Multiplexed detection of febrile infections using CARMEN

Abstract Detection and diagnosis of bloodborne pathogens are critical for patients and for preventing outbreaks, yet challenging due to these diseases’ nonspecific initial symptoms. We advanced CRISPR-based Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN) technology for simultaneous detection of pathogens on numerous samples. We developed three specialized panels that target viral hemorrhagic fevers, mosquito-borne viruses, and sexually transmitted infections, collectively identifying 23 pathogens. We used deep learning to design CARMEN assays with enhanced sensitivity and specificity, validating them and evaluating their performance on synthetic targets, spiked healthy normal serum samples, and patient samples for Neisseria gonorrhoeae in the United States and for Lassa and mpox virus in Nigeria. Our results show multiplexed CARMEN assays match or outperform individual assay RT-PCR in sensitivity, with matched specificity. These findings underscore CARMEN’s potential as a highly effective tool for rapid, accurate pathogen detection for clinical diagnosis and public health surveillance.