Diafiltration MALDI-TOF mass spectrometry method for culture-independent detection and identification of pathogens directly from urine specimens.

Abstract

With the aim of rapid, culture-independent identification of microorganisms directly from urine specimens, we developed a diafiltration matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. In this procedure, urine specimens are desalted, fractionated, and concentrated prior to MS analysis. The analytic performance characteristics of the diafiltration method were assessed in a prospective trial whereby 100 fresh urine specimens were processed using diafiltration MALDI-TOF MS. Concomitant with this, conventional culture was performed with results blinded to the MS operator. The diafiltration method correctly identified urine specimens positive for uropathogens (Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) and correctly classified all specimens negative for clinically relevant bacteriuria, including a subset of contaminated urine specimens and a subset with growth of clinically insignificant flora. The sensitivity and specificity of the assay were 67% and 100%, respectively. The detection limit of this method was 10(5) to 10(6) colony-forming units/mL. Using the diafiltration method, we were able to improve the turnaround time for microorganism identification from 24 to 48 hours (for conventional culture) to 2 to 3 hours. Although methodological refinements are under way to further improve the clinical sensitivity and turnaround time, the 100% positive predictive value of this method suggests that it could be used to guide the selection of antimicrobial agents.