Tuberculosis (TB) remains a significant human health issue. More effective biomarkers for use in tuberculosis prevention, diagnosis, and treatment, including markers that can discriminate between healthy individuals and those with latent infection, are urgently needed. To identify a set of such markers, we used Solexa sequencing to examine microRNA expression in the serum of patients with active disease, healthy individuals with latent TB, and those with or without prior BCG inoculation. We identified 24 microRNAs that are up-regulated (2.85–1285.93 fold) and 6 microRNAs that are down-regulated (0.003–0.11 fold) (P<0.05) in patients with active TB relative to the three groups of healthy controls. In addition, 75 microRNAs were up-regulated (2.05–2454.58 fold) and 11 were down-regulated (0.001–0.42 fold) (P<0.05) in latent-TB infected individuals relative to BCG- inoculated individuals. Of interest, 134 microRNAs were differentially-expressed in BCG-inoculated relative to un-inoculated individuals (18 up-regulated 2.9–499.29 fold, 116 down-regulated 0.0002–0.5 fold), providing insights into the effects of BCG inoculation at the microRNA level. Target prediction of differentially-expressed microRNAs by microRNA-Gene Network analysis and analysis of pathways affected suggest that regulation of the host immune system by microRNAs is likely to be one of the main factors in the pathogenesis of tuberculosis. qRT-PCR validation indicated that hsa-miR-196b and hsa-miR-376c have potential as markers for active TB disease. The microRNA differential-expression profiles generated in this study provide a good foundation for the development of markers for TB diagnosis, and for investigations on the role of microRNAs in BCG-inoculated and latent-infected individuals.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis , the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III‐A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA‐seq approaches, we show it is active in invader defense and has features atypical of type III‐A systems: mature CRISPR RNA (crRNA) in its crRNA‐CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3′‐end processing after Cas6 cleavage of repeat RNA 8 nt from its 3′ end. crRNAs generated resemble mature crRNA in type I systems, having both 5′ (8 nt) and 3′ (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3′ hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis , providing a foundation for investigating the potential of a type III‐A–based genome editing system.—Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.‐E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III‐A CRISPR/Cas system crRNA and its maturation have atypical features. FASEB J. 33, 1496–1509 (2019). www.fasebj.org

The most prevalent strains of Mycobacterium tuberculosis (M.tb) in Beijing belong to the Beijing genotype family. The influence of Beijing genotype prevalence on the development of drug resistance, and the association of infection with Beijing genotype M.tb with population characteristics, in Beijing, however, are still unclear. In this retrospective study, 1189 isolates were subjected to drug susceptibility testing (DST) and molecular epidemiological analysis, and differences in the percentage of drug resistance between Beijing and non-Beijing genotype strains were compared. The association between the occurrence of drug resistance and the prevalence of Beijing genotype M.tb was analyzed using statistical methods. The Beijing genotype family was the dominant genotype (83.3%) among the 1189 M.tb isolates. Beijing genotype M.tb strains were more likely to spread among males [p = 0.018, OR (95% CI):1.127(1.004–1.264)] and people in the 45–64 age group [p = 0.016, OR (95% CI): 1.438 (1.027–2.015)]. On the contrary, non-Beijing genotype M.tb strains were more probably disseminated among the over 65 [p = 0.005, OR (95% CI):0.653 (0.474–0.9)] and non-resident population [p = 0.035, OR (95% CI):1.185(0.985–1.427)]. DST results showed that 849 (71.4%) strains were fully sensitive to first-line drugs, while 340 (28.6%) strains were resistant to at least one drug, and 9% (107/1189) were MDR-TB. The frequency of INH-resistance among Beijing genotype strains was significantly lower than that among non-Beijing genotype strains (p = 0.032). In addition, the Beijing genotype family readily formed clusters. Our findings indicate that male and middle-aged people were more probably be infected by Beijing genotype M.tb, older people and non-residents were more probably be infected by non-Beijing genotype M.tb. The high percentage of resistance to INH occurring in non-Beijing genotype strains suggested that non-Beijing genotype strains should be given much more interest in Beijing.

Objective: Tuberculosis diagnosis requires rapid, simple and highly sensitive methods. Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated protein (Cas) systems are increasingly being used for clinical diagnostic applications, due to their high flexibility, sensitivity and specificity. We developed a sensitive Mycobacterium tuberculosis (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens.Methods: The conserved MTB IS1081 sequence was used to design CRISPR-derived RNAs (crRNAs) and T7 promoter sequencing-containing PCR primers for use in the CRISPR-MTB assay, then assay performance was evaluated using 401 clinical specimens.Results: The CRISPR-MTB assay provided a low limit of detection of 1 target sequence copy/μL and excellent specificity. Furthermore, use of the assay to detect MTB in bronchoalveolar lavage fluid (BALF), sputum and pus samples provided superior sensitivity (261/268, 97.4%) as compared to sensitivities of acid-fast bacilli (130/268, 48.5%) and mycobacterial culture (192/268, 71.6%) assays, and comparable or greater sensitivity to that of GeneXpert MTB/RIF (260/268, 97.0%).Conclusion: The CRISPR-MTB assay, which provides excellent sensitivity and specificity for MTB detection in sputum, BALF and pus samples, is a viable alternative to conventional tests used to diagnose TB in resource-limited settings.

Thirteen extensively drug-resistant tuberculosis isolates which were highly resistant to a broad spectrum of antituberculosis drugs were identified from 1,926 clinical isolates in China. They had highly diverse mycobacterial interspersed repetitive-unit-variable-number tandem-repeat patterns. Most, but not all, of the drug target genes had mutations contributing to resistance to the corresponding drug.

The predominant prevalent Mycobacterium tuberculosis (M. tb) lineage was the Beijing genotype family in Beijing. There has been no systematic study on the association between drug resistance and Beijing genotype. Here we collected 268 M. tb strains, analyzed the background information and the bacteriological characteristics. The mean age of the cases was 40.12 years; male patients were almost three times than female patients. After genotyping analyzation, 81.7% (219/268) strains were categorized as Beijing genotype; no significant difference was observed between Beijing and non-Beijing genotype in gender, age and treatment history. Drug susceptibility testing (DST) analyzation demonstrated that 172 (64.2%) strains were fully sensitive to all drugs (Isoniazid, Rifampin, Streptomycin, and Ethambutol), while 96 (35.8%) strains were resistant to at least one of the drugs. Beijing genotype strains exhibited a significantly higher clustering rate. However, no significant association relationship was observed between drug resistance and Beijing genotype family. The study provided insights into the genotype diversity and revealed that the frequencies of drug-resistance of Beijing genotype strains.It would be helpful for the establishment of the efficient tuberculosis (TB) prevention and control strategy in Beijing.