1. Introduction
It is estimated that there are as many as 80,000 fungal species in the fungal kingdom, but less than 1% of these species are involved in infection. Even among these few species, the classification of fungi has long been confusing, but in recent years, with the introduction of DNA sequencing, the isolation of pathogenic fungi has made remarkable progress. The significance of isolation in the field of medical mycology is the contribution it makes to diagnosis and treatment by identifying the veritable causative organisms.
The genus Candida is the most important fungus in medical mycology [1] , and the incidence of candidiasis has been on the rise in recent years [2] . Candidiasis is mainly caused by four Candida species, i.e., C. albicans, C. glabrata, C. parapsilosis and C. tropicalis [3] [4] . Candida albicans is the most virulent and commonly isolated species from clinical samples, even though the reports of non-albicans species are increasing [5] [6] . These microorganisms frequently cause fungemia [7] and vulvovaginal infections [8] . Moreover, some species have been regarded as important nosocomial pathogens in newborns [9] , elderly people [10] , transplant recipients [11] , and immunocompromised patients [12] .
Some Candida species have been reclassified due to the description of new genetically related species. These microorganisms are currently divided into four complexes of cryptic species [13] . C. albicans complex comprises C. albicans, C. dubliniensis [14] , and C. africana [15] . C. glabrata complex comprises C. glabrata [16] , and C. nivariensis [17] . C. parapsilosis complex comprises C. parapsilosis, C. orthopsilosis, and C. metapsilosis [18] . A fourth complex of cryptic species called C. haemulonii complex includes C. haemulonii, C. haemulonii var. vulnera, and C. duobushaemulonii [19] . Other phylogenetically closely related species to C. haemulonii complex have been also registered, i.e., C. pseudohaemulonii, C. auris and others [20] [21] .
An accurate identification of cryptic species in the clinical setting is required in epidemiology and medicine. It is also important to better understand the evolution of antifungal resistance. The most notable example of the importance of identifying cryptic species might be the emergence and rapid diffusion of C. auris. This organism is considered a serious threat to public health worldwide due to frequent relapses and treatment failures [22] [23] . The emergence of new cryptic species of Candida poses a challenge for clinical laboratories because it is not always possible to have updated methodologies for their correct identification, particularly in low-income countries. Conventional methods based on carbohydrate assimilation or chromogenic media are designed to identify the most common yeast species but cannot detect all cryptic species. The introduction of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) in the clinical laboratory has greatly improved the identification of fungi [24] . As a result of the implementation of this approach, it is possible to identify most cryptic species, however, this technology is still expensive, requires constant database updates, and its use is limited to high-income countries or third/fourth-level hospitals.
C. haemulonii complex (C. haemulonii, C. haemulonii var. vulnera, and C. duobushaemulonii), C. pseudohaemulonii and C. auris are rarely detected in human specimens; however, it remains unclear whether those are the human resident microorganisms or not, and where the source of infection is. The purpose of the present study was to design species-specific primers in order to identify and detect five Candida species, i.e., C. haemulonii, C. haemulonii var. vulnera, C. duobushaemulonii, C. pseudohaemulonii and C. auris, using a multiplex PCR.
2. Materials and Methods
2.1. Fungal Strains and Culture Conditions
Fungal strains were obtained from the Japan Collection of Microorganisms (JCM; Japan) and Medical Mycology Research Center, Chiba University (IFM; Japan). The following bacterial strains were used in the present study: C. haemulonii JCM 3762, C. duobushaemulonii IFM 64590, C. pseudohaemulonii JCM 12453, and C. auris JCM 15448. C. haemulonii var. vulnera NUM-CHV 1010 was isolated with a non-selective medium, i.e., BHI-Y, from the human sample. These strains were maintained by cultivating them on BactTM Brain Heart Infusion (BHI, Becton, Dickinson and Co., Sparks, MD, USA) and 1.5% agar (BHI agar), adjusted to pH 7.2. These microorganisms were cultured at 30˚C overnight under an aerobic condition.
2.2. Design of Species-Specific Primers for Five Candida Species
Design of species-specific primers for five Candida species, i.e., C. haemulonii, C. haemulonii var. vulnera, C. duobushaemulonii, C. pseudohaemulonii and C. auris was performed as described previously [11] . Briefly, the 26S rRNA gene sequences of C. haemulonii (accession no. AY267823), C. haemulonii var. vulnera (JX459789), C. duobushaemulonii (JX459765), C. pseudohaemulonii (AB118792), and C. auris (AB375773) and the 18S rRNA gene sequences of C. haemulonii (AY500375), C. haemulonii var. vulnera (JX459688), C. duobushaemulonii (LC317494), C. pseudohaemulonii (MT974625), and C. auris (AB375772) and the internal transcribed spacer (ITS) region sequences of C. haemulonii (JX459660), C. haemulonii var. vulnera (JX459686), C. duobushaemulonii (JX459666), C. pseudohaemulonii (AB118792), and C. auris (EU884177) and the largest subunit of RNA polymerase II (RPB1) gene sequences of C. haemulonii (JX459692), C. haemulonii var. vulnera (JX459719), C. duobushaemulonii (JX459698), C. pseudohaemulonii (JX459704), and C. auris (JX459712) were obtained from the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/services.html, Mishima, Japan), and a multiple sequence alignment analysis was performed with the CLUSTAL W program; i.e., each gene sequence of five Candida species was aligned and analyzed, respectively. Homology among the primers selected for each Candida species and their respective gene sequences was confirmed by a BLAST search.
