A comparison of routine blood culture methods and multiplex quantitative PCR for detecting pathogens in simulated polymicrobial blood cultures

Introduction. Polymicrobial bacteraemia, the simultaneous presence of multiple bacterial species in the bloodstream, complicates diagnosis and treatment and is linked to poorer patient outcomes. Accurate detection is essential for effective clinical management. Gap Statement. Despite being the diagnostic gold standard, conventional blood culture may fail to detect all pathogens in polymicrobial infections, particularly when species differ in growth rate or abundance. The relative performance of culture versus molecular methods under these conditions remains poorly characterized. Aim. To compare the performance of conventional blood culture and quantitative PCR (qPCR) for detecting pathogens in simulated polymicrobial blood cultures containing fast- and slow-growing bacteria at varying ratios and concentrations. Methodology. Four clinically relevant polymicrobial mixtures were prepared using seven bacterial species. Human blood was spiked with bacterial suspensions at varying ratios and inoculated into BACTEC blood culture bottles. After incubation, samples were analysed using standard culture techniques and an in-house multiplex qPCR assay. Results. Routine culture detected both organisms in only 42% of samples, while qPCR identified both pathogens in 83%. Differences in bacterial growth rates significantly influenced culture outcomes, with slower-growing or less abundant species frequently missed. Notably, Staphylococcus aureus was not detected when co-cultured with Escherichia coli at ratios of 1:1 to 25:1, and only visible on Gram stain when S. aureus was initially 50 times more abundant. Conclusion. These findings highlight a key limitation of conventional methods in detecting polymicrobial infections and underscore the need for more sensitive diagnostics. qPCR offers improved detection, particularly when organism abundance and growth rates vary. Incorporating molecular tools alongside routine culture may enhance diagnostic accuracy and provide a more complete understanding of polymicrobial bacteraemia.