Translation elongation factor 1-alpha gene as a marker for diagnosing of Candida onychomycosis

Background and Purpose: Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species. Materials and Methods: For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens. Results: The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively. Conclusion: Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.


Introduction
andida species are common human commensal and important human yeast pathogens that cause a wide spectrum of diseases ranging from less severe superficial lesions to lifethreatening systemic conditions in a vast spectrum of immunocompromised patients [1]. The incidence of infections due to Candida species has increased markedly as a result of a growing number of immunocompromised and critically ill patients [2]. Candida contains about 150 species. However, only a few of these species, including C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, C. dubliniensis, and C. krusei, are implicated in human pathogenesis.
Superficial candidiasis may involve one or more mucocutaneous tissues, including the skin, mucous membrane, and nails [3]. Nail infections caused by Candida species known as candidal onychomycosis are normally associated with paronychia, onychia, or chronic mucocutaneous candidiasis. Further comprehensive studies have pointed out that Candida is the second most common etiology for onychomycosis after dermatophytes (tinea unguium) [4]. Conventional laboratory techniques for the diagnosis of candidal onychomycosis, such as direct microscopic investigation and mycological culture, are time consuming and laborious. Recent studies have shown that advances in molecular technologies are useful in detecting microbial infections without the need for microbial isolation [5,6].
Nuclear ribosomal DNA (rDNA) regions, including ITS1 and ITS2, have been the most commonly used targets for the detection/identification of Candida C 16 Curr Med Mycol, 2020, 6(1): 15-21 species [7,8]. Nevertheless, the description and characterization of new genetic markers for Candida species can clarify its taxonomy and might be helpful for detection/identification goals. The protein-coding genes (e.g., translation elongation factor 1-alpha [TEF-1α]) have been proven to be a powerful tool for the species delimitation of the taxonomically complex Fusarium species, considered an alternative to rDNA for species identification [9,10]. Likewise, the gene is useful for developing robust phylogenetic inferences for other groups of pathogenic fungi, such as zygomycetes [11], dermatophytes [12], and Aspergillus species [13]. Since TEF-1α gene has not been used in Candida taxonomy and detection, our aim was to investigate TEF-1α as a new genetic marker to evaluate a pan-Candida PCR assay in the diagnosis of candidal onychomycosis.

Standard strains and clinical specimens
The specificity and sensitivity of the designed PCR system were determined against 17 standard strains of fungi. These strains included C. albicans ( A total of 137 nail scrapings collected from patients who referred to the Medical Mycology Laboratory of Razi Hospital in Tehran [14] were used as clinical samples. Nail specimens from 10 healthy volunteers were used as negative clinical controls to verify the diagnostic specificity of the tests. All specimens were examined by both direct microscopy and culture, and a portion of each sample was stored at -20°C for use in the PCR assay. This research was approved by the Ethics Committee of the Shiraz University of Medical Science (IR.SUMS.REC.1398.020).

Mycological examination
A part of the samples was observed with 20% KOH, and the other part was inoculated onto three different points on Sabouraud dextrose agar (SDA) plate (Merck, Darmstadt, Germany) containing chloramphenicol (0.05 mg/l), with and without actidione (500 mg/l) (Merck, Darmstadt, Germany). The inoculated specimens were incubated at 28°C for 1-4 weeks. The culture plates were examined for fungal growth twice a week.

Primer design
The sequences of TEF-1α in Candida species and other causative agents of onychomycosis, such as dermatophytes and non-dermatophytic molds, were downloaded from the National Center for Biotechnology Information (NCBI) (https://www.ncbi. nlm.nih. gov/pubmed/). The number of sequences for Candida species is limited in NCBI (a total of 13 TEF-1α sequences representing 5 species of Candida). Region of sequences that were conserved within Candida species but different from other fungi responsible for onychomycosis was chosen manually to design a pan-Candida primer pair (Table 1) considering the criteria specified by the Primer Biosoft [15] and optimized using the Multiple Primer Analyzer )https://www.thermofisher.com). The primers were also synthesized by the Bioneer Company (Bioneer, Korea).

