Rapid and Accurate Identification of C. albicans Isolates using PNA FISH Flow ACCEPTED

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1 JCM Accepts, published online ahead of print on 20 February 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 NOTE PREPARED FOR JOURNAL OF CLINICAL MICROBIOLOGY Rapid and Accurate Identification of C. albicans Isolates using PNA FISH Flow Jan Trnovsky 1*, William. Merz 2, Phyllis Della-Latta 3, Fann Wu 3, Maiken Cavling Arendrup 4 and Henrik Stender 1. 1 AdvanDx Inc., Woburn, MA 2 The Johns Hopkins Medical Institutes, Baltimore, MD 3 Columbia University Medical Center, New York, NY 4 Statens Serum Institut, Copenhagen, Denmark Running Title: Identification of C. albicans using PNA FISH Flow * Corresponding author: Jan Trnovsky, AdvanDx, Inc., 10A Roessler Road, Woburn, MA 01801, Tel.: , jtr@advandx.com Key words: in situ hybridization; FISH; flow cytometry; fluorescence microscopy; C. albicans; yeast isolates. 17 ABSTRACT We have developed a simple, rapid (1 hour) and accurate PNA FISH Flow method for identification of Candida albicans. The method exploits unique in solution in situ 1

2 hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques: flow cytometry and fluorescence microscopy. Candida albicans is a common cause of severe healthcare-associated bloodstream infections. In the routine diagnostic microbiology laboratory, C. albicans can be identified presumptively with simple, rapid and inexpensive methods such as germ tube or colorimetric tests, as well as selective chromogenic agar media (1-4). Germ tube test is often used to exclude C. albicans before applying other yeast species level identification schemes. However, up to 5% of the C. albicans isolates have been reported as germ tube negative (5) and non-c. albicans isolates can be misinterpreted as germ tube positive (5, 6). Definitive identification of C. albicans can be accomplished with commercially available automated systems such as API Candida (biomerieux), MicroScan WalkAway System (Dade Behring) or Vitek (biomerieux), Auxacolor (Sanofi Diagnostic Pasteur) or Yeast Star (CLARC Laboratories). However, a limitation to these identification systems is the prolonged time to obtain final results, averaging hours (7-10). Therefore there is a clinical need for an accurate assay to identify C. albicans from culture in real time and differentiate it from other yeast species that are often more resistant to antifungal agents. 2

3 We developed a simple, rapid (1 hour) and accurate PNA FISH Flow method aimed for definitive identification of C. albicans isolated from solid or liquid media (excluding blood cultures). This method facilitates the use of two scoring techniques: flow cytometry and fluorescence microscopy. The main goal of this pre-clinical study was to determine the feasibility, specificity and sensitivity of the C. albicans PNA FISH Flow method using appropriate laboratory strains and clinical isolates. The Peptide Nucleic Acid (PNA) probe used in this study was previously rigorously tested using glass side-based FISH assays (11-13), which are now FDA cleared for in vitro diagnostic (IVD) use. Clinical application of this test led to substantial cost saving for hospitals (14, 15). The C. albicans PNA FISH Flow kit (prototype kit, AdvanDx, Woburn, MA) was used to analyze samples by the PNA FISH Flow method. One small (0.5 mm) colony or 20 µl of liquid culture was placed in the microcentrifuge tube containing 0.2 ml C. albicans PNA Flow reagent (combined hybridization and fixation solution containing C. albicansspecific fluorophore-labeled PNA probe). The contents were mixed by vortexing and the samples were incubated at 55 C for 30 min. After 5 min centrifugation (10,000xg), the supernatant was removed and the pellet was resuspended in 0.5 ml of Wash Buffer Flow. Samples were incubated at 55 C for 10 min and the cells were repelleted, re-suspended in 0.5 ml of Wash Buffer Flow and heated (55 C, 10 min). Samples were then analyzed by flow cytometry and fluorescence microscopy after a 5 min cooling period. Flow cytometry scoring: Guava EasyCyte (Guava Technologies, Hayward, CA) equipped with 488 nm laser line, forward (FSC) and side (SSC) scatter detectors and 3

