JOURNAL OF CLINICAL MICROBIOLOGY, June 2002, p. 2182–2186 0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.6.2182–2186.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 40, No. 6
Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles Susan Rigby,1 Gary W. Procop,2 Gerhard Haase,3 Deborah Wilson,2 Geraldine Hall,2 Cletus Kurtzman,4 Kenneth Oliveira,1 Sabina Von Oy,3 Jens J. Hyldig-Nielsen,1 James Coull,1 and Henrik Stender1* Applied Biosystems, Bedford, Massachusetts1; Section of Clinical Microbiology, Cleveland Clinic Foundation, Cleveland, Ohio2; Institute of Medical Microbiology, University Hospital RWTH Aachen, Aachen, Germany3; and National Center for Agricultural Utilization Research, Peoria, Illinois4 Received 10 December 2001/Returned for modification 28 January 2002/Accepted 26 March 2002
A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n ⴝ 72), C. dubliniensis (n ⴝ 58), C. glabrata (n ⴝ 5), C. krusei (n ⴝ 2), C. parapsilosis (n ⴝ 4), and C. tropicalis (n ⴝ 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. laboratory for final identification. Final identification of the organisms in yeast-positive blood cultures may therefore take 4 to 7 days, during which time patients are treated empirically. Only a few methods for the identification of C. albicans directly from yeast-positive blood cultures have been described. These methods are mainly based on molecular techniques, such as fluorescence in situ hybridization (FISH) (6) and PCR (17). Peptide nucleic acid (PNA) probes are DNA probe mimics with an uncharged, neutral backbone which provide the PNA probes with improved hybridization characteristics such as high degrees of specificity, strong affinities, and rapid kinetics, as well as an improved ability to hybridize to highly structured targets such as rRNA (3, 18). In addition, the relatively hydrophobic character of PNA probes compared to the character of DNA enables PNA probes to penetrate the hydrophobic cell wall following preparation of a standard smear (19). FISH with PNA probes that target rRNA (the PNA FISH method) is a novel technique that combines the unique performance characteristics of PNA probes with the advantages of using rRNA as a target. It has recently been applied to the culture identification of both bacteria and yeasts (9, 11, 13, 20), including the identification of Staphylococcus aureus directly from blood culture bottles (10). In the study described here, we designed a PNA probe that targets C. albicans 26S rRNA and used the same assay format described previously for the identification of S. aureus for the rapid and specific identification of C. albicans directly from blood cultures.
Candidemia is presumptively diagnosed by the detection of yeasts by the Gram staining of the contents of positive-bloodculture bottles (herein referred to as yeast-positive blood culture bottles) (2). Unfortunately, definitive identification by traditional methods is time-consuming, and often, empirical antibiotic therapy is prescribed for patients whose blood cultures are positive for yeast. Most Candida species, including Candida albicans, the most common cause of candidemia, are generally susceptible to common antifungals, such as fluconazole. However, the increasing prevalence of other Candida species, such as C. glabrata and C. krusei, with different resistance patterns has necessitated more rapid and accurate methods for the identification of the yeast species in blood cultures (14, 16). Current standard methods for the presumptive identification of C. albicans from yeast-positive blood culture bottles rely on subculture on Sabouraud glucose or potato dextrose agar followed by germ tube analysis or subculture onto CHROMagar (22). Final identification requires subsequent subculture followed by a series of biochemical or carbon assimilation tests (e.g., API 20C, IDS RapID Yeast plus, or Vitek Yeast Biochemical Card) (4). Often, the presumptive identification of C. albicans is performed as part of the routine workup of positive blood cultures in the clinical microbiology laboratory, and non-C. albicans isolates are then sent to a specialized mycology * Corresponding author. Mailing address: Applied Biosystems, Bedford, MA 01730. Phone: (781) 280-2845. Fax: (781) 280-2940. E-mail:
