A Nested PCR-Based Point of Care Testing of Multiplex Pathogens Associated with Bloodstream Infection

Bloodstream infections (BSI) carry high mortality, but traditional blood culture is too slow for urgent clinical needs. This study aims to develop a rapid point-of-care testing assay based on one-tube multiplex nested polymerase chain reaction (PCR) (OM-NPCR-POCT) for early diagnosis of three pathogens in bloodstream infection patients: <i>Escherichia coli</i> (ECO), <i>Klebsiella pneumoniae</i> (KPN), and <i>Streptococcus pneumoniae</i> (SPN). The analytical sensitivity of the one-tube multiplex nested PCR (OM-NPCR) was evaluated using recombinant bacterial plasmids. The analytical sensitivity of the OM-NPCR-POCT assay was assessed using simulated samples. The clinical performance was evaluated in 90 clinical blood samples, with results compared to conventional quantitative PCR (qPCR). Finally, the agreement between the two detection methods was assessed via Kappa analysis. The limits of detection (LODs), calculated from serial dilution experiments, were 4, 2, and 1 copies/μL for plasmids ECO, KPN, and SPN, respectively. The OM-NPCR-POCT assay achieved LODs of 20, 10, and 10 CFU/mL for ECO, KPN, and SPN in simulated samples, with a total testing time of approximately 2 h. The clinical evaluation of OM-NPCR-POCT demonstrates consistency with conventional qPCR while exhibiting higher sensitivity. This method has potential as a rapid diagnostic tool for early bloodstream infection detection.