Tuberculosis (TB) remains the primary cause of death from an infectious disease worldwide. The MPT64 antigen assay is an expedited diagnostic method that is increasingly adopted in many TB diagnostic laboratories and clinical settings worldwide. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63bp deletion variants within the mpt64 gene, and the potential association of this variant with the lineage or sub-lineage of Mycobacterium tuberculosis complex (MTBC) remains unverified. In this study, we analyzed the MPT64 antigen assay using isolates from the major lineage and sub-lineage of MTBC combined with whole-genome sequencing. Notably, we found a significant association between the 63bp deletion variant in the mpt64 gene and M. tuberculosis Lineage 4.2.2 (L4.2.2) globally. Phylogenetic analysis revealed that L4.2.2 isolates were divided into two clusters (Clades A and B). Importantly, all isolates within Clade A exhibited the 63bp deletion variant in the mpt64 gene. Furthermore, using the Bayesian Skyline population model, we observed that the effective population size of Clade A has remained relatively constant. These findings highlight that MPT64-based testing should be cautiously used, and MPT64 negative results should be validated using an alternative assay in regions where L4.2.2 isolates are prevalent, such as China and Vietnam. These L4.2.2 isolates with the 63bp deletion variant in mpt64 warrant greater consideration during the development of diagnostic tests and study of their immunomodulatory role. Overall, this study provides valuable insights into a better understanding of the virulence and diagnosis of MTBC isolates based on the MPT64 protein. IMPORTANCE: To date, rapid diagnostic methods based on the MPT64 antigen assay are increasingly utilized to differentiate between non-tuberculous mycobacteria and TB disease in clinical settings. Furthermore, numerous novel techniques based on the MPT64 release assay are continuously being developed and applied for the identification of both pulmonary and extrapulmonary TB. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63 bp deletion variants within the mpt64 gene. To our knowledge, this is the first report on the association between the 63 bp deletion variant in mpt64 and Mycobacterium tuberculosis L4.2.2 globally, which highlights the need for the cautious utilization of MPT64-based testing in regions where L4.2.2 isolates are prevalent, such as China and Vietnam, and MPT64 negative results should be confirmed with another assay. In addition, further studies on vaccine development and immunology based on MPT64 should consider these isolates with 63 bp deletion variant.

Objectives: To investigate epidemiological/drug-resistance characteristics and identify potential factors related to drug-resistant and clustered tuberculosis in Sichuan. Methods: A total of 295 Mycobacterium tuberculosis (MTB) isolates were collected from surveillance sites in Sichuan from 2019 to 2021. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole-genome sequencing (WGS) analysis. Results: Of 268 MTB isolates with both WGS and drug-susceptibility testing (DST) results, 159 (59.3%, 159/268) strains belonged to the Beijing lineage (L2). Isoniazid had the highest resistance rate (15.3%, 41/268), followed by rifampicin (9.3%, 25/268). The sensitivity of WGS to predict drug resistance varied from 75% to 97.6%, and the specificity was above 96.0% for all. rpoB Ser450Leu (41.7%, 10/24) and katG Ser315Thr (70%, 28/40) were the most frequent mutations in rifampicin and isoniazid resistance isolates, respectively. The clustering rate in Sichuan was 9.3% (25/268), and patients ≤ 24 years old (aOR = 11.697; 95% CI: 0.817–167.463) had a greater risk of clustering. Conclusions: Our findings prove that WGS is a promising tool for predicting drug resistance to isoniazid, rifampicin, ethambutol, moxifloxacin and levofloxacin in Sichuan. The higher resistance rate to isoniazid emphasizes the urgent need for susceptibility testing surveillance and application management. Improving the diagnosis, treatment and management of patients ≤ 24 years old may reduce the transmission of MTB in Sichuan.

Background: Tuberculosis (TB) remains a severe public health problem globally, and it is essential to comprehend the transmission pattern to control tuberculosis. Herein, we evaluated the drug-resistant characteristics, recent transmission, and associated risk factors of TB in Golmud, Qinghai, China. Methods: In this study, we performed a population-based study of patients diagnosed with TB in Golmud from 2013 to 2018. Drug-susceptibility testing and whole-genome sequencing were performed on 133 Mycobacterium tuberculosis strains. The genomic clustering rate was calculated to evaluate the level of recent transmission. Risk factors were identified by logistic regression analysis. Results: Our results showed that 46.97% (62/132) of strains were phylogenetically clustered and formed into 23 transmission clusters, suggesting a high recent transmission of TB in the area. 12.78% (17/133) strains were multidrug-resistant/rifampicin tuberculosis (MDR/RR-TB), with a high drug-resistant burden. Based on drug resistance gene analysis, we found 23 strains belonging to genotype MDR/RR-TB, where some strains may have borderline mutations. Among these strains, 65.2% (15/23) were found within putative transmission clusters. Additionally, risk factor analysis showed that recent transmission of TB happened more in patients with Tibetan nationality or older age. Conclusion: Overall our study indicates that the recent transmissions of MTB strains, especially genotypic MDR/RR strains, drive the tuberculosis epidemic in Golmud, which could contribute to developing effective TB prevention and control strategies.

Clostridium perfringens is an anaerobic, toxin-producing pathogen responsible for enteric and systemic disease in livestock, yet its ecology in high-altitude yaks remains poorly understood. We conducted a comprehensive investigation covering eight counties of the Xizang Autonomous Region between July 2021 and October 2024. Serological screening of 922 unvaccinated yaks revealed an individual-level antibody prevalence of 0.76% (95% CI: 0.4–1.6) and a herd-level prevalence of 25% (95% CI: 12.7–43.4). Multivariable logistic regression identified residence below 4000 m as the sole significant risk factor (adjusted OR = 8.75; 95%CI: 1.3–56.8; P = 0.04). From seven seropositive animals, one representative isolate CPTibet-Y1 was obtained and subjected to detailed phenotypic, genomic and virulence analyses. Phenotypic profiling confirmed classic biochemical traits of C. perfringens, while 16 S rRNA, Toxin gene PCR typing and whole-genome sequencing classified the strain as toxin type A, harboring Toxin gene cpa, but lacking cpb, etx, iap, cpe and netB. Antimicrobial resistance profiling revealed broad-spectrum resistance encompassing sulfonamides, lincosamides and fluoroquinolones, underpinned by six acquired resistance determinants. In a murine infection model, intraperitoneal challenge with 5 × 10⁷ CFU resulted in 100% lethality within 48 h accompanied by severe intestinal necrosis, hemorrhage and systemic pathology. This study provides unequivocal evidence for the presence of multidrug-resistant, hypervirulent type A strains on the Qinghai-Tibet Plateau, underscoring the need for enhanced surveillance and targeted control strategies in yak-farming communities.