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VOL. 40, 2002 MATERIALS AND METHODS Reference strains and clinical isolates. Thirteen C. albicans reference strains and nine other reference strains representing phylogenetically related Candida species and Lodderomyces elongisporus, mainly within the C. albicans clade, were selected from the Agricultural Research Service Culture Collection (NRRL), Peoria, Ill. One Saccharomyces cerevisiae strain was obtained from the American Type Culture Collection (ATCC), Manassas, Va. Fifty-eight C. dubliniensis clinical isolates and 39 C. albicans clinical isolates were collected at the Institute of Medical Microbiology, University Hospital, Aachen, Germany. The C. dubliniensis isolates were mainly from human immunodeficiency virus-positive patients (21) and from respiratory specimens of patients with cystic fibrosis (12). Clinical isolates of C. albicans were from various clinical specimens, including blood, and were chosen to represent different strains, i.e., serotype A, C. albicans bv. stellatoidea, and phenotypically aberrant isolates such as a red-pigmented strain (7) and strains that failed to assimilate glucosamine and N-acetylglucosamine (15). All strains and isolates were identified by D1-D2 26S ribosomal DNA (rDNA) sequence analysis as described previously (8). For PNA FISH analysis, reference strains and clinical isolates were inoculated into yeast-malt broth (Difco Laboratories, Detroit, Mich.) and incubated overnight at 35°C. Furthermore, 33 C. albicans isolates and 18 other isolates representing clinically significant yeast species obtained from various clinical specimens, including blood (Clinical Microbiology Laboratory, Cleveland Clinic Foundation, Cleveland, Ohio), were spiked into FAN BacT/Alert medium (Organon Teknika, Durham, N.C.) and incubated in the BacT/Alert Microbial detection system (Organon Teknika) until the system indicated that they were positive. The 18 non-C. albicans Candida isolates comprised C. glabrata (n ⫽ 5), C. tropicalis (n ⫽ 3), C. krusei (n ⫽ 2), C. parapsilosis (n ⫽ 4), C. lusitaniae (n ⫽ 3), and C. zeylanoides (n ⫽ 1). Clinical specimens. A total of 33 yeast-positive blood culture bottles (FAN BacT/Alert; Organon Teknika) from routine testing at the Clinical Microbiology Laboratory, Cleveland Clinic Foundation, were included in this study. In addition, 25 blood culture bottles (FAN BacT/Alert, Organon Teknika) that were negative following 7 days of incubation were artificially spiked with yeast species. The bottles were inoculated with just a few CFU of strains representing clinically significant non-C. albicans Candida species and comprised C. glabrata (n ⫽ 2), C. lusitaniae (n ⫽ 4), C. tropicalis (n ⫽ 4), C. guilliermondii (n ⫽ 1), C. krusei (n ⫽ 3), C. parapsilosis (n ⫽ 4), C. famata (n ⫽ 2), and C. norvegensis (n ⫽ 4) and Cryptococcus neoformans (n ⫽ 1). These strains included reference strains from ATCC and the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, as well as recent clinical isolates from the Institute of Medical Microbiology, University Hospital RWTH Aachen, Aachen, Germany. The blood culture bottles were reincubated in the BacT/Alert Microbial detection system (Organon Teknika) until the system indicated that they were positive. Preparation of smears. For each smear, one drop of phosphate-buffered saline with 1% (vol/vol) Triton X-100 (Aldrich) was placed in a well (diameter, 14 mm) of a ClearCell microscope slide (Erie Scientific, Portsmouth, N.H.) and a small drop of resuspended culture taken directly from the blood culture bottle was gently mixed in. The slide was then flame fixed or placed on a slide warmer at 55 to 60°C for 20 min. Subsequently, the smears were disinfected by immersion into 96% (vol/vol) ethanol for 5 to 10 min and air dried. Selection of probe sequence. Sequence processing was performed with computer software from DNASTAR (Madison, Wis.) and the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). The alignments of the D1-D2 26S rDNA sequences of closely related yeast species (8) were evaluated with Megalign (version 4.03) software. The sequences were from 11 species within the Candida clade as well as 10 other clinically relevant yeast species, mainly of the Candida genus. From these alignments, a specific probe sequence (AGAGAGCAGCATGCA) complementary to the 26S rRNA of C. albicans was identified. The probe was designed to minimize any secondary structure within the probe by use of the PrimerSelect (version 4.03) program. In addition, the target sequence was checked for specificity by comparison with sequences in the GenBank database by using both GeneMan (version 3.30) software and an Advanced BLAST search of the GenBank database (www.ncbi.nlm.nih.gov /blast) (1). Synthesis of fluorescein-labeled PNA probes. The PNA probes were synthesized at Boston Probes (now Applied Biosystems), Bedford, Mass., as described previously (20) with an Expedite 8909 Nucleic Acid Synthesis system with a PNA probe option and reagents from Applied Biosystems, Foster City, Calif. C. albicans PNA FISH method. The C. albicans PNA FISH method was performed as described previously (10). Briefly, fixed smears were covered with approximately 20 l of hybridization solution containing 10% (wt/vol) dextran sulfate (Sigma Chemical Co., St. Louis, Mo.), 10 mM NaCl (J. T. Baker Chemical Co.), 30% (vol/vol) formamide (Sigma), 0.1% (wt/vol) sodium pyrophosphate
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(Sigma), 0.2% (wt/vol) polyvinylpyrrolidone (Sigma), 0.2% (wt/vol) Ficoll (Sigma), 5 mM disodium EDTA (Sigma), 1% (vol/vol) Triton X-100 (Aldrich), 50 mM Tris-HCl (pH 7.5), and 250 nM fluorescein-labeled PNA probe targeting C. albicans 26S rRNA. Coverslips were placed on the smears to ensure even coverage with hybridization solution, and the slides were subsequently placed on a slide warmer with a humidity chamber (Slidemoat, Boeckel, Germany) and incubated for 90 min at 55°C. Following hybridization, the coverslips were removed by submerging each slide in approximately 20 ml of prewarmed 5 mM Tris (pH 10), 15 mM NaCl (J. T. Baker Chemical Co.), and 0.1% (vol/vol) Triton X-100 (Aldrich) in a water bath at 55°C; the slides were then washed for 30 min. Each smear was finally mounted with 1 drop of IMAGEN mounting fluid (DAKO, Ely, United Kingdom), and a coverslip was applied. Microscopic examination was conducted with a fluorescence microscope (Optiphot; Nikon Corporation, Tokyo, Japan) equipped with a ⫻60/1.4 oil objective (Nikon), an HBO 100-W mercury lamp, and a fluorescein isothiocyanate (FITC)-Texas Red dual-band filter set (Chroma Technology Corp., Brattleboro, Vt.). C. albicans cells were identified as bright green fluorescent yeast cells in multiple fields of view. Images were obtained with a color charge-coupled device camera (Diagnostic Instruments, Inc., Sterling Heights, Mich.) connected to a computer system. Identification of yeasts. The contents of yeast-positive blood culture bottles were subcultured onto Sabouraud glucose or potato dextrose agar and were typically incubated for 24 to 48 h at 25°C. The colonies were tested by germ tube analysis to rule in C. albicans or C. dubliniensis. If the result was negative, a cornmeal agar plate was set up to view pseudohyphae, blastoconidia, arthroconidia, and chlamydospores; and a yeast card (bioMerieux Vitek, Hazelwood, Mo.) was set up for identification. Occasionally, an API 20C test (bioMerieux Vitek) was applied. Isolates that are germ tube positive but that test negative by the C. albicans PNA FISH method with a sample from a blood culture bottle should be tested by D1-D2 26S rDNA sequence analysis (8), as these may be C. dubliniensis isolates.
RESULTS Theoretical sensitivity and specificity. The BLAST search and sequence alignments showed that the target sequence for the C. albicans-specific PNA probe is not present in rRNA sequences other than those of 14 C. albicans strains. However, the rRNA target sequences of three C. albicans strains, including NRRL Y-12983 and NRRL Y-81, differed from the probe target sequence by a single base. There were no C. albicans rRNA target sequences that differed from the probe target sequence by more than a single base. Analytical sensitivity and specificity. Initially, the sensitivity and specificity of the PNA probe were determined by the C. albicans PNA FISH method with reference strains representing the Candida genus (Table 1). Ten C. albicans reference strains were detected as bright green fluorescent yeast cells, whereas three C. albicans reference strains gave weaker but clearly positive fluorescent signals. Two of those strains were NRRL Y-12983 and NRRL Y-81, whose target sequences, according to the BLAST search, differed from the probe target sequence by a single base. We did make the PNA probe sequence complementary to the published rRNA sequences for NRRL Y-12983 and NRRL Y-81, but it did not result in a brighter signal. The sensitivity and specificity of the C. albicans PNA FISH method were further examined by using 148 clinical isolates representing C. albicans and other clinically relevant yeast species (Table 2). The assay correctly identified all C. albicans isolates and gave negative results for all other isolates. The detection limit for the C. albicans PNA FISH method was determined to be approximately 105 CFU per ml by use of serial dilutions of a culture positive for C. albicans.
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TABLE 1. Results of C. albicans PNA FISH method with 23 reference strains representing C. albicans and phylogenetically related species Species
Strain
C. albicans PNA FISH method resulta
C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. dubliniensis C. glabrata C. maltosa C. tropicalis C. viswanathii C. lodderae C. parapsilosis C. sojae L. elongisporus S. cerevisiae
NRRL Y-107 NRRL Y-12983 NRRL Y-17967 NRRL Y-17968 NRRL Y-17974 NRRL Y-17976 NRRL Y-302 NRRL Y-477 NRRL Y-6359 NRRL Y-6943 NRRL Y-79 NRRL Y-81 NRRL YB-3898 NRRL Y-17841 NRRL Y-65 NRRL Y-17677 NRRL Y-12968 NRRL Y-6660 NRRL Y-17317 NRRL Y-12969 NRRL Y-17909 NRRL Y-7681 ATCC 4098
⫹ ⫹b ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹b ⫹b ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
a b
Identification
Negative
9 0 0 0 0 0 0 0 0 0 0
0 11 10 5 4 9 2 4 1 2 1
DISCUSSION
TABLE 2. Results of C. albicans PNA FISH method with 148 isolates representing clinically relevant Candida yeast species
albicans dubliniensis glabrata tropicalis krusei parapsilosis lusitaniae zeylanoides
Positive
PNA probe appear reddish. This clear visual discrimination between positive and negative samples is due to the use of an FITC-Texas Red double filter consisting of a band-pass FITC filter and a long-pass Texas Red filter.
Diagnostic sensitivity and specificity. The diagnostic performance of the C. albicans PNA FISH method was evaluated directly with samples from 33 yeast-positive blood culture bottles, and the results were compared to the results obtained by standard methods. The cultures comprised 9 C. albicans cultures and 24 non-C. albicans cultures representing six to seven different species. Furthermore, the specificity was tested with 25 simulated blood culture bottles with nine different species. The results are summarized in Table 3 and show 100% agreement between the results of the C. albicans PNA FISH method and those of standard methods, supporting a 100% diagnostic sensitivity and 100% diagnostic specificity of the C. albicans PNA FISH method. That is equivalent to positive and negative predictive values of 100% each. Representative images of a positive and a negative test result are shown in Fig. 1. C. albicans cells appear as bright green fluorescent cells on a reddish smear background, whereas cells not detected by the
C. C. C. C. C. C. C. C.
No. of blood cultures with the following result by the C. albicans PNA FISH method:
C. albicans C. glabrata C. parapsilosis C. tropicalis C. krusei C. lusitaniae C. famata C. norvegensis C. guilliermondii C. neoformans Other yeast, not identified
⫹, positive; ⫺, negative. Weakly positive.
Candida species
TABLE 3. Results of C. albicans PNA FISH method with yeast-positive blood cultures, comprising 33 real blood culture bottles and 25 artificially spiked blood culture bottles
No. of isolates with the following result by the C. albicans PNA FISH method: Positive
Negative
72 0 0 0 0 0 0 0
0 58 5 3 2 4 3 1
The PNA FISH method with PNA probes that target the rRNA of C. albicans was used for the identification of C. albicans from yeast-positive blood culture bottles in a time frame not possible by conventional methods. The test was performed with smears made directly with the contents of the blood culture bottles, and interpretation of the results was conducted by microscopy, such that the PNA FISH procedure simply added the high degree of specificity of PNA probes to standard microbiological procedures (i.e., smear preparation and microscopy) to provide a definitive identification. The C. albicans-specific PNA probe showed a very high degree of specificity not only when it was used to test clinical isolates but also when it was further challenged by use of strains from the C. albicans clade. These findings are ascribed to the high degrees of specificity of PNA probes combined with the use of the D1-D2 region of 26S rRNA, a region that has been used for systematic studies of yeasts, as the target (8). These data are yet another example of how molecular diagnostic methods with rRNA sequences as the target can replace classic phenotype-based microbiological identification methods. In fact, several atypical C. albicans strains were correctly identified by the C. albicans PNA FISH method (7, 15). Also, C. dubliniensis, a recently described Candida species that is almost indistinguishable from C. albicans by standard methods, tested negative by the C. albicans PNA FISH method. This study did not include rarely occurring mixtures of organisms in cultures, but other studies have shown that mixtures of isolates in cultures can be detected by the PNA FISH method (5; K. Oliveira et al., Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. C-236, 2002). These smears will typically have fewer positive cells. In fact, a recent study (Oliveira et al., Abstr. 102nd Gen. Meet. Am. Soc. Microbiol.) indicates that the PNA FISH method is more sensitive than conventional
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FIG. 1. Images of two routine yeast-positive blood culture smears analyzed by the C. albicans PNA FISH method. The organism in panel A was identified as C. glabrata by standard methods, and the organism in panel B was identified as C. albicans by standard methods. C. albicans cells detected by the C. albicans PNA FISH method appear as bright green fluorescent yeast cells on a reddish smear background, whereas C. glabrata cells are slightly visible as reddish yeast cells infiltrated in the smear.
methods for the detection of mixtures of isolates in blood cultures. The C. albicans PNA FISH method provides a means for the rapid and specific identification of C. albicans. No confirmatory testing is required for identification, thus allowing the appropriate patient treatment to be administered more quickly, i.e., immediate treatment with fluconazole, since almost all isolates of C. albicans are susceptible to this drug. Moreover, yeastpositive blood cultures that test negative by the C. albicans PNA FISH method can be immediately submitted to a mycology laboratory for identification of the organism and the physician can be informed that fungemia is caused by a non-C. albicans yeast with potentially reduced susceptibility to commonly prescribed antifungals, i.e., fluconazole. In fact, recent data on the species distributions of Candida bloodstream isolates show that approximately 50% of non-C. albicans isolates detected in patient blood belong to species with reduced susceptibilities to fluconazole, such as C. glabrata, C. tropicalis, and C. krusei (14, 16). Rapid testing for C. albicans thus provides important information pertaining to the optimal antifungal therapy for candidemia caused by both C. albicans and non-C. albicans isolates. The C. albicans PNA FISH method supplements the previously described S. aureus PNA FISH method in that it provides a new diagnostic concept for the rapid and definitive identification of organisms in blood cultures in a routine clinical microbiology laboratory. Also, germ tube-negative C. albicans isolates as well as other phenotypically atypical C. albicans isolates could be reliably identified by this technique. In the future, the C. albicans PNA FISH method may be complemented by tests with a palette of PNA probes that target other Candida species, such as C. glabrata and C. krusei, for the potential identification of the organisms in yeast-positive blood cultures that test negative for C. albicans. In this way, fungemia caused by Candida yeasts resistant to common antibiotics can be ruled out in a more timely fashion.
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