2017 Volume 91 Issue 5 Pages 752-758
Bacterial sepsis is a leading cause of death worldwide. Rapid identification of causative pathogens is particularly important for treatment success, but conventional methods such as blood culture are time consuming. In this study, we developed and evaluated a combined cell-direct PCR/nucleic acid lateral flow (cdPCR/NALF) assay for rapid detection of bacterial DNA from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterobacter cloacae which are often isolated from blood culture. We focused on the process that neutrophils ingest bacteria during the early stage of infection. First of all, we prepared leukocyte samples which ingested one of the seven target bacterial strains in vitro and applied them to the cdPCR/NALF assay. By using the cdPCR assay, the bacterial DNA was directly amplified from monolayers of leukocytes coated on PCR tube. The species of bacteria were exactly identified by the NALF assay in a species-specific manner. Next, we compared the detection limit of a culture method and the cdPCR/NALF assay using blood incubated with E. coli or S. aureus in vitro. With the cdPCR/NALF assay, bacterial DNA in neutrophils was directly detected from whole blood within 4.5 h. Positive result with the cdPCR/NALF assay was also obtained from some culture-negative samples (＜10 colony forming unit/mL). Finally, the number of bacteria in the neutrophils was estimated with a realtime PCR assay. Positive result from the cdPCR/NALF assay was obtained when approximately 101 cells of bacteria were present in 200,000 leukocytes. These results indicated that the cdPCR/NALF assay was able to detect bacterial DNA in neutrophils with high sensitivity and specificity. The proposed assay is expected to have broad applicability in many clinical laboratories for identifying causative pathogens in sepsis.