Identification of Pathogenic Rare Yeast Species in Clinical Samples: Comparison between Phenotypical and Molecular Methods

Abstract

Species identification using both phenotypic and molecular methods and antifungal susceptibility tests was carried out with 60 uncommon clinical yeasts. Our data show that phenotypic methods were insufficient for correct identification (only 25%) and that most of the wrongly identified strains showed a resistant antifungal profile.

Although Candida and Aspergillus species are the most common causes of invasive fungal infection (IFI) in debilitated individuals, almost all yeasts are potential pathogens, causing great morbidity and mortality in those patients (11). More than 90% of infections due to yeasts are attributed to only six species—Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Cryptococcus neoformans—but the list of reported species continues to grow as laboratories are pushed to provide identification to the species level as an aid to optimize the treatment of Candida and other yeast infections (19). Some of these new pathogens (Candida orthopsilosis, Candida metapsilosis, Candida nivariensis, or Candida bracarensis) have been well characterized recently thanks to molecular methods such as PCR-based procedures and sequencing (1, 5, 25). Several reports have addressed the difficulty of identifying yeast strains to the species level by conventional methods, since they are highly dependent on variables such as growth medium and temperature. In addition, databases are limited only to common species, and in general terms, their use is time-consuming. On the other hand, molecular methods based on DNA sequencing have been shown to improve strain characterization (15, 16), which is critical to ensure early and effective antifungal therapy, since differences in susceptibility have been reported between members of the same genus. This paper compares two methods of yeasts identification, molecular and conventional, in a collection of rare yeast isolates from clinical samples. The susceptibility profiles of nine antifungal agents against these isolates were also evaluated in order to provide some insight into their clinical management.

A total of 60 uncommon yeasts were included in this study. They were defined as strains belonging to species which account for less than 1% of the total number of isolates in the yeast collection of the Spanish Mycology Reference Laboratory (SMRL). These strains were received at our institution over a period of 17 years, from 1992 to 2009. They were obtained from clinical samples, most of them from deep sites (Table 1), and identified by using physiological and morphological tests, including some of the following: morphology on CMA (cornmeal agar), assimilation of sugars commercial kits (AuxaColor; Bio-Rad, Madrid, Spain, and API 20 AUX ID32 C galleries; bioMérieux, Madrid, Spain), fermentation of several carbon sources, growth on nitrogen sources, growth at various temperatures, and ability to hydrolyze urea (14).

TABLE 1.

DNA-based identities, conventional identification, and clinical sources of 60 uncommon yeasts analyzed

