Comparison between IRIS iq 200ELITE and microscopy for urinalysis and evaluation of performance in predicting outcome of urine cultures

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1 Article Reprint Comparison between IRIS iq 200ELITE and microscopy for urinalysis and evaluation of performance in predicting outcome of urine cultures S. Ledru, J.P. Canonne Laboratoire de microbiologie, Centre hospitalier Schaffner, Lens Annales de Biologie Clinique, Volume 66, Number 5, September-October 2008 Article Received 2 June 2008, Accepted 4 September 2008

2 Comparison between IRIS iq 200ELITE and microscopy for urinalysis and evaluation of performance in predicting outcome of urine cultures S. Ledru, J.P. Canonne, Laboratoire de microbiologie, Centre hospitalier Schaffner, Lens Annales de Biologie Clinique, Volume 66, Number 5, September-October 2008, Article Received 2 June 2008, Accepted 4 September 2008 Key words: urinary tract infection, urine cell analyzer, urinalysis, comparative study Summary Since the marketing of the urinary strips, various automated instruments have appeared using the principle of flow cytometry for analysis of urinary elements; Sysmex UF-100, UF-50 and UF Recently a new analyzer, that uses an innovative method, the Iris iq 200ELITE, has been introduced that analyzes urine specimens by photographic analysis of cells passing in a laminar flow in front of a microscopic objective. Five hundred microscopic fields are photographed and analyzed. Each particle of the microscopic field is separated and classified using Auto- Particle Recognition software. The possibilities of visualization by the operator permits us to avoid confusion between various artifacts identified on flow cytometry analyzers (for example: yeasts confused with red blood cells, spermatozoa interpreted like bacilli, etc). Apart from any interpretation Iris iq200elite has compared to the culture a sensitivity of 68%, a specificity of 80%, a positive predictive value of 60% and a negative predictive value of 86%. By combining the various parameters provided by Iris iq200elite, we can eliminate 43% from the direct microscopic examinations and to predict the negativity of 32% of the cultures with a sensitivity and a negative predictive value of 100%, which quantitatively optimizes this most important work station. Introduction UTI s are the most frequent pathologies found in Private Laboratories and in hospitals. For its diagnosis, the gold standard remains a direct cytological examination followed by a culture 1. In our hospital, cytodiagnostic urinalysis is the number one analysis done in the laboratory between 20 and 25% of the occurrence which generates an important investment in materials and human resources. However, a majority of cytodiagnostic urinalysis are negative (70% in our series), but we cannot accept any tolerance compared to the bacteria culture because of the risks of chronic morbidity that a failure of diagnosis can involve. Various automated flow cytometry instruments (Sysmex UF-50, Sysmex UF-100, Sysmex UF-1000) were put on the market with the goal of screening urines to reduce the cost of those tests with more or less success 2-4. Here are the results of an analyzer which is comparable with the usual cytodiagnostic test: an analysis of pictures of cells passing in front of a microscope objective. Materials and Methods Fresh urine specimen samples arriving at the laboratory were analyzed in a consecutive way from February 18th to March 28th, of each sample was carried out on routine tests and an analysis was run on the Iris iq200elite (Iris Diagnotics France, Baillet-en-France, France). The routine analysis consisted of a cytological examination on Slide Fast-Read 102 (Biosigma, S.R.L., Italy) (same system as KOVA slides) with numeration of red blood cells and leucocytes, direct microscopic examination and a culture with AGAR plates. The direct microscopic examination was carried out in the following way: a gauged amount of 10 μl of urine was deposited and spread out in a circle traced with an engraver. The slide was dried and then it was colored by Gram stain and the full slide was observed under microscope at objective 100 with immersion oil. The results were reported in semi-quantitative grades according to Table 1. Presence of bacteria was performed using plates of BCP media (Bio-Rad ), CNA Colombia media (Oxoid ) and chromogene media for yeast (CAND BD ) if there was presence of yeasts with the microscopic examination. Cultures were interpreted according to the European recommendations 5. The urine was tested in parallel on the Iris iq200elite. The Iris iq200elite is an automated microscope. A known volume of urine passes in front of a microscope objective. A camera takes 500 fields per sample. Within each field, images of the particles are individualized and isolated. The particles are classified using a model of a neural network used by the software: Auto-Particle Recognition (APR ). In the majority of the tests, automatic classification can be realized in all confidence without any operator help. For samples with very abnormal particles, a user can visually confirm the identification of those particles according to their morphological details seen on the screen (Figure 1). The minimum size of the recognized particles is 3 μm; that is sufficient for bacilli and Gram positive cocci chains. Bacteria smaller than 3µm are classified in All Small Particles, which makes this parameter important to look at in correlation with leucocytes concentration and the presence of bacteria. Table 1: Interpretation of the direct microscopic examination. Observation 0 Absence < 1 in 10 fields Rare 1/10 fields to 1/field Occasional 1/field to 10/field 10/field to 100/field Semi-quantitative results Few Moderate > 100/field Many 1

