Introduction
Candida albicans,
C. glabrata, and
C. parapsilosis were reported to be the three most clinically important
Candida species [
1]. Due to the inclusion of newly designated species through taxonomic studies, these three
Candida species are now considered cryptic complex species, including
C. albicans,
C. africana, and
C. dubliniensis (as
C. albicans complex) [
2,
3],
C. glabrata,
C. nivariensis, and
C. bracarensis (as
C. glabrata complex) [
4,
5], and
C. parapsilosis,
C. orthopsilosis, and
C. metapsilosis (as
C. parapsilosis complex) [
6]. Despite the fact that for more than a decade these cryptic species have been introduced, still limited knowledge exists on their distribution, pathogenicity, and antifungal susceptibility pattern. Moreover, from an evolutionary standpoint, identification and discrimination of these cryptic species complexes could shed light on pathogenicity acquisition, as Pryszcz et al. (2015) found
C. metapsilosis as a highly heterozygous opportunistic pathogen arose from a two parental lineages that were not pathogenic [
7].
Biochemical and morphological tests failed to unequivocally identify and differentiate these cryptic species, which is attributed to the presence of similar phenotypic properties among the complexes [
8]. Accordingly, various methodologies, including amplified fragment length polymorphism (AFLP) [
9], matrix-assisted laser desorption-time of flight (MALDI-TOF) [
10], sequencing of ITS rDNA [
11] have been used to tackle this problem. Nowadays, due to affordability and the high reproducibility of PCR, myriad aspects of biology have been revolutionized throughout the world [
12,
13]. In line with this, identification and differentiation of
Candida albicans,
Candida glabrata, and
Candida parapsilosis complexes representatives through fast and simple conventional PCR by targeting hyphal wall protein 1(HWP1) gene [
14], large ribosomal protein 31 (RPL31) gene [
15], and vacuolar ATPase (VMA) gene [
16], have been addressed. However, the simultaneous detection of all of aforementioned nine medically important cryptic species in a single reaction was not described, previously.
Consequently, the aim of this study was to develop an easy-to-perform, low-cost, highly specific and accurate, multiplex PCR assay capable of differentiation of all these nine medically important Candida species, without tedious DNA extraction steps and directly through mixing of pure colonies into the PCR master mix.
Discussion
Representatives of the cryptic species of
Candida albicans,
Candida glabrata, and
Candida parapsilosis complexes account for the majority of candidiasis cases [
21]. Appropriate identification and differentiation of cryptic species complexes is clinically relevant, as not only there are differences on virulence and antifungal susceptibility patterns among species within the same complex, but also contradictory observations for antifungal susceptibility patterns among different studies have been reported [
5,
22‐
25]. Due to facing these contradictory results along with the limited epidemiological data for cryptic species complexes, the genuine distribution and antifungal susceptibility profiles of these species in different geographical locations remained unclear. Moreover, as antifungal susceptibility profile of cryptic species within the same complex are varied, identification down to the species level is imperative to establish the appropriate antifungal therapy [
24,
26,
27]. In line with this, providing epidemiologists and small laboratories with a fast, accurate, specific, and cheap means of identification to disclose the prevalence and antifungal susceptibility profiles of isolates belonging to cryptic
Candida species. Accordingly, we have developed and validated an inexpensive, reliable, accurate, specific, and user friendly multiplex PCR assays capable of identifying nine cryptic species in one assay.
From different perspectives including, time needed to finish the experiments, required expenses and need of trained technicians our assays is comparable to other PCR-based assay and platforms such as MALDI-TOF.