2.3. Development of a Multiplex PCR Method Using Designed Primers
Bacterial cells were cultured in BHI supplemented with 0.5% yeast extract for 24 h, and 1 ml of the samples were then collected in microcentrifuge tubes and resuspended at a density of 1.0 McFarland standard (approximately 107 colony-forming units (CFU)/ml) in 1 ml of sterile distilled water. A total of 5.6 μl of the suspension was then used as a PCR template. The detection limit of PCR was assessed by serially diluting known numbers of bacterial cells in sterile distilled water and then subjecting each suspension to PCR. The multiplex PCR mixture contained 0.2 μM of each primer, 10 μl of 2 × MightyAmp Buffer Ver.3 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 5.6 μl of the template in a final volume of 20 μl. PCR reactions were performed in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler; Applied Biosystems, CA, USA). PCR conditions included an initial denaturation step at 98˚C for 2 min, followed by 30 cycles consisting of 98˚C for 10 s and 68˚C for 1 min. PCR products were analyzed by 2.0% agarose gel electrophoresis before being visualized by electrophoresis in 1 × Tris-borate-EDTA on a 2% agarose gel stained with ethidium bromide. A 100-bp DNA ladder (Takara Biomed, Shiga, Japan) was used as a molecular size marker. All experiments were performed in triplicate.
3. Results
3.1. Primer Design
Ten specific primers covering the upstream regions of the 26S rRNA gene, 18S rRNA gene, ITS region and RPB1 gene sequences of five Candida species were designed in the present study (Figures 1-4). The specific forward primers were designated as CAUF for C. auris, CPSF for C. pseudohaemulonii, CH + CHVF for C. haemulonii var. vulnera and C. haemulonii, CDUF for C. duobushaemulonis, and CHF for C. haemulonii, whereas the specific reverse primers were designated as CAUR for C. auris, CPSR for C. pseudohaemulonii, CH + CHVR for C. haemulonii var. vulnera and C. haemulonii, CDUR for C. duobushaemulonis, and CHR for C. haemulonii. The amplicon sizes of C. duobushaemulonis, C. pseudohaemulonii, C. auris and C. haemulonii var. vulnera were 106 bp, 260 bp, 346 bp, 420 bp, respectively. That of C. haemulonii was 248 bp and 420 bp.
3.2. Multiplex PCR
Our multiplex PCR method for identifying and detecting five Candida species, i.e., C. auris, C. pseudohaemulonii, C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera successfully amplified DNA fragments of the expected size for each species and produced no extra bands at all (Figure 5). Moreover, no amplicons were produced from any of representative Candida species other
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Figure 1. Locations and sequences of species-specific primers for the 26S rRNA gene of Candida auris. The nucleotide sequence of each primer has been underlined.
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Figure 2. Locations and sequences of species-specific primers for the 18S rRNA gene of Candida pseudohaemulonii. The nucleotide sequence of each primer has been underlined.
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Figure 3. Locations and sequences of species-specific primers for the RPB1 gene of C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonis. The nucleotide sequence of each primer has been underlined.
than five fungal species. In addition, similar results were obtained using several different models of DNA thermal cyclers (data was not shown).
The detection limit was assessed in the presence of titrated bacterial cells, and the sensitivity of the PCR assay was between 5 × 1 and 5 × 10 CFU per PCR template (5.0 μl) for the C. auris-specific primer set with strain JCM 15448, the C. pseudohaemulonii-specific primer set with strain JCM 12453, the C. duobushaemulonis-specific primer set with strain IFM 64590, the C. haemulonii-specific primer set with strain JCM 3762 and the C. haemulonii var. vulnera-specific primer set with strain NUM-CHV 1010 (data was not shown).
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Figure 4. Locations and sequences of species-specific primers for the ITS region of C. haemulonii. The nucleotide sequence of each primer has been underlined.
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Figure 5. PCR assay for identifying five Candida species. The primer mixture contained CAUF, CAUR, CPSF, CPSR, CH + CHVF, CH + CHVR, CDUF, CDUR, CHF and CHR. Lanes: 1, C. duobushaemulonis IFM 64590; 2, C. pseudohaemulonii JCM 12453; 3, C. auris JCM 15448; 4, C. haemulonii var. vulnera NUM-CHV 1010; 5, C. haemulonii JCM 3762; 6, Candida lusitaniae JCM 1814; 7, Candida krusei JCM1609; 8, Candida glabrata JCM3761; 9, Candida tropicalis JCM 1541; 10, Candida parapsilosis JCM 1612; 11, Candida dubliniensis IFM 54605; 12, Candida guilliermondii JCM 1539; 13, Candida albicans JCM 1537; 14, Candida kruisii JCM 1779; 15, Candida orthopsilosis JCM 1784; 16, Candida kefyr JCM 9556; 17, Candida aaseri JCM 1689; 18, Candida inconspicua JCM 9555; M, molecular size marker (100-bp DNA ladder).