Preparation of genomic DNA
DNA was extracted and purified from the fungal colonies using the boiling method [16]. Briefly, a small amount of fresh colony was suspended in 200 µL of distilled water, boiled at 95C for 15 min, and centrifuged for 4 min at 7,000 rpm; subsequently, the supernatant was used for PCR. For clinical specimens, DNA was extracted by means of a commercial kit (Yekta Tajhiz Azma, Iran) according to the manufacturer's protocol after being treated by 500 μL of a digestion buffer (containing 400 mM Tris-HCL [pH=8], 60 mM EDTA [pH=8], 150 mM NaCl, and 1% sodium dodecyl sulfate) for 30 min and crushed for 3 min by a mechanical grinder.

Polymerase chain reaction
The PCR reactions were performed using 2X PCR Master Mix (Amplicon, Denmark), 0.5 μM of each primer, 4 μL of DNA template, and sufficient distilled water to reach a final volume of 25 μL. The amplification was accomplished using one cycle at 95°C for 3 min, as well as 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec, followed by a final extension step at 72°C for 3 min. Negative (i.e., sterile distilled water) and positive (i.e., C. parapsilosis [ATCC 4344]) controls were included in each extraction run. Furthermore, 5 μL of the aliquots of the amplicons was electrophoresed using a 1.1% agarose gel in Tris-Borate-EDTA buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, and pH of 8.3) and visualized under ultraviolet irradiation after ethidium bromide staining. A 100-bp DNA ladder was used as a molecular size marker.

DNA Sequence Analysis
The PCR product of standard Candida species examined in this study was purified and sequenced unilaterally with the forward primer on an automated DNA sequencer (ABI PrismTM 3500 Genetic Analyzer, Genetic Group) and edited with the Geneious (http://www.geneious.com) software. The sequences were compared with those in the Genebank database using the web-based software BLAST, (http://www.ncbi.nlm.nih.gov/BLAST). The sequence Curr Med Mycol, 2020, 6(1): 15-21 17 of each sample was subjected to ClustalW pairwise and multiple alignments using the MEGA6 software, and a phylogenetic tree was built using the maximumlikelihood algorithm with the Tamura-Nei parameter.

Analytical sensitivity of primers
To determine the detection limit of this system, fungal inocula were prepared from culture for C. albicans (ATCC 5982). The inocula were adjusted to a turbidity of 0.5 on the McFarland scale, with approximately 1-5×10 6 CFU/ml, counted in a hemocytometer. The inoculum was diluted with sterile saline at a concentration of 10 1 -10 6 CFU/ml. Subsequently, 200 μL of pool nail samples made from healthy nails added to each dilution. Commercial kit (Yekta Tajhiz Azma, Iran) was applied for DNA extraction from the nails treated with fungi. Eventually, PCR was performed on DNAs extracted from six serial dilutions and positive and negative controls.

Statistical analysis
The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for the pan-Candida PCR against the culture (as the reference method). The method of chi square test was used for categorical data. Statistically, the Curr Med Mycol, 2020, 6(1): 15-21 P-value<.05 was considered as significant. The data were analyzed using SPSS software Version 22.0.

