Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis

Abstract

BackgroundMicrobiological diagnosis of sepsis relies primarily on blood culture data. This study compares four diagnostic methods, i.e. those developed by us: nested, multiplex, qPCR (qPCR) and FISH with commercial methods: SeptiFast (Roche) (SF) and BacT/ALERT® 3D blood culture system (bioMérieux). Blood samples were derived from adult patients with clinical symptoms of sepsis, according to SIRS criteria, hospitalized in the Intensive Care Unit.ResultsUsing qPCR, FISH, SF, and culture, microbial presence was found in 71.8%, 29.6%, 25.3%, and 36.6% of samples, respectively. It was demonstrated that qPCR was significantly more likely to detect microorganisms than the remaining methods; qPCR confirmed the results obtained with the SF kit in all cases wherein bacteria were detected with simultaneous confirmation of Gram-typing. All data collected through the FISH method were corroborated by qPCR.ConclusionsThe qPCR and FISH methods described in this study may constitute alternatives to blood culture and to the few existing commercial molecular assays since they enable the detection of the majority of microbial species, and the qPCR method allows their identification in a higher number of samples than the SF test. FISH made it possible to show the presence of microbes in a blood sample even before its culture.

Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia

“…Similar results obtained using SeptiFast test (Roche) demonstrated the presence of bacteria in 75 % of analyzed blood samples [17]. Gosiewski et al, thanks to the nested PCR method, indicated that 71.8 % of analyzed samples tested positive for bacterial presence [8]. …”

“…In addition, blood culture method detects only viable bacterial cells. There are also few molecular, culture-independent methods, such as PCR or FISH (Fluorescent In Situ Hybridization), that enable the detection of selected species of bacteria based on the presence of their DNA [7, 8]. These techniques are very sensitive and allow for quick detection of even a very low number of microorganisms in the samples.…”

Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing

“…In this respect, the safety of the blood supply, as well as our understanding of the involvement of the blood microbiome in health and disease, may benefit from sensitive and exhaustive methods to detect bacteria in blood such as quantitative polymerase chain reaction (qPCR) and high‐throughput targeted metagenomics sequencing. These methods, which are already used in some cases to screen for sepsis in which a single bacterial strain is highly present in blood, are much more challenging to apply to blood from healthy donors. Not only is the blood microbiome highly diverse but, in addition, the overall bacterial quantity present in the blood of healthy individuals is obviously much lower than in the case of sepsis.…”

Improving molecular diagnosis of neonatal septicaemia: Comparative evaluation of different protocols for extraction of bacterial and fungal DNA from blood samples

“…[13] Lowest sensitivity reported was 10 1 CFU/ml for each of the four studied species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus). [14] This difference in LOD may be due to various factors like method of DNA extraction, primersprobes used, reagents and PCR reaction used.…”