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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 293-298
 

Evaluation of matrix assisted laser desorption ionisation-time of flight mass spectrometry in direct identification of bacteriuria from urine samples


1 Department of Medical Microbiology, PGIMER, Chandigarh, India
2 Department of Microbiology, Dr YSPGMC, Nahan, Himachal Pradesh, India
3 Department of Microbiology, Apollo Hospitals, Chennai, Tamil Nadu, India

Date of Submission15-May-2020
Date of Decision13-Jul-2020
Date of Acceptance31-Aug-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Neelam Taneja
Department of Medical Microbiology, PGIMER, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_206

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 ~ Abstract 


Objective: The use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI) directly on urine can significantly improve turnaround time for diagnosing urinary tract infection (UTI). The present study was planned to compare the performance of MALDI directly on urine samples with conventional urine culture and evaluate an algorithm using a combination of significant pyuria and MALDI directly on urine samples as a screening method for diagnosing UTI. Materials and Methods: A total of 1000 urine samples from patients with symptoms suggestive of UTIs were analysed. Urine microscopy, urine culture and MALDI were performed simultaneously on all the samples. Results: MALDI correctly identified the microorganisms in 73.83% monomicrobial samples. In culture showing a mixed growth of two and more than three organisms, MALDI was able to identify one microorganism in 27.58% and 15.78% of samples, respectively. There were no peaks by MALDI in 93.53% of 464 sterile samples. The sequential algorithm using urine microscopy and MALDI could correctly identify UTI in 66.23% cases. Conclusion: MALDI can be utilised to rule out bacteriuria in >93% of sterile urine samples. The combination of significant pyuria and direct MALDI as screening method to diagnose UTI (whether monomicrobial or polymicrobial) was not found to be superior than using direct MALDI on urine samples alone.


Keywords: Direct identification, matrix-assisted laser desorption/ionisation-time of flight, turnaround time, uropathogens


How to cite this article:
Mohan B, Gautam N, Sethuraman N, Kaur H, Taneja N. Evaluation of matrix assisted laser desorption ionisation-time of flight mass spectrometry in direct identification of bacteriuria from urine samples. Indian J Med Microbiol 2020;38:293-8

How to cite this URL:
Mohan B, Gautam N, Sethuraman N, Kaur H, Taneja N. Evaluation of matrix assisted laser desorption ionisation-time of flight mass spectrometry in direct identification of bacteriuria from urine samples. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 24];38:293-8. Available from: https://www.ijmm.org/text.asp?2020/38/3/293/299817





 ~ Introduction Top


Urinary tract infections (UTIs) are the most common bacterial infections, causing considerable economic and public health burden.[1] The routine diagnosis of UTI involves the semi-quantitative culture, which is considered the 'gold standard'. However, the results of culture and antibiotic susceptibility are available after 48 h. Therefore, clinicians often initiate empiric antibiotic therapy. Direct identification methods from urine are capable of significantly improving turnaround time (TAT) and patient management. These methods need high sensitivity to identify microorganisms present in urine and must be able to differentiate between UTI-positive and UTI-negative specimens. UTI-negative specimens encompass specimens with no microorganism growth, clinically insufficient growth and contaminated (polymicrobial) specimens.[2] Screening methods like microscopy either as wet mount or Gram stain and dipsticks giving information about nitrites and leucoesterase give only preliminary identification and cannot guide antimicrobial therapy. Recently, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF-MS) has emerged as a quick and reliable method for bacterial identification based on their typical protein profiles. MALDI-TOF (abbreviated as MALDI hereafter) can significantly decrease TAT despite the requirement of the growth of distinct colonies on solid media. This test has been applied directly on samples to further decrease the TAT for identification. This is especially useful in positive blood culture bottles having growth of single organism.[3] In case of urine samples, large specimen volume and high bacterial concentration facilitates direct MALDI-mediated identification of urinary pathogens. Few earlier studies have shown a higher sensitivity of MALDI in diagnosing monomicrobial UTI.[4],[5]

The present study was planned to compare the performance of MALDI directly on urine samples with conventional urine culture for diagnosing UTI. We also aimed to evaluate an algorithm using significant pyuria and direct MALDI on urine samples can be used as a screening method for diagnosing UTI.


