Title Detection of Pathogens by a Novel User-Developed Broad-Range BR 16S PCR rRNA Polymerase Chain Reaction/Gene Sequencing Assay: Multiyear Experience in a Large Canadian Healthcare Zone

Between 2015 and 2022, we evaluated a novel BR 16S PCR rDNA PCR/Sanger sequencing assay to improve diagnosis of invasive infections in culture-negative specimens. Using dual-priming oligonucleotide (DPO), this assay analyzed ribo-somal DNA from sterile fluids or tissues. A total of 772 specimens from 661 patients were studied: 425 specimens (61%) had negative culture and BR 16S PCR tests; 245 specimens (35%) had negative cultures but positive BR 16S PCR tests; and 29 specimens (4%) had negative cultures with indeterminate BR 16S PCR results. Excluding indeterminate results, the assay showed sensitivity, specificity, PPV, NPV, and accuracy of 93.9%, 99.77%, 99.64%, 95.97%, and 97.38%, re-spectively. Gram stain purulence predicted the BR 16S PCR result better (69.4%) than organisms (24.6%) but the latter had a higher PPV (78.5%). Increased peripheral WBC (86.1%) or CRP (71.8%) predicted positive BR 16S PCR results. Our DPO BR 16S PCR assay improved pathogen detection with minimal contamination.