Results
By aligning the TEF-1α gene sequences of most of the common Candida species, a highly conserved region was used for designing a new pan-Candida pair primer for PCR detection. The primers included CEFF: 5'-GGACAAAAACAGATTTGAA-3' as forward primer and CEFR: 5'-TTGCAATGGCAATCTCAAT-3' as reverse. The primers amplified an approximately 270bp Candida-specific fragment in all tested Candida species, except for C. glabrata and reacted neither with other fungi nor with E. coli DNAs (Figure 1 left).
The sequence difference count matrix showed poor intraspecies differences among the species belonging to Candida (0-2 nt insertions/deletions). Pairwise and multiple alignment rates were also evaluated among the sequences. The mean similarity of two sequences for 9 Candida isolates was obtained as 94.6%, and the overall similarity of the sequences was estimated at 86.6%. Figure 2 depicts the multiple DNA sequence alignment of EF-1a sequences in the most common pathogenic Candida tested in this study. The consensus nucleotide sequence data determined in this study were deposited in the GenBank, under the accession numbers of MK568517-MK568521 and MN231082-MN231085.
Phylogenetic tree of TEF-1α sequences in Candida species indicated that the nine strains tested in this study (i.e., C. parapsilosis clinical isolate Ci 78, C. tropicalis CBS 94, C. dubliniensis Ci 390, C. albicans ATCC 5982, C. albicans ATCC 2730, C. albicans ATCC 562, C. albicans 1912, C. albicans ATCC 4344, and C. albicans ATCC 64553) were most closely related with strains derived from the gene bank and shared a common branch. In the phylogenetic tree, the studied species were divided into two clades. Clad one consisted of two clusters of C. albicans and C. dublinensis and clad two contained three species of C. parapsilosis, C. tropicalis, and C. glabrata (Figure 3).
For the determination of the analytical detection limit of the designed PCR reaction, serially diluted DNAs were assayed by pan-Candida PCR (Figure 1 right). According to the results, 10 1 cells/mL was the minimum number for reliable detection.     Out of the 137 tested nail samples suspected of onychomycosis, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the specimens were positive for yeast elements by direct microscopy examination, culture, and pan-Candida PCR, respectively ( Table 2).
The proportion of patients with positive pan-Candida PCR was higher than that of the patients with positive culture for Candida species (30.7% vs. 40.1%, respectively). This difference was statistically significant (P<0.001). Considering culture as the reference method to assess the performance of the molecular method used in this study, the PPV, NPV, specificity, and sensitivity were calculated as 63.3%, 91.4%, 78.9%, and 83.3% for pan-Candida PCR test, respectively.

Discussion
Rapid and accurate detection of Candida species in nail specimens is essential for the initiation of proper antifungal treatments. Among the routine methods, KOH preparation is easy to perform and economic; however, its sensitivity is inconsistent and influenced by such factors as inadequate sample volume, different sampling methods, and experience of the lab workers. Although fungal culture can identify specific pathogens, it takes a long incubation period and its sensitivity is much lower than that of KOH. In this regard, the false-negative rate of culture for the diagnosis of onychomycosis was approximately 30%, and the sensitivity was about 60% [17].
DNA-based diagnostic tests are increasingly being employed in the clinical microbiological laboratory for the detection of the etiological agent directly from the clinical specimens. The sensitivity of the detection molecular methods depends on various factors, one of the most important of which is the selection of the target DNA for amplification. Over the last two decades, the use of molecular diagnostic study for the detection of a specific sequence of Candida to investigate intra-species and inter-species differences has been reviewed [18,19]. In such studies, when the main purpose was to design pan-Candida primers, the designed primer pair usually amplified common environmental contaminants, such as Aspergillus. The

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Curr Med Mycol, 2020, 6(  [21]. In our study, 7 out of 42 samples which were positive in culture showed negative results in pan-Candida PCR. The DNA from these samples was diluted, and in another reaction, was exposed to panfungal primers (ITS1 and ITS4); however, the results stayed negative in the PCR. The positive and negative controls in all experiment runs ruled out the possibility of experimental error. The few negative results can be due to the fact that positive material was not included in the subsample set used for molecular testing.
The pan-Candida PCR detected Candida in 20 samples identified negative by culture. This nonconformity that reduce the specificity (78.9%) and PPV (63.3%) of pan-Candida PCR has been linked rather to false-negative results of the culture method because PCR is more sensitive than culture methods and can detect all pathogenic fungi, both viable nonviable types.
Our pan-Candida primers facilitated the detection of DNA for all tested Candida species, except for C. glabrata. This finding is in the same line with the results obtained by Zhang et al. evaluating four pan-Candida primer sets by conventional PCR assays [22].
The lower limit of detection in this assay was 10 1 cells/mL which could be sufficient to find the low number of fungal elements causing candidal onychomycosis in a small volume of nail samples although this would require a highly efficient extracted template DNA. Candida DNA was detected in a few nail samples obtained from healthy volunteers, suggesting that this represented commensal fungi living on the nails. Accordingly, such methods as quantitative real-time PCR can be important for the appropriate evaluation of clinical samples containing both pathogenic and commensal fungi.

Conclusion
As the results of the present study indicated, pan-Candida PCR assay showed good performance characteristics by increasing the detection rates of Candida species and drastically reducing the clinical turnaround time, compared to culture. However, it is required to perform further research using more types of standard isolates and larger clinical samples to define the practical value of such an approach in the mycology laboratory.