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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2013 Jan;51(1):117–124. doi: 10.1128/JCM.01686-12

Candida guilliermondii and Other Species of Candida Misidentified as Candida famata: Assessment by Vitek 2, DNA Sequencing Analysis, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Two Global Antifungal Surveillance Programs

Mariana Castanheira a,✉, Leah N Woosley a, Daniel J Diekema b, Ronald N Jones a, Michael A Pfaller a,b
PMCID: PMC3536252  PMID: 23100350

Abstract

Candida famata (teleomorph Debaryomyces hansenii) has been described as a medically relevant yeast, and this species has been included in many commercial identification systems that are currently used in clinical laboratories. Among 53 strains collected during the SENTRY and ARTEMIS surveillance programs and previously identified as C. famata (includes all submitted strains with this identification) by a variety of commercial methods (Vitek, MicroScan, API, and AuxaColor), DNA sequencing methods demonstrated that 19 strains were C. guilliermondii, 14 were C. parapsilosis, 5 were C. lusitaniae, 4 were C. albicans, and 3 were C. tropicalis, and five isolates belonged to other Candida species (two C. fermentati and one each C. intermedia, C. pelliculosa, and Pichia fabianni). Additionally, three misidentified C. famata strains were correctly identified as Kodomaea ohmeri, Debaryomyces nepalensis, and Debaryomyces fabryi using intergenic transcribed spacer (ITS) and/or intergenic spacer (IGS) sequencing. The Vitek 2 system identified three isolates with high confidence to be C. famata and another 15 with low confidence between C. famata and C. guilliermondii or C. parapsilosis, displaying only 56.6% agreement with DNA sequencing results. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) results displayed 81.1% agreement with DNA sequencing. One strain each of C. metapsilosis, C. fermentati, and C. intermedia demonstrated a low score for identification (<2.0) in the MALDI Biotyper. K. ohmeri, D. nepalensis, and D. fabryi identified by DNA sequencing in this study were not in the current database for the MALDI Biotyper. These results suggest that the occurrence of C. famata in fungal infections is much lower than previously appreciated and that commercial systems do not produce accurate identifications except for the newly introduced MALDI-TOF instruments.

INTRODUCTION

Candida famata (formerly Torulopsis candida; teleomorph, Debaryomyces hansenii) is an ascomycetous yeast commonly found in foods, including dairy products (1, 2). C. famata is a rare human pathogen that has been the putative etiologic agent in cases of bloodstream infection (BSI) (3–9) peritonitis (10, 11) and ocular (12, 13) and bone (14) infections. In the 10.5-year ARTEMIS DISK survey (15), C. famata was ranked ninth among 31 different species.

Recent publications from reference laboratories have suggested that isolates initially identified as C. famata by phenotypic methods were found to include strains of C. guilliermondii, C. lusitaniae, C. fermentati, C. intermedia, and C. palmioleophila when subjected to molecular identification (5, 16). Such findings suggest that C. famata may be much less common as an etiologic agent of invasive candidiasis (IC) than previously reported (5, 16). Our recent experience has been similar in that five isolates originally identified as C. famata in the 2010 SENTRY antimicrobial surveillance program were found by molecular methods to be three different species of Candida (C. guilliermondii, C. lusitaniae, and C. parapsilosis) (17). With this background, we investigated the true prevalence of C. famata among 53 isolates originally submitted as such in two very large global surveillance programs, the ARTEMIS and SENTRY programs, for the time interval from 2005 through 2011.

MATERIALS AND METHODS

Yeast isolates.

Fifty-three clinical isolates from individual patients previously identified as C. famata and submitted to the ARTEMIS (24 isolates) or the SENTRY (29 isolates) program were included in the study. The yeast identification methods employed in the submitting laboratories included the Vitek yeast identification system (32 isolates [60%]), MicroScan (4 isolates [8%]), API (4 isolates [8%]), and AuxaColor (2 isolates [4%]). The identification system was not specified for 11 isolates (20%). The isolates represented 33 different study sites in 22 countries; 34 were obtained from blood or deep tissue sites of infection. These isolates represented all (100.0%) of the C. famata isolates submitted to the University of Iowa (Iowa City) and JMI (North Liberty, IA) reference laboratories during the time period from 2005 through 2011. Isolates were subcultured upon receipt and stored until further testing.

