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

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.

Clinical Utility of Molecular Tests for Guiding Therapeutic Decisions in Bloodstream Staphylococcal Infections: A Meta-Analysis

“…Supplementary Table 1 summarizes the studies reviewed, includin why these studies were excluded (see Supplementary Table 1 ). Essentially, 33 studies were included in subsequent analyses that met all the inclusion criteria ( 21 , 32 – 63 ).…”

“…Table 1 shows the baseline features of the studies included ( 21 , 32 – 63 ). Twenty-six studies were performed in countries with high incomes and seven in countries with lower-middle incomes.…”

“…Eight studies ( 21 , 32 , 38 , 48 , 51 , 55 , 57 , 59 ) met the inclusion criteria for comparing NAAT with a microbiological culture among children consisting of a total of 4,089 samples for MSSA detection in suspected BSI patients. The sensitivity and specificity of NAAT for MSSA detection ranged from 0.25 (95% CI 0.0–0.94) to 1.0 (95% CI, 0.85–1.0), whilst our search did not result in any MRSA studies ( Figure 3 ).…”

Species-specific bacterial detector for fast pathogen diagnosis of severe pneumonia patients in the intensive care unit

“…Conventional culture test (CCT) is the most commonly used and golden standard identification method of pathogenic microorganisms in most countries. However, it showed two critical limitations: long time-consuming (2 to 5 days) and low sensitivity (30-50%), which limited the application of this method in the ICU [6, 7]. To overcome this bottleneck, several new tools were developed and showed significant improvement in time consumption and accuracy.…”

Pathogenicity and drug resistance of animal streptococci responsible for human infections

“…MALDI-TOF MS is a technique widely used in microbiology, based on analysis of the so-called fingerprints of the tested bacterial isolate’s proteome. This method allows us to obtain an identification in a short time, provides better accuracy and does not require multiplication of the microorganism [21, 22].…”