Rapid Tuberculosis Diagnosis Using Reporter Enzyme Fluorescence

• Type of Material: Article
• Authors: Preeti Sule, Texas A&M Health Science Center; Ronak Tilvawala, Texas A&M Health Science Center; Torifq Mustapha, Texas A&M Health Science Center; Hany Hassounah, Texas A&M Health Science Center; Aneesa Noormohamed, Texas A&M Health Science Center; Suprateek Kundu, Emory University; Edward A. Graviss, Houston Methodist Research Institute; Grant K. Walkup, Agios Pharmaceuticals; Ying Kong, University of Tennessee Health Science Center; Jeffrey D. Cirillo, Texas A&M Health Science Center
• Language: English
• Date: 2019-12-01
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2019 Sule et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1128/JCM.01462-19
• Title of Journal or Parent Work: Journal of Clinical Microbiology
• ISSN: 0095-1137
• Volume: 57
• Issue: 12
• Grant/Funding Information: This work was supported by the National Heart, Lung, and Blood Institute and the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under grants HL115463 to Y.K. and AI104960 to J.D.C.; and by grant 48523 to J.D.C. from the Bill & Melinda Gates Foundation; and Wellcome Trust translation award 100598 to J.D.C.
• Keywords: CHALLENGES; BIOMARKERS; MYCOBACTERIUM-TUBERCULOSIS; biomarker; lactamase; BlaC; Mycobacterium tuberculosis; Life Sciences & Biomedicine; Microbiology; point of care; BETA-LACTAMASE; Science & Technology; INFECTION; IN-VITRO; LABORATORY DIAGNOSIS; CEPHALOSPORIN; GENE-EXPRESSION; RESISTANCE
• Research Categories: Biology, Biostatistics; Health Sciences, Pathology; Biology, Microbiology

Abstract

Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until 3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise. This assay utilizes the Mycobacterium tuberculosis-specific biomarker BlaC in reporter enzyme fluorescence (REF) that has been optimized for clinical samples, designated REFtb, along with a more specific fluorogenic substrate, CDG-3. We report the first evaluation of clinical specimens with REFtb assays in comparison to the gold standards for tuberculosis diagnosis, culture and smear microscopy. REFtb assays allowed diagnosis of 160 patients from 16 different countries with a sensitivity of 89% for smear-positive, culture-positive samples and 88% for smear-negative, culture-positive samples with a specificity of 82%. The negative predictive value of REFtb for tuberculosis infection is 93%, and the positive predictive value is 79%. Overall, these data point toward the need for larger accuracy studies by third parties using a commercially available REFtb kit to determine whether incorporation of REFtb into the clinical toolbox for suspected tuberculosis patients would improve case identification. If results similar to our own can be obtained by all diagnostic laboratories, REFtb would allow proper treatment of more than 85% of patients that would be missed during their initial visit to a clinic using current diagnostic strategies, reducing the potential for further spread of disease.