JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2008, p. 1537–1540 0095-1137/08/$08.00⫹0 doi:10.1128/JCM.00030-08 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 4
Rapid and Accurate Identification of Candida albicans Isolates by Use of PNA FISHFlow䌤 Jan Trnovsky,1* William Merz,2 Phyllis Della-Latta,3 Fann Wu,3 Maiken Cavling Arendrup,4 and Henrik Stender1 AdvanDx, Inc., Woburn, Massachusetts1; The Johns Hopkins Medical Institutes, Baltimore, Maryland2; Columbia University Medical Center, New York, New York3; and Statens Serum Institut, Copenhagen, Denmark4 Received 7 January 2008/Returned for modification 22 January 2008/Accepted 6 February 2008
We developed the simple, rapid (1 h), and accurate PNA FISHFlow method for the identification of Candida albicans. The method exploits unique in solution in situ 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. The C. albicans PNA FISHFlow kit (prototype kit; AdvanDx, Woburn, MA) was used to analyze samples by the PNA FISHFlow method. One small (0.5-mm) colony or 20 l of liquid culture was placed in a microcentrifuge tube containing 0.2 ml of C. albicans PNAFlow reagent (combined hybridization and fixation solution containing C. albicans-specific 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 (10,000 ⫻ g), the supernatant was removed, and the pellet was resuspended in 0.5 ml of Wash BufferFlow. Samples were incubated at 55°C for 10 min, and the cells were repelleted, resuspended in 0.5 ml of Wash BufferFlow, and heated (55°C, 10 min). The samples were then analyzed by FC and FM after a 5-min cooling period. FC scoring. Guava EasyCyte (Guava Technologies, Hayward, CA) equipped with 488-nm laser line, forward-scatter (FSC) and side-scatter (SSC) detectors and a fluorescence (Fl-1) detector with 525/30 filter was used for FC scoring. Microbial cells were first visualized by 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 the FL-1 scale (as shown in Fig. 1A and C). Positive results were reported for samples which had ⱖ60% of events in upper left quadrant; negative results were reported for samples which had ⱖ60% events in the lower left quadrant. Cell populations with ⬍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 Fig. 1A and C, respectively. FM scoring. Aliquots of processed samples (⬃5 l) 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 charge-coupled device camera), fluorescein isothiocyanate/Texas Red dualband filter, and a ⫻60 (oil immersion) objective lens. Microscopic observations were scored as no fluorescence (negative) or bright green fluorescence (positive). Typical FM signals for
Candida albicans is a common cause of severe health careassociated 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 the use of selective chromogenic agar media (4, 9–11). A 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 (15), and non-C. albicans isolates can be misinterpreted as germ tube positive (6, 15). Definitive identification of C. albicans can be accomplished with commercially available automated systems such as API Candida (bioMerieux), the MicroScan WalkAway System (Dade Behring), 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 24 to 48 h (2, 12, 16, 17). 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. We developed the simple, rapid (1 h), and accurate PNA FISHFlow method for the 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 (FC) and fluorescence microscopy (FM). The main goal of this preclinical study was to determine the feasibility, specificity, and sensitivity of the C. albicans PNA FISHFlow method using appropriate laboratory strains and clinical isolates. The peptide nucleic acid (PNA) probe used in the present study was previously rigorously tested by using glass side-based fluorescence in situ hybridization (FISH) assays (8, 14, 18), which are now approved by the U.S. Food and Drug Administration for in vitro diagnostic use. Clinical application of this test led to substantial cost savings for hospitals (1, 3). * Corresponding author. Mailing address: AdvanDx, Inc., 10A Roessler Road, Woburn, MA 01801. Phone: (781) 376-0009. Fax: (781) 376-0111. E-mail:
[email protected]. 䌤 Published ahead of print on 20 February 2008. 1537
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FIG. 1. Detection of C. albicans with PNA FISHFlow. Exponentially growing liquid cultures of C. albicans (A and B) and C. tropicalis (C and D) were collected with plastic inoculating loops and added into 5 drops (0.2 ml) of C. albicans PNAFlow placed in a microcentrifuge tube. The content was mixed by vortexing (5 s) and analyzed by the PNA FISHFlow method. Microscopic images (B and D) are positioned next to the corresponding FC dot plots.
positive (C. albicans) and negative (C. tropicalis) cultures are shown in Fig. 1B and D, respectively. The C. albicans PNA FISHFlow method was tested with 29 reference strains representing phylogenetically related and clinically significant yeast species. All strains were obtained either from the American Type Culture Collection (ATCC), Rockville, MD, or from the Agricultural Research Service Culture Collection (NRRL), Peoria, IL. Of 29 laboratory strains, 9 were C. albicans (ATCC 14053, NRRL Y-12983, Y-17967, Y-17968, Y-17974, Y-22735, Y-27022, Y-7873, and Y-7976), 12 were Candida species (Candida tropicalis ATCC 750; Candida lodderae NRRL Y-17317; Candida sojae NRRL Y-17909 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-11827, Saccharomyces cerevisiae ATCC 9763, Picchia norwegensis NRRL Y-7651, Lodderomyces elongisporus NRRL Y-7681 and Y-27304, Kluyveromyces delphensis ATCC 24205, Cryptococcus neoformans ATCC 204092, and Issatchenkia orientalis ATCC 6258). Microorganisms were growing on YM agar plates (Teknova, Hollister, CA), on chromogenic agar plates (HardyCHROM Candida; Hardy Diagnostics, Santa Maria, CA), or in YM broth (Teknova). The strains were analyzed after 24 h of incubation at 35°C (solid media) or after 16 h (liquid media). Samples were processed immediately after collection.
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TABLE 1. C. albicans PNA FISHFlow results obtained with reference strains No. of isolatesb Reference strain
a
FC score
FM score
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 no.
9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 2 1 4 1 1 1 2 1 1 1 1 1 1 2 1 1 2 1 1 1
9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1
0 2 1 4 1 1 1 2 1 1 1 1 1 1 2 1 1 2 1 1 1
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TABLE 2. C. albicans PNA FISHFlow results obtained with clinical isolates No. of isolatesa
Total no. of isolates
9 2 1 4 1 1 1 2 1 1 1 1 1 1 2 1 1 2 1 1 1
Clinical isolate
FC score
FM score
Positive Negative Positive Negative
Candida albicans Candida glabrata Candida tropicalis Candida parapsilosis Candida krusei Candida dubliniensis Candida rugosa Candida intermedia Candida kefyr Candida lusitaniae Candida guillermondii Saccharomyces cerevisiae Cryptococcus neoformans Cryptococcus albidus Rhodotorula rubra Trichosporon asahii Total no. a
49 1* 0 0 0 1* 0 0 0 0 0 1* 0 0 0 0
0 32 11 18 9 5 2 1 2 2 1 5 5 1 3 1
49 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 33 11 18 9 6 2 1 2 2 1 6 5 1 3 1
Total no. of isolates
49 33 11 18 9 6 2 1 2 2 1 6 5 1 3 1 150
FC and FM scoring was done as described in the text. *, Inconclusive result.
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a
Strain descriptions were taken over from collection agencies. The names reflect either an anamorphic or a teleomorphic state. b FC and FM scoring was done as described in the text.
The C. albicans PNA FISHFlow 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 Laboratory, Baltimore, MD (51 samples); and the Statens Serum Institute, Copenhagen, Denmark (49 samples). The isolates were grown on Sabouraud dextrose agar plates for 24 h or on chromogenic agar plates. Colonies were placed in C. albicans PNAFlow reagent and shipped from clinical sites to the AdvanDx laboratory, 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: chromogenic agar; an identification (JHH) system that utilizes germ tube production; methyl-␣-Dglucoside (MDG) assimilation; urease, morphology, and phenoloxidase on corn meal-Tween 80-caffeic acid agar; pattern of seven (glucose, maltose, sucrose, lactose, galactose, trehalose, and cellobiose) carbohydrate fermentation assays; temperature growth studies; and the commercially available automated identification systems API 20C (bioMerieux), the MicroScan WalkAway System (Dade Behring, Sacramento, CA), Yeast BICHRO-DUBLI and Glabrata RTT (Fumouze Diagnostics, Simoco, Denmark), and ATB ID32C (bioMe´rieux, Marcy l’Etoile, France). C. albicans PNA FISHFlow correctly identified all (9 of 9) tested C. albicans reference strains and all non-C. albicans yeast (20 of 20) species (Table 1). Therefore, both the sensitivity and the specificity for the method were 100%. We tested colonies collected from agar plates and liquid cultures with
which we obtained identical results. For reference strains, the PNA FISHFlow results were not affected by the presence of chromophors generated as a result of growth on chromogenic plates (data not shown here). The results of clinical isolate testing, in which the PNA FISHFlow method was compared to standard routine identification methods, are shown in Table 2. When three inconclusive FC results were excluded from the analysis, the specificity and the sensitivity of C. albicans PNA FISHFlow method were 100% (49 of 49) and 100% (101 of 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 of 101) if 3 FC inconclusive results are included (as false positives) in the analysis. Three inconclusive FC results were obtained with isolates grown on chromogenic agar and originating at the Staten Serum Institute in Copenhagen, Denmark (one C. glabrata, one C. dubliniensis, and one 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 that scored negative for C. albicans with PNA FISHFlow 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 it was retested with real-time PCR. Conclusion. The present study demonstrates that identification of C. albicans isolates can be accomplished reliably with the PNA FISHFlow method (using either FC or FM as the scoring systems) and in considerably less time than with commercially available automated identification systems (1 h versus 24 to 48 h). An advantage of the PNA FISHFlow method compared to similar FC-based methods (5, 7) is the use of a
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combined hybridization and fixation reagent. Application of this reagent leads to a significant decrease in time and complexity of sample preparation (allowing us to increase testing throughput), particularly compared to other FC methods that need cell fixation and subsequent washing steps. FC scoring allows additional increase in testing throughput due to fast acquisition times (⬃1 s for analyses of colonies) and nonsubjective 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 FISHFlow method for analyses of blood cultures. Preliminary results indicate that both FC and FM scoring are possible when non-charcoal blood culture (BD Bactec) bottles are used. With the appropriate PNA probes, the PNA FISHFlow format can be easily adapted for the detection of other clinically relevant Candida species (C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei) as accomplished previously with a glass slide-based format (13). These assays could replace complicated systems that use a combination of multiple methods for yeast species level identification. None of the authors from the clinical sites held any financial interest in AdvanDx, Inc. The kits and disposables for this study were provided by AdvanDx, Inc. We thank Cletus Kurtzman at the Agricultural Research Service Culture Collection, Peoria, IL, for yeast strains. REFERENCES 1. Alexander, B. D., E. D. Ashley, L. B. Reller, and S. D. Reed. 2006. Cost savings with implementation of PNA FISH testing for identification of Candida albicans in blood cultures. Diagn. Microbiol. Infect. Dis. 54:277–282. 2. Fenn, J. P., H. Segal, B. Barland, B. D. Denton, J. Whisenant, H. Chun, K. Christofferson, L. Hamilton, and K. Carrol. 1994. Comparison of updated Vitek yeast biochemical card and API 20C yeast identification systems. J. Clin. Microbiol. 32:1184–1187. 3. Forrest, G. N., K. Mankes, M. A. Jabra-Rizk, E. Weeks, J. K. Johnson, D. P. Lincalis, and R. A. Venezia. 2006. Peptide nucleic acid fluorescence in situ hybridization-based identification of Candida albicans and its impact on mortality and antifungal therapy costs. J. Clin. Microbiol. 44:3381–3383. 4. Freydiere, A. M., R. Guinet, and P. Boiron. 2001. Yeast identification in the clinical microbiology laboratory: phenotypical methods. Med. Mycol. 39: 9–33.
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