Phenotypic characterization of isolates.

Isolates were subcultured on CHROMagar (Becton, Dickinson and Company), and colony morphology and color were observed after 48 h of incubation at 35°C. Species identification was performed using the Vitek 2 yeast identification card (bioMérieux, Hazelwood, MO) according to the manufacturer's instructions. Isolates were assessed for the presence or absence of pseudohyphae after growth for 24 h at 35°C on cornmeal agar (Remel, Lenexa, KS). The MIC values for amphotericin B, flucytosine, fluconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin were determined by broth microdilution as described in Clinical and Laboratory Standards Institute (CLSI) document M27-A3 (18).

DNA sequencing-based identification.

Isolates were subjected to DNA extractions using the QIAquick extraction kit (Qiagen, Hilden, Germany) in the QiaCube automated system (Qiagen). Amplification and sequencing were performed for the intergenic transcribed spacer (ITS) region for all strains (19). Additionally, the 28S ribosomal subunit (D1/D2) (19, 20) was used for assisting in the identification of C. fabianii and C. intermedia/pseudointermedia, and the intergenic spacer (IGS) was sequenced for Debaryomyces spp. (21). Amplicons were sequenced on both strands and compared with available reference sequences through BLAST. Results were considered acceptable if homology with other entries in the databases used for comparison was >99.5%. Available sequences that were considerably different from the majority of entries for one species were considered outliers and discarded in the analysis. Additionally, if no match was found in the database, the identification was based on species complex, genus, family, or order, according to the most current classifications systems.

MALDI-TOF MS.

All isolates were tested via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using the Bruker Daltonik MALDI Biotyper (Fremont, CA) by following the manufacturer's instructions and previously described guidelines for yeast identification (22, 23).

RESULTS AND DISCUSSION

DNA-sequencing identification results compared to those of Vitek 2 and MALDI-TOF MS (MALDI).

Among the 53 isolates originally identified as C. famata by the submitting laboratory, the majority (33/53; 62.3%) (Table 1) were identified as C. guilliermondii (19 isolates) or C. parapsilosis (14 isolates), with two identified as the cryptic species C. orthopsilosis (one isolate) and C. metapsilosis (one isolate), by ITS sequencing. An additional seven Candida species were identified among 17 remaining isolates, with five being C. lusitaniae, four C. albicans, three C. tropicalis, and two C. fermentati and three isolates belonging to other species. The latter three isolates included one strain each of C. intermedia/pseudointermedia, C. pelliculosa, and C. (Cyberlidnera/Pichia) fabianni. Additionally, one Kodomaea ohmeri isolate was identified using ITS sequencing, and two isolates displaying low discrimination on the ITS and 28S rRNA sequencing between D. hansenii and D. nepalensis or D. castellii were identified as Debaryomyces nepalensis and Debaryomyces fabryi using IGS sequencing (21).

Table 1.

Identification by DNA sequencing methods, the Vitek 2, and mass spectroscopy for 53 clinical isolates sent to the ARTEMIS and/or SENTRY surveillance program between 2005 and 2011 as Candida famata