Strain	Identification		Clinical sample type
	Conventional methoda	Sequencing
CL-4637	Candida parapsilosis	Candida orthopsilosis	Blood
CL-4710	Candida parapsilosis	Candida orthopsilosis	Blood
CL-5198	Candida parapsilosis	Candida orthopsilosis	Blood
CL-5362	Candida parapsilosis	Candida orthopsilosis	Blood
CL-5372	Candida parapsilosis	Candida orthopsilosis	Blood
CL-6822	Candida parapsilosis	Candida orthopsilosis	Blood
CL-6823	Candida parapsilosis	Candida orthopsilosis	Blood
CL-5720	Candida parapsilosis	Candida orthopsilosis	Blood
CL-4438	Candida parapsilosis	Candida metapsilosis	Blood
CL-5144	Candida parapsilosis	Candida metapsilosis	Blood
CL-7098	Unidentifiable	Candida metapsilosis	Skin wound
CL-6329	Candida parapsilosis	Candida metapsilosis	Unknown
CL-5221	Candida parapsilosis	Candida metapsilosis	Blood
CL-7106	Candida parapsilosis	Candida metapsilosis	Blood
CL-4886	Candida parapsilosis	Candida metapsilosis	Blood
CL-4926	Candida parapsilosis	Candida metapsilosis	Blood
CL-4638	Candida parapsilosis	Candida metapsilosis	Blood
CL-5897	Candida sake	Candida dubliniensis	Bronchial secretion
CL-7124	Candida sake	Candida dubliniensis	Urine
CL-7022	Candida albicans	Candida dubliniensis	Oropharyngeal exudate
CL-7028	Candida dubliniensis	Candida dubliniensis	Blood
CL-6838	Candida dubliniensis	Candida dubliniensis	Bronchoalveolar lavage fluid
CL-5390	Candida dubliniensis	Candida dubliniensis	Blood
CL-5418	Unidentifiable	Candida dubliniensis	Sputum
L06-390	Unidentifiable	Debaryomyces hansenii	Skin
CL-6240	Candida sake	Lodderomyces elongisporus	Bronchial secretion
CL-6877	Unidentifiable	Lodderomyces elongisporus	Urine
L07-121	Candida pelliculosa (Pichia anomala)	Pichia anomala	Nail
CL-343	Pichia anomala	Pichia anomala	Nail
CL-6620	Unidentifiable	Pichia fermentans	Unknown
CL-7027	Candida rugosa	Pichia fermentans	Sputum
CL-6542	Unidentifiable	Pichia membranifaciens	Sputum
CL-7074	Pichia jadinii	Pichia fabianii	Blood
CL-6710	Candida pelliculosa	Pichia fabianii	Blood
L06-338	Unidentifiable	Candida ciferri	Nail
CL-7030	Candida glabrata	Candida bracarensis	Unknown
CL-3905	Candida kefyr	Kluyveromyces lactis	Blood
CL-6301	Kluyveromyces lactis (Candida sphaerica)	Kluyveromyces lactis	Retropharyngeal abscess
CL-6194	Candida intermedia	Candida intermedia	Bronchial biopsy
CL-6800	Unidentifiable	Candida haemulonii	Skin Wound
CL-4640	Unidentifiable	Candida haemulonii	Unknown
CL-4641	Unidentifiable	Candida haemulonii	Unknown
CL-7073	Candida sake	Candida haemulonii	Cutaneous exudate
CL-4642	Unidentifiable	Candida haemulonii	Unknown
CL-6149	Candida rugosa	Candida rugosa	Vaginal exudate
L06-34	Candida rugosa	Candida rugosa	Blood
CL-6932	Candida norvegensis	Candida inconspicua	Unknown
CL-6946	Candida inconspicua	Candida inconspicua	Sputum
CL-6150	Candida rugosa	Candida inconspicua	Bronchial secretion
CL-6867	Unidentifiable	Candida inconspicua	Blood
CL-7156	Unidentifiable	Candida inconspicua	Urine
CL-6598	Kodamaea ohmeri	Kodamaea ohmeri	Blood
CL-6272	Kodamaea ohmeri	Kodamaea ohmeri	Blood
CL-7143	Kodamaea ohmeri	Kodamaea ohmeri	Tracheal aspirate
CL-6744	Unidentifiable	Kodamaea ohmeri	Bile
CL-7006	Unidentifiable	Issatchenkia terricola	Blood
CL-6878	Unidentifiable	Issatchenkia terricola	Blood
CL-6574	Pichia membranifaciens	Issatchenkia terricola	Blood
CL-2026	Kloeckera apiculata (Hanseniaspora uvarum)	Hanseniaspora uvarum	Blood
CL-6749	Kloeckera apiculata (H. uvarum)	Hanseniaspora uvarum	Nail

^aConventional identification includes physiological and morphological testing. Unidentifiable, inconclusive results by biochemical and morphological identification.

For molecular identification, genomic DNA was prepared directly from a single yeast colony (17). DNA fragments, comprising the internal transcribed spacer 1 (ITS1) and ITS2 regions, were amplified and sequenced using universal primers (26). For these analyses, we used the sequence database designed by the SMRL, which holds 5,000 strains belonging to 270 different fungal species and contains a large number of sequences from the reference database (Table 2). All phylogenetic analyses were conducted with InfoQuest FP software version 4.50 (Bio-Rad Laboratories, Madrid, Spain), using maximum parsimony clustering methodology. An identity of 96 to 100% to the respective type/validated strain has been proposed for species identification in the study (2). In some particular species (Candida ciferri, Candida rugosa, and Issatchenkia terricola), the nearest CBS or GenBank match was used for final identification. Susceptibility testing followed the recommendations proposed by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) for fermentative yeasts (23). The antifungal agents used were amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (POS), caspofungin (CAS), micafungin (MCF), and anidulafungin (AND). Interpretative breakpoints proposed by EUCAST for FLC and VRC were used (20, 21). For AMB, ITC, and POS, the breakpoints were defined based on the wild-type distribution of MICs determined by a EUCAST method based on preliminary studies of correlation in vitro/in vivo with strains causing oropharyngeal candidosis in AIDS patients and on pharmacokinetic/pharmacodynamic (PK/PD) bibliographic data (AMB, >1.0 mg/liter; ITC, >0.125 mg/liter; and POS, >0.125 mg/liter) (6, 7, 22). In the case of echinocandins, breakpoints proposed by the CLSI were used to interpret susceptibility results (18).

TABLE 2.

