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: Escherichia coli (ECO), Klebsiella pneumoniae (KPN), and Streptococcus pneumoniae (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.

Background: Klebsiella pneumoniae bloodstream infection (KP-BSI) represents a global clinical threat, while local prognostic data are scarce in Hebei Province, China. This study aimed to identify risk factors for carbapenem-resistant Klebsiella pneumoniae (CRKP) infection and in-hospital mortality in KP-BSI patients. Methods: A retrospective observational audit was conducted at a provincial tertiary hospital. Adult non-repeat KP-BSI episodes (2022–2025) were divided into CRKP (n = 156) and carbapenem-susceptible Klebsiella pneumoniae (CSKP, n = 357), as well as survivor and non-survivor subgroups. Clinical, laboratory and microbiological data were extracted from electronic medical records. Univariate and multivariate logistic regression analyses were performed. Results: Among 513 KP-BSI cases, CRKP detection rate was 30.4% and the overall in-hospital mortality was 25.0%. CRKP isolates showed extensive multidrug resistance, with low resistance to colistin (3.5%) and tigecycline (49.0%). Advanced age (per 10-year increment), male sex, invasive procedures, empirical antimicrobial therapy and pre-existing respiratory disease were independent risk factors for CRKP infection. quick Pitt bacteremia score (qPitt score) ≥ 2 and intensive care unit (ICU) admission independently predicted in-hospital mortality; CRKP bacteremia was not an independent mortality predictor after confounder adjustment. Multisite KP isolation occurred in 44.4% of patients, most frequently from the respiratory tract. Conclusions: CRKP-BSI imposes a considerable resistance burden. Infection control should target high-risk patients: elderly males with respiratory comorbidities undergoing invasive procedures. Mortality depends on disease severity (qPitt score, ICU admission), not carbapenem-resistance status. qPitt-based risk stratification and antimicrobial stewardship are essential to improve patient outcomes.