Feasibility of a novel approach for rapid detection of simulated bloodstream infections via enzymatic template generation and amplification (ETGA)-mediated measurement of microbial DNA Polymerase activity.

Bloodstream infections (BSIs) caused by bacteria and fungi are associated with significant morbidity and mortality. Currently, blood culture is the gold standard for confirming a suspected BSI, but has the drawback of lengthy time-to-detection (TTD) required for indicating the presence of microbes. Detection of conserved microbial nucleic acid sequences within blood culture samples via PCR has been demonstrated to offer potential for reducing the TTD of BSI; however, these approaches have various other limitations. We report a novel approach toward rapid detection of microbes from simulated BSI via differential hematopoietic cell lysis followed by enzymatic template generation and amplification (ETGA)-mediated measurement of microbial DNA polymerase extension activity. The differential cell lysis procedure effectively reduced the level of detectable DNA polymerase extension activity associated with human-derived hematopoietic cells present in blood culture samples taken from healthy donors. After treatment with the differential cell lysis procedure, the ETGA assay detected a panel of clinically prevalent bacteria and Candida albicans from spiked blood culture samples. The ETGA blood culture method also reduced by threefold the TTD required for simulated BSI, compared with a continuous-monitoring blood culture instrument. In summary, these findings demonstrate the feasibility of an innovative approach toward a rapid, sensitive, and universal screen for microbes within blood culture samples. Potential for clinical application and automation are also addressed.