Accelerated bacterial detection in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). 2019

Xiao Xuan Huang, and Nadezda Urosevic, and Timothy J J Inglis
Department of Microbiology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, Western Australia, Australia.

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 generic detection of bacteria 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 at a low starting concentration of 10 CFU/mL: Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa. 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 culture incubation of 5 to 10 hours. Using either PCR-based FilmArray 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 for rapid bacterial detection in BC with significantly improved turnaround time over currently used laboratory techniques.

UI MeSH Term Description Entries
D007711 Klebsiella pneumoniae Gram-negative, non-motile, capsulated, gas-producing rods found widely in nature and associated with urinary and respiratory infections in humans. Bacillus pneumoniae,Bacterium pneumoniae crouposae,Hyalococcus pneumoniae,Klebsiella pneumoniae aerogenes,Klebsiella rhinoscleromatis
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D011550 Pseudomonas aeruginosa A species of gram-negative, aerobic, rod-shaped bacteria commonly isolated from clinical specimens (wound, burn, and urinary tract infections). It is also found widely distributed in soil and water. P. aeruginosa is a major agent of nosocomial infection. Bacillus aeruginosus,Bacillus pyocyaneus,Bacterium aeruginosum,Bacterium pyocyaneum,Micrococcus pyocyaneus,Pseudomonas polycolor,Pseudomonas pyocyanea
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000071997 Blood Culture Test to determine the presence of blood infection (e.g. SEPSIS; BACTEREMIA). Blood Culture Test,Blood Culture Tests,Blood Cultures,Culture Test, Blood,Culture Tests, Blood,Culture, Blood,Cultures, Blood,Test, Blood Culture,Tests, Blood Culture
D001419 Bacteria One of the three domains of life (the others being Eukarya and ARCHAEA), also called Eubacteria. They are unicellular prokaryotic microorganisms which generally possess rigid cell walls, multiply by cell division, and exhibit three principal forms: round or coccal, rodlike or bacillary, and spiral or spirochetal. Bacteria can be classified by their response to OXYGEN: aerobic, anaerobic, or facultatively anaerobic; by the mode by which they obtain their energy: chemotrophy (via chemical reaction) or PHOTOTROPHY (via light reaction); for chemotrophs by their source of chemical energy: CHEMOLITHOTROPHY (from inorganic compounds) or chemoorganotrophy (from organic compounds); and by their source for CARBON; NITROGEN; etc.; HETEROTROPHY (from organic sources) or AUTOTROPHY (from CARBON DIOXIDE). They can also be classified by whether or not they stain (based on the structure of their CELL WALLS) with CRYSTAL VIOLET dye: gram-negative or gram-positive. Eubacteria
D016470 Bacteremia The presence of viable bacteria circulating in the blood. Fever, chills, tachycardia, and tachypnea are common acute manifestations of bacteremia. The majority of cases are seen in already hospitalized patients, most of whom have underlying diseases or procedures which render their bloodstreams susceptible to invasion. Bacteremias
D017404 In Situ Hybridization, Fluorescence A type of IN SITU HYBRIDIZATION in which target sequences are stained with fluorescent dye so their location and size can be determined using fluorescence microscopy. This staining is sufficiently distinct that the hybridization signal can be seen both in metaphase spreads and in interphase nuclei. FISH Technique,Fluorescent in Situ Hybridization,Hybridization in Situ, Fluorescence,FISH Technic,Hybridization in Situ, Fluorescent,In Situ Hybridization, Fluorescent,FISH Technics,FISH Techniques,Technic, FISH,Technics, FISH,Technique, FISH,Techniques, FISH

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