Reference strains used for comparison of ITS regions and their GenBank accession numbers

Yeast species	CBS strain	ATCC straina	GenBank accession no.
C. orthopsilosis		ATCC 96139T	AJ698048
C. metapsilosis		ATCC 96144T	AJ698049
L. elongisporus	CBS 2606		AY391845
P. anomala		ATCC 8168	U96720.1
P. fabianii	CBS 5640		AF335967
C. bracarensis	CBS 1054		M589573.2
C. sphaerica	CBS 6170		AY338967
C. intermedia	CBS 572T		AF218968
C. haemulonii	CBS 5149T		DQ898168
C. rugosa		ATCC 10571T	AF335927
P. fermentans		ATCC 24750	AF336843.1
P. membranifaciens	CBS 5516		DQ198964.1
K. ohmeri	CBS 9452		EF196810
D. hansenii	CBS 161		AF210327
H. uvarum	CBS 2584		AJ512428
I. terricola	CBS 5259
C. dubliniensis	CBS 7987		AB035589
C. inconspicua	CBS 180T		AB179767
C. ciferri	CBS 5295		AY493435

^aT, type strain.

All strains were easily identified by molecular methods. However, 44 could be identified by biochemical means (16 out of 60 were classified as unidentifiable since they were not properly discriminated by phenotyping), and only 15 of those 44 matched the molecular identification (Table 1).

It should be noted that conventional identification was not discriminatory enough to characterize the most recently described species, such as C. orthopsilosis, C. metapsilosis, C. bracarensis, and Lodderomyces elongisporus. Notably, most Candida haemulonii isolates (4 out 5 isolates) and most Issatchenkia terricola isolates (2 out of 3 included) were not classified by conventional methods. MIC distributions are shown in Table 3. Most strains were considered susceptible in vitro to AMB, as defined by a MIC of <1 mg/liter. It is worth noting that 4 out 5 of the C. haemulonii strains showed high MICs to AMB (1 to 4 mg/liter).

TABLE 3.

MIC ranges and geometric means (GMs) of MICs for 57 isolates included in the study

Species (no. of isolates)a	MIC (GM)b
	AMB	5FC	FLC	ITC	VRC	POS	CAS	MCF	AND
C. orthopsilosis (8)	0.03-0.12 (0.077)	0.12 (0.12)	0.5 (0.5)	0.03-0.06 (0.038)	0.015-0.03 (0.021)	0.03-0.06 (0.032)	0.12-0.5 (0.24)	0.12-0.5 (0.22)	0.12-1 (0.38)
C. haemulonii (5)	0.5-4 (1.14)	0.12-2 (0.49)	8->64 (42.22)	0.06->8 (1.712)	0.12->8 (4.55)	0.5->8 (4.59)	0.12-16 (0.65)	0.03-0.06 (0.042)	0.06 (0.06)
C. inconspicua (5)	0.25-0.5 (0.42)	1-16 (2.82)	32->64 (90.50)	0.12 (0.12)	0.12-1 (0.24)	0.06-0.12 (0.071)	0.25 (0.25)	0.03 (0.03)	0.03 (0.03)
C. metapsilosis (9)	0.06-0.12 (0.088)	0.12-0.25 (0.13)	0.5-8 (1.16)	0.015-0.06 (0.034)	0.015-0.06 (0.03)	0.015-0.12 (0.018)	0.015-0.3 (0.33)	0.12-1 (0.24)	0.06-1 (0.18)
C. dubliniensis (7)	0.03-0.06 (0.036)	0.12 (0.12)	0.12-0.5 (0.163)	0.015 (0.015)	0.015 (0.015)	0.015 (0.015)	0.03-0.5 (0.174)	0.03 (0.03)	0.03 (0.03)
K. ohmeri (4)	0.03-0.12 (0.05)	0.12-1 (0.29)	2-8 (3.36)	0.03-0.06 (0.035)	0.03-0.06 (0.030)	0.015-0.06 (0.02)	0.5->16 (4)	0.03-16 (0.144)	0.03-4 (0.144)
I. terricola (3)	0.03-0.06 (0.037)	0.5-1 (0.629)	8-16 (12.69)	0.015-0.03 (0.018)	0.03-0.12 (0.06)	0.015-0.03 (0.018)	1 (1)	0.03-0.12 (0.047)	0.03 (0.03)
L. elongisporus (2)	0.03-0.12	0.12-0.25	0.12	0.015-0.03	0.015	0.015-0.03	0.06	0.03	0.03
P. anomala (2)	0.03	0.12	4	0.12	0.12	0.5	0.12	0.03	0.03
P. fermentans (2)	0.03	0.5-2	>64	0.25-0.5	1-4	0.25-0.5	0.12-0.5	0.03	0.03
P. membranifaciens	0.25	16	>64	1	1	0.5	0.25	0.03	0.03
P. fabianii (2)	0.06-0.25	0.12	0.5-1	0.03-0.12	0.015	0.03-0.06	0.12-0.5	0.03	0.03
C. ciferri	0.25	2	>64	0.5	0.25	0.5
C. bracarensis	0.25	0.5	2	0.06	0.03	0.12	0.25	0.03	0.03
C. sphaerica (2)	0.06-0.12	0.12-0.25	0.25-0.5	0.03-0.06	0.015-0.03	0.015	0.12	0.03	0.03
C. intermedia	0.03	0.12	0.25	0.015	0.015	0.015	0.12	0.03	0.03
C. rugosa (2)	0.25	0.12-0.25	1-2	0.015	0.015	0.015	1->16	0.03	0.03-0.25

