Performance comparison of phenotypic and molecular methods for detection and differentiation of Candida albicans and Candida dubliniensis

Candida albicans is the predominant pathogenic Candida species in susceptible human host. Infections caused by non-albicans Candida spp. have, however, also increased in recent years and may account for up to 60% of all episodes of candidemia or invasive candidiasis at some centers [1, 2]. Candida dubliniensis, first described in 1995 from oral cavities of human immunodeficiency virus-infected individuals [3], is increasingly being reported from patients with candidemia in recent years [1, 2, 4]. C. dubliniensis shares several phenotypic characteristics, such as ability to form chlamydospores on cornmeal agar and germ tubes in serum with C. albicans. Since germ tube test is routinely used for the differentiation of C. albicans from other Candida species, identification based solely on this test leads to misidentification of some C. albicans isolates in routine diagnostic laboratories [5]. Accurate identification is warranted since C. dubliniensis exhibits increased adherence to buccal epithelial cells and is more likely to develop resistance against fluconazole and other azoles [6, 7].

Several phenotypic tests based on colony morphology and physiological assimilation tests have been developed to distinguish C. albicans from C. dubliniensis. Commercially available yeast identification systems (Vitek 2 ID-YST, API 20C and ID32C) based on utilization of various compounds have been used for differentiating various Candida spp., however, these methods are expensive and require at least 1–2 days to report results [8]. Formation of rough/fringed colonies by germ tube positive isolates on differential media such as Niger seed agar [9], simplified sunflower seed agar (SSA) [10], or tobacco agar [11], or inability to grow in a hypertonic Sabouraud dextrose broth containing 6.5 M NaCl [12] are often used to differentiate C. albicans from C. dubliniensis. A commercial latex agglutination test (Bichro-Dubli Fumouze) is also available [13]. However, prior presumptive identification of C. albicans/C. dubliniensis is required before phenotypic methods could be used for their differentiation. Also, variations in growth conditions (incubation temperature, repeated subculturing and storage) may impede accurate identification of these two species by phenotypic tests. Unambiguous identification can be established only by molecular techniques and PCR-based methods are mostly employed [5]. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has also been exploited recently for identification and differentiation of cultured yeast isolates including even closely related Candida spp. [14]. However, the high cost of the equipment is a major impediment for its widespread use in resource-limited settings. In this study, we have developed a novel duplex PCR assay by using primers derived from unique rDNA sequences for rapid detection and differentiation of C. albicans and C. dubliniensis. The performance of duplex PCR was compared with three phenotypic methods for identification of clinical isolates as C. albicans or C. dubliniensis.

Duplex PCR amplification with CALF + CALR + CDUF + CDUR primers yielded an amplicon of ~100 bp and ~325 bp only with DNA from reference strains of C. albicans and C. dubliniensis, respectively, but not from other Candida species, as expected (data not shown). Of 134 clinical Candida spp. isolates, 122 were positive for germ tube formation and 12 were germ tube negative. Vitek 2 ID-YST system identified the isolates as C. dubliniensis (n = 67), C. albicans (n = 55), C. parapsilosis (n = 2), C. glabrata (n = 2), C. tropicalis (n = 2), C. lusitaniae (n = 2), C. krusei (n = 2) and C. kefyr (n = 2). Duplex PCR correctly identified all 67 C. dubliniensis and 55 C. albicans isolates (data from 11 selected isolates are shown in Figure 1) while negative results were obtained for the remaining 12 germ tube negative isolates (Table 1). Growth on SSA correctly identified 67 of 122 germ tube positive isolates as C. dubliniensis. Bichro-Dubli test was positive for 64 of 67, weakly positive for 2 of 67 and negative for 1 of 67 isolates identified as C. dubliniensis by Vitek 2 ID-YST system and ITS region sequencing. Bichro-Dubli test was negative for 55 of 122 germ tube positive isolates identified as C. albicans by Vitek 2 ID-YST system as well as 12 germ tube negative isolates of six other Candida species (Table 1). Thus, duplex PCR accurately identified all 55 C. albicans and 67 C. dubliniensis strains among a panel of 134 well-characterized Candida spp. isolates with 100% sensitivity and specificity.