Strain Identification by:
 Pseudohyphae Country Specimen type
DNA sequencing Vitek 2 MALDI-TOF (score value if <2.00)
1 C. albicans C. albicans C. albicans NTa Brazil Blood
2 C. albicans C. albicans C. albicans NT Poland Blood
3 C. albicans C. albicans C. albicans NT Italy Unknown
4 C. albicans C. albicans C. albicans Positive Germany Blood
5 C. fabianii Unidentified No reliable identification Negative Slovakia Blood
6 C. fermentati Low-discrimination C. famata/C. guilliermondii C. guilliermondii (1.70) Negative United States Blood
7 C. fermentati Low-discrimination C. guilliermondii/C. famata No reliable identification Negative Colombia Blood
8 C. guilliermondii Low-discrimination C. famata/C. guilliermondii C. guilliermondii Negative Italy Blood
9 C. guilliermondii Low-discrimination C. famata/C. guilliermondii C. guilliermondii Positive Mexico Blood
10 C. guilliermondii Low-discrimination C. famata/C. guilliermondii C. guilliermondii Negative Turkey Unknown
11 C. guilliermondii Low-discrimination C. famata/C. guilliermondii C. guilliermondii Negative Brazil Blood
12 C. guilliermondii Low-discrimination C. guilliermondii/C. famata C. guilliermondii Negative United States Blank
13 C. guilliermondii Low-discrimination C. guilliermondii/C. famata C. guilliermondii Positive United States Bronchoalveolar lavage fluid
14 C. guilliermondii Low-discrimination C. guilliermondii/C. famata No reliable identification Negative Argentina Blood
15 C. guilliermondii Low-discrimination C. guilliermondii/C. famata C. guilliermondii Negative France Other
16 C. guilliermondii Low-discrimination C. guilliermondii/C. famata C. guilliermondii Negative United States Blood
17 C. guilliermondii Low-discrimination C. guilliermondii/C. famata C. guilliermondii Positive Argentina Blood
18 C. guilliermondii Low-discrimination C. guilliermondii/C. famata C. guilliermondii Negative Italy Unknown
19 C. guilliermondii C. guilliermondii/C. famata C. guilliermondii Positive Switzerland Unknown
20 C. guilliermondii C. guilliermondii C. guilliermondii Positive Brazil Blood
21 C. guilliermondii C. guilliermondii C. guilliermondii Positive Colombia Urine
22 C. guilliermondii C. guilliermondii C. guilliermondii Positive Australia Blood
23 C. guilliermondii C. guilliermondii No reliable identification Negative Argentina Blood
24 C. guilliermondii C. guilliermondii C. guilliermondii Positive Australia Blood
25 C. guilliermondii C. famata C. guilliermondii Positive South Korea Blood
26 C. guilliermondii C. famata C. guilliermondii Positive Germany Blood
27 C. intermedia/C. pseudointermedia C. intermedia C. intermedia (1.97) Positive China Blood
28 C. lusitaniae C. lusitaniae C. lusitaniae Negative Mexico Unknown
29 C. lusitaniae C. lusitaniae C. lusitaniae Positive Mexico Unknown
30 C. lusitaniae C. lusitaniae C. lusitaniae Positive Mexico Unknown
31 C. lusitaniae C. lusitaniae C. lusitaniae Negative France Other
32 C. lusitaniae C. lusitaniae C. lusitaniae Negative Hungary Tissue
33 C. metapsilosis C. parapsilosis No reliable identification Positive United States Blood
34 C. orthopsilosis C. parapsilosis C. orthopsilosis Positive Venezuela Abscess
35 C. parapsilosis Low-discrimination C. parapsilosis/C. famata C. parapsilosis Positive South Africa Blood
36 C. parapsilosis C. parapsilosis C. parapsilosis Positive Mexico Blood
37 C. parapsilosis C. parapsilosis C. parapsilosis Positive Turkey Unknown
38 C. parapsilosis C. parapsilosis C. parapsilosis Positive Brazil Blood
39 C. parapsilosis C. parapsilosis C. parapsilosis Positive Mexico Blood
40 C. parapsilosis C. parapsilosis C. parapsilosis Positive Ireland Unknown
41 C. parapsilosis C. parapsilosis C. parapsilosis Positive Italy Blood
42 C. parapsilosis C. parapsilosis C. parapsilosis Positive Turkey Unknown
43 C. parapsilosis C. parapsilosis C. parapsilosis Positive Mexico Unknown
44 C. parapsilosis C. parapsilosis C. parapsilosis Positive Brazil Unknown
45 C. parapsilosis C. parapsilosis C. parapsilosis Negative Brazil Blood
46 C. parapsilosis C. parapsilosis C. parapsilosis Positive South Africa Blood
47 C. pelliculosa C. pelliculosa C. pelliculosa Negative South Korea Blood
48 C. tropicalis C. tropicalis C. tropicalis NT United States Blood
49 C. tropicalis C. tropicalis C. tropicalis NT Brazil Tissue
50 C. tropicalis Low-discrimination C. tropicalis/C. parapsilosis C. tropicalis Positive Israel Blood
51 Debaryomyces fabryi C. sphaerica No reliable identification Negative France Blood
52 Debaryomyces nepalensis C. famata No reliable identification Negative China Unknown
53 Kodamaea ohmeri Kodamaea ohmeri C. guilliermondii (1.78) Positive Brazil Unknown
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a

NT, not tested.