3 Table 3: Diagnostic performance of the Iris iq200elite compared to direct microscopic examination (ED) and to bacteria cultures of the 400 urine samples. Comparison %FP 1 (n) %FN 2 (n) Se% 3 Spe 4 % PPV 5 % NPV 6 % ED Iris 7 /Culture 40 (56) 14 (38) ED 8 / Culture 30 (41) 9 (23) ED Iris 7 / ED 8 25 (34) 13 (34) Percentage of False Positive (number); 2 Percentage of False Negative (Number); 3 Sensitivity; 4 Specificity; 5 Positive Predictor Value; 6 Negative Predictor Value; 7 Result of the detection of bacteria by the analyzer; 8 Result of the detection of bacteria by the direct microscopic examination Figure 1: Particle images from iq 200ELITE. The user can visually confirm the identification of particles according to their morphological details seen on the screen. Table 2: Distribution of the various bacterial pathogens isolated from urine samples. Bacteria Escherichia coli 73 Enterococcus 23 Proteus mirabilis 10 Klebsiella pneumoniae 10 Number Table 4: Combinations of various values provided by Iris iq200elite and the direct microscopic examination (ED). Comparison of the parameters of Iris iq200elite to direct microscopic examination ED Iris Negative 7 and WBC 8 < 5 ED Iris Negative 7 and WBC 8 > 40 ED Iris Negative 7 and WBC 8 < 40 and ASP 9 < 10,000 %FP 1 (n) %FN 2 (n) Se 3 % Spe 4 % PPV 5 % NPV 6 % 58 (190) 0 (0) (58) 36 (115) (102) 8 (5) (a) Percentage of False Positive (number); 2 Percentage of False Negative (Number); 3 Sensitivity; 4 Specificity; 5 Positive Predictor Value; 6 Negative Predictor Value; 7 Absence of bacteria detected by the analyzer; 8 Leukocytes; 9 All Small Particles; (a) 8 cultures not significant (6 negative, 2 bacteria in insufficient concentration). Enterobacter aerogenes 6 Other Enterobacteriaceae 9 Candida albicans 8 Torulopsis glabrata 2 Pseudomonas aeruginosa 3 Staphylococcus aureus 3 Staphylococcus with negative coagulase enzyme 3 Streptococcus agalactiae 2 2

4 Leukocytes 40/µL 5/µL N = 54 ED Mandatory N = 98 ED Negative = 90 ED Positive = 8 8 Cultures not significant N = 70 ED = Negative Negative Culture 10,000/µL N = 26 ED Mandatory N = 12 ED Mandatory N = 2 ED = Negative Negative Culture All Small Particles Figure 2: Opportunity of doing a direct microscopic examination (ED) according to the parameters provided by Iris iq200elite in case of an absence of bacteria (N = 262). Direct on Analyzer (N = 400) Negative (N=262) Positive (N=138) Leukocytes Leukocytes 10/µL (N=124) > 10/µL (N=138) 100/µL (N=57) > 100/µL (N=81) All Small Particles Direct and Culture Mandatory Direct Direct and Culture Mandatory < 10,000/µL No Direct No Culture (Sterile) (N=119) Negative No Culture (Sterile) (N=3) 10,000/µL Direct Positive Culture Mandatory (N=2) Negative (N=16) Culture (Sterile) Positive (N=41) Culture Mandatory Figure 3: Decisional algorithm with a negative predictive value of 100% to reduce of the number of cultures and give faster results of negative cultures on patient s report. 3