4. Discussion
Some Candida species have been reclassified due to the description of new genetically related species. The independence of C. dubliniensis from C. albicans is all too well known; C. dubliniensis is primarily isolated from the oral cavity of HIV patients [25] . This microorganism is genetically very close to C. albicans. Other fungal species such as C. guilliermondii, C. parapsilosis, C. famata, and C. haemulonii can be taxonomically subdivided into several fungal species, but the isolation frequency of atypical fungal species from clinical material is very low and their antifungal susceptibilities do not differ significantly. An accurate identification of cryptic species in the clinical setting is required in epidemiology and medicine. It is also important to better understand the evolution of antifungal resistance. The most notable example of the importance of identifying cryptic species might be the emergence and rapid diffusion of C. auris. This organism is considered a serious threat to public health worldwide due to frequent relapses and treatment failures [26] [27] .
To develop a PCR-based technique more applicable for clinical use than conventional PCR, we established a multiplex PCR system for identifying and detecting simultaneously four medically important Pseudomonas species, using only one PCR tube per sample. A multiplex-PCR method is a rapid tool that allows for the simultaneous amplification of more than one sequence of target DNA in a single reaction, thereby saving time and reagents [15] . The most significant problem with regard to this method is the possibility of hybridization among the different sequences of primers. A multiplex PCR for the detection of five Candida species has not ever been developed. Therefore, a reliable identification method is needed to accurately assess the prevalence of five Candida species, i.e., C. auris, C. pseudohaemulonii, C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera.
It is estimated that there are about 80,000 fungal species in the fungal kingdom, but less than 1% of them are involved in infection. Even among these few species, there has long been confusion over their classification, but recent years have seen remarkable progress in the taxonomy of pathogenic fungi with the introduction of DNA sequencing. For example, Malassezia furfur has been implicated as an aggravating factor in atopic dermatitis, but recent studies have shown that this organism is not a major fungal species [28] . This is due to the fact that M. furfur was a complex of five fungal species. It was DNA sequencing that revealed this taxonomic heterogeneity. Similar examples apply to C. albicans/C. dubliniensis and Trichosporon cutaneum/T. asahii. Almost all pathogenic fungi can be identified by sequencing the D1/D2 26S rDNA or ITS region.
Genes used for fungal taxonomic identification must be present in all fungi and show a moderate evolutionary rate. A widely used gene for both bacteria and fungi is rRNA. Fungal rRNA genes have four subunits: 18S (small subunit), 5.8S, 26S (large subunit), and 5S, as well as an ITS region between 26S and 18S and an IGS (intergenic spacer) region between 26S and 18S. The lengths of the four subunits are almost the same regardless of fungal species. On the other hand, the lengths of the ITS and IGS vary markedly among fungal species. For example, the total length of the ITS in C. albicans is about 300 bp, whereas in the same genus, C. glabrata, it is more than twice as long. In general, partial sequences of the 26S subunit (about 600 bp long in the Domain 1 and 2 regions) and the ITS1/2 region are suitable for classification and identification. When attempting to identify between variants or at the strain level, analysis of the IGS region between 26S and 18S, or RPB1 gene is an excellent tool. In the present study, the 26S rRNA gene, 18S rRNA gene, RPB1 gene and ITS region sequences were used in order to design species-specific primers to selectively and simultaneously detect five Candida species.
In the present study, we designed species-specific primers with the already mentioned means, for the identification of C. auris, C. pseudohaemulonii, C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera with a PCR method. These primers were able to distinguish each Candida species and did not display cross-reactivity with each other. Moreover, similar results were obtained using several different models of DNA thermal cyclers, indicating that the multiplex method developed in this study is highly reproducible. In addition, we developed a one-step multiplex PCR method with the ability to identify and differentiate five Candida species using only one PCR tubes per sample.
Our multiplex PCR method is easy because the use of MightyAmp DNA Polymerase Ver.3 (Takara) means that DNA extraction may be avoided, and the subspecies identification and detection using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence of C. haemulonii complex (Candida haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) and two genetically close species (C. pseudohaemulonii and C. auris) and their involvement in the various infections, to be fully clarified in future studies.
5. Conclusion
Our developed multiplex PCR method enables the reliable identification of five clinically important Candida species. Its simplicity means that it can be employed readily in most laboratories, where it might contribute to a better understanding of the epidemiology and clinical significance of the most important Candida species, i.e., Candida haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera, C. pseudohaemulonii and C. auris.
Authors’ Contributions
Fuchigami M, Tsuzukibashi O, Fukatsu A, Takahashi Y, Yamamoto H, Komine C, Hagiwara-Hamano M and Iizuka Y corrected the data. Fuchigami M, Tsuzukibashi O, Hayashi S, Umezawa K, Fukatsu A, Wakami M, Murakami H, Kobayashi T and Fukumoto M drafted and wrote the manuscript. The concept of this manuscript was devised by Fuchigami M. All authors read and approved the final manuscript.