Accuracy of the InnowaveDX MTB/RIF test for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre study.

Early and accurate diagnosis of tuberculosis (TB) is necessary to initiate proper therapy for the benefit of the patients and to prevent disease transmission in the community. In this study, we developed the InnowaveDX MTB/RIF (InnowaveDX) to detect Mycobacterium tuberculosis (MTB) and rifampicin resistance simultaneously. A prospective multicentre study was conducted to evaluate the diagnostic performance of InnowaveDX for the detection MTB in sputum samples as compared with Xpert and culture. The calculated limit of detection (LOD) for InnowaveDX was 9.6 CFU/ml for TB detection and 374.9 CFU/ml for RIF susceptibility. None of the other bacteria tested produced signals that fulfilled the positive TB criteria, demonstrating a species-specificity of InnowaveDX. Then 951 individuals were enrolled at 7 hospitals, of which 607 were definite TB cases with positive culture and/or Xpert results, including 354 smear-positive and 253 smear-negative cases. InnowaveDX sensitivity was 92.7% versus bacteriologically TB standard. Further follow-up revealed that 61 (91.0%) out of 67 false-positive patients with no bacteriological evidence met the criteria of clinically diagnosed TB. Among 125 RIF-resistant TB patients diagnosed by Xpert, 108 cases were correctly identified by InnowaveDX, yielding a sensitivity of 86.4%. Additionally, the proportion of very low bacterial load in the discordant susceptibility group was significantly higher than in the concordant susceptibility group (P = 0.029). To conclude, we have developed a novel molecular diagnostic with promising detection capabilities of TB and RIF susceptibility. In addition, the discordant RIF susceptibility results between InnowaveDX and Xpert are more frequently observed in samples with very low bacterial load.

Structural and Biochemical Characterization of the Cognate and Heterologous Interactions of the MazEF-mt9 TA System.

Toxin-antitoxin (TA) modules widely exist in bacteria, and their activities are associated with the persister phenotype of the pathogen Mycobacterium tuberculosis (M. tb). M. tb causes tuberculosis, a contagious and severe airborne disease. There are 10 MazEF TA systems in M. tb that play important roles in stress adaptation. How the antitoxins antagonize toxins in M. tb or how the 10 TA systems crosstalk to each other are of interest, but the detailed molecular mechanisms are largely unclear. MazEF-mt9 is a unique member among the MazEF family due to its tRNase activity, which is usually carried out by the VapC toxins. Here, we present the cocrystal structure of the MazEF-mt9 complex at 2.7 Å. By characterizing the association mode between the TA pairs through various techniques, we found that MazF-mt9 bound not only its cognate antitoxin but also the noncognate antitoxin MazE-mt1, a phenomenon that could be also observed in vivo. Based on our structural and biochemical work, we propose that the cognate and heterologous interactions among different TA systems work together in vivo to relieve the toxicity of MazF-mt9 toward M. tb cells.

Structure of the MazF-mt9 toxin, a tRNA-specific endonuclease from Mycobacterium tuberculosis.

Tuberculosis (TB) is a severe disease caused by Mycobacterium tuberculosis (M. tb) and the well-characterized M. tb MazE/F proteins play important roles in stress adaptation. Recently, the MazF-mt9 toxin has been found to display endonuclease activities towards tRNAs but the mechanism is unknown. We hereby present the crystal structure of apo-MazF-mt9. The enzyme recognizes tRNALys with a central UUU motif within the anticodon loop, but is insensitive to the sequence context outside of the loop. Based on our crystallographic and biochemical studies, we identified key residues for catalysis and proposed the potential tRNA-binding site.

[Forward genetic screening for zebrafish mutants defective in erythropoiesis].

OBJECTIVE: To screen and identify zebrafish mutants with erythropoiesis defects by N-ethyl-N-nitrosourea (ENU) mutagenesis and large-scale forward genetic screening using beta e 1 as the marker. METHODS: The chemical mutagen ENU was used to treat healthy wild-type male fish (AB strain, F0). The surviving ENU-treated fish were mated with wild-type female fish to generate F1, and further F2 family was generated by F1 family intercross. The adult F2 fish were intercrossed within each F2 family and the resulting F3 embryos from each crossing were subjected to whole mount in situ hybridization (WISH) with the beta e 1 probe. Mutagenesis was performed by treating the male zebrafish with ENU to induce mutations in pre-meiotic germ cells to generate the founders, which were outcrossed to obtained the F1 fish. The F1 fish from different founders were mated to generate the F2 families. F3 embryos from the sibling cross in the F2 family were examined by whole mount in situ hybridization using beta e 1-globin probe. The putative mutants were then characterized with different hematopoiesis markers. RESULTS AND CONCLUSION: We identified 4 beta e 1-deficient mutants with erythropoiesis defects, including two with specific erythiod lineage defects and two with concurrent lymphopoiesis defects.