Only three isolates (5.7%) were identified as C. famata with high confidence using the Vitek 2 yeast identification card (Table 1). However, these isolates were confirmed neither by ITS sequencing nor by MALDI-TOF MS. The most frequent Vitek 2 result was a low discrimination between C. famata and C. guilliermondii (14 isolates), for which 12 were identified as C. guilliermondii and two as C. fermentati by ITS sequencing. MALDI was not able to produce reliable results for two C. guilliermondii strains or for any of the C. fermentati strains (one was identified as C. guilliermondii with a low score value [1.70], and one had no reliable identification) (Table 1).

Among 13 isolates that were identified with high confidence as C. parapsilosis by the Vitek 2 and one that had low discrimination between C. famata and C. parapsilosis, 11 were confirmed as belonging to this species by both ITS sequencing and MALDI. Two isolates were identified as the cryptic species C. orthopsilosis and C. metapsilosis (one each) by sequencing, but among those the MALDI was able to produce reliable results only for C. orthopsilosis (Table 1).

The Vitek 2 system identified five of the putative C. famata isolates as C. lusitaniae, five as C. guilliermondii, and one as C. pelliculosa. These results were confirmed by ITS sequencing and MALDI-TOF MS, except for one C. guilliermondii isolate which was not identified by the MALDI Biotyper. One isolate had low discrimination in the Vitek 2 between C. tropicalis and C. parapsilosis, but ITS sequencing and MALDI identified it as C. tropicalis. Four isolates received as C. famata were identified as C. albicans and two as C. tropicalis by all methods used.

One isolate showed low discrimination among C. intermedia/C. pseudointermedia using ITS sequencing analysis (95.0 to 98.5% homology). The Vitek 2 and the MALDI identified this strain as C. intermedia, with very good confidence and a score value of 1.97, respectively. One C. sphaerica isolate identified by the Vitek 2 had high ITS homology with Debaryomyces spp. and was identified as Debaryomyces fabryi by IGS sequencing. No reliable identification was produced using the MALDI.

The Vitek 2 showed a good identification for a Kodamaea ohmeri isolate that was in agreement with ITS sequencing, and this isolate was identified with a score of 1.78 as C. guilliermondii by the MALDI. One isolate was not reliably identified by the Vitek 2 and MALDI and was identified as C. fabianii by ITS sequencing.

Thus, within the entire collection of 53 putative C. famata isolates, none were confirmed as such by DNA-sequencing-based methods. The Vitek 2 system gave C. famata as a possible identification for a total of 18 isolates (three C. famata isolates and 15 isolates with low discrimination between C. famata and C. guilliermondii/C. parapsilosis), but the MALDI Biotyper did not identify any of these isolates as C. famata.

Whereas Jensen and Arendrup (16) demonstrated that the type strain of C. famata (CBS 796) did not form pseudohyphae on cornmeal agar and produced light red pigmented colonies on CHROMagar, our collection of putative C. famata isolates showed variable production of pseudohyphae (55%) and a colony color on CHROMagar that ranged from white to green to blue and purple. None of the strains in the present collection produced a red or light red colony on CHROMagar.

Accuracy of Vitek 2 and MALDI versus sequencing-based identification.

Clearly, this collection of predominantly non-C. albicans Candida species poses problems for yeast identification systems. Using ITS and/or 28S ribosomal gene sequencing, this collection of 53 isolates previously identified in clinical laboratories worldwide as C. famata was shown to contain 14 different species (Table 2). By comparison, the Vitek 2 yeast identification card identified eight different species, and MALDI identified six species. Several of these species are distinctly uncommon as causes of IC and are quite difficult to identify with any degree of accuracy (17, 24). Among the more common species as determined by reference sequencing (C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, and C. lusitaniae), 58.1% (25/43) were correctly identified by the Vitek 2 and 88.4% (38/43) were correctly identified by MALDI (Table 2). The accuracy of the Vitek 2 for the identification of these five species is considerably lower than reported previously (89.8% [one choice] and 99.1% [including low discrimination]) (25), but this would improve to 97.6% in the present study if the low-discrimination results of C. famata/C. guilliermondii and C. famata/C. parapsilosis were considered acceptable for the identification of C. guilliermondii and C. parapsilosis, respectively (Table 2). Likewise, the cryptic species C. fermentati, C. metapsilosis, and C. orthopsilosis are not expected to be differentiated from C. guilliermondii and C. parapsilosis, respectively, by the Vitek 2 or any other phenotypic identification system.