^aHanseniaspora uvarum(2) and Debaryomyces hansenii(1) isolates were not include because these isolates were not able to grow on RPMI media.

^bThe GM was not calculated if the number of isolates was <3. MICs are measured in mg/liter. AMB, amphotericin B; 5FC, 5-flucytosine; FLC, fluconazole; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; CAS, caspofungin; MCF, micafungin; AND, anidulafungin.

The azole agents showed a broad range of activity against these isolates. A total of 33.34% (19/57) of the strains were resistant to FLC (MIC of >4 mg/liter). In addition, ITC (MIC of >0.125 mg/liter), VRC (MIC of >0.125 mg/liter), and POS (MIC of >0.125 mg/liter) showed reduced activity for 10.52% (6/57), 19.29% (11/57), and 17.54% (10/57) of the isolates, respectively. Candida ciferri, C. haemulonii, Pichia anomala, Pichia membranifaciens, and Pichia fermentans showed high MICs or resistance in vitro to all azole compounds tested.

Most strains revealed patterns of susceptibility to echinocandins (MIC range of between 0.015 and 2 mg/liter). Three out of four strains of Kodamaea ohmeri showed high MICs to CAS (4 to >16 mg/liter) but showed different susceptibility profiles to AND (0.03 to 4 mg/liter) and MCF (0.03 to 16 mg/liter). One of two C. rugosa strains had a CAS MIC value of 16 mg/liter but showed a MIC value of <2 mg/liter for MCF and AND. It should be noted that only 8 out of 27 of the antifungal-resistant isolates were correctly classified by phenotyping, as follows: Candida inconspicua, 1 out of 5 strains; K. ohmeri, 3 out of 4 strains; C. rugosa, 2 out of 2 strains, and P. anomala, 2 out of 2 strains.

In our study, a high percentage of uncommon yeasts were not identified using some commercial kits (44 out of 60; 73%), and only a few of them showed some correlation between conventional and molecular methods (15 out of 60; 25%). In general terms, commercial kits are designed to identify common yeasts in the clinical laboratory; however, they usually fail to characterize those less frequent strains (15). Recently described yeast species such as C. orthopsilosis, C. metapsilosis, C bracarensis, C. haemulonii, and C. nivariensis have been identified and classified only by molecular methods (1, 5, 13, 25), since they are not included in the currently available commercial databases. However, sequencing of the ITS region is an effective tool for differentiating the rare species most frequently confused, bearing in mind that reliable sequence databases should be used. Indeed, given the inability of standard phenotypic methods to distinguish some of these rare yeasts species, it is possible that molecular methods may ultimately become the primary means of identification of clinically important yeast isolates.

In addition, our study stated that most of the uncommon yeasts wrongly identified using conventional identification methods showed a resistant antifungal profile (C. haemulonii, C. ciferri, P. anomala, P. membranifaciens, P. fermentans, K. ohmeri, and C. rugosa), as well as the fact that their mistaken identification could imply inappropriate treatment and clinical management (3, 4, 6, 8, 10, 12, 24). In other cases, although different susceptibility profiles among species have not been demonstrated to be clinically relevant yet, there is no doubt that a precise standard of species identification is necessary to monitor changes in fungal infection epidemiology and antifungal susceptibility (9).

For labs with no experience using molecular identification methods, or where this service is unavailable, susceptibility testing must be performed, since some of the antifungal agents available are inactive against most of these species (12, 13, 24). At least susceptibility results may provide valuable information to physicians for patient management.

We suggest submitting strains to reference laboratories as a cost-effective alternative to using currently available tests for the identification of problem cases and rare yeast species which cannot be easily identified using biochemical tests.

In summary, we have presented data demonstrating that sequencing the ITS region is a robust procedure, identifying many clinically relevant yeast isolates. This is a quick and accurate method for better definition of both the epidemiology of the fungal infection and the prevalence of antifungal-resistant species of yeasts. In conclusion, because of the emergence of rare yeast pathogens and their resistant susceptibility patterns, it is of paramount importance that the identification methods available provide the highest possible degree of precision.