Comparison of CHROMagar, polymerase chain reaction-restriction fragment length polymorphism, and polymerase chain reaction-fragment size for the identification of Candida species

Background and Purpose: The epidemiological alteration in the distribution of Candida species, as well as the significantly increasing trend of either intrinsic or acquired resistance of some of these fungi highlights the need for a reliable method for the identification of the species. Polymerase chain reaction (PCR) is one of the methods facilitating the quick and precise identification of Candida species. The aim of this study was to compare the efficiency of CHROMagar, PCR-restriction fragment length polymorphism (PCR-RFLP), and PCR-fragment size polymorphism (PCR-FSP) assays in the identification of Candida species to determine the benefits and limitations of these methods. Materials and Methods: This study was conducted on 107 Candida strains, including 20 standard strains and 87 clinical isolates. The identification of the isolates was accomplished by using CHROMagar as a conventional method. The PCR-RFLP assay was performed on the entire internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), and the consequent enzymatic digestion was compared with PCR-FSP results in which ITS1 and ITS2 regions were separately PCR amplified. In both molecular assays, yeast identification was carried out through the specific electrophoretic profiles of the PCR products. Results: According to the results, the utilization of CHROMagar resulted in the identification of 29 (33.3%) Candida isolates, while the PCR-RFLP and PCR-FSP facilitated the identification of 83 (95.4%) and 80 (91.9%) clinical isolates, respectively. The obtained concordances between CHROMagar and PCR-RFLP, between CHROMagar and PCR-FSP, as well as between PCR-RFLP and PCR-FSP were 0.23, 0.20, and 0.77, respectively. Conclusion: The recognition of the benefits and limitations of PCR methods allows for the selection of the most efficient technique for a fast and correct differentiation. The PCR-RFLP and PCR-FSP assays had satisfactory concordance. The PCR-FSP provides a rapid, technically simple, and cost-effective method for the identification of Candida species. Nevertheless, to accurately differentiate among the taxonomically related species, PCR-RFLP should be implemented.


Introduction
andida species usually reside as commensals at mucosal membranes in healthy individuals and can be detected in approximately 50% of the population in a non-virulent form [1]. However, these species can become pathogenic in case the host's normal flora is disrupted or the immunity is impaired. Among the human pathogenic fungi, genus Candida has a dominant role in afflicting the hospitalized patients with systemic life-threatening infections [2]. Candida species has been the fourth most common microorganisms causing nosocomial blood stream infection in hospitalized patients [3]. This genus includes at least 30 species of medical importance that are involved in human candidiasis [4]. During the past several decades, the incidence of infections caused by genus Candida has substantially increased due to the expansion of immunosuppressive situations. Moreover, those species once thought to be non-pathogenic are currently considered as opportunistic pathogens [5,6]. The current changes in the epidemiology of candidiasis highlight a shift in the prevalence of Candida species so that a reduced proportion of C. albicans and an increase in nonalbicans Candida species Can be seen [7]. Based on a recent review, while in the 1980s, C. albicans accounted for more than 80% of all Candida isolates recovered from nosocomial yeast infection [8], currently, C. albicans constitutes less than 50% of all Candida blood isolates [9].
Considering the variation of Candida species in susceptibility to antifungal agents, the rapid and accurate identification of the species may assist in finding an appropriate therapy for candidiasis [10]. On the other hand, there is a need for recognizing the main source of the infections and determine whether it is endogenous or acquired exogenously from other patients or even health care workers [11,12]. Therefore, the precise identification of the strains at species and sub-species levels is highly demanded to perform epidemiological investigations and control the outbreaks.
There are many assays targeted toward the identification of Candida species, which could be divided into two phenotypic and genotypic groups. Phenotypic assays, such as yeast colony morphologies on malt extract agar or chromogenic culture media, sugar absorption and fermentation tests, and commercial kits (e.g., API), can be time-consuming. Furthermore, the reliance of these techniques on the variable expression of phenotypic characteristics can lead to inconsistent results.
In contrast, genotypic assays that are mostly DNAbased approaches are more accurate and less vulnerable to variations due to growth condition and phenotypic switching. Among the various molecular techniques, the best known methods include specific primers in polymerase chain reaction (PCR) and multiplex PCR [13], specific probes for each species [14], PCR-restriction fragments length polymorphism (PCR-RFLP) [15], sequencing of specific regions of genome [16], real-time PCR [17], and PCR-fragment size polymorphism (PCR-FSP) [18].
There are several studies confirming PCR-RFLP [19][20][21] and PCR-FSP [18,22] assays as simple, rapid, inexpensive, and highly valuable tools, which can be used to differentiate the Candida species. Therefore, in this present study, we aimed to compare the efficiency of these PCR assays and CHROMagar assay as a conventional method in terms of performance, accuracy, speed, and cost.

