Direct Identification of Urinary Tract Pathogens from Urine Samples, Combining Urine Screening Methods and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

Early diagnosis of urinary tract infections (UTIs) is essential to avoid inadequate or unnecessary empirical antibiotic therapy. Microbiological confirmation takes 24 to 48 h. The use of screening methods, such as cytometry and automated microscopic analysis of urine sediment, allows the rapid prediction of negative samples. In addition, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a widely established technique in clinical microbiology laboratories used to identify microorganisms. We evaluated the ability of MALDI-TOF MS to identify microorganisms from direct urine samples and the predictive value of automated analyzers for the identification of microorganisms in urine by MALDI-TOF MS. A total of 451 urine samples from patients with suspected UTIs were first analyzed using the Sysmex UF-1000i flow cytometer, an automatic sediment analyzer with microscopy (SediMax), culture, and then processed by MALDI-TOF MS with a simple triplecentrifuged procedure to obtain a pellet that was washed and centrifuged and finally applied directly to the MALDI-TOF MS plate. The organisms in 336 samples were correctly identified, mainly those with Gram-negative bacteria (86.10%). No microorganisms were misidentified, and no Candida spp. were correctly identified. Regarding the data from autoanalyzers, the best bacteriuria cutoffs were 1,000 and 200 U/l for UF-1000i and SediMax, respectively. It was concluded that the combination of a urine screening method and MALDI-TOF MS provided a reliable identification from urine samples, especially in those containing Gram-negative bacteria. U rinary tract infections (UTIs) are among the most common nosocomial and community-acquired bacterial infections (1). The etiology is varied, but in approximately 90% of cases, enteric bacteria are implicated, especially Escherichia coli, which produces Ͼ70% of these infections. Other urinary tract pathogens are Klebsiella spp., Enterobacter spp., Proteus spp., Pseudomonas spp., Enterococcus spp., and Staphylococcus saprophyticus (2).A rapid diagnosis of UTIs has a significant beneficial impact on patient health, since it reduces unnecessary or inadequate empirical antimicrobial therapy (3). Urine culture is still the gold standard for the microbiological confirmation of UTIs; however, it takes 24 to 48 h to provide results. The use of screening methods, such as traditional Gram staining or other methods for counting urine particles, like flow cytometry or automated microscopic urine sediment analysis, allow the prompt prediction of negative samples and a preliminary identification of microorganisms in positive samples (4-7). Nevertheless, this information obtained from these methods is insufficient and requires culture and other biochemical tests.In recent years, proteomic techniques have achieved a relevant role in the identification of microorganisms in the field of clinical microbiology. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been suggested as a...

Prospective Evaluation of Light Scatter Technology Paired with Matrix-Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry for Rapid Diagnosis of Urinary Tract Infections

“…First, our pediatric population had a median age of 7 years, which might make the data difficult to generalize to adult populations. However, our percent culture positivity and distribution of uropathogens were comparable to the same data reported from adult settings (13). Second, the majority of positive urine cultures in this study were monomicrobial infections with E. coli (n ϭ 57; 70.4% of monomicrobial infections).…”

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

“…As reported previously by others, the analysis of polymicrobial cultures did not provide reliable results (15,20). In our study, nine polymicrobial urine samples provided the correct identification of one microorganism, which is similar to Inigo et al who showed that 75% of the polymicrobial urine samples provided invalid results (15).…”

“…The most appropriate volumes of the urine sample, formic acid, acetonitrile (15 ml, 3 l, and 3 l, respectively) and 3 l of supernatant spotted onto the target plate showed LODs of 1.0 ϫ 10 5 , 4.0 ϫ 10 5 , and 1.52 ϫ 10 5 CFU/ml for E. coli, K. pneumoniae, and E. faecalis, respectively, which were equivalent to those previously reported on the Bruker Biotyper system (17). 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).…”

“…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.…”