 ~ Materials and Methods Top


A total of 1000 urine samples submitted to the Enteric Laboratory, Department of Medical Microbiology, PGIMER, from January 2015 to June 2015 from outpatients and inpatients with symptoms suggestive of UTIs were included in the study.

Urine microscopy and culture:

Fifty microliters of a well-mixed, un-centrifuged urine specimen was placed on a clean glass slide using a sterile micropipette and covered with 22 mm × 22 mm coverslip avoiding bubbles. The wet mount was examined for the presence of pus cells, bacteria, epithelial cells and yeast cells. More than one pus cell/7 high power fields corresponded to significant pyuria (>104 pus cells/ml of urine).[6] Semi-quantitative urine culture (1 μl loop) was performed using standard methods on cysteine lactose electrolyte deficient medium. The isolates were identified using Bruker MALDI Biotyper 3.1 mass spectrometer, where a score of 2.300–3.000 and 2.000–2.299 signifies highly probable and probable species, respectively.

Direct matrix-assisted laser desorption/ionisation-time of flight on urine samples

Four millilitre of urine was taken and centrifuged at 2000 g for 30s to remove leukocytes. Thereafter, the supernatant was centrifuged at 15,500 g for 5 min to collect bacterial pellet. The pellet was washed twice with deionised water and applied to the MALDI plate in a thin film, overlaid with 1 μl of matrix solution (saturated solution of α-cyano-4-hydroxy cinnamic acid in the organic solvent [50% acetonitrile and 2.5% trifluoroacetic acid]) and air-dried. Spectra were generated and analysed on the MALDI-TOF MS Bruker Biotyper Microflex LT using the FlexControl operating system and the BrukerBiotyper (version 3.0) software and taxonomy library. MALDI-TOF MS analysis was performed in automatic mode. MALDI-TOF MS identifications were classified using modified score values as proposed by the manufacturer: a score of ≥2 indicates species identification; a score between 1.7 and 1.9 indicates genus identification and a score of <1.7 indicates no identification. The results of conventional culture were correlated with MALDI, as shown in [Table 1].
Table 1: Comparison of matrix-assisted laser desorption/ionization-time of flight with conventional culture

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Urine samples were classified into five categories according to colony enumeration results in conventional culture, as shown in [Figure 1]. Group 1 was further divided into three groups according to the number of bacterial or yeast species grown on culture and their counts (CFU/ml). A sequential algorithm comprising of urine microscopy and MALDI was evaluated as a screening test where significant pyuria along with MALDI positive sample was taken as 'True UTI', no significant pyuria but MALDI positive sample was defined as 'colonisation' and no significant pyuria along with MALDI negative sample was considered 'No UTI'.
Figure 1: Categorization of urine samples

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Statistical analysis

Diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values were calculated using MedCalc version 12.1.4 (MedCalc Software bvba, Mariakerke, Belgium).


 ~ Results Top


Out of 1000 urine samples analysed, 388 were positive by culture (Group 1). [Table 1] Of these 388 isolates, 328 were Gram-negative bacilli (GNB), 26 were Gram-positive cocci (GPC) and 34 samples showed growth of yeast spp. [Table 2]. The most frequently isolated organisms were Escherichia coli (57.47%), Klebsiella pneumonia (10.56%) and Pseudomonas aeruginosa (4.38%). [Table 3] provides a detailed result of Group 1. In Group 2, of the 58 samples reported as 'Mixed growth of doubtful significance', MALDI identified at least one of the two organisms grown in culture in 16 (27.58%) samples whereas in 42 (72.41%) samples, there were no significant peaks. In Group 3, of 38 samples, which had growth of three or more organisms. MALDI was able to pick up one organism in 6 samples. In the remaining 32 samples there was no reliable identification by MALDI (either no peaks or score <1.7). In Group 4, MALDI identified an organism known to cause UTI in three (5.76%) samples, normal flora in four samples (7.64%), whereas in the remaining 45 samples (86.54%), there were no peaks. In the last group, of the 464 samples sterile by culture, MALDI gave no peaks in 434 (93.53%) samples. There was a positive identification in 30 (6.47%) samples by MALDI. [Table 4] shows the microorganisms reported by MALDI in urine samples, which were sterile by conventional culture.
Table 2: Organisms grown in conventional culture Group 1