Materials and Methods
This study was conducted on a total of 107 Candida strains, including 20 standard strains and 87 clinical isolates. The standard strains were supplied by Teikyo University Institute of Medical Mycology (TIMM), Tokyo, Japan. These strains included C. albicans (TIMM 1768), C. krusei (TIMM 3404), C. kefyr (TIMM 0300), C. tropicalis (TIMM 0313), C. guilliermondii (TIMM 3400), C. lusitaniae (TIMM 1439), and C. rugosa (TIMM 3411). The clinical strains used in the study were part of a large collection already isolated from various clinical specimens obtained from different hospitals in Tehran, Iran [23].
The isolates were subcultured on Sabouraud Dextros agar (Difco, Detroit, MI, USA) containing Chloramphenicol (Merck, Germany) and incubated for 2 days at 30°C to obtain Candida colony. For the identification of Candida strains by means of CHROMagar, each isolate was subcultured on a CHROMagar Candida (CHROMagar, Paris, France) plate and incubated at 35°C for 48 h. Candida species were identified based on the colony color according to the manufacturer's instructions. For DNA extraction, the genomic DNA of each yeast was extracted by boiling assay. Briefly, a single colony was re-suspended in 50 μL sterile water, heated for 10 min at 95°C, and centrifuged for 3 min at 2000 g. The obtained supernatant was preserved at -20°C until use.
Subsequently, 3 μL of the PCR-RFLP products and each PCR-FSP amplicons were separated on 2% agarose gel electrophoresis in TBE buffer (Tris 90mM, Boric acid 90mM, EDTA 2mM) for about 2.5 h at 80 V. Species identification was based on the unique pattern of each species. The created bands were detected by staining with 0.5 μg/mL of ethidium bromide, and then photographed. Candida species were identified by the two PCR methods according to the expected band size obtained from in silico sequencing analysis (Table 1).

Statistical analysis
In order to measure the agreement between the assays, the samples were classified into two groups ( Table 2). One group was comprised of the species that could be identified by both genotypic and phenotypic approaches, and the other group contained the species identified only by the molecular assays. The agreement was analyzed using the Cohen's kappa (k) coefficient. Data analysis was performed in SPSS, version 16.0.

Results
The standard strains, including a vast variety of yeast species, were subjected to species identification using CHROMagar, PCR-RFLP, and PCR-FSP assays. Although there was no disagreement among the assays regarding the identification capability, CHROMagar was unable to identify many species. The frequency rates of identification of the 87 clinical samples by the three investigated assays are presented in Table 3. When it was not possible to determine the species of a strain, the result was classified as Candida species.
According to the results, CHROMagar could identify 29 (33.3%) Candida isolates, while PCR-RFLP and PCR-FSP identified 83 (95.4%) and 80 (91.9%) isolates, respectively. The PCR-RFLP was able to identify the most diverse species (10 species), followed by PCR-FSP and CHROMagar (9 and 3 species, respectively). Figure 1 demonstrates an example of the variation of Candida species identified by PCR-RFLP after digestion by the MSP1 enzyme. The agarose gel electrophoresis of mixed ITS1 and ITS2 PCR amplicons in PCR-FSP is illustrated in Figure 2.
The comparison of the agreement between the assays is described in Table 2. Considering the species types in Group 1, namely C. albicans, C. tropicalis, C. krusei, and Candida species, the agreement values between CHROMagar and PCR-RFLP, CHROMagar and PCR-FSP, and PCR-RFLP and PCR-FSP were 0.89, 0.87, and 0.97, respectively. Therefore, all assays had a satisfactory concordance. In Group 2, which included all species identified by the two molecular assays, there were slight (κ=0.23), considerable (κ=0.20), and substantial (κ=0.77) concordances between CHROMagar and PCR-RFLP, between CHROMagar and PCR-FSP, as well as between PCR-RFLP and PCR-FSP, respectively.
The minimum time needed for the identification of 10 samples of yeast species in our study included approximately 2 days for CHROMagar, 8 h for PCR-RFLP, and 6 h for PCR-FSP.