Table 2.

Species identification of isolates previously identified as C. famata by the Vitek 2 and MALDI-TOF methods in comparison with reference molecular methodology

Reference identification (no. of isolates) Test method No. (%) correct Discrepancies (no. of isolates)
C. guilliermondii (19) Vitek 2 5 (26.3) C. famata/C. guilliermondii low discrimination (12), C. famata (2)
MALDI 17 (89.5) C. guilliermondii with low score value (1), no reliable identification (1)
C. parapsilosis (12) Vitek 2 11 (91.7) C. famata/C. parapsilosis low discrimination (1)
MALDI 12 (100.0)
C. lusitaniae (5) Vitek 2 5 (100.0)
MALDI 5 (100.0)
C. albicans (4) Vitek 2 4 (100.0)
MALDI 4 (100.0)
C. tropicalis (3) Vitek 2 2 (66.7) C. tropicalis/C. parapsilosis low discrimination (1)
MALDI 3 (100.0)
C. fermentati (2) Vitek 2 0 (0.0) C. famata/C. guilliermondii low discrimination (2)
MALDI 0 (0.0) C. guilliermondii with low score value (1), no reliable identification (1)
C. fabianii (1) Vitek 2 0 (0.0) Unidentified (1)
MALDI 0 (0.0) No reliable identification (1)
C. intermedia (1) Vitek 2 1 (100.0)
MALDI 0 (0.0) C. intermedia with low score value (1)
C. metapsilosis (1) Vitek 2 0 (0.0) C. parapsilosis (1)
MALDI 0 (0.0) No reliable identification (1)
C. orthopsilosis (1) Vitek 2 0 (0.0) C. parapsilosis (1)
MALDI 1 (100.0)
C. pelliculosa (1) Vitek 2 1 (100.0)
MALDI 1 (100.0)
D. nepalensis (1) Vitek 2 0 (0.0) C. sphaerica (1)
MALDI 0 (0.0) No reliable identification (1)
D. fabryi (1) Vitek 2 0 (0.0) C. famata (1)
MALDI 0 (0.0) No reliable identification (1)
K. ohmeri (1) Vitek 2 1 (100.0) K. ohmeri (1)
MALDI 0 (0.0) C. guilliermondii (1)
Total (53) Vitek 2 30 (56.6)
MALDI 43 (81.1)
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An incorrect species identification (major error) occurred for two isolates (3.8%) with the MALDI Biotyper (one C. fermentati and one K. ohmeri misidentified as C. guilliermondii); however, the misidentification of the C. fermentati can be explained by the fact that this organism, a cryptic species within the C. guilliermondii species complex (26), was not in the current database. Generally, MALDI has the advantage of having a “no reliable result” response rather than making an erroneous identification (Table 2). Among the 10 discrepancies or low-score results observed between MALDI and the molecular reference method, six were due to a report of no reliable identification by the MALDI Biotyper, two of which were species not included in the MALDI database (C. fermentati and C. fabianii). C. metapsilosis and C. orthopsilosis, newly recognized as members of the C. parapsilosis species complex, are frequently misidentified by phenotypic methods as C. parapsilosis sensu stricto (27). Previous studies show that these two species can be identified by MALDI (28, 29); however, in two instances (2, 7, 11, 13, 28, 29) the original manufacturer spectral database library was secondarily modified to include these rare yeast species. Interestingly Hendrickx et al. (30) were able to identify both of these cryptic species by MALDI-TOF MS without any database modifications. Overall, our results are comparable with those obtained in previously published studies where MALDI was compared to a reference molecular identification method (22, 29, 31).

In vitro susceptibility profile of C. famata and other species of Candida identified by molecular methods.

