Real-time PCR versus MALDI-TOF MS and culture-based techniques for diagnosis of bloodstream and pyogenic infections in humans and animals

Aims This study was applied to evaluate the usefulness of a high-throughput sample preparation protocol prior to the application of quantitative real-time PCR (qPCR) for the early diagnosis of bloodstream and pyogenic infections in humans and animals compared to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and classical culture. Methods and Results Saponin-mediated selective host cell lysis combined with DNase-1 was applied for processing of whole blood and pus clinical samples collected from suspected cases of septicaemia and pyogenic infections in humans and animals. The pre-PCR processing strategy enabled the recovery of microbial cells with no changes in their colony forming units immediately after the addition of saponin. DNase-1 was efficient for removing the DNAs from the host cells as well as dead cells with damaged cell membranes. The metagenomic qPCR and MALDI-TOF MS could identify the bacterial community of sepsis at species level with a concordance of 97 center dot 37% unlike the conventional culture. According to qPCR results,Staphylococcus aureus(24 center dot 24%) was predominated in animal pyogenic infections, whereasKlebsiella pneumonia(31 center dot 81%) was commonly detected in neonatal sepsis. Conclusions Saponin combined with DNase-1 allowed the efficient recovery of microbial DNA from blood and pus samples in sepsis using qPCR assay. Significance and Impact of the Study Metagenomic qPCR could identify a broad range of bacteria directly from blood and pus with more sensitivity, higher discriminatory power and shorter turnaround time than those using MALDI-TOF MS and conventional culture. This might allow a timely administration of a prompt treatment.

Automated summary

This study demonstrated that a high-throughput sample preparation protocol using saponin and DNase-1 enabled efficient recovery of microbial DNA from blood and pus samples for the early diagnosis of bloodstream and pyogenic infections. Metagenomic qPCR showed better sensitivity, discriminatory power, and turnaround time compared to MALDI-TOF MS and conventional culture, which could lead to timely administration of treatment.