Discussion
The rapid and accurate identification of the clinical isolates of Candida species can affect the mortality rate, cost of treatment, and hospitalization duration for the invasive infections. In this study, the chromogenic medium CHROMagar, PCR-RFLP, and PCR-FSP were compared regarding their performance, accuracy, speed, and cost in identifying the Candida species.
We found that although CHROMagar is a straightforward assay, it was unable to recognize more than three species. Accordingly, this method could only identify 33.3% of all the isolates at the species level, while 4.6% and 8.0% of the samples tested by PCR-RFLP and PCR-FSP, respectively, were not identified. Accordingly, there are several similar studies reporting an inconsistency between the results of molecular and phenotypic assays [24,25].
The chromogenic medium CHROMagar has been provided for both isolation and identification of Candida species, based on the pigmentation of the colonies with different colors, which is due to different enzyme activities in Candida species. This technique has the advantage of being inexpensive and less difficult in comparison with other conventional assays, such as API systems and Vitek 2 ID system. However, this method is time-consuming in comparison to molecular assays, such as PCR-RFLP and PCR-FSP. This medium is able to detect the presence of mixed cultures by giving different colored colonies on a plate at the same time. However, as CHROMagar is designed only for the differentiation of three species )i.e., C. albicans [green], C. tropicalis [metallic blue with a pink halo], and C. krusei [pink with velvety appearance]), it fails to clearly identify other species [26]. Such misclassification of species was also reported in the studies carried out by Estrada et al. and Souza et al. [27,28].
The ability of molecular biology assays to detect fungal pathogens is more reliable than that of the traditional phenotyping assays. The analysis of concordance between PCR-RFLP and PCR-FSP in this study revealed almost a substantial concordance between the two assays (kappa=0.77). In PCR-RFLP assay, considering the size of the fragments obtained from the restriction digestion of the PCR products by an enzyme, the common and also some uncommon or rare pathogenic Candida were differentiated.
The PCR-FSP is also a DNA-based assay for the identification of uncommon pathogenic Candida species as well as the common ones by differing in size across one or both ITS1 and ITS2 regions. Nonetheless, the ability of this method to differentiate between taxonomically related speciesis under dispute because it cannot provide sufficient discriminatory power for these species. As mentioned before [18], this approach could not easily distinguish between C. albicans and C. tropicalis, which are the common causes of candidiasis.
Moreover, the storage and transfer of the enzyme needed for RFLP could be more expensive. However, PCR-FSP has been demonstrated to be an easy to handle procedure.
On the other hand, the interpretation of the results in PCR-RFLP are fast, easy, and clear, whereas in PCR-FSP assay, it is rather complicated and timeconsuming and requires standard conditions for electrophoresis, including having a good marker size and multiple reference controls. In PCR-RFLP, the digestion of the PCR products with restriction enzymes increases the time required to identify Candida species, while PCR-FSP does not need post-PCR procedures, such as sequencing, enzymatic digestion, or application of probe. This feature in PCR-FSP would give the clinicians valuable time to decide on the treatment of candidiasis before the antifungal sensitivity reports are available.
While PCR-RFLP requires an enzyme, which may be expensive, PCR-FSP is a cost-effective molecular assay, which requires only basic usual equipment used for PCR and electrophoresis. Nonetheless, if you need to identify new species by these two PCR methods, adding data to cover its identifications is almost easy in both methods.

Conclusion
We have described the benefits and limitations of three assays for the identification of Candida species. It was concluded that CHROMagar is an excellent assay for the identification of C. albicans; nonetheless, additional tests are required for non-albicans species other than C. tropicalis and C. krusei. The PCR assays are more efficient in the identification of other Candida species than the CHROMagar; however, the use of each PCR assay has its own advantages and disadvantages.
Species identification through PCR-FSP is a rapid, technically simple, and cheap procedure as compared to the time-consuming, technically demanding, and expensive PCR-RFLP. The PCR-FSP can be reliably used for the identification of common and also some uncommon species. Nevertheless, the identification of taxonomically related species requires the implementation of PCR-RFLP.