In general the MIC values obtained for the eight antifungal agents tested conform to the wild-type (WT) MIC distributions for C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, C. lusitaniae, C. orthopsilosis, and C. pelliculosa described previously for each species (32–38). Notable exceptions include decreased susceptibility to flucytosine (MIC > WT) among C. guilliermondii (MIC > 1 μg/ml [one isolate]) and C. lusitaniae (MIC > 0.5 μg/ml [two isolates]) strains, to fluconazole among C. guilliermondii (MIC > 8 μg/ml [three isolates]) and C. parapsilosis (MIC > 2 μg/ml [two isolates]) strains, to posaconazole among C. guilliermondii (MIC > 0.5 μg/ml [six isolates]), C. albicans (MIC > 0.06 μg/ml [one isolate]), and C. tropicalis (MIC > 0.12 μg/ml [one isolate]) strains, and to voriconazole among C. guilliermondii (MIC > 0.25 μg/ml [two isolates]) and C. tropicalis (MIC > 0.06 μg/ml [one isolate]) strains (Table 3).

Table 3.

Selected in vitro susceptibility results for 53 isolates of Candida identified by molecular methods as determined by 24-h CLSI broth microdilution methods

Organism (no. of isolates) MIC (μg/ml)
Amphotericin B Flucytosine Anidulafungin Caspofungin Micafungin Fluconazole Posaconazole Voriconazole
C. guilliermondii (19) 1 ≤0.5 2 0.12 1 2 0.5 0.06
1 ≤0.5 4 0.5 2 16 1 0.25
1 ≤0.5 2 0.12 1 2 0.5 0.12
0.5 ≤0.5 1 0.12 1 2 0.5 0.06
0.5 ≤0.5 2 0.5 1 8 0.25 0.12
1 4 2 0.12 8 1 2
0.5 ≤0.5 2 1 1 2 0.5 0.06
0.5 ≤0.5 2 0.12 2 2 1 0.12
1 ≤0.5 2 0.12 1 2 0.5 0.06
1 ≤0.5 1 0.06 0.5 8 1 0.25
1 ≤0.5 2 0.25 1 2 0.25 0.06
0.5 ≤0.5 1 0.12 1 2 0.25 0.03
1 ≤0.5 1 0.25 1 16 1 0.5
1 ≤0.5 2 0.25 2 2 0.5 0.12
0.5 ≤0.5 2 0.5 1 4 0.5 0.12
0.5 ≤0.5 2 0.12 1 2 0.25 0.06
0.5 ≤0.5 2 0.25 1 2 0.25 0.06
0.5 ≤0.5 2 0.12 1 2 0.5 0.06
1 ≤0.5 2 0.25 1 16 1 0.25
C. parapsilosis (12) 1 ≤0.5 2 0.12 1 0.25 0.12 0.015
1 ≤0.5 2 0.5 1 0.5 0.12 ≤0.008
1 ≤0.5 4 0.5 2 16 0.25 0.25
1 ≤0.5 1 0.25 1 0.5 0.25 0.03
1 ≤0.5 2 0.25 1 0.5 0.25 0.03
1 ≤0.5 2 0.5 2 0.5 0.25 0.03
1 ≤0.5 2 0.5 2 0.5 0.12 0.03
1 ≤0.5 2 0.25 2 0.5 0.12 0.015
1 ≤0.5 0.5 0.25 1 1 0.25 0.03
1 ≤0.5 2 0.25 1 2 0.25 0.06
0.5 ≤0.5 2 0.5 1 2 0.25 0.06
1 ≤0.5 2 1 1 8 0.12 0.5
C. lusitaniae (5) 0.5 ≤0.5 0.5 0.12 0.25 0.5 0.12 0.015
1 ≤0.5 0.25 0.12 0.25 0.5 0.12 0.015
0.5 ≤0.5 0.12 0.06 0.12 1 0.12 0.03
1 16 0.5 0.25 0.5 0.12 0.12 ≤0.008
1 4 0.5 0.25 0.25 0.25 0.12 0.015
C. albicans (4) 0.5 ≤0.5 0.015 0.12 0.03 ≤0.5 ≤0.06 ≤0.06
1 ≤0.5 0.03 0.12 0.06 ≤0.06 0.12 0.015
0.25 ≤0.5 0.03 0.03 0.03 ≤0.06 0.015 ≤0.008
1 ≤0.5 ≤0.008 0.015 0.015 0.12 0.03 0.015
C. tropicalis (3) 1 ≤0.5 0.015 0.06 0.015 0.5 0.12 0.03
0.5 ≤0.5 0.015 0.03 0.12 0.5 0.06 0.03
1 ≤0.5 0.03 0.03 0.03 2 0.5 0.12
C. fermentati (2) 1 ≤0.5 2 0.25 0.5 8 0.5 0.25
1 ≤0.5 1 0.25 1 2 0.5 0.12
C. fabianii (1) 0.5 4 0.03 0.03 0.03 4 1 0.12
C. intermedia/pseudointermedia (1) 0.5 ≤0.5 ≤0.008 ≤0.008 0.12 0.25 0.06 0.015
C. methapsilosis (1) 1 ≤0.5 2 0.12 0.5 1 0.25 0.06
C. orthopsilosis (1) 1 ≤0.5 0.25 0.25 0.5 1 0.25 0.03
C. pelliculosa (1) 0.5 ≤0.5 0.015 0.03 0.06 2 0.5 0.12
Debaryomyces fabryi (1) 2 ≤0.5 0.25 0.25 0.25 0.5 0.25 0.015
Debaryomyces nepalensis (1) 2 2 0.06 0.25 0.25 16 1 0.12
Kodamaea ohmeri (1) 0.5 1 1 0.5 0.5 4 0.25 0.06
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None of the 53 isolates were confirmed to be C. famata (D. hansenii); however, the two clinical isolates that were confirmed to be Debaryomyces spp. (D. nepalensis and D. fabryi) displayed low MIC values against echinocandins (MIC values of 0.25 and 0.06 μg/ml for anidulafungin, 0.06 μg/ml for caspofungin, and 0.25 μg/ml for micafungin) and were less susceptible to amphotericin B (MIC, 2 μg/ml), flucytosine (MIC range, ≤0.5 to 2 μg/ml), fluconazole (MIC range, 0.5 to 16 μg/ml), and posaconazole (MIC range, 0.25 −1 μg/ml). In contrast to the other triazoles, voriconazole was quite active (MIC range, 0.015 to 0.12 μg/ml) against these species (Table 3). Notably, the two species in this collection that were most frequently misidentified as C. famata, C. parapsilosis and C. guilliermondii, both show elevated echinocandin MIC values.