5 Results During this period, 400 urines were consecutively analyzed. The specimen analyzed consists of 255 women and 145 men age 0 to 99 years (average age: 53). The distribution between hospital departments was: 161 patients from medical department, 88 from gynecology-obstetrics department, 41 of surgery department, 34 from pediatric department, 29 from geriatric department, 28 from emergencies, 13 from general-purpose and 6 from psychiatry department. The incidence of positive cultures was 30% (120 of 400). Only one pathogen was isolated in 88 from the 120 positive bacteria cultures, 2 pathogens in 27 bacteria cultures and finally 3 pathogens in 5 cultures. The distribution of the bacteria is presented in Table 2. The direct microscopic examination and culture remaining the reference method 1, were initially compared to results of the analyzer (Table 3). One notes a good correlation between the Iris iq200elite results and direct microscopic examination, and also between results on the Iris iq200elite and bacteria cultures. However, as for the microscopic direct examination, a negative result in bacteria and yeasts on Iris iq200elite does not make it possible to omit the culture (more sensitive and more specific). To improve the productivity of the cytodiagnostic urinalysis job, we searched for combinations of various parameters from the Iris iq200elite (numeration of the leucocytes, numeration of All Small Particles) in order to eliminate direct examinations (time-saver) in the case of absence of bacteria detected by the Iris iq200elite (Table 4). When leucocytes are lower than 5/μL, whatever the number of All Small Particles, the direct microscopic examination is always negative. When leucocytes are more than 40/μL, whatever the number of All Small Particles, the direct microscopic examination is mandatory because of the possibility of false negative (36%). When the leucocytes concentration is between 5/μL and 40/μL, we must take into consideration the All Small Particles count. If the number of All Small Particles is lower than 10,000/μL, the direct examination is negative in 92% of the cases and the 8% of the cases when it was positive, the cultures were not significant. However, if the number of All Small Particles is higher than 10,000/μL, one cannot bypass direct examination because of the possibility of presence of Gram positive cocci colonies. All of the possible situations are summarized in Figure 2. In our series, that allows us to eliminate 43% of the direct microscopic examinations with a sensitivity and a negative predictive value of 100% on direct microscopic examination. On the same principle, we searched for the combinations which could enable us to predict the negativity of cultures (Figure 3). In case of absence of bacteria and yeasts, with a number of leucocytes lower or equal to 10/µL and of a number of small particles less than 10,000/µL, the direct microscopic examination is always negative and culture is sterile (sensitivity = 100%, specificity = 43%, PPV = 43%, NPV = 100%). We can also predict the negativity of the cultures with a sensitivity and a NPV of 100% if the direct examination on the instrument is negative, the number of leucocytes lower or equal to 10/µL, the number of small particles equal to or higher than 10,000/µL and a negative direct microscopic examination or if the direct examination on the analyzer is positive, and the number of leucocytes is lower or equal to 100/μL and the direct microscopic examination is negative. This algorithm would allow us, if the French health reimbursement mode should change, to eliminate 32% of the cultures with a sensitivity and a negative predictive value of 100%. Discussion The goal of this study was to evaluate the performance of Iris iq200elite in order to decrease the number of microscopic examinations and to anticipate the negativity of the cultures with a sensitivity and a negative predictive value from 100% on one of the most important work stations in microbiology laboratory: cytodiagnostic and microbiological urinalysis. Other authors already studied the performances of the strips and analyzers which use the flow cytometry technology. Some authors are satisfied this analysis by accepting a percentage of false negatives. It is the case of Evans, et al. 6 and Kim, et al. 7 for the UF-100 (Sysmex, Japan). Other authors are not satisfied with those results and do cultures on all urines because of the possibility of false negatives 4. Iris iq200elite has a major advantage over others systems of detection: that is the possibility of pictures of urine particles. Compared to the other systems, that allows the avoidance of several pitfalls. According to Steinmetz, et al. 8, UF-100 over-estimates the presence of leucocytes and red blood cells. This over-estimation can be due to the presence of bacteria or yeasts which are counted like red blood cells 9, oxalate crystals 10, amorphous phosphate, yeast or spermatozoa 11,12. It is very easy on Iris iq200elite to eliminate those artifacts. Thanks to this innovative process, we can visualize the elements and thus eliminate artifacts (Figure 1). 4