4 fluorescence (Fl-1) detector with 525/30 filter was used for FC scoring. Microbial cells were first visualized using FSC and SSC profiles and the visualized cell population was gated. The gated population was analyzed further in FSC/FL-1 dot blots. Border lines of 1,000 (FSC axis) and 100 (FL-1 axis) were established so that the population of C. albicans cells (positive control) was above 100 and the population of C. tropicalis (negative control) was below 100 on FL-1 scale (as shown in Figure 1A and C). Positive results were reported for samples which had 60% of events in upper left quadrant; negative results were reported for samples which had 60 % events in the lower left quadrant. Cell populations with less than 60 % of the cells in culture identifying quadrants were reported as inconclusive. Typical positive (C. albicans) and negative (C. tropicalis) results obtained by FC scoring are shown in Figure 1A and 1C, respectively. Fluorescent microscopy scoring: Aliquots of processed samples (~5 µl) were added on the surface of glass slides and dried. The dried spots were covered with mounting medium (AdvanDx) and a coverslip before examining with the OLYMPUS BX-51 microscope equipped with OLYMPUS DP70 (digital color CCD camera), FITC/Texas Red dual band filter, and 60X (oil immersion) objective. Microscopic observations were scored as no fluorescence (negative) or bright green fluorescence (positive). Typical FM signals for positive (C. albicans) and negative (C. tropicalis) cultures are shown in Figure1B and D, respectively. The C. albicans PNA FISH Flow method was tested with 29 reference strains representing phylogenetically related and clinically significant yeast species. All strains 4

5 were obtained either from American Type Culture Collection (ATCC), Rockville, MD or from Agricultural Research Service Culture Collection (NRRL), Peoria, IL. Out of 29 laboratory strains 9 strains were C. albicans (ATCC 14053, NRRL Y-12983,Y-17967, Y , Y-17974, Y-22735, Y-27022, Y-7873, and Y-7976), 12 strains were Candida species (Candida tropicalis ATCC 750, Candida lodderae NRRL Y-17317, Candida sojae NRRL Y and Y-27145, Candida utilis ATCC 9950, Candida viswanathii NRRL Y-27370, Candida krusei ATCC 14243, Candida glabrata ATCC 2001 and 22875, Candida guillermondii NRRL Y-324, Candida parapsilosis ATCC 22019, NRRL Y-12969, Y-543 and YB-415, Candida kefyr ATCC 4135, Candida dubliniensis NRRL Y-27201, and Candida zeylanoides NRRL Y-1774) and 8 were yeast species (Debaromyces hansenii var. fabryi NRRL Y-17914, Clavispora lusitaniae NRRL Y , Saccharomyces cerevisiae ATCC 9763, Picchia norwegensis NRRL Y-7651, Lodderomyces elongisporus NRRL Y-7681 and Y-27304, Kluyveromyces delphensis ATCC 24205, Cryptococcus neoformans ATCC , and Issatchenkia orientalis ATCC 6258). Microorganisms were growing on YM agar plates (TEKNOVA Hollister, CA), chromogenic agar plates (HardyCHROM Candida, HARDY DIAGNOSTICS, Santa Maria, CA) or in YM broth (TEKNOVA Hollister, CA). The strains were analyzed after 24 hr incubation at 35 C (solid media), or 16 hr (liquid media). Samples were processed immediately after collection. The C. albicans PNA FISH Flow method was also tested with 150 clinical yeast isolates obtained from Columbia University Medical Center, New York-Presbyterian Hospital, New York, NY (50 samples), The Johns Hopkins Hospital Clinical Mycology 5

6 Laboratory, Baltimore, MD (51 samples) and Statens Serum Institute, Copenhagen, Denmark (49 samples). The isolates were grown on Sabouraud s Dextrose agar plates for 24 hr or on chromogenic agar plates. Colonies were placed in C. albicans PNA Flow reagent and shipped from clinical sites to the AdvanDx laboratory (Woburn, MA), where they were processed and analyzed. All isolates were tested in a blind fashion, and results were compiled at the end of the study for analysis. At clinical sites the isolates were identified by the following standard routine identification methods (SRIMs): chromogenic agar, identification (JHH) system which utilizes germ tube production, methyl-α-d-glucoside (MDG) assimilation, urease, morphology and phenoloxidase on corn meal/tween 80/caffeic acid agar, pattern of seven (glucose, maltose, sucrose, lactose, galactose, trehalose, cellobiose) carbohydrate fermentation assays, temperature growth studies and commercially available automated identification systems API 20 C (biomerieux), the MicroScan WalkAway System (Dade Behring, Sacramento, CA),Yeast BICHRO-DUBLI and Glabrata RTT (Fumouze Diagnostics, Simoco, Denmark) and ATB ID32C (biomérieux, Marcy l Etoile, France). C. albicans PNA FISH Flow correctly identified all (9/9) tested C. albicans reference strains and all non-c. albicans yeast (20/20) species (Table 1). Therefore both the sensitivity and specificity for the method was 100%. We tested colonies collected from agar plates and liquid cultures with which we obtained identical results. For reference strains, the PNA FISH Flow results were not affected by the presence of chromophors generated as a result of growth on chromogenic plates (the data not shown here). The results of clinical isolates testing, in which the PNA FISH Flow method was 6

