RAPID FLOW CYTOMETRIC IDENTIFICATION AND ANALYSIS OF STAPHYLOCOCCUS SPP. FROM PERITONEAL DIALYSATE

K MULRONEY1, T INGLIS2, A CHAKERA1, 3,

1Translational Renal Research Group, Harry Perkins Institute of Medical Research, Perth, Western Australia; 2Department of Microbiology; PathWest Laboratory Medicine, Perth, Australia; 3Renal Unit, Sir Charles Gairdner Hospital, Perth, Western Australia

Aim: To assess the utility of an anti-Staphylococcal antibody for flow cytometry detection of Staphylococcal spp from peritoneal dialysate.

Background: Staphylococci are a common cause of peritonitis. Clinical outcomes for patients are directly impacted by the speed with which appropriate antimicrobial therapy can be administered. Current identification and antibiotic sensitivity testing for causative organisms leads to delays between 48 and 72 hours. We present a technique for identification of Staphylococci, to the genus level, within 3 hours of dialysate collection.

Methods: Bacterial control strains and dialysate from 10 cases of peritonitis were analysed. Suspensions were incubated with a monoclonal anti-Staphylococcus antibody, and exposed to a secondary antibody conjugated to AlexaFluor® 647. Samples were washed, then stained with 10µM SYTO® 9 and 2x Live/DEADTM Fixable Violet. Samples were acquired using an Attune NxT flow cytometer. Data were analysed with FlowJo.

Results: 75-95% of all events in samples of S. aureus and S. epidermidis were positive for the presence of the antibody, with an increase in antibody-specific mean fluorescence intensity between 1-2 orders of magnitude. In E. coli and K. pneumoniae samples, 5% – 7% of events had a fluorescence intensity above unstained cells. Approximately 90% of events for Strep. pneumoniae samples were positive. Of the 10 clinical isolates tested, all samples containing Staphylococcus spp. were positive and matched results from conventional culture and MALDI-Biotyper analysis.

Conclusions: Use of an antibody specific for Staphylococcus spp. in a flow cytometry assay provides a rapid measure of identifying and enumerating Staphylococcal cells from clinical samples, but currently provides a false positive for Streptococcus pneumoniae. Further development will be required to improve specificity, and target range, of the assay.

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