Automated Sequencing for Culture-free Diagnosis of Drug Resistant TB

PROJECT SUMMARY While transformative, all-oral treatment regimens have recently been endorsed by World Health Organization (WHO) for the treatment of drug resistant TB (DR -TB), the efficacy of these new regimens is dependent on prior knowledge of the susceptibility profile of key drugs before treatment initiation. Companion molecular diagnostics for drug susceptibility testing (DST) of the new and repurposed anti-tuberculosis drugs is the most likely path to achieving practical and timely DST for these new regimens. However, there are currently no commercially available molecular diagnostics to detect mutations conferring resistance to bedaquiline, pretomanid, linezolid or delamanid, nor is there an easy developmental path forward using existing nucleic acid amplification test (NAAT) approaches given the complexity of the molecular targets for these drugs. Culture- free, next generation sequencing (NGS) has the greatest potential for delivering a comprehensive diagnostic solution, but existing workflows are highly complex, expensive, and rely on highly skilled staff to run them. To achieve broad adoption of NGS approaches, particularly in low resource settings, these workflows must be simplified substantially to reduce both their complexity and cost. Our goals for the proposed study are to bring culture-free NGS closer to patient care by simplifying NGS workflows for DR-TB diagnosis to the point that NGS can be run mostly hands-free by any laboratorian who can run a NAAT and to reduce costs by removing several enzymatic processes. Our objective is to demonstrate that automated, single amplification sequencing (ASAS) can be used to accurately diagnose resistance to all legacy and new/repurposed drugs for which there are recognized molecular targets (i.e., isoniazid, rifampicin, pyrazinamide, amikacin, moxifloxacin, bedaquiline, clofazimine, linezolid, and delamanid). We will achieve this objective by completing the following three aims; Aim 1) Expand with additional gene targets an existing single amplification targeted NGS assay and describe the assay performance; Aim 2) Integrate an existing Akonni sputum extraction/PCR workstation with our novel single amplification sequencing assay and sequence on Illumina iSeq100; and Aim 3) Evaluate the accuracy of the ASAS solution for detection of drug resistance against a phenotypic and genotypic reference standard in sputum samples from patients at risk for DR-TB under field conditions at clinical laboratories in India, South Africa, and Moldova. The results of this project will demonstrate key characteristics of the ASAS workflow and will provide a solid developmental foundation for the application of this tool in clinical settings, reducing cost and time-to-result for comprehensive DST without culturing.