Direct application of duplex PCR on a panel of 239 clinical isolates of yeast-like fungi correctly identified all 148 C. albicans and 50 C. dublininsis strains. The remaining isolates representing other Candida spp. or other yeast-like fungi yielded negative results. The results were confirmed by direct DNA sequencing of ITS region of rDNA.

The results of duplex PCR assay described here were completely concordant with species-specific identification of C. dubliniensis and C. albicans strains by Vitek 2 ID-YST system and colony characteristics (fringed/rough colonies) on SSA. However, both, Vitek 2 ID-YST system and growth on SSA usually require 24–48 hours before results are available. Furthermore, identification of C. dubliniensis strains on SSA requires prior testing for germ tube formation while Vitek 2 ID-YST system is expensive and thus, is not readily available in resource-poor settings [10]. The requirement for minimal amount of genomic DNA for duplex PCR allows the whole procedure to be completed within 4 hours. Furthermore, since duplex PCR employs species-specific primers for both, C. albicans and C. dubliniensis, it can be directly applied on clinical isolates of yeast and yeast-like fungi. Indeed, direct application of duplex PCR on a panel of 239 clinical isolates of yeast-like fungi correctly identified all 50 C. dublininsis and 148 C. albicans strains.

Although several PCR-based methods using rDNA as target have been described previously, they either involve two separate PCR reactions for each strain [16, 20, 21] or further manipulations (such as restriction digestion to generate restriction fragment length polymorphism or DNA sequencing) are needed for species-specific identification [16, 22‐24]. These additional steps add to the cost of the test and/or consume additional time thus delaying results. Other investigators have used PCR-based methods targeting intronic sequences for differentiation of C. albicans from C. dubliniensis[25‐27]. However, these approaches may lead to misidentification of some isolates since intronic sequences may vary significantly [27]. A real-time PCR assay using melting point analysis was described recently for detection and differentiation of C. albicans and C. dubliniensis strains but involves separate reactions for each Candida spp. and expensive probe primers [28]. Although two real-time PCR assays using SYBR Green dye have also been described, these assays either require prior presumptive identification of C. albicans/C. dubliniensis strains by another (such as germ tube) test and/or two rounds of real-time PCR assays are needed [29, 30]. Furthermore, the difference in T _m values for C. albicans and C. dubliniensis strains in the two reports was either small (<0.5°C) or negligible which may lead to misidentification of some isolates.

Performance of another rapid (Bichro-Dubli Fumouze latex agglutination) test was also compared with duplex PCR results. The 98.5% sensitivity and specificity of 100% of Bichro-Dubli test is in agreement with the reported sensitivity (97% to 100%) and specificity (100%) values suggested by the kit manufacturer. Similar values have also been reported in other studies for identification of C. dubliniensis isolates [31, 32]. Although Bichro-Dubli test is also a rapid method for identification of C. dubliniensis, the test can only be applied on isolates previously identified as C. albicans/C. dubliniensis either by germ tube formation of by application of another (Bichro Latex Albicans) latex agglutination test [13, 32]. This increases both, the cost and the time to report results for Bichro-Dubli test. The use of chromogenic differential media such as CHROMagar® Candida has also been implemented in many clinical microbiology laboratories for identification and differentiation of both C. albicans and C. dubliniensis, thus diminishing the need for germ tube test. However, the production of light green (for C. albicans) or dark green (for C. dubliniensis) color is inconsistent when large number of isolates are screened, thus requiring further cultivation on a bird-seed agar medium for their accurate identification [33].

A duplex PCR assay has been developed and extensively evaluated for the detection and differentiation of clinical isolates as C. dubliniensis or C. albicans. The method may be applied directly on clinical yeast isolates for their identification as C. dubliniensis or C. albicans as it does not require prior testing for germ tube formation or other such tests and is suitable for resource-limited clinical microbiology laboratories equipped with basic PCR technology. A limitation of the study is that other Candida and yeast-like organisms will require additional testing for their species-specific identification.