7 compared to SRIMs, are shown in Table 2. When 3 inconclusive FC results were excluded from the analysis, the specificity and the sensitivity of C. albicans PNA FISH Flow method was 100% (49/49) and 100% (101/101), respectively. Exclusion of inconclusive results from the analysis is warranted because FM scoring identified those isolates correctly. The specificity of the method would drop to 97% (98/101) if 3 FC inconclusive results are included (as false positives) in the analysis. Three inconclusive FC results were obtained from isolates grown on chromogenic agar and originated at the Staten Serum Institute in Copenhagen, Denmark (1 C.glabrata, 1 C. dubliniensis, and 1 S. cerevisiae). These isolates contained noticeable aggregates (observed by microscopy). Multiple clinical isolates of these same species all gave the correct results by FC. The other 46 samples sent from Denmark produced the correct FC results. Further investigation is needed to corroborate inconclusive FC results with shipment conditions. One clinical isolate sample which scored negative for C. albicans with PNA FISH Flow assay was initially incorrectly identified as C. albicans with conventional routine identification methods at the clinical site, but this sample was later identified as C. dubliniensis at that site after re-testing with Real-Time PCR. Conclusion: This study demonstrates that identification of C. albicans isolates can be accomplished reliably with the PNA FISH Flow method (using either flow cytometry or fluorescence microscopy as scoring systems) and in considerably less time than with commercially available automated identification systems (1 hour versus hours). An advantage of the PNA FISH Flow method compared to similar flow cytometry-based methods (16, 17) is the use of a combined hybridization and fixation reagent. Application 7

8 of this reagent leads to a significant decrease in time and complexity of sample preparation (allowing to increase testing throughput), particularly when compared to other flow cytometry methods which need cell fixation and subsequent washing steps. FC scoring allows additional increase in testing throughput due to fast acquisition times (~1 sec for analyses of colonies) and non-subjective mode, predisposing the method for use in automated formats. FM scoring can be used for low volume testing, to confirm FC results or to supplement morphological information. Work is in progress to adapt the PNA FISH Flow method for analyses of blood cultures. Preliminary results indicate that both FC and FM scoring is possible when using non-charcoal blood culture (BD BACTEC) bottles. Using appropriate PNA probes, the PNA FISH Flow format can be easily adapted for detection of other clinically relevant Candida species (C. parapsilosis, C. tropicalis, C. glabrata and C. krusei) as accomplished previously with glass slide-based format (18). Those assays would have a potential to replace complicated systems, which use a combination of multiple methods for yeast species level identification Acknowledgements None of the authors from the clinical sites held any financial interest in AdvanDx, Inc. Kits and disposables for this work were provided by AdvanDx, Inc. The authors thank Dr. Cletus Kurtzman at Agricultural Research Service Culture Collection, Peoria, Illinois for yeast strains. 8

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13 Table 1 C. albicans PNA FISH Flow results obtained with reference strains Microorganism*1 FC Score*2 FM Score*3 Total number of isolates Reference Strains Positive Negative Positive Negative Candida albicans Candida glabrata Candida tropicalis Candida parapsilosis Candida krusei Candida dubliniensis Candida lodderae Candida sojae Candida kefyr Candida utilis Candida viswanathii Candida guillermondii Candida zeylanoides Debaromyces hansenii Clavispora lusitaniae Saccharomyces cerevisiae Cryptococcus neoformans Lodderomyces elongisporus Kluyveromyces delphensis Picchia norwegensis Issatchenkia orientalis Total number 36 Notes: *1) Description of strains was taken over from collection agencies. The names reflect either anamorphic or teleomorphic state. *2) Scoring is described in the text under Flow cytometry scoring *3) Scoring is described in the text under Fluorescence microscopy scoring 13

14 Table 2 C. albicans PNA FISH Flow results obtained with clinical isolates Microorganism FC Score FM Score Total number of isolates Clinical Isolates Positive Negative Positive Negative Candida albicans Candida glabrata 1* Candida tropicalis Candida parapsilosis Candida krusei Candida dubliniensis 1* Candida rugosa Candida intermedia Candida kefyr Candida lusitaniae Candida guillermondii Saccharomyces cerevisiae 1* Cryptococcus neoformans Cryptococcus albidus Rhodotorula rubra Trichosporon asahii Total number 150 Note: * denotes inconclusive result 14

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