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Table 3: Detailed results of Group 1

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Table 4: Organisms identified by matrix-assisted laser desorption/ionization in samples reported as sterile

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To define the diagnostic performance of the MALDI for mono-microbial samples, all groups except Groups 1b, 2 and 3 were included in the final calculations [Figure 2] MALDI's diagnostic performance was: sensitivity 73.84 (95% confidence interval [CI] 68.85%–78.4%), specificity 92.83 (95% CI 90.25%–94.90%), positive likelihood ratio 10.3 (CI 95% 7.5–14.13), negative likelihood ratio 0.28 (95% CI 0.24–0.34), positive predictive value 87.29 (CI 95% 83.34%–90.41%) and negative predictive value 84.18 (CI 95% 81.65%–86.42%).
Figure 2: Evaluation of results

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Significant pyuria was present in 590 urine samples (59%), of which 279 samples were culture positive. MALDI gave the correct identification in 267 of these 279 samples. Therefore, 'True UTI' was identified in 68.81% of the culture-positive samples by using this algorithm. In the absence of significant pyuria, MALDI identified a UTI causing organism in 102 samples classifying them as 'colonisation'. In 308 samples without significant pyuria, MALDI showed no peaks indicating 'no UTI'. The correlation of results of conventional culture with MALDI in samples having significant pyuria is depicted in [Table 5].
Table 5: Correlation of results of conventional culture with matrix-assisted laser desorption/ionization in samples having significant pyuria (n=590)

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 ~ Discussion Top


Although semi-quantitative urine culture forms the standard method of diagnosis of UTI, the results are available in 2–3 days. Many authors have attempted multiple tests to speed up the diagnosis with variable results. Nitrite test has sensitivity and specificity, ranging from 41% to 64% and 85%–98%, respectively.[7],[8],[9],[10] Leucocyte esterase tests have sensitivity and specificity from 48% to 86% and 17%–93%, respectively, and the sensitivity of leucocyte (pus cells) count in urine varies from 67% to 80% and specificity ranges from 82% to 90%.[7],[8],[9],[10] Even the combination of such tests has a false negative rate of around 10%. MALDI has shown significant success in identifying the organism directly from clinical samples like blood cultures.[11],[12],[13] However, limited data with variable results are available regarding the direct use of MALDI on urine samples. [Table 6] shows a comparison of results obtained in some of the studies evaluating the use of MALDI for direct identification in urine samples.
Table 6: Comparative analyses of studies evaluating the use of matrix-assisted laser desorption/ionization-time of flight for direct identification in urine samples

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The present study was attempted to evaluate the utility of MALDI directly on urine samples for diagnosing UTI. Our results reiterate that micro-organisms in urine can be identified by MALDI within 30 min, especially in mono-microbial samples with ≥105 CFU/ml organisms. The importance of identifying the causative pathogen early would aid inguiding antibiotic therapy, especially in extended-spectrum beta-lactamase producing Enterobacteriaceae, non-fermenting GNB or Staphylococcus aureus causing acute pyelonephritis.[4] MALDI identified Gram-negative bacteria more reliably as compared to GPC. The concurrence rate of semi-quantitative culture and MALDI for GNB was 71.14% at the species level and 74.69% at the genus level. The correlation between conventional identification and MALDI identification in infected urine samples varied from 58% to 67% in previous studies[5],[14],[15],[16] The sensitivity is comparatively higher in mono-microbial samples varying from 66.2% to 87.2%.[4],[5] Our concordance is quite low compared to the report by Ferreira et al. where the concurrence rate was 93.7% and 94.6% at species and genus level, respectively.[5] However, they arrived at this concurrence, taking into consideration only those samples where the colony count was ≥105 CFU/ml. The concurrence rate was lower for Gram-positive microorganisms (9/26) in our study, which may be either due to the resistant Gram-positive cell wall or to the overlap of some Gram-positive proteomic spectra.[2]

MALDI missed the identification in 96 (24.74%) samples showing bacterial growth. In 61 of these 96 samples, the bacterial counts were ≥105/ml, which is within the detection limit of MALDI in urine samples.[5] A possible reason for missing these samples could be the interference by other non-bacterial proteins present in urine like defensins or a high concentration of cells (pus cells, red blood cells or epithelial cells), which were not removed effectively by centrifugation steps used in our protocol.[15] In the remaining 34 samples, the bacterial counts were <105 CFU/ml could explain the non-detection by MALDI. In Group 2 (mixed growth of doubtful significance) and Group 3 (Gross contamination), MALDI was able to identify one microorganism in 27.58% and 15.78% of samples, respectively. In a recent study by Veron et al., MALDI identified at least one microorganism in all (six) mixed samples tested, whereas in a previous study MALDI gave no reliable identification in all mixed samples tested by De Marco and Burnham.[14],[16] Wang et al. detected that the identification of two types of the bacterium was possible only if they were present in ratios of 1:1 or 1:2.[17]

In our study, 93.53% (434/464) of sterile samples and 94.23% (45 + 4/52) of samples reported as having 'growth of no significance' were correctly classified as negative (no identification) by MALDI analysis. At our tertiary care hospital, approximately 200 urine specimens are submitted daily for culture examination, and around 60%–80% of these turn out to be sterile. Our results show that the direct method of detection by MALDI-TOF would benefit the management of those patients falling in Group 1a, 1c, 4 and 5, which are either mono-microbial or sterile. But when the sample is polymicrobial or the sample collection is not proper, the results given by MALDI performed directly on samples could be misleading. Therefore, MALDI can be used as a reliable and cost-effective technique for diagnosing UTI only if the sample collection is impeccable.

Using a sequential algorithm, we were able to correctly identify 'True UTI' in 68.81% of cases and 'No UTI' correctly in 66.17% of cases. Therefore, relying on this algorithm, we missed 33.77% positive cases and over-diagnosed UTI in 33.84% cases. Hence, we did not find this algorithm superior to using MALDI directly on samples.

Though MALDI is a rapid and economical technique that is easily implemented, there are limitations to this technique. The major hurdle to identification is the insufficient bacterial concentration in the urine sample. The threshold of microorganisms required for MALDI detection varies among the different species. Thus, sample selection is a prerequisite which could be done using simple microscopy as Gram stain. However, UTI occurs at counts under 105 CFU/ml. Currently available rapid screening tests are not precise enough for samples with low bacterial counts.[7],[18],[19] Therefore, in symptomatic patients, children, patients of obstructive uropathy, diabetics, catheter/suprapubic/percutaneous nephrostomy samples/patients on antibiotics and diuretics where lower CFUs are significant, pre-selection should not be done and all such samples should be processed for culture. However, these screening tests have a high negative predictive value and, therefore, should be continued as majority of the samples are sterile.[5]


 ~ Conclusion Top


MALDI can be effectively utilised to exclude bacteriuria in 93.8% of sterile urine samples. Effective utilisation of these tests can greatly control costs and effort required for culture. This, in turn, will lead to prompt management of patients before the culture results are available.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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2.
DeMarco ML, Ford BA. Beyond identification: Emerging and future uses for MALDI-TOF mass spectrometry in the clinical microbiology laboratory. Clin Lab Med 2013;33:611-28.  Back to cited text no. 2
    
3.
Lagacé-Wiens PR, Adam HJ, Karlowsky JA, Nichol KA, Pang PF, Guenther J, et al. Identification of blood culture isolates directly from positive blood cultures by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry and a commercial extraction system: Analysis of performance, cost, and turnaround time. J Clin Microbiol 2012;50:3324-8.  Back to cited text no. 3
    
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Burillo A, Rodríguez-Sánchez B, Ramiro A, Cercenado E, Rodríguez-Créixems M, Bouza E. Gram-stain plus MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) for a rapid diagnosis of urinary tract infection. PLoS One 2014;9:e86915.  Back to cited text no. 4
    
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Ferreira L, Sánchez-Juanes F, González-Avila M, Cembrero-Fuciños D, Herrero-Hernández A, González-Buitrago JM, et al. Direct identification of urinary tract pathogens from urine samples by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2010;48:2110-5.  Back to cited text no. 5
    
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Collee JG, Marr W. Culture of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. New York: Churchill Livingstone; 2008. p. 113-29.  Back to cited text no. 6
    
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Devillé WL, Yzermans JC, van Duijn NP, Bezemer PD, van der Windt DA, Bouter LM. The urine dipstick test useful to rule out infections. A meta-analysis of the accuracy. BMC Urol 2004;4:4.  Back to cited text no. 7
    
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Williams GJ, Macaskill P, Chan SF, Turner RM, Hodson E, Craig JC. Absolute and relative accuracy of rapid urine tests for urinary tract infection in children: A meta-analysis. Lancet Infect Dis 2010;10:240-50.  Back to cited text no. 9
    
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Clerc O, Prod'hom G, Vogne C, Bizzini A, Calandra T, Greub G. Impact of matrix-assisted laser desorption ionization time-of-flight mass spectrometry on the clinical management of patients with Gram-negative bacteremia: A prospective observational study. Clin Infect Dis 2013;56:1101-7.  Back to cited text no. 11
    
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Huang AM, Newton D, Kunapuli A, Gandhi TN, Washer LL, Isip J, et al. Impact of rapid organism identification via matrix-assisted laser desorption/ionization time-of-flight combined with antimicrobial stewardship team intervention in adult patients with bacteremia and candidemia. Clin Infect Dis 2013;57:1237-45.  Back to cited text no. 12
    
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Vlek AL, Bonten MJ, Boel CH. Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry improves appropriateness of antibiotic treatment of bacteremia. PLoS One 2012;7:e32589.  Back to cited text no. 13
    
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Demarco ML, Burnham CA. Diafiltration MALDI-TOF mass spectrometry method for culture-independent detection and identification of pathogens directly from urine specimens. Am J Clin Pathol 2014;141:204-12.  Back to cited text no. 14
    
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Köhling HL, Bittner A, Müller KD, Buer J, Becker M, Rübben H, et al. Direct identification of bacteria in urine samples by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and relevance of defensins as interfering factors. J Med Microbiol 2012;61:339-44.  Back to cited text no. 15
    
16.
Veron L, Mailler S, Girard V, Muller BH, L'Hostis G, Ducruix C, et al. Rapid urine preparation prior to identification of uropathogens by MALDI-TOF MS. Eur J Clin Microbiol Infect Dis 2015;34:1787-95.  Back to cited text no. 16
    
17.
Wang XH, Zhang G, Fan YY, Yang X, Sui WJ, Lu XX. Direct identification of bacteria causing urinary tract infections by combining matrix-assisted laser desorption ionization-time of flight mass spectrometry with UF-1000i urine flow cytometry. J Microbiol Methods 2013;92:231-5.  Back to cited text no. 17
    
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Broeren MA, Bahçeci S, Vader HL, Arents NL. Screening for urinary tract infection with the sysmex UF-1000i urine flow cytometer. J Clin Microbiol 2011;49:1025-9.  Back to cited text no. 18
    
19.
Giesen CD, Greeno AM, Thompson KA, Patel R, Jenkins SM, Lieske JC. Performance of flow cytometry to screen urine for bacteria and white blood cells prior to urine culture. Clin Biochem 2013;46:810-3.  Back to cited text no. 19
    


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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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