Status: Funded - Open
BACKGROUND: In southwestern Nigeria, diagnosis of neonatal sepsis is currently via blood culture method that although cost effective, has a turn-around time of five days and a low sensitivity rate that often results in false negatives. This increases morbidity and mortality rates, and promotes indiscriminate use of antibiotics and amplification of antimicrobial resistance (AMR) in bacterial strains. GAP: There is need to develop novel, or strengthen existing diagnostic infrastructure in this setting. Here we propose to optimize a CRISPR-Cas9 enrichment assay for detection of Enterobacterales (Escherichia coli and Klebsiella pneumoniae) from blood specimens. Our assay is designed to give clinicians information on 1speciation of infectious pathogen(s), 2identification of AMR mechanisms of the infectious pathogen and 3analysis of genetic relatedness of the infectious pathogens to identify potential transmission clusters. HYPOTHESIS: We hypothesize that our CRISPR-Cas9 enrichment assay will successfully detect E. coli and K. pneumoniae species, including associated AMR genes, in a more rapid, highly sensitive and specific manner compared to blood culture. METHODS: Custom CRISPR-cas9 guides were designed using K. pneumoniae strain SGH10. Evaluation of our assay will involve assessment of the analytical sensitivity and specificity using spiked blood specimens, assay precision, and clinical sensitivity and specificity using clinical specimens. RESULTS: Pending. IMPACT: What is attainable with our assay is rapid and targeted diagnostics that can be coupled with an affordable and easily deployable platform such as the Oxford MinION (approximately $1000) for use in low resource regions for public health investigations.