Development of a new protocol for rapid bacterial identification and susceptibility testing directly from urine samples

Abstract

The current gold standard method for the diagnosis of urinary tract infections (UTI) is urine culture that requires 18-48 h for the identification of the causative microorganisms and an additional 24 h until the results of antimicrobial susceptibility testing (AST) are available. The aim of this study was to shorten the time of urine sample processing by a combination of flow cytometry for screening and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for bacterial identification followed by AST directly from urine. The study was divided into two parts. During the first part, 675 urine samples were processed by a flow cytometry device and a cut-off value of bacterial count was determined to select samples for direct identification by MALDI-TOF-MS at ≥5 × 10(6) bacteria/mL. During the second part, 163 of 1029 processed samples reached the cut-off value. The sample preparation protocol for direct identification included two centrifugation and two washing steps. Direct AST was performed by the disc diffusion method if a reliable direct identification was obtained. Direct MALDI-TOF-MS identification was performed in 140 urine samples; 125 of the samples were positive by urine culture, 12 were contaminated and 3 were negative. Reliable direct identification was obtained in 108 (86.4%) of the 125 positive samples. AST was performed in 102 identified samples, and the results were fully concordant with the routine method among 83 monomicrobial infections. In conclusion, the turnaround time of the protocol described to diagnose UTI was about 1 h for microbial identification and 18-24 h for AST.

New and developing diagnostic technologies for urinary tract infections

“…Flow cytometry is a good system for selecting samples for further analysis, and has been used to identify pathogen-positive urine for further complex testing, such as mass spectrometry analysis. Initial screening of urine samples by flow cytometry might improve clinical laboratory workflow by reducing the number of samples sent for further analysis; however, flow cytometry is only a screen for bacteriuria as it does not provide species identification or AST for definitive diagnosis 48–50 .…”

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm

“…Inigo et al established 2 ml as the optimum volume, as it was easily manipulated in Eppendorf tubes and centrifuges (15). Zboromyrska et al used 10 ml of urine for MALDI-TOF MS and obtained higher score values of direct identification (16). In order to improve the LOD, Sánchez-Juanes et al added a pretreatment with SDS to enhance cell lysis and the release of microorganisms, and Veron et al compared different methods, concluding that previous short-culture and dual-filtration methods provided the best results (18,19).…”

“…Three studies have been reported to combine MALDI-TOF MS with rapid UA (4,15,16). In order to evaluate the performance of the combination of UA and MALDI-TOF MS, only samples that were granulocyte esterase positive or nitrate reduction test positive or had bacterial counts that were greater than 500/l were combined with MALDI-TOF MS.…”

“…Much effort has been spent to improve urine processing and to enhance the identification capability of MALDI-TOF MS by coupling it with rapid urine analysis (4,10,12,(14)(15)(16). This study aimed to establish and optimize a urine processing method to improve the analytical sensitivity and specificity of MALDI-TOF MS.…”

Proteomics progresses in microbial physiology and clinical antimicrobial therapy

“…The MS identification of Candida species directly from blood culture bottles within 30 min was concordant with the conventional culture-based method for 95.9% of C. albicans and 86.5% of Candida non- albicans [72]. Moreover, mass cytometry, namely flow cytometry coupled with mass spectrometry, has been applied to rapidly process urine samples [77, 78]. …”