Dataset Information

CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis.

ABSTRACT: Rapid and simple-to-use diagnostic methods for tuberculosis are urgently needed. Recent development has unveiled the diagnostic power of the CRISPR system in the detection of viral infections. However, its potential use in detecting the Mycobacterium tuberculosis complex (MTB) remained unexplored. We developed a rapid CRISPR-based assay for TB detection and conducted a retrospective cohort study of 179 patients to evaluate the CRISPR-MTB test for identifying MTB in various forms of direct clinical samples. Its diagnostic performance was compared, in parallel with culture and the GeneXpert MTB/RIF assay (Xpert). The CRISPR-MTB test is highly sensitive with a near single-copy sensitivity, demands less sample input and offers shorter turnaround time than Xpert. When evaluated in the clinical cohort of both pulmonary and extra-pulmonary tuberculosis, the CRISPR-MTB test exhibited an overall improved sensitivity over both culture (79% vs 33%) and Xpert (79% vs 66%), without comprise in specificity (62/63, 98%). The CRISPR-MTB test exhibits an improved overall diagnostic performance over culture and Xpert across a variety of sample types, and offers great potential as a new diagnostic technique for both pulmonary and extra-pulmonary tuberculosis.

SUBMITTER: Ai JW

PROVIDER: S-EPMC6758691 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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CRISPR-based rapid and ultra-sensitive diagnostic test for <i>Mycobacterium tuberculosis</i>.

Ai Jing-Wen JW Zhou Xian X Xu Teng T Yang Minling M Chen Yuanyuan Y He Gui-Qing GQ Pan Ningp N Cai Yuwei Y Li Yongjun Y Wang Xiaorui X Su Hang H Wang Ting T Zeng Weiqi W Zhang Wen-Hong WH

Emerging microbes & infections 20190101 1

Rapid and simple-to-use diagnostic methods for tuberculosis are urgently needed. Recent development has unveiled the diagnostic power of the CRISPR system in the detection of viral infections. However, its potential use in detecting the <i>Mycobacterium tuberculosis</i> complex (MTB) remained unexplored. We developed a rapid CRISPR-based assay for TB detection and conducted a retrospective cohort study of 179 patients to evaluate the CRISPR-MTB test for identifying MTB in various forms of direct ...[more]

PMID: 31522608

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Project description:Infection with the Mycobacterium bovis (M. bovis) causes a disease referred to as bovine tuberculosis (bTB), which affects a wide range of mammal hosts. Many countries have implemented control and eradication plans that have resulted in variable levels of efficacy and success. Although bTB is a notifiable disease in Argentina, and a control plan that targets cattle herds has been in place for decades, M. bovis is still prevalent in cattle, swine, and certain wild species. The aim of the paper here was to assess the sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV) of PCR from tissue, which is a test for rapid M. bovis detection in swine. Bacteriological culture was also performed for comparison purposes. A Bayesian approach was applied to estimate the accuracy of the diagnostic tests, PCR and bacteriological culture, in 266 swine samples with bTB-like lesions recovered during routine official inspections at slaughterhouses. A one-population model, assuming conditional dependence between test results, and incorporating prior information on the performance of the tests obtained from the literature, was used to estimate the tests Se and Sp. The accuracy of the combined (in parallel) application of both tests was also estimated. The Se of the PCR (82.9%) was higher than the Se of the bacteriological culture (79.9%), whereas the Sp of both tests was similar (88.5 and 89.0%, respectively). Furthermore, when both techniques were assessed in parallel, the Se of the diagnostic system increased substantially (Se = 96.6%) with a moderate Sp loss (Sp = 78.8%; PPV = 92.8%; NPV = 89%). Results suggest that the PCR, or the combined application of bacteriological culture and PCR, may serve as an accurate diagnostic tool to confirm bTB in swine samples. Results here will help the design and implementation of effective surveillance strategies for the disease in swine of Argentina and other settings in which the disease is prevalent.

Project description:Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Dataset Information

CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis.

Publications

CRISPR-based rapid and ultra-sensitive diagnostic test for <i>Mycobacterium tuberculosis</i>.

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