RAPID ASSESSMENT OF BACTERIAL LOAD AND RESISTANCE PHENOTYPE IN PERITONEAL DIALYSIS EFFLUENT BY FLOW CYTOMETRY

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 use flow cytometry to identify bacterial load and antimicrobial resistance in a patient with peritonitis who had received antibiotics prior to sample collection.

Background: Culture-negative peritonitis cases are common, with rates as high as 20% in some centres. We have developed a novel flow cytometry application allowing identification and quantification of bacterial load, as well as direct determination of antimicrobial sensitivity using our in-house flow-assisted susceptibility testing (FAST) assay. We present a case study using the FAST assay to assess a dialysate sample received after empirical antibiotic therapy had been administered, invalidating standard microbiology techniques.

Methods: Dialysate was analysed by flow cytometry on an Attune NxT flow cytometer. 10 μM SYTO® 9 and 2× Fixable Near-IR amine-reactive viability dye were used for specific identification of cellular (host and bacterial) populations. An enriched sub-specimen was subjected to our FAST assay. Transmission electron microscopy was used to confirm the morphology of the bacterial population found in the dialysate.

Results: A leucocyte population (CD45 positive) at a density of 1.12 x 106 /mL, and a bacterial population at 1.2 x 106 / mL, were detected within 2 hours of sample collection. The FAST assay, gave an invalid result consistent with previous antibiotic exposure, with results available within 4 hours. Transmission electron microscopy studies showed cell carcasses consistent with a Gram positive, spore forming bacillus, with the characteristic features of Micrococcus spp.

Conclusions: We present evidence for the substantial time-benefits of assessment of PD effluent by flow cytometry over traditional culture-based testing, demonstrate a workflow for validating the findings of bacterial flow assays, and highlight some of the difficulties with adopting this technology for routine clinical use.

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