The MIC values for the triazoles and the echinocandins against the very rare species of Candida were generally low (<2 μg/ml and <0.5 μg/ml, respectively), with the exception of C. fermentati (fluconazole, anidulafungin, and micafungin), C. fabianii (fluconazole), and C. orthopsilosis (anidulafungin).

The results of this study are similar to the previous reports by Desnos-Ollivier et al. (5) and Jensen and Arendrup (16) in that the vast majority of isolates identified as C. famata by conventional phenotypic methods cannot be confirmed as such by either molecular or proteomic methods of identification. Among 26 isolates sent to the laboratory of Desnos-Ollivier (Paris, France) with an identification of C. famata (D. hansenii), only three were confirmed as such using ITS sequence analysis; six were found to be C. fermentati (P. caribbica), 10 were C. guilliermondii (P. guilliermondii), two were C. haemulonii, two were C. lusitaniae, and three were C. palmioleophila. Jensen and Arendrup (16) analyzed 11 isolates in a Danish collection of clinical isolates previously identified as C. famata and found that none could be confirmed using either ITS sequencing or MALDI. They found that four isolates were C. palmioleophila, one was C. guilliermondii, three were C. lusitaniae, and two were C. intermedia. In the present study, we found isolates of C. guilliermondii, C. fermentati, C. lusitaniae, and C. intermedia, as well as seven additional species (Tables 1 and 2), all misidentified as C. famata. Clearly these studies demonstrate that the vast majority of isolates identified as C. famata by phenotypic methods are likely to be a different species of Candida, and they call into question numerous reports of this species as an etiologic agent of IC.