Application of cryptic species complexes strains isolated in clinical settings with our 9-plex PCR assay and its comparison with results obtained by Sanger sequencing of D1/D2 domain of LSU rDNA, revealed 100% consistency between these techniques. However, due to the high similarity between
C. albicans and
C. africana in the sequences of the D1/D2 and ITS rDNA fragments (99.3–100%), distinguishing these two species is difficult [
28]. Consistently, the sequencing of LSU rDNA fragment in
C. africana showed > 99% similarity with
C. albicans. Importantly, application of our 9-plex PCR could discriminate these two species. Like DNA microarray [
29] and pyrosequencing [
30], Sanger sequencing requires highly trained technicians and more turn-around time [
8], while our multiplex PCR assay is straightforward and running of the whole application (from master mix preparation to visualization on the gel) only consumes four hours. In terms of expenses, only 0.75–1 euro is enough to finalize the results on the gel electrophoresis, however, sequencing require specific devices and is more expensive. However, in order to resolve the issue of discrepancy between the length of predicted and actual PCR products we suggest to use either our in-house ladder or to run the amplicons of each control species individually in a separate lane.
Our multiplex PCR successfully identified all
C. africana isolates and, hence, improved the results obtained from MALDI-TOF. Except for
C. africana strains (
n = 3), Bruker MALDI-TOF MS could identify the rest of cryptic species complexes, resulting in 98.93% agreement with our multiplex PCR assay. Variability in accuracy of commercial MALDI-TOF MS database for identification of uncommon and cryptic
Candida species [
9,
31,
32], inability of the Bruker MALDI-TOF to distinguish
C. africana from
C. albicans, and incompetence of VITEK MS systems for identification of
C. bracarensis,
C. nivariensis, and
C. orthopsilosis and low cut-off value of Bruker MS systems (< 1.700) for identification of
C. bracarensis complex reinforced the urge for molecular identification tools [
22,
33]. On the other hand, unlike the domination of PCR even in developing countries [
12,
13], MALDI-TOF is a newer introduced platform, mainly restricted to large or reference laboratories [
34‐
36].
Unlike, AFLP [
9] and RFLP [
37], there is no need for restriction of the PCR products, several visualization steps and reading of the results are straightforward. Muriel Cornet et al. (2011) used PCR-RFLP, to identify all eight cryptic species except for
C. africana. However, this method used three primers targeting ribosomal intergenic spacer (IGS), tedious post-PCR restriction and required electrophoretic visualization twice making this experiment daunting, time consuming, and expensive [
37]. Additionally, digestion of PCR products with restriction enzymes generated multiple fragments, in turn, makes the interpretation difficult when compared to banding pattern of reference strains.
The successful identification of all nine cryptic species in one tube, complements previous studies identifying members of the species complexes of
Candida albicans [
14],
Candida glabrata [
15], and
Candida parapsilosis [
16] using three separate tubes, and, hence, is time- and cost-saving.
Application of broad diversity of yeast species, five
Aspergillus species, and human DNA for specificity testing and inclusion of an extensive number of CBS reference strains and clinical isolates, showed that our 9-plex PCR is 100% specific. As a result, our assay circumvents the imperfections of phenotypic assays (CHROMagar, VITEK 2 and ID 32C) that suffer from the lack of specificity [
38,
39]. Additionally, subjecting various populations of the same species to phenotypic assay could showed various results [
32,
40,
41]. Accordingly, as suggested by Griseo et al. (2015), small clinical laboratories can take advantage of specific phenotypic methods supplemented with easy-to-perform PCR-based approaches to identify and report isolated cryptic
Candia species [
8].
Conclusion
Due to the problems with the identification and discrimination of cryptic Candida species from their closest relatives, there is still uncertainty and unclarity about their epidemiology, pathogenicity and antifungal susceptibility pattern [43,44]. Consequently, developing reliable, specific, cost and labor effective methods is necessary. Successful testing of specificity and validation using a broad range of CBS reference strains and clinical isolates, revealed the potential of this assay to be implemented in routine diagnostics and epidemiological studies. As C. albicans, C. glabrata, and C. parapsilosis complex species constitute 80–90% of candidiasis cases, identification of all nine cryptic species within one multiplex PCR assay, could be of a great assistance.