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Comparative Study
. 2021 May;130(5):1630-1644.
doi: 10.1111/jam.14862. Epub 2020 Oct 18.

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

N K Abd El-Aziz  1 , A A Gharib  1 , E A A Mohamed  1 , A H Hussein  2
Affiliations
Comparative Study

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

N K Abd El-Aziz et al. J Appl Microbiol. 2021 May.

Abstract

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 97·37% unlike the conventional culture. According to qPCR results, Staphylococcus aureus (24·24%) was predominated in animal pyogenic infections, whereas Klebsiella pneumonia (31·81%) 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.

Keywords: MALDI-TOF MS; blood culture; real-time PCR; sepsis.

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