Empowering global disease surveillance with CURED: a tool for rapid identification of unique genomic biomarkers.

Rapid tracking of emerging pathogenic microorganisms is crucial for designing effective treatment, infection control, and prevention strategies. While whole-genome sequencing (WGS) offers the necessary granularity to track emerging clones, it remains prohibitively expensive at the scales needed to monitor with high resolution in real time. We present CURED (Classification Using Restriction Enzyme Diagnostics), which uses a training set of sequenced genomes to identify unique k-mers with restriction sites specific to a clonal lineage. CURED enables fast and inexpensive PCR-based diagnostic tests for surveillance or outbreak investigations with minimal use of WGS. Benchmarking against existing tools, CURED compares favorably and scales more efficiently than other k-mer search strategies. We validated and tested CURED in five distinct data sets: (i) previously identified biomarkers described for a methicillin-resistant Staphylococcus aureus (MRSA) clone in Rio de Janeiro, (ii) diagnostic alleles for different lineages in the USA300 MRSA clone, (ii) the extensively drug-resistant Acinetobacter baumannii Global Clone 1 lineage, (iv) toxigenic versus non-toxigenic Clostridioides difficile, and (v) circulating S. aureus clones in a neonatal intensive care unit (NICU). We implemented CURED as part of NICU infection prevention efforts and report the test's speed, sensitivity, and specificity in a real-world setting. CURED is a scalable, multithreaded, memory-, and cost-efficient pipeline tailored for rapid clone detection and restriction site analysis. While particularly impactful for localized outbreak investigations and targeted surveillance, our preliminary work at the global scale suggests broader implementation is feasible. CURED is freely available at https://github.com/microbialARC/CURED.IMPORTANCETimely and cost-effective detection of emerging microbial clones is essential for infection prevention and public health surveillance. While whole-genome sequencing remains the gold standard for tracking microbial evolution and transmission, its cost, infrastructure requirements, and turnaround time limit its scalability, especially in resource-limited settings. CURED addresses this gap by enabling the development of inexpensive, PCR-based diagnostic assays informed by genomic data, without requiring further sequencing. By identifying lineage-specific restriction sites through a scalable and memory-efficient k-mer pipeline, CURED enables the translation of genome-scale insights into actionable diagnostics. This tool supports broader implementation of genomic-informed diagnostics in both local and global pathogen surveillance efforts.