Enrichment of bacterial DNA for the diagnosis of blood stream infections

Abstract

BackgroundBlood cultures are commonly employed to identify bacterial pathogens causing sepsis. PCR assays to diagnose septicemia require extraction of bacterial DNA from blood samples and thus, delay the initiation of appropriate antimicrobial treatment. The presence of abundant human DNA may hamper the sensitivity of PCR in the detection of bacteria.MethodsWe used serial dilutions of E. Coli spiked pseudo-blood-sepsis samples to develop a simple method that combines the use of a polar detergent solvent and adjustment of the basic pH to remove human DNA. A 16S rRNA gene-based screening algorithm was established to differentiate Gram-positive and Gram-negative groups of bacteria and the family of Enterobacteriaceae. A stringent validation with appropriate controls was implemented. The method of human DNA removal was then applied on 194 sepsis blood samples and 44 cerebrospinal fluid (CSF) samples by real-time PCR.ResultsThis uncomplicated and straightforward approach allows to remove up to 98 % of human DNA from peripheral blood of septic patients. The inhibitory effect of human DNA is efficiently prevented and the detection limit of real-time PCR is increased to 10 E. Coli CFUs/ml. This sensitivity is 10 times higher compared to conventional real-time PCR assays. The classical blood culture detected 58/194 (30 %) of sepsis and 9/44 (21 %) of CSF samples. Out of the 194 blood samples tested, the conventional real-time PCR targeting 13 common sepsis causing pathogens correctly detected the bacterial DNA in 16/194 (8 %) only and 14/44 (32 %) in cerebrospinal fluid samples. Our newly established approach was able to provide correct diagnoses in 78 (40 %) of the 194 blood samples and in 14 (32 %) of the CSF samples. The combination of both blood cultures and our technique raised the rate of sepsis diagnoses to 112/194 (58 %). Of the total group tested positive, 46 (24 %) cases showed overlap with the classical methodology.ConclusionWe report a simple optimized in-house protocol for removal of human DNA from blood sepsis samples as a pre-analytical tool to prepare DNA for subsequent PCR assays. With the detection increase of our in-house DNA removal approach, subsequent PCR assays can reach detection limits of 10 E. coli CFUs/ml and significantly improve the diagnostic rate in blood sepsis cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1568-1) contains supplementary material, which is available to authorized users.

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“…The homogenate obtained will be treated with collagenase D (Sigma-Aldrich) (2 mg/mL final concentration) at 37°C for 15 min, followed by 10 min at 2000 × g at room temperature (RT). The pellet will be resuspended in 2–5 mL (depending on the initial sample weight) of mammalian cell lysis buffer (MCLB), [24] and repeatedly vortexed for 5 min at RT. The reaction will be stopped with adding an equal volume of neutralisation buffer.…”

MagicplexTM Sepsis real-time test for the rapid diagnosis of bloodstream infections in adults

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