Rapid Species-Level Diagnosis and Treatment Monitoring of NTM Infections Using a CRISPR-Droplet Platform

Nontuberculous mycobacteria (NTM) infections, particularly those caused by Mycobacterium avium complex (MAC) and Mycobacterium abscessus (Mab), represent an urgent and growing clinical concern due to their rising prevalence, species-specific drug resistance, and the inadequacies of current diagnostic methods. Traditional diagnostic workflows rely on time-consuming cultures and lack the resolution to differentiate among over 30 clinically significant NTM species, often delaying effective treatment. To address these limitations, we propose the development and clinical validation of CRISPR-Assisted, Nanodroplet-paired Differentiation test for NTM (CANDi-NTM) platform. This culture-free assay is designed for rapid, sensitive, and species-specific detection of NTM DNA directly from sputum or blood samples, with minimal equipment requirements and high-throughput compatibility. CANDi-NTM combines optimized species-specific guide RNAs, universal primers, and a dropletbased color-coding system to enable robust, orthogonal detection across diverse NTM species. We will: (1) establish and validate the diagnostic accuracy of CANDi-NTM using retrospective clinical samples from multiple biobank cohorts; (2) evaluate its ability to monitor treatment response using a longitudinal clinical cohort and murine infection model; and (3) perform real-world prospective validation to assess turnaround time, usability, and impact on treatment decisions. The assay’s high sensitivity, rapid results, and capacity to track dynamic changes in pathogen burden will enable earlier diagnosis, support individualized treatment strategies, and reduce reliance on prolonged culture-based methods. By bridging the gap between assay development and clinical validation, this study aims to provide a fast, reliable, and minimally invasive blood-based diagnostic solution for NTM infections, ultimately improving patient outcomes and guiding timely therapeutic interventions.