung and pleural effusion/extrapulmonary sites were collected. Whole-genome sequencing (WGS) was used for drug resistance prediction, and genetic diversity (π value) was calculated as well as differential genes screening. Statistical analysis included paired t-tests and χ2 tests to compare clinical, bacteriological and genetic diversity features among groups.Results:The simple pulmonary tuberculosis group exhibited significantly higher rates of retreatment (71.7%, 27/38), cavitation (70.4%, 19/27), and multidrug-resistant or rifampicin-resistant (MDR/RR) (60.5%, 23/38) compared to the tuberculous pleurisy group (retreatment 11.9%, 5/42; cavitation 11.9%, 5/42; MDR/RR 16.3%, 7/43) and extrapulmonary tuberculosis group (retreatment 9.1%, 2/22; cavitation 18.2%, 4/22; MDR/RR 13.6%, 3/22) ( P<0.05). The overall π values of the MTB strain genomes in lung [(5.94±3.93)×10 ?5], pleural effusion[(6.22±3.51)×10 ?5], and extrapulmonary tissues [(5.83±3.54)×10 ?5] showed no significant differences ( H=0.10, P=0.94). Differential gene diversity analysis revealed that π value alternating genes related to respiration and intermediate metabolism were prominently high [tuberculous pleurisy 32.4% (11/34) and extrapulmonary tuberculosis groups 31.4% (32/102)], while cell wall-associated genes dominated in the simple pulmonary tuberculosis group (42.9%, 6/14). Drug resistance profiles and mutation spectra were identical across isolates from different sites within the same patient. Conclusion:WGS revealed the MTB diversity among different types of tuberculosis. Difference between pulmonary and extrapulmonary environments may impel the adaptive alternations of the bacterial strains to maintain survival with higher overall genome stability. Drug resistance testing of lung-derived isolates may provide references on extrapulmonary tuberculosis treatment.

To evaluate the clinical value of targeted next generation sequencing (tNGS) in diagnosing rifampicin and rifabutin resistance in tuberculosis patients. In this retrospective cohort study, 119 culture-positive Mycobacterium tuberculosis (MTB) strains from tuberculosis patients in Shenzhen Third People′s Hospital from 2020 to 2023 were collected, then tNGS was performed to detect mutations of rpoB gene. Fourteen different types of rpoB gene mutation were detected in 46 mutation MTB strains, including 43 resistance related mutations and 3 synonymous mutations at codon 529. Using the phenotypic drug susceptibility results of rifampicin and rifabutin as the reference standard, the sensitivities of tNGS for detecting resistance to rifampicin and rifabutin were 100%, and the specificities were 96.2% and 89.4% respectively, therefore, tNGS showed good diagnostic performance. Mutations at positions 531 and 526 of rpoB were highly associated with resistance to rifampicin and rifabutin. Moreover, the results of tNGS from the clinical specimens were consistent with those from the corresponding culture strains. tNGS analysis was performed on 83 MTB strains from 18 patients with multiple positive cultures. The results showed that 2 patients with no mutations in the initial MTB strains were subsequently detected with rpoB gene mutation and their phenotypic drug susceptibilities changed from sensitive to resistant. In summary, using tNGS to detect rpoB mutations can reduce false positive results caused by synonymous mutations, and have satisfactory performance for the diagnosis of rifampicin and rifabutin resistance. tNGS can directly detect clinical sputum samples, and also can be used to dynamically monitor the molecular resistance profiles of MTB, therefore it has extremely broad clinical application prospects.

