Diagnosis of Oral candidiasis in Patients under 12 Years: 18S rRNA as a Marker of Molecular Characterization of Candida tropicalis

1. Introduction

Candida tropical has been identified as the most prevalent pathogenic yeast species from the group Candida-non-albicans, but despite this, it is taxonomically close to C. albicans and shares many pathogenic traits and itis one of the most widespread pathogens and colonizers, causing many human diseases in tropical countries ( 1 ), this disease vary depending on the location where the species colonizes such as oropharyngeal candidiasis, balanoposthitisoral thrush or oral candidiasis and vulvovaginal candidiasis ( 2 ).

The most prevalent mucocutaneous mycosis of the oral cavity is oral candidiasis, brought on by the species Candida spp. As a common commensal organism, Candida is present in the oral cavity of the general population in about 53% of cases. Around 150 species in this genus have been isolated from the oral cavity, and Candida albicans is the most prevalent species, accounting for 80% of the isolates. When combined with Candidaglabrata or Candida tropicalis, Candida albicans can colonize the oral cavity. This combination is seen in 7% of healthy individuals and 80% of candidiasis cases ( 3 ).

The pathogenicity or virulence factors of C. tropicalis due to its ability to form a biofilm, produce lytic enzymes like protease and phospholipase enzymes, adhere to epithelial cells, and switch from bud to hyphae (phenotypic switching) and the filamentous growth of C. tropicalis only have the ability to invade and colonize oral epithelial cells ( 4 , 5 ).

C. tropicalis needs some cofactors to enter and cause infection in the host; once attached to the host′s cells, excretion of extracellular enzymes known as protease enzymes facilitate the penetration of the C. tropicalis and permit it to interfere with the host defense system. Produced aspartic proteases (sap) support the ability of C. tropicalis to penetrate deep into the host tissue and attach to affect the organs because proteases hydrolyze peptide bonds. Phospholipases hydrolyze phospholipids and help to destroy the structure of the epithelial cell membrane allowing the tip of a hypha to enter the cytoplasm ( 6 ). Moreover, hwp1 is another virulence factor that was shown to have a considerable role in hyphae production, host tissue injury, and biofilm formation, respectively ( 7 ).

C. tropicalis is essential for many diseases like oral candidiasis, so molecular methods to determine some. C. tropicalis virulence factors necessary to control disease, wherefore this study aims to investigate some virulence genes (hwp1, sap1, cph1, plb1) in C. tropicalis that may increase pathogenicity for it.

2. Materials and Methods

2.1. Samples Collection from Oral Swabs

Randomly, 150 Oral swabs samples were collected from children infected with oral thrush. Their ages ranged from newborns to 12 years who attended the consulting clinic at Al-Rifai General Hospital and Mohammad Al- Mousawi Children′s Hospital in AL-Nasiriyah City, Thi-Qar province, south of Iraq. The current study was conducted from the beginning of January to the end of December 2020. All Oral swabs were cultivated on sabouraud dextrose agar to diagnose Candida spp. that may grow.

2.2. C. tropicalis Isolates

The phenotype of fungal colonies was checked and identified after the cultivation and incubation on SDA depending on, microscopic and traditional tests (Germ tube formation, Sugar fermentation, Chlamydospore formation, and Differential medium CHROM agar Candida) were used to identify Ctropicalis ( 8 ).

2.3. Genomic DNA Extraction

Using EZ-10 Spin Column Fungal Genomic DNA Mini-Preps Kit, genomic DNA of Candida spp. was extracted according to the produced company protocol, and then DNA was checked with Nanodrop-spectrophotometer. Finally, the extracted DNA was stored at -20˚C until use.

