Accelerated detection of Gram negative bacteria in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)

Abstract

Bacteraemia is a risk factor for subsequent clinical deterioration and death. Current reliance on culture-based methods for detection of bacteraemia delays identification and assessment of this risk until after the optimal period for positively impacting treatment decisions has passed. Therefore, a method for rapid detection and identification of bacterial infection in the peripheral bloodstream in acutely ill patients is crucial for improved patient survival through earlier targeted antibiotic treatment. The turnaround time for current clinical laboratory methods ranges from 12 to 48 hours, emphasizing the need for a faster diagnostic test. Here we describe a novel assay for accelerated detection of bacterial infection in blood culture (BC) using peptide nucleic acid fluorescence in situ hybridization enhanced acoustic flow cytometry (PNA-FISH-AFC). For assay development, we used simulated blood cultures (BCs) spiked with one of three bacterial species: Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa at a low concentration of 10 CFU/mL. Under current clinical settings, it takes a minimum of 12 hours incubation to reach positivity on the BacTEC system, corresponding to a bacterial concentration of 107-109 CFU/mL optimal for further analyses. In contrast, our PNA-FISH-AFC assay detected 103 - 104 CFU/mL bacteria in BC following a much shorter incubation of 5 to 10 hours in culture. Using either PCR-based FilmArray(R) assay or MALDI-TOF for bacterial detection, it took 7-10 and 12-24 hours of incubation, respectively, to reach the positive result. These findings indicate a potential time advantage of PNA-FISH-AFC assay over currently used laboratory techniques for rapid bacterial detection in BC with significantly improved turnaround time.