Development of a Next-generation Rapid Phenotypic Assay for Drug-Resistant M. tuberculosis

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PROJECT SUMMARY The long turnaround time and need for specialised laboratories for conventional culture-based Mycobacterium tuberculosis drug-susceptibility testing (DST) regularly commits patients with drug-resistant tuberculosis (DR- TB) to months of potentially ineffective treatments. Current commercial molecular tuberculosis (TB) tests can rapidly identify DR-TB but are limited to testing resistance to a small number of drugs, excluding any new drugs. In theory, next-generation sequencing (NGS) could comprehensively identify resistance profiles, but is currently prohibitively expensive for routine use in high-burden, low-resource settings, and knowledge on drug-resistance mutations is still incomplete. To address this barrier, a fluorescence-based phenotypic assay will be developed to identify with high specificity resistance to drugs contained in the so-called “BPaLM” regimen, in clinical specimens. The overall goal of the current K43 application is to advance a novel phenotypic solution to transform the clinical management of DR-TB in high burden settings. The objectives of the K43 proposal are to 1) provide critical career advancing training and 2) advance and evaluate a rapid phenotypic workflow requiring minimal hands-on time within a high throughput TB laboratory. The sample collections and technical staff from an existing NIH-funded R01 (R01AI153213, PI: John Metcalfe, US primary mentor on this proposal) cluster randomized controlled trial evaluating the programmatic outcomes associated with benchtop NGS sequencing (the “TS Eliot study”) will be leveraged. Dr. Rob Warren, Distinguished Professor at Stellenbosch University and the Unit Director of the South African Medical Research Council Centre for Tuberculosis Research will serve as the LMIC mentor. Both Dr. Metcalfe and Dr. Warren have vast experience in molecular epidemiology, drug-resistant TB, and development of diagnostic tests. The project will have the following specific aims: 1) Advance a fluorescence- based assay as a rapid, high-throughput pDST, and 2) Perform preliminary diagnostic accuracy evaluation of the assay to detect phenotypic resistance directly in clinical TB specimens. In this study rapid, comprehensive DST methods will be established, which will continue to inform molecular assays for testing of new and repurposed drugs. At the end of this K43-funded mentored program, the applicant is expected to be able to lead a research project independently in the low-resource setting of South Africa. In future research projects based on the preliminary data generated in the current project, she will be able to answer key questions related to drug- resistant TB.