The diversity of MIC values for the triazoles, and especially the echinocandins, in this so-called C. guilliermondii/C. famata group (16) is impressive and again emphasizes the importance of accurate species identification if one chooses to initiate therapy based on such information. Whereas C. famata, along with C. albicans, C. tropicalis, C. fabianii, C. intermedia, and C. pelliculosa, is shown to be highly susceptible to the echinocandins (Table 3), isolates within the C. parapsilosis and C. guilliermondii species complexes are well known to show reduced susceptibility to echinocandins due to a naturally occurring polymorphism within the FKS locus that reduces susceptibility (39, 40). Echinocandins are not recommended as first-line agents for the treatment of the latter two species but would likely be effective in the treatment of IC due to the more highly susceptible species, including C. famata (41).

The difficulty in differentiating C. famata from C. guilliermondii by phenotypic methods is well known (5, 16, 42, 43). This is shown very clearly to be the case for the Vitek 2 yeast identification card in the present study. Within this collection of 53 clinical isolates previously identified as C. famata, 19 (35.8%) were found to be C. guilliermondii by ITS sequence analysis, of which only 5 showed an acceptable result by the Vitek 2 (Table 2). The remaining 14 isolates were classified either as having low discrimination between C. famata and C. guilliermondii (12 isolates) or as having excellent identification for C. famata (two isolates). The Vitek 2 identified three isolates as C. famata with either a good or excellent result, and these results were not confirmed by sequencing. At this time, given the extremely low prevalence of C. famata as an etiologic agent of IC relative to that of C. guilliermondii, coupled with the inability of this identification method to differentiate between the two, it would be prudent for the Vitek 2 software to be reconfigured to automatically select C. guilliermondii whenever the biochemical profile indicates C. famata or C. famata/C. guilliermondii. Based on the data shown in the present study, the probability of an erroneous result using this strategy would be very remote.

Previous studies have shown MALDI to be rapid, accurate, and cost-effective in the identification of both common and uncommon species of Candida (16, 31, 44). The present study confirms the capabilities of this technology against a very challenging collection of Candida species. MALDI showed a good degree of accuracy (88.4%) against the five most common species in this collection, including a number of isolates of C. guilliermondii that were unable to be definitively identified by the Vitek 2. Although the very rare species in this collection were not identified by the MALDI Biotyper, a result of “no reliable identification” was given rather than an incorrect organism identification. Future supplementation of the existing MALDI database with additional strains of these rare species should result in improved performance.

In conclusion, C. famata appears to be far less common as a cause of IC than was previously understood, to the extent that an identification of C. famata from a phenotypically based system is almost certainly incorrect. Although C. guilliermondii and C. parapsilosis are the species most likely to be misidentified as C. famata, we and others have now demonstrated that a wide range of Candida species may be similarly misidentified. The application of molecular and proteomic methods of fungal identification provide rapid, powerful and, in the case of proteomics, cost-effective alternatives to the currently applied phenotypic techniques.

ACKNOWLEDGMENTS

The global antifungal surveillance programs which served as the source of data used in the preparation of the manuscript were supported in part by Pfizer Inc. and by Astellas.

We acknowledge the excellent technical assistance of R. Prochaska, S. Messer, and M. Konrardy in performing the susceptibility and MALDI-TOF testing and of S. Benning in the preparation of the manuscript.

JMI Laboratories, Inc., has received research and educational grants in 2009 to 2011 from Achaogen, Aires, American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, bioMérieux, Cempra, Cerexa, Cosmo Technologies, Contrafect, Cubist, Daiichi, Dipexium, Enanta, Furiex, GlaxoSmithKline, Johnson & Johnson (Ortho McNeil), LegoChem Biosciences Inc., Meiji Seika Kaisha, Merck, Nabriva, Novartis, Paratek, Pfizer (Wyeth), PPD Therapeutics, Premier Research Group, Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, Shionogi USA, The Medicines Co., Theravance, ThermoFisher, TREK Diagnostics, Vertex Pharmaceuticals, and some other corporations. Some JMI employees are advisors/consultants for Astellas, Cubist, Pfizer, Cempra, Cerexa-Forest, J&J, and Theravance. In regard to speaker bureaus and stock options, we have none to declare. D. J. Diekema has received research funding from Merck, Astellas, Schering, Pfizer. Cerexa, BioMeriuex, Innovative Biosensors, and PurThread Technologies.

Footnotes

Published ahead of print 24 October 2012

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