2.4. PCR Amplification

In the study, the 18S rRNA gene was used to identify C. tropicalis isolates, according to Fatima, Bashir ( 9 ). The conventional PCR technique was also used to determine virulence genes (hwp1, plb1, sap1, cph1) in all C. tropicalis isolates. The primers were designed online with the NCBI database and Primer 3. They were provided by Bioneer Company (Korea) (Table 1). According to the AccuPower^®PCR PreMix kit, the PCR master mix (Bioneer, Korea). The PCR master mix reaction components were inserted in conventional PCR tubes and included 5 μl of DNA template, 10 pmol of each F and R primer, and 12μl of PCR water. The PCR thermocycler's settings were 95 °C for five minutes of initial denaturation, followed by 30 cycles of 95 °C for 20 seconds, 60 °C for 20 seconds, and 72 °C for one minute. The final extension was at 72 °C for 5 min. PCR products of each gene were electrophoresed using 1% agarose gel with 3µL of ethidium bromide. In each comb well, 10 µl of PCR product was added, and 5 µl of the ladder (100bp) was added in one well. Next, the gel tray was fixed and filled with a (1X) TBE buffer inside the chamber. After that, the current was set at 100 volts and 80 mA for 1 hr. Finally, PCR amplification products were imaged with a UV transilluminator.

2.5. Phylogenetic Analysis

The gene samples were purified using a purification kit, and one sample was sent to Macrogene Company (Korea) for sequencing and deposited into GenBank for accession numbers. The recorded nucleotide sequences of C .tropicalis isolates were compared with C .tropicalis strains in NCBI GenBank to detect mismatching between gene sequences using the NCBI-Blast, (). Phylogenetic tree analysis has based on molecular evolutionary genetics analysis using Mega V. 6.

2.6. Statistical Analysis.

Analysis of findings was done by excel 2010 and statistical packages for social science-version 23 (SPSS). Chi-squared was used to assess these significant differences at P≤0.05 ( 10 ).

3. Results

3.1. Diagnosis of Candida spp.

The study found that 106 (70.66 %) of 150 isolates were positive for Candida spp. Five different species of this genus were identified, as follows: 71 (66.98 %) C. albicans, 14 (13.21%) C. tropicalis, C. krusie 10 (9.43%), 8 (7.55%) C. parapsilosis, and 3 (2.83%) C. glabrata are all included in table 2. The study also confirmed the existence of significant differences between the isolated fungal species.

3.2. Molecular Study of Candida tropicalis

The isolates of Candida tropicalis, which were identified by biochemical tests and CHROM gen agar Candida have supported their diagnosis by a detective the presence of 18SrRNA gene at 415bp (Figure 1A). All isolates of this genus were positive for this gene. All Candida tropicalis isolate positive to hwp1 and cph1gene (100%) at 598 bp and 224 bp, respectively (Figure 1B and 1C). In 11 (78.5%) Candida tropicalis isolates, the sap1 gene was found at 460 bp, and in 10 (71.4%) of these isolates, the plb1 gene was found at 489 bp (Figure 1D and 1E).

Figure 1. Agarose gel electrophoresis of C.tropicalis. Where Marker ladder (100-2000bp), lane (NTC): Non template negative control. A: Lanes (1-10) positive for 18S rRNA gene (A) at 415bp, B:cph1 gene at 224bp, C:hwp1 adhesion gene at 589bp, D:sap1 proteinase gene at 460bp and E:plb1 gene at 489bp, at 100 volts and 80 mA for 1 hr. Finally

3.3. DNA Sequence results for Candida tropicalis

Among all C. tropicalis isolates containing virulence genes in their genome, one isolate contained virulence genes; it was sent for sequencing for multiple alignment analysis. Nevertheless, the nucleotide sequence of the 18S rRNA gene showed slight genetic variation between the local C. tropicalis18Sr RNA gene and NCBI BLAST C.tropicalis isolates. Phylogenetic tree analysis was intended to reveal the extent of genetic similarity between this isolate and the registered strains globally, where local C. tropicalis isolate (MZ025916) has shown a genetic similarity to NCBI-Blast strains (MF925001.1/China/2018) with Identity: 99.00 %at total genetic change (0.006-0.001) (Figure 2A).

