Multicenter Evaluation of a Candida albicans Peptide Nucleic Acid Fluorescent In Situ Hybridization Probe for Characterization of Yeast Isolates from Blood Cultures

Abstract

We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a Candida albicans peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.

Yeasts, long known to cause thrush and vulvovaginal infections, have emerged as a significant cause of nosocomial infections (18, 21). Although Candida albicans usually is the most frequently isolated organism from patients with fungemia, other species of Candida are also important pathogens in hospitalized patients (18, 21). Furthermore, some of the Candida species other than C. albicans are inherently resistant to or display dose-dependent susceptibility to fluconazole, the antifungal agent that is most commonly used to treat infections caused by C. albicans (18, 23).

The first evidence of fungemia often is the presence of yeasts with or without pseudohyphae in a blood culture bottle that signals positive in an automated system (1). A variety of tests have been developed for the rapid characterization of yeast isolates. These include the rapid assimilation of trehalose by Candida glabrata, the detection of rapid urease production or the detection of phenoloxidase by the caffeic acid disk test for Cryptococcus neoformans, and the germ tube test or the detection of a colonial fringe on primary isolation media for the identification of C. albicans (2, 4-6, 20). A subculture onto specialized CHROMagar is another means of differentiating yeasts (19, 22). All of these tests, however, require subculture rather than direct assessment of the yeast in the positive blood culture bottle. The subcultures from positive blood culture bottles are often not examined and tested until the next day, which may be 18 to 24 h after the blood culture bottle first signaled positive. This delay in identification may affect patient care, since some Candida species other than C. albicans are much more likely to be resistant to fluconazole; Candida krusei is inherently resistant to fluconazole, and C. glabrata, a commonly encountered clinical isolate, does not have predictable fluconazole susceptibility and may be resistant or demonstrate dose-dependent susceptibility.

Fluorescent in situ hybridization (FISH) is a well-established diagnostic tool in molecular pathology laboratories and has been used to detect and differentiate microorganisms in histologic sections (9, 10). This technology has been applied to culture and specimens in the clinical microbiology laboratory. Examples include the direct, species-specific identification of bacteria and yeasts from positive blood cultures and the direct identification of the common bacterial pathogens from respiratory specimens of patients with cystic fibrosis (3, 12-14). The rapid differentiation of Candida species that are commonly encountered in the hospital setting has been accomplished with traditional fluorescent in situ hybridization (12-14). A peptide nucleic acid (PNA) FISH probe specific for Candida albicans has also been demonstrated to be potentially useful on aliquots taken directly from positive blood culture bottles to differentiate C. albicans from non-C. albicans yeasts (16). This technology has been also been developed and tested for the rapid differentiation of Staphylococcus aureus from non-S. aureus bacteria that form cocci in clusters (3, 15, 17). We performed a multi-institutional evaluation of the Candida albicans PNA FISH assay (AdvanDx, Woburn, MA) on blood culture bottles that signaled positive and demonstrated yeasts on the Gram stain.

The blood culture systems used were the BD BACTEC 9600 and 9240 systems (Becton Dickinson, Sparks, MD) and the BacT/ALERT system (bioMérieux, Durham, NC). The media used were the BD BACTEC resin, standard (aerobic and anaerobic), and the Lytic 10, as well as BacT/ALERT FA, FN, SN, SA, and plastic SN and BacT/ALERT PF (plastic; for investigational use only). More than one blood culture isolate was not accepted from the same patient, unless 7 days or more had elapsed from the detection of the prior isolate. One hundred ten of the positive blood cultures that contained yeasts were collected at the Cleveland Clinic Foundation, where the bioMérieux BacT/ALERT system was used. The remaining 134 blood cultures were collected at Duke University Medical Center and Mayo Clinic. Mayo Clinic provided samples from the BD BACTEC system, whereas Duke University provided samples from both the BD BACTEC and the BacT/ALERT systems.

At Mayo Clinic, yeast isolates were first assessed after subculture using the germ tube test. If positive, the yeast was identified as C. albicans; if negative the yeast was assessed with the API20Caux (bioMérieux, Durham, NC) in conjunction with morphology on cornmeal agar. At the Cleveland Clinic, macroscopic morphology was assessed after subculture onto sheep blood agar for the presence of a colonial fringe. If present, the isolate was characterized as C. albicans; the fringe was used as a surrogate for a positive germ tube test. If a colonial fringe was absent, the germ tube test was performed. Yeasts that produced germ tubes were characterized as C. albicans, whereas yeasts that did not produce germ tubes were sent to the mycology laboratory and characterized by the Vitek YBC yeast identification card (bioMérieux, Durham, NC) in conjunction with their microscopic morphology on cornmeal agar. At Duke University Medical Center, yeast colonies from positive blood culture bottles were first tested with the C. albicans screen (Remel, Lenexa, KS). Isolates that produced a positive reaction according to the manufacturer's guidelines were characterized as C. albicans. Additionally, isolates that produced a negative reaction were sent to the mycology laboratory for testing with the API20Caux (bioMérieux), by four sugar fermentation reactions, and by morphological assessment on cornmeal agar.

When yeasts were detected by routine Gram staining of positive blood culture bottles, duplicate slides were made for C. albicans PNA FISH analysis. The slides from all three institutions were submitted for C. albicans PNA FISH testing to one author (D.A.W.), in a blinded fashion. The yeast isolates were kept as stock cultures after routine identification for further testing in case of discrepancies between conventional and FISH results.

The C. albicans PNA FISH assay (AdvanDx, Inc., Woburn, MA) was performed according to the manufacturer's instructions. Briefly, a 10-μl aliquot from the blood culture bottle that signaled positive and contained yeasts in the Gram stain was mixed with 1 drop of fixation solution. After fixation on a slide warmer for 20 min, slides were immersed briefly in 95% ethyl alcohol. One drop of the C. albicans PNA probe was placed on the slide and hybridized at 55°C for 90 min in a humidified atmosphere. The slides were washed in buffer heated to 55°C for 30 min, mounting media were applied, and a coverslip was applied to each slide. The entire PNA FISH procedure required approximately 2.5 h. Slides were read using a fluorescence microscope with both fluorescein isothiocyanate (FITC) and a dual FITC/Texas Red filter(s); this was done to compare filters. The manufacturer, however, recommends only the dual FITC/Texas Red filter. In such preparations, C. albicans was expected to appear as fluorescent green yeast cells (Fig. 1), whereas non-C. albicans yeasts were nonfluorescent. Positive (C. albicans) and negative (C. glabrata) control slides were included in each run.

FIG. 1.

Candida albicans demonstrated bright green fluorescence when hybridized with the Candida albicans PNA FISH probe. Magnification, ×400. An FITC filter was used.

The results of the C. albicans PNA FISH test were first compared with the results of the routine screening tests used by the three laboratories, as previously described. Concordant results between C. albicans PNA FISH and the initial routine identification of the yeast as C. albicans or non-C. albicans were not investigated further. Any discrepancies were retested with the C. albicans PNA FISH test and the API20Caux system (11). If discrepancies could not be clarified, the identification of the isolates was made at Mayo Clinic by DNA sequencing of the D2 region of the large ribosomal subunit, as previously described (8). The collaborator at the Mayo Clinic (L.S.H.), who performed the DNA sequencing, did not know the results of the phenotypic assays or the PNA FISH test. The results of the DNA sequence analysis, if needed, were considered as the definitive identification.

Two hundred forty-four blood cultures from 228 patients that contained yeasts on Gram stain were evaluated by C. albicans PNA FISH (Table 1). One hundred one of the 244 positive blood cultures were initially reported to contain C. albicans as identified by routine laboratory methods, whereas only 96 were positive by the C. albicans PNA FISH test. The repeat C. albicans PNA FISH test on aliquots from the five cultures with discrepant results yielded one PNA FISH-positive culture, whereas four remained PNA FISH negative. These four isolates were analyzed with the API 20Caux system for further resolution of the discrepancies. The one isolate that was initially interpreted as PNA FISH negative but was PNA FISH positive on repeat testing was determined to be C. albicans by testing with the API20Caux. This isolate was also submitted for DNA sequencing (out of interest), which confirmed the identity as C. albicans. The four isolates that were identified as C. albicans by the routine laboratory methods but were PNA FISH negative were identified as Candida dubliniensis (two isolates), Candida parapsilosis, and C. albicans using the API20Caux (Table 2). The single isolate that was PNA FISH negative but identified as C. albicans by the API20Caux was submitted for DNA sequencing, which revealed it to also be C. dubliniensis.

TABLE 1.

Initial Candida albicans PNA FISH compared to identification by routine laboratory methods

Yeast identification by routine methods	Initial C. albicans PNA FISH result	No. of blood cultures
Candida albicans	Positive	93
C. albicans and C. glabrata	Positive	1
C. albicans and C. parapsilosis	Positive	2
C. albicansa	Negative	5
C. glabrata	Negative	74
C. glabrata and C. parapsilosis	Negative	1
C. parapsilosis	Negative	18
C. tropicalis	Negative	21
C. krusei	Negative	12
Cryptococcus neoformans	Negative	6
Saccharomyces cerevisiae	Negative	2
S. cerevisiae and C. glabrata	Negative	1
Other non-C. albicans fungib	Negative	8

^aSee Table 2 for final identification.

^bThese were all read as yeasts in the Gram stain, and species were determined as follows: 1 each of Candida guilliermondii, Candida kefyr, Candida lusitaniae, Pichia ohmeri, Rhodotorula glutinis, Fusarium sp., Trichosporon sp., and Exophiala dermatitidis.

TABLE 2.

Discrepancy resolution

Discrepant isolate	Initial identification	Repeat C. albicans FISH	API20Caux identification	DNA sequencing identification
1	C. albicans	Negative	C. parapsilosis	NAa
2	C. albicans	Negative	C. dubliniensis	NA
3	C. albicans	Negative	C. dubliniensis	NA
4	C. albicans	Negative	C. albicans	C. dubliniensis
5	C. albicans	Positive	C. albicans	C. albicans

^aNA, not applied.

The C. albicans isolates highlighted with the C. albicans PNA FISH probe were readily demonstrable with both the FITC and dual FITC/Texas Red filters. The yeasts were slightly more obvious when viewed with the dual FITC/Texas Red filter, since the background was red rather than light green.

This multicenter evaluation of the C. albicans PNA FISH assay (AdvanDx) demonstrated that this method is an accurate means of differentiating C. albicans from non-C. albicans species present in blood culture bottles. Only five discrepancies were observed with the routine screening methods, four of which were initially characterized as C. albicans by routine testing, but as non-C. albicans by the Candida albicans PNA FISH assay. Discrepancy resolution showed that three of these isolates were C. dubliniensis, an organism known to produce germ tubes and easily misidentified by this test as C. albicans when this test is used alone. Therefore, yeasts that are negative by the C. albicans PNA FISH probe but traditionally identified as C. albicans may represent C. dubliniensis (7). The fourth discrepancy was caused by C. parapsilosis, which was thought to have a colonial fringe in the subculture on sheep blood agar. This isolate was examined on a plate after approximately 24 hours of incubation. Candida parapsilosis, like many of the other Candida species, may produce pseudohyphae after extended incubation, which may explain why this isolate was initially mischaracterized as C. albicans. One culture of C. albicans that was initially interpreted as negative by the PNA FISH test had to be considered as a false-negative result in this study, even though the repeat PNA FISH test was positive, since repeat testing would not commonly be done in a routine clinical laboratory. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the combined routine screening methods used at the various institutions were 100%, 97.3%, 96.0%, and 100%, respectively.

This assay was assessed using a wide variety of the blood culture media that are commercially available and performed well. The C. albicans isolates that appeared positive with the C. albicans PNA FISH probe were categorized similarly with both the FITC and dual FITC/Texas Red filters. Subjectively it was felt that it was easier to see the fluorescent apple-green yeast cells against the red background displayed with the FITC/Texas Red filter than against the light green background produced by the single FITC filter.

Although the differentiation of C. albicans from non-C. albicans is useful, additional probes that identify non-C. albicans isolates that are or could be resistant to fluconazole, such as C. krusei or C. glabrata, respectively, would be useful. In addition, the detection of the presence of C. albicans in the blood culture does not exclude the possibility, although rare, of the presence of a second yeast. Five of the 244 blood culture bottles examined in this study contained two species of yeasts. Only 97/244 (39.8%) of the bottles tested contained C. albicans, which reiterates the increasing importance of non-C. albicans yeasts as causes of fungemia. In summary, the C. albicans PNA FISH assay compares well with routine methods of screening for C. albicans but has the advantage of not requiring subculture and overnight incubation prior to analysis.