6 Conclusion The Iris iq200elite enabled us to significantly increase the productivity of the work station of the cytodiagnostic and microbiology urinalysis. It allows us to eliminate 43% of direct examinations of urine specimens per day. This automation allows a standardization of results and precision of the direct examinations. More, the possibility of connection to LIS decreases the risks of transcription errors and optimizes the ergonomics of this work station. Automation enables us to predict during the current day 32% of the urinary cultures which will be negative with a sensitivity and a negative predictive value of 100%. References 1. Kouri T, Fogazzi G, Gant V, Hallander H, Hofmann W, Guder WG. European urinalysis guidelines. ECLM-European urinalysis group. Scand J Clin Lab Invest 2000 ; 60(Suppl.) : Boucaud-Maitre Y, Thoinet S, Fuhrmann C, Coronel B, Freney J. Evaluation des performances et de la praticabilité de l automate de cytobactériologie Sysmex UF-100TM pour la prédiction de l infection urinaire. Rev Fr Lab 2001 ; 338 : Okada H, Sakai Y, Miyazaki S, Arakawa S, Hamagushi Y, Kamidono S. Detection of significant bacteriuria by automated urinalysis using flow cytometry. J Clin Microbiol 2000 ; 38 : Zaman Z, Roggeman S, Vaerhaegen J. Unsatisfactory performance of flow cytometer UF-100 and urine strips in predicting outcome of urine cultures. J Clin Microbiol 2001 ; 39 : Aspevall O, Hallander H, Gant V, Kouri T. European guidelines for urinalysis : a collaborative document produced by European clinical microbiologists and clinical chemists under ECLM (European Confederation for Clinical Microbiology and Infectious Diseases). Clin Microbiol Infection 2001 ; 7 : Evans R, Davidson MM, Sim LW, Hay A. Testing by Sysmex UF-100 flow cytometer and with bacterial culture in a diagnostic laboratory : a comparison. J Clin Pathol 2006 ; 59 : Kim SY, Kim YJ, Hwang SH, Kim HH, Son HC, Lee E. Evaluation of the Sysmex UF-100 urine cell analyser as a screening test to reduce the need for urine cultures for community-acquired urinary tract infection. Am J Clin Pathol 2007 ; 128 :

7 8. Steinmetz J, Henny J, Gueguen R. Etablissement de limites de référence des sédiments urinaires mesurés sur UF-50TM Sysmex. Ann Biol Clin 2004 ; 62 : Fenili D, Pirovano B. The automation of sediment urinalysis using a new urine flow cytometer (UF-50TM). Clin Chem Lab Med 1998 ; 36 : Hannemen-Pohl K, Kampf SC. Automation of urine sediment examination : a comparison of the Sysmex UF-100 automated flow cytometer with routine manual diagnosis (microscopy, test strips, and bacterial culture). Clin Chem Lab Med 1999 ; 37 : Langlois MR, Delanghe JR, Steyaert SR, Everaert KC, De Buyzerz ML. Automated flow cytometry compared with an automated dipstick reader for urinalysis. Clin Chem 1999 ; 45 : Lun A, Zoebog R, Priem F, Filler G. Routine workflow for use of urine strip and urine cytometer UF-100 in the hospital laboratory. Clin Chem 2001 ; 45 :

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