A study on factors influencing delayed sputum conversion in newly diagnosed pulmonary tuberculosis based on bacteriology and genomics.

Conversion of sputum from positive to negative is one of the indicators to evaluate the efficacy of anti-tuberculosis treatment (ATT). We investigate the factors associated with delayed sputum conversion after 2 or 5 months of ATT from the perspectives of bacteriology and genomics. A retrospective study of sputum conversion in sputum positive 1782 pulmonary tuberculosis (PTB) was conducted from 2021 to 2022 in Beijing, China. We also designed a case-matched study including 24 pairs of delayed-sputum-conversion patients (DSCPs) and timely-sputum-conversion patients (TSCPs), and collect clinical isolates from DSCPs before and after ATT and initial isolates of TSCPs who successfully achieved sputum conversion to negative after 2 months of ATT. A total of 75 strains were conducted drug sensitivity testing (DST) of 13 anti-TB drugs and whole-genome sequencing (WGS) to analyze the risk factors of delayed conversion and the dynamics changes of drug resistance and genomics of Mycobacterium tuberculosis (MTB) during ATT. We found TSCPs have better treatment outcomes and whose initial isolates show lower levels of drug resistance. Clinical isolates of DSCPs showed dynamically changing of resistance phenotypes and intra-host heterogeneity. Single nucleotide polymorphism (SNP) profiles showed large differences between groups. The study provided insight into the bacteriological and genomic variation of delayed sputum conversion. It would be helpful for early indication of sputum conversion and guidance on ATT.

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Transmission and resistome of extremely drug-resistant tuberculosis in Beijing, China: A retrospective population-based epidemiological study.

BACKGROUND: In this study, we utilized whole genome sequencing (WGS) of clinical extremely drug-resistant tuberculosis (EDR-TB) strains collected during 2014-2020 in Beijing to detect clustered strains. METHODS: A retrospective cohort study was conducted by inclusion of EDR-TB patients with positive cultures in Beijing between 2014 and 2020. RESULTS: A total of 95 EDR-TB patients were included in our analysis. Up on the WGS based genotyping, 94 (94/95, 98.9%) out of 95 were identified as lineage 2 (East Asia). The pairwise genomic distance analysis identified 7 clusters, ranging in size from 2 to 5 isolates. The clustering rate of EDR-TB was 21.1%; while no patients had significantly higher odds of clustering. All isolates harbor rpoB RRDR mutations that confer RIF resistance and katG or inhA promoter mutations that confer INH resistance. Of 95 EDR-TB isolates, a total of 15 mutation types were recorded in the transcriptional regulator mmpR5. In vitro susceptibility testing results revealed that 14 (14/15, 93.3%) out of 15 mutation types were resistant to CFZ; whereas only 3 (3/15, 20.0%) showed resistance to BDQ. Interestingly, 12 isolates harbored mutations within rrl locus, whereas only mutations at positions 2294 and 2296 conferred CLA resistance. Favorable outcomes of EDR-TB patients were positively associated with more effective drugs in the regimes. CONCLUSION: WGS data demonstrate limited transmission of EDR-TB in this metropolis city. WGS-based drug susceptibility predictions will bring benefits to EDR-TB patients to formulate optimal therapeutic regimens.

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates using four molecular methods in China.

To evaluate the relationship between mutations in rpsL or rrs genes and streptomycin (SM) resistance, we compared four molecular methods for their clinical value in the detection of SM resistance. Genotypic analysis of SM resistance in 167 M. tuberculosis clinical strains isolated from Chinese patients was performed by direct DNA sequencing, SSCP, RFLP, and reverse dot-blot hybridization (RDBH) assays. Of the 98 SM-resistant isolates, 78 (79.6%) had missense mutations in codon 43 or 88 of rpsL resulting in a Lys to Arg substitution, 6 (6.1%) had mutations of the rrs gene at positions 513 A to C or T or 516 C to T, and 14 (14.3%) had the wild-type sequence. None of the 69 SM-susceptible isolates examined had alterations in rpsL or rrs. The results of the SSCP, RFLP, and RDBH analyses for these mutations and wild-type sequences were completely consistent with DNA sequencing data. Five distinct single-nucleotide substitutions in codon 43 or 88 of rpsL gene or in position 513 or 516 of rrs gene were correctly identified in 84 of 98 (85.7%) phenotypically SM-resistant isolates by RDBH assay. Molecular analyses of the rpsL and rrs genes are useful for rapid prediction of SM resistance in most clinical strains of M. tuberculosis. Reverse dot-blot hybridization assay is a rapid, simple, and reliable method for the detection of drug resistance.

Identification of Mycobacterium marinum 65 kD heat shock protein gene by polymerase chain reaction restriction analysis from lesions of swimming pool granuloma.

BACKGROUND: Nontuberculous mycobacterium (NTM) had been reported to cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. Among NTM infections, Mycobacterium marinum (M. marinum) are mostly seen to cause skin infection. It is therefore important to establish a rapid approach for detection and identification of M. marinum from lesions of patients with suspected M. marinum infections. METHODS: Specimens were obtained from 5 patients with swimming pool granuloma. DNA was extracted and polymerase chain reaction (PCR) was performed. PCR products were digested with Hae III and BstE II, then analysed by pattern restriction analysis to detect heat shock protein (hsp) 65 kD gene. RESULTS: The 65 kD hsp gene was found in all specimens from patients with swimming pool granuloma. PCR restriction analysis (PRA) identified all 5 samples to be M. marinum infections, and the result was consistent with that of routine bacteriological identification. The lesions subsided or markedly improved upon treatment. CONCLUSIONS: PRA is a sensitive, specific and rapid method in identification of mycobacteria. Application of this method will be helpful for early diagnosis of mycobacterial skin infections.

[Molecular epidemical characteristics of Mycobacterium tuberculosis strains from the nationwide random survey for the epidemiology of tuberculosis in China, 2000].

OBJECTIVE: To explore the epidemical distribution characteristics of Mycobacterium tuberculosis isolated from China. METHODS: The M. tuberculosis strains were gained by multi-stratified grouping random sampling method from the nationwide random survey for the epidemiology of tuberculosis in China, 2000, and analyzed by IS6110-based RFLP and DR-based Spoligotyping DNA fingerprinting. These fingerprinting patterns were transferred into digital data and compared each other, and clustered by Gel compar4.1 Software. The clustering values in different TB patients were compared by chi(2) test and the risk factors for recent transmission were calculated by Odd Ratios. RESULTS: Two hundreds and four of 402 M. tuberculosis strains from this survey were determined to be "Beijing Genotype" M. Tuberculosis strain by DNA fingerprinting analysis. Three clusters M. tuberculosis strains sharing identical fingerprint pattern were found, including one cluster belong to a same family. There were significant difference between female and male, and younger (< 40 years old) and elder (>or= 40 years old) (P < 0.05). Odds Ratio 1 showed youth [1.68 95% CI (1.01 - 2.80)] and male [2.04 95% CI (1.07 - 3.96)], but Odds Ratio 2 showed youth [0.3 95% CI (0.2 - 0.44)] and male [4.96 95% CI (2.77 - 9.00)]. CONCLUSION: M. tuberculosis strains of Beijing Genotype are prevailing in China at present. Male and elder were shown to be significant risk factors for recent transmission. The infection source of M. tuberculosis could be traced by DNA fingerprinting.