Rapid and accurate diagnosis of bloodstream infections: A multiplex real-time PCR assay with selective microbial enrichment for direct whole-blood detection and positive blood culture confirmation.

BACKGROUND: Bloodstream infections (BSIs) remain a critical clinical challenge with high morbidity and mortality, yet conventional blood culture-based diagnostics delayed timely antimicrobial therapy due to long turnaround times. This study evaluated a rapid BSI diagnostic method combining selective microbial enrichment with multiplex real-time fluorescent polymerase chain reaction (PCR), with a focus on direct whole-blood detection as the primary innovation. METHODS: The enrichment-PCR assay targets 16 common BSI pathogens. Technical performance was confirmed using 479 Gram-stained positive blood culture bottles (May-October 2025, two centers), with conventional culture + MALDI-TOF MS as the reference standard. Direct whole-blood detection was then prospectively evaluated in 50 patients with suspected BSI, using a composite reference standard of blood culture and metagenomic next-generation sequencing (mNGS). Clinical impact was assessed by analyzing time savings and antimicrobial therapy adjustments. RESULTS: In positive blood culture bottles, enrichment-PCR generated results in 2 h (vs. 18-24 h for conventional methods), achieving 97.48% overall concordance with reference standards (95% CI: 95.65%-98.56%). In the 50-patient direct whole-blood cohort, enrichment-PCR identified all 14 culture-confirmed cases (100% concordance) and detected 6 additional culture-negative infections that were validated by mNGS. Direct testing reduced the median time to pathogen identification from blood collection to approximately 4 h, compared with a median of 19.6 h to blood culture positivity. Among the 6 culture-negative, PCR-positive cases, early identification led to targeted antimicrobial adjustments in 5 of 6 patients. One false-positive result was observed. The enrichment step reduced Ct values by 3-4 cycles (10-fold sensitivity gain; P < 0.001), achieving a limit of detection of 100 CFU/mL. CONCLUSION: This enrichment-PCR assay enables rapid, accurate BSI diagnosis, with the direct whole-blood application providing a meaningful clinical time advantage over culture-based methods. Preliminary data suggest potential for detecting culture-negative BSI and guiding early antimicrobial decisions, although validation in larger cohorts is needed.