Retrospective analysis of clinical data associated with patients enrolled in a molecular diagnostic feasibility study highlights the potential utility for rapid detection of bloodstream infection.

BACKGROUND: Measurement of pathogen DNA polymerase activity by enzymatic template generation and amplification (ETGA) has shown promise in detecting pathogens in bloodstream infection (BSI). We perform an in-depth analysis of patients with clinical BSI enrolled in ETGA feasibility experiments. METHODS: In addition to hospital blood cultures, 1 study aerobic culture bottle was drawn from patients with suspected BSI. The study bottle was split into 2 bottles and was additionally subjected to ETGA analysis. Enzymatic template generation and amplification sensitivity/specificity for BSI detection was determined against the Centers for Disease Control BSI definition. When split cultures were both positive, time course analysis was performed to determine time to detection. The records of patients with BSI were reviewed for presence of systemic inflammatory response syndrome, antibiotic timing and appropriateness, and organism identification. RESULTS: Of 307 enrollees, 38 met the Centers for Disease Control BSI definition. Seventy-four percent met systemic inflammatory response syndrome criteria on admission. Antibiotic coverage was adequate in 76% of patients. Antibiotics were more often delayed in afebrile patients (odds ratio, 5). Twenty-seven of the split study culture bottles were positive in at least 1 sample, and ETGA detected microbes within all samples (sensitivity/specificity, 70.3%/99.3%). Of these, 22 were culture positive in both split study bottles and underwent ETGA time course analysis. Enzymatic template generation and amplification detected microbes within these 3-fold faster than culture. CONCLUSIONS: Patients with BSI often have diagnostic and treatment delays. Enzymatic template generation and amplification provides clinically meaningful data more rapidly than cultures. Future development should focus on real-time application of assays that detect microbes at the molecular level.

A novel approach for rapid detection of bacterially contaminated platelet concentrates via sensitive measurement of microbial DNA polymerase activity.

BACKGROUND: Transfusion of bacterially contaminated platelet concentrates (PCs) can result in serious health consequences for the affected patient. Before being released from blood banking facilities, PCs are routinely screened for bacterial contamination by culture-based tests. However, culture-based PC screening methods require extended holding and incubation periods and are prone to false-negative results due to sampling error. Screening PCs closer to the time of transfusion using rapid point-of-issue tests represents an alternative approach; however, FDA-approved assays generally suffer from a lack of sensitivity. STUDY DESIGN AND METHODS: Presented herein is the feasibility of a novel approach toward rapid, sensitive, and universal detection of bacterially contaminated PCs via selective measurement of microbial DNA polymerase activity. This approach is achieved using a differential cell lysis procedure in combination with enzymatic template generation and amplification (termed ETGA-PC assay). RESULTS: Serial dilution spiking experiments revealed an approximate sensitivity of 30 to 200 colony-forming units (CFUs)/mL (mean, 85 CFUs/mL) for Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. An additional 22 clinically relevant strains of bacteria were also detected below 200 CFUs/mL after spiking into PC aliquots. Furthermore, the ETGA-PC assay was able to accurately monitor the presence and growth of seven clinically relevant bacterial species that were spiked into PC units. CONCLUSION: Together, the data presented here demonstrate that the ETGA-PC assay is a feasible approach for rapid and sensitive detection of bacterially contaminated PCs. Experiments, aimed at simplification and/or automation of the assay procedure, are under way.

Measurement of microbial DNA polymerase activity enables detection and growth monitoring of microbes from clinical blood cultures.

Surveillance of bloodstream infections (BSI) is a high priority within the hospital setting. Broth-based blood cultures are the current gold standard for detecting BSI, however they can require lengthy incubation periods prior to detection of positive samples. We set out to demonstrate the feasibility of using enzymatic template generation and amplification (ETGA)-mediated measurement of DNA polymerase activity to detect microbes from clinical blood cultures. In addition to routine-collected hospital blood cultures, one parallel aerobic blood culture was collected and immediately refrigerated until being transported for ETGA analysis. After refrigeration holding and transport, parallel-collected cultures were placed into a BACTEC incubator and ETGA time-course analysis was performed. Of the 308 clinical blood cultures received, 22 were BACTEC positive, and thus were initially selected for ETGA time course analysis. The ETGA assay detected microbial growth in all 22 parallel-positive blood cultures in less time than a BACTEC incubator and also yielded genomic DNA for qPCR-based organism identification. In summary, feasibility of detecting microbes from clinical blood culture samples using the ETGA blood culture assay was demonstrated. Additional studies are being considered towards development of clinically beneficial versions of this methodology.

Feasibility study demonstrating that enzymatic template generation and amplification can be employed as a novel method for molecular antimicrobial susceptibility testing.

BACKGROUND: Antimicrobial Susceptibility Testing (AST) is a methodology in which the sensitivity of a microorganism is determined via its inability to proliferate in the presence of an antimicrobial agent. Results are reported as minimum inhibitory concentrations (MICs). The present study demonstrates that measurement of DNA polymerase activity via Enzymatic Template Generation and Amplification (ETGA) can be used as a novel means of determining the MIC of a microbe to an antibiotic agent much sooner than the current standardized method. METHODS: Time course analysis of ETGA is presented from bacterial cultures containing antibiotic agents and compared to the end-point results of standard macrobroth method AST. RESULTS: MIC determinations from ETGA results at 4, 6, and 22 hours are compared to the MICs from the standard method and the results are shown to be in agreement. Additionally, reliable AST analysis using ETGA can be performed on bacteria harvested directly from spiked blood cultures. CONCLUSIONS: AST analysis with ETGA is shown to be equivalent to AST analysis using gene-specific qPCR assays against the measured microbe. Future development of this novel method for performing AST in a clinical setting is discussed.

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.

Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes.

During the past 50 years, in vitro measurement of DNA polymerase activity has become an essential molecular biology tool. Traditional methods used to measure DNA polymerase activity in vitro are undesirable due to the usage of radionucleotides. Fluorescence-based DNA polymerase assays have been developed; however, they also suffer from various limitations. Herein we present a rapid, highly sensitive and quantitative assay capable of measuring DNA polymerase extension activity from purified enzymes or directly from microbial lysates. When tested with purified DNA polymerase, the assay detected as little as 2 × 10(-11)U of enzyme (∼ 50 molecules), while demonstrating excellent linearity (R(2)=0.992). The assay was also able to detect endogenous DNA polymerase extension activity down to less than 10 colony forming units (cfu) of input Gram-positive or Gram-negative bacteria when coupled to bead mill lysis while maintaining an R(2)=0.999. Furthermore, preliminary evidence presented here suggests that DNA polymerase extension activity is an indicator of microbial viability, as demonstrated by the reproducibly strong concordance between assay signal and bacterial colony formation. Together, the innovative methodology described here represents a significant advancement toward sensitive detection of potentially any microorganism containing active DNA polymerase within a given sample matrix.