Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

Background and Objective: This study was performed to investigate the association of peripheral T lymphocyte subsets with disseminated infection (DI) by Mycobacterium tuberculosis (MTB) in HIV-negative patients. Methods and Materials: The study included 587 HIV-negative tuberculosis (TB) patients. Results: In TB patients with DI, the proportion of CD4+ T cells decreased, the proportion of CD8+ T cells increased, and the ratio of CD4+/CD8+ T cells decreased. According to univariate analysis, smoking, alcohol consumption, rifampicin-resistance, retreatment, and high sputum bacterial load were linked to lower likelihood of developing MTB dissemination. Multivariate analysis indicated that after adjustment for alcohol use, smoking, retreatment, smear, culture, rifampicin-resistance, and CD4+/CD8+, the proportion of CD8+ T cells (but not CD4+ T cells) was independently and positively associated with the prevalence of DI in HIV-negative pulmonary TB (PTB) patients. Conclusions: Examining T lymphocyte subsets is of great value for evaluating the immune function of HIV-negative TB patients, and an increase in the CD8+ T cell proportion may be a critical clue regarding the cause of DI in such patients.

Background: China is a high‐burden country for multidrug‐resistant tuberculosis/rifampin‐resistant tuberculosis (MDR/RR‐TB). Fluoroquinolones (FQs) are key drugs for the treatment of patients with MDR/RR‐TB. However, research on the resistance of FQs in Beijing is limited.Methods: We collected clinical isolates from all patients with pulmonary TB in Beijing from January 2016 to December 2021, conducted drug‐sensitivity tests and sequencing for levofloxacin (LFX) and moxifloxacin (MFX), and collected the treatment plans and outcomes of the patients.Results: A total of 8512 clinical isolates were collected from patients with pulmonary TB, and 261 RR‐TB strains were screened. The proportions of drug‐sensitive and drug‐resistant strains significantly differed by age group and treatment history. The rates of LFX and MFX resistance were 27.6% (72/261) and 36.4% (95/261), respectively. The detection rates of MDR‐TB and pre‐extensively drug‐resistant TB (pre‐XDR‐TB) were 73.2% (191/261) and 36.4% (95/261), respectively, and the trends were significant (χ² trend = 9.995, p = 0.002; χ² trend = 12.744, p = 0.026). Among the 261 RR‐TB strains, 14.9% (24/261) were sensitive to LFX but resistant to MFX. Among the four patients with LFX‐resistant TB who received LFX treatment failed in three patients(Fisher's exact test, p = 0.009). The common mutation sites were 94 and 90 in gyrA. A novel mutation Ala90Ser was discovered.Conclusions: FQs resistance trends in RR‐TB patients in Beijing are striking. Strains showed incomplete cross‐resistance to LFX and MFX. Testing for FQs resistance and developing a reasonable treatment plan are recommended. Attention should be given to the changing trends in MDR‐TB and pre‐XDR‐TB.

Direct sputum targeted next-generation sequencing (tNGS) offers rapid resistance profiling without culture, but its concordance with isolate-based whole-genome sequencing (WGS) and phenotypic drug susceptibility testing (pDST) remains unclear. We compared tNGS (direct sputum) and WGS (cultured isolates) for 14 drugs’ resistance prediction in 68 culture-positive tuberculosis specimens, using pDST as reference. Performance for lineage concordance was also assessed. tNGS showed the highest rifampicin (RIF) sensitivity (90.9%) but the lowest specificity (65.2%); WGS achieved the best overall agreement (86.8%). For isoniazid, tNGS sensitivity was 82.6% vs. WGS 78.3%, but WGS specificity was higher (91.1% vs. 75.6%). tNGS outperformed WGS for ethambutol (EMB) sensitivity (80.0% vs. 40.0%). Both methods performed poorly for pyrazinamide (PZA) (agreement ~40%). Among 68 specimens, 51.5% had fully concordant resistance profiles; tNGS-only variants outnumbered WGS-only variants 2:1. Crucially, tNGS and WGS on the same 24 cultured isolates yielded identical results, proving that discrepancies arise from culture-driven clonal selection, not technical differences. Direct sputum tNGS suggests broader within-host resistance diversity that may be missed by culture, whereas WGS offers superior specificity. The two methods are complementary, with culture bias being the primary source of discordance.

This study evaluated broth microdilution (BMD) and fluorescence PCR melting curve analysis (MeltPro MTB/PZA, targeting pncA mutations) for detecting pyrazinamide (PZA) resistance-associated mutations in rifampicin-resistant tuberculosis (RR-TB). PZA susceptibility was assessed in RR-TB isolates from patients at TB prevention and control institutions and at designated hospitals in Beijing. Whole-genome sequencing (WGS) was used as a genotypic comparator to assess the agreement of BMD at critical concentrations (CCs) of 100 and 200 μg/mL and of the MeltPro assay. BMD showed resistance rates of 63.6% (70/110) at 100 μg/mL and 45.5% (50/110) at 200 μg/mL. Relative to WGS-defined genotypes (wild-type vs. mutation), BMD at 100 μg/mL showed 96.1% agreement for isolates with mutations and 64.4% for wild-type ( κ = 0.590); at 200 μg/mL, agreement was 92.2% for mutations and 94.9% for wild-type ( κ = 0.872). The MeltPro assay showed 89.7% agreement for isolates with mutations and 98.4% for wild-type ( κ = 0.882). These findings indicate that using BMD at 200 μg/mL as the critical concentration achieves closer agreement with WGS-defined genotypes than 100 μg/mL, and that the MeltPro MTB/PZA assay shows high concordance with WGS for rapid detection of pncA mutations in RR-TB isolates. Further validation using phenotypic reference methods and clinical outcomes is warranted.