Polymerase chain reaction detection of sepsis-inducing pathogens in blood using SepsiTest™

Background

PCR enables the identification of bacterial DNA in culture-negative samples from patients with suspected infection, allowing the confirmation of, for example, meningitis and septic arthritis. Gross discrepancies in the incidence of positive results between culturing and PCR have been reported, the latter corresponding better to bacterial loads observed by immunofluorescence microscopy and inflammatory response measurements. The goals of PCR assaying of clinical samples for pathogens are improved disease surveillance, early guidance on appropriate antibiotic therapy and patient management.

Methods

SepsiTest™ is a new PCR test for the presence of bacterial and yeast pathogens in whole blood samples. The test combines sample preparation, the directed extraction of pure pathogen DNA from 1 ml blood, with PCR assays for the universal detection of bacteria and yeasts based on the amplification and monitoring of 16S and 18S rDNA sequences, respectively. Blood from septic patients was extracted and analysed, using SepsiTest™ together with sequencing of amplicons from positive samples and online BLASTN analysis for the identification of pathogens.

Results

The test was validated by the determination of the limits of detecting pathogens in blood (spiking experiments, >95% sensitivity, n = 6), including (colony-forming units/ml) Staphylococcus epidermidis (20), Staphylococcus aureus (40), Streptococcus pneumoniae (40), Escherichia faecalis (120), Escherichia coli (150), Klebsiella pneumoniae (110), Enterobacter aerogenes (210), Pseudomonas aeruginosa (460) and Candida albicans (400). In total, samples from 55 patients with systemic inflammatory response syndrome criteria were analysed in an ongoing study. Compared with blood culturing, the preliminary data showed a diagnostic sensitivity of 60%, specificity of 98%, negative predictive value of 91% and positive predictive value of 86%.

Conclusion

The data are discussed with respect to the significance of the molecular test for the diagnosis of sepsis. Special emphasis is put on the clinical data available supporting the finding of PCR-positive but culture-negative results.