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Between 2015 and 2022, we evaluated a novel broad-range (BR) 16S PCR rDNA PCR/Sanger sequencing assay to improve diagnosis of invasive infections in culture-negative specimens. Using dual-priming oligonucleotides (DPO), this assay analyzed ribosomal DNA from sterile fluids or tissues. A total of 762 specimens were analyzed from 661 patients: 61% had negative cultures and BR 16S PCR tests; 35% had negative cultures but positive BR 16S PCR tests; and only 4% had negative cultures with indeterminate BR 16S PCR results. After resolution of indeterminate BR 16S PCR results (i.e., 29 negative, 1 false-positive, and 1 positive) the assay showed a sensitivity of 98.26% (95% CI = 96.00–99.43%), specificity of 99.79% (95% CI: 99.82–99.99%), positive predictive value of 99.65% (95% CI: 97.56–99.95%), negative predictive value of 98.94% (95% CI: 97.51–99.55%), and accuracy of 99.21% (95% CI: 98.28–99.71%) for a disease prevalence of 38.10% (95% CI: 34.62–41.66%). 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 over culture and minimized contamination. Broad range 16S rDNA PCR/sequencing (BR 16S PCR) is an important diagnostic technique in cases with invasive infection due to fastidious or uncultivatable pathogens. However, appropriate case selection, the quality of clinical specimen, and the specific assay primers affect its performance. Our novel BR 16S PCR assay uses unique dual-priming oligonucleotides (DPO) primers and fast protocols for rapid, optimal detection of bacterial pathogens, while minimizing contamination. Fast BR 16S PCR assay reports occurred within 24–48 h. BR 16S PCR and culture analyzed a diverse range of clinical specimens from patients with invasive infections. BR 16S PCR demonstrated a high performance for accurately detecting pathogens, ruling out infections, and minimizing contamination. BR 16S PCR detection of a pathogen allowed the appropriate clinical management of one-third of patients in this cohort. BR 16S PCR is an essential tool for the clinical management of patients with invasive infection when primary cultures are negative or contaminated. Full article