To evaluate the clinical value of targeted next generation sequencing (tNGS) in diagnosing rifampicin and rifabutin resistance in tuberculosis patients. In this retrospective cohort study, 119 culture-positive Mycobacterium tuberculosis (MTB) strains from tuberculosis patients in Shenzhen Third People′s Hospital from 2020 to 2023 were collected, then tNGS was performed to detect mutations of rpoB gene. Fourteen different types of rpoB gene mutation were detected in 46 mutation MTB strains, including 43 resistance related mutations and 3 synonymous mutations at codon 529. Using the phenotypic drug susceptibility results of rifampicin and rifabutin as the reference standard, the sensitivities of tNGS for detecting resistance to rifampicin and rifabutin were 100%, and the specificities were 96.2% and 89.4% respectively, therefore, tNGS showed good diagnostic performance. Mutations at positions 531 and 526 of rpoB were highly associated with resistance to rifampicin and rifabutin. Moreover, the results of tNGS from the clinical specimens were consistent with those from the corresponding culture strains. tNGS analysis was performed on 83 MTB strains from 18 patients with multiple positive cultures. The results showed that 2 patients with no mutations in the initial MTB strains were subsequently detected with rpoB gene mutation and their phenotypic drug susceptibilities changed from sensitive to resistant. In summary, using tNGS to detect rpoB mutations can reduce false positive results caused by synonymous mutations, and have satisfactory performance for the diagnosis of rifampicin and rifabutin resistance. tNGS can directly detect clinical sputum samples, and also can be used to dynamically monitor the molecular resistance profiles of MTB, therefore it has extremely broad clinical application prospects.

Adult ; Anemia, Hemolytic ; diagnosis ; drug therapy ; Anti-Bacterial Agents ; therapeutic use ; Humans ; Male ; Mycoplasma pneumoniae ; pathogenicity ; Pneumonia, Mycoplasma ; diagnosis ; drug therapy

Objective To investigate the distribution and susceptibility patterns of common uropathogens causing community-acquired urinary tract infection (UTI) in Beijing.MethodsA total of 300non-duplicate isolates were randomly collected from 3 hospitals in Beijing between Jan,1 2010 and Mar,312011.Minimal inhibitory concentrations (MICs) were determined by the broth microdilution methods,which were performed and interpreted according to the guidelines established by the Clinical and Laboratory Standards Institute (CLSI).A panel of 8 antimicrobial agents were tested:amikacin,cefaclor,cefepime,cefoperazone/sulbactam,ciprofloxacin,levofloxacin,gentamicin and nitrofurantoin. Fosfomycin trometamol MICs were determined by the agar-dilution method in cation-adjusted MH agar supplemented with glucose 6-phosphate at a concentration of 25 mg/L as detailed in the guidelines issued by 2010 CLSI. All the Escherichia coli,Klebsiella pneumoniae and Proteus mirabilis strains were screened and confirmed by double-disk synergy test for extended-spectrum β-lactamase (ESBLs).Results Among the organisms cultured,E.coli wasthepredominantpathogen(65.0％ ), followedby Enterococcus(11.7％ ),Staphylococcus( 6.3％ ), Klebsiella pneumoniae( 5. 3％ ), Proteus mirabilis( 4. 7％ ), and Pseudomonas aeruginosa (3.0％).Lower susceptibility rates to ciprofloxacin and levofloxacin (31.4％ -47.4％ ) were observed among all the stains.Amikacin,cefoperazone/sulbactam,nitrofurantoin and fosfomycin trometamol were the most active drugs (92.1％,92.1％,88.4％ and 87.9％ susceptible strains,respectively) among the Gram-negative strains.Isolates of Staphylococcus were highly sensitive to amikacin ( 100.0％ ),cefoperazone/sulbactam (94.7％),nitrofurantoin ( 100.0％ ).Higher susceptibility rates to nitrofurantoin (91.4％) and fosfomycin trometamol (90.0％) were observed in Enterococcus.ESBLs-producing strains accounted for 52.3％ (102/195) in E.coli,43.8％ (7/16) in K.pneumoniae and 14.3％(2/14) in P.mirabilis,respectively.ConclusionsResistance is most common to ciprofloxacin and levofloxacin of all the stains.Currently,the most appropriate agents for the empirical management of uncomplicated UTI seems to be amikacin,cefoperazone/sulbactam,nitrofurantoin and fosfomycin trometamol.