A multicentre study investigating parameters which influence direct bacterial identification from urine

Abstract

Rapid diagnosis is one of the best ways to improve patient management and prognosis as well as to combat the development of bacterial resistance. The aim of this study was to study parameters that impact the achievement of reliable identification using a combination of flow cytometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS).The study was carried out in nine hospitals in Spain and included 1,050 urine samples with bacterial counts of 5x106 bacteria/ml. MALDI-ToF-MS-based identification was performed according to a previously described protocol. Valid identification by direct MALDI-ToF-MS was obtained in 72.8% of samples, in 80.3% of samples found to be positive by culture, 32.2% of contaminated samples, and 19.7% of negative samples. Among the positives samples with a valid identification the concordance at the species level was 97.2%. The parameters related to success of direct identification were: high bacterial count, the presence of Escherichia coli as a pathogen and rod-bacteria morphology provided by flow cytometry. The parameters related to failure were a high epithelial cell (EC) count, a high white blood cell (WBC) count and urine samples obtained from in-patients. In summary, this multicentre study confirms previously published data on the usefulness and accuracy of direct MALDI-ToF-MS-based identification of bacteria from urine samples. It seems important to evaluate not only the bacterial count, but also other parameters, such as EC and WBC counts, bacterial species and morphology, and the health care setting, to decide whether the sample is suitable for direct identification.

Rapid Identification and Antimicrobial Susceptibility Testing for Urinary Tract Pathogens by Direct Analysis of Urine Samples Using a MALDI-TOF MS-Based Combined Protocol

“…While Zboromyrska et al used 10 mL of urine with bacterial counts of ≥5,000 bacteria/μL for MALDI-TOF MS direct identification and obtained high scores (Zboromyrska et al, 2016). Moreover, these parameters were confirmed by a multicentre study that followed soon after (Zboromyrska et al, 2018).…”

“…For this study, 1,638 urine samples submitted to our clinical microbiology laboratory with sufficient volumes were screened for bacteria through a UF-1000i cytometer. Based on a previously established bacterial counts cut-off value of ≥5,000 bacteria/μL (Zboromyrska et al, 2016, 2018), 307 (18.74%) out of 1,638 samples were positive and used for further direct identification. Among these positive samples, 265 had a significant growth of a single microorganism by urine culture, 16 had a significant growth of two microorganisms, 22 were contaminated, and 4 were negative (Table 1).…”

“…In the past years, matrix-assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) has been considered as a rapid and trustable technique for identifying microorganisms from culture plates, positive blood cultures, and different clinical samples, such as urine (Ferreira et al, 2011; Clark et al, 2013). Previous studies have shown that urine itself can be used for direct identification by MALDI-TOF MS; besides, several studies have been carried out to improve sample processing and MALDI-TOF MS identification capacity (Ferreira et al, 2010; Wang et al, 2013; Burillo et al, 2014; Íñigo et al, 2016; Zboromyrska et al, 2016; Huang et al, 2017; Zboromyrska et al, 2018). However, these studies have focused on direct identification only, while not having addressed direct AST from urine, especially to accurately determine minimum inhibitory concentrations (MICs).…”

Application and Perspectives of MALDI–TOF Mass Spectrometry in Clinical Microbiology Laboratories

“…Before extraction with formic acid and acetonitrile, both are subjected to low-speed centrifugation to eliminate human cells [ 10 , 17 , 36 ]. For the processing of urine samples specifically, an initial flow cytometric scan is recommended to eliminate negative urine and limit direct processing to urine with counts greater than 10 5 colony forming units per millilitre (CFU/mL) [ 17 , 24 , 37 ]. Recently, various research groups have proposed different extraction methods based on differential centrifugations [ 37 , 38 ], ultrafiltration [ 39 ], or a simple pre-treatment of samples with sodium-dodecyl-sulfate (SDS) [ 40 , 41 ].…”

“…For the processing of urine samples specifically, an initial flow cytometric scan is recommended to eliminate negative urine and limit direct processing to urine with counts greater than 10 5 colony forming units per millilitre (CFU/mL) [ 17 , 24 , 37 ]. Recently, various research groups have proposed different extraction methods based on differential centrifugations [ 37 , 38 ], ultrafiltration [ 39 ], or a simple pre-treatment of samples with sodium-dodecyl-sulfate (SDS) [ 40 , 41 ]. A recent study conducted by Veron and colleagues [ 42 ] compared centrifugation (68.4% of correct MALDI identification), urine filtration (78.9%) and 5 h bacterial cultivation on solid culture media (84.2%), which demonstrated that a short culture step is the most straightforward and efficient sample preparation method for fast and reliable identification of uropathogens.…”