Figure 2. Phylogenetic analysis using Maximum Likelihood method (MEGA 6.0 version). A:18S rRNA gene sequencing of local C. tropicalis (MZ025916) isolate at total genetic changes (0.006-0.001). , B:cph1 gene partial sequence in local C. tropicalis (MZ032148) isolate at total genetic changes (1.5 -0.5), C:hwp1 gene partial sequence in local C. tropicalis (MZ032147) isolate at total genetic changes (0.6 - 0.1 %), D:sap1 gene partial sequence in local C. tropicalis(MZ032149) isolate at total genetic changes (0.004 -0.001%), E:plb1 gene partial sequence in local C. tropicalis(MZ032146)isolate at total genetic changes (0.007 -0.001%)

Depending on the multiple alignment analysis, the sequencing of partial virulence gene cph1gene for local C. tropicalis isolate (MZ032148) showed (99.15 %)homology identity to NCBI- BLAST isolate (XM_002549816.1/USA /2018) (Table 3) and by using the phylogenetic tree analysis between C.tropicalis isolates the genetic variation was (1.5 -0.5%) (Figure 2B). AS for hwp1 gene of local C. tropicalis isolate (MZ032147) recorded simple genetic variation. It was near NCBI- BLAST isolate (EU616624.1/Iran/2018), with identity: 99.38 % at genetic change (0.6 - 0.1 %) (Figure 2C). Phylogenetic tree of sap1 gene of local C. tropicalis isolate (MZ032149) showed genetically similarity related to NCBI-Blast C. tropicalis isolate (MF925001.1/China/2018) (Identity: 99.09 %) at total genetic change (0.004 -0.001 %), (Figure 2D). While local C. tropicalis isolate of plb1gene (MZ032146) showed homology identity to NCBI-BLAST isolate (AY394565.1/Malaysia/2005) (Identity: 98.58 %) at total genetic change was (0.007 -0.001 %) (Figure 2E).

4. Discussion

The colonization of the NAC species in the oral cavity was progressively increasing. The determination of non-C. albicans species in oral cavity candidiasis is essential where some of these species resist the azole compounds used to treat this infection. Many species of non-C .albicans have been registered in this study. According to the study of Candida tropical (13.21 %), the results were comparable to those of Ambe, Longdoh ( 11 ), who found a prevalence of 9.4% for C. tropicalis in patients with oral candidiasis. Other studies report that 6.06% of C. tropicalis that isolated from the oral cavity of a patient with oral candidiasis ( 1 ), the study by Muadcheingka and Tantivitayakul ( 12 ) found that C. tropicalis recorded (10.4%) when have been isolated from the oral cavity. According to Costa, Passos ( 13 ), oral isolates were created when Candida tropicalis and Candida albicans. They observed that compared to C. albicans, C. tropicalis showed a more remarkable ability to adhere to laminin and fibronectin.

In the current study, C. tropicalis was identified using the 18SrRNA gene, and the results revealed that all isolates have this gene at 415pb. Traditional diagnostic techniques that depend on biochemical studies are not very reliable; in contrast, molecular techniques like conventional PCR are highly accurate ( 14 ). The most popular approach to differentiating different fungus species is based on 18S rRNA sequences ( 14 - 16 ). In the current study, some virulence genes have been found in the genome of C. tropicalis. Several therapeutic bacteria use lytic enzymes, including proteinases, phospholipases, and hemolysins, to alter or destroy the host membranes, causing the host cells to malfunction or lacerate to encourage invasions of the host tissues ( 17 ).

sap1 gene was detected In this study with 11 isolates (78.5%) at 460pb. Several secreted aspartyl protease superfamily members have been considered virulence factors in the genus Candida. Among them Candida tropicalis ( 18 ). According to Dabiri, Shams-Ghahfarokhi ( 19 ), the sap expression is dependent on strain and source, and that plays a significant role in infections with Candida spp; high proteinase activity belongs to oral candidiasis .sap1-4 genes have been identified in species C. tropicalis and sap1 is the prevalent enzyme that produced in vitro. At the same time, the expression level of the residual 3 sap genes is low and can only be measured by RT-PCR ( 20 ). Das, Mangayarkarasi ( 21 ) found that all isolates positive to sap1 genes 100% (3-3) that isolated from clinical vulvovaginal candidiasis symbiotic and (4-4) 100% asymptotic. On the contrary, Costa, Passos ( 13 ) estimated proteinase enzyme activity of 15 isolates of Candida albicans and 15 isolates of Candida tropicalis isolated from the saliva of dental inflammation patients. Also, Compared to C. albicans isolates, all C. tropicalis isolates had increased enzymatic secretion. These findings go opposite to the majority of other research findings that C. albicans has stronger ( 20 - 22 )

Many studies refer to fungal phospholipases that may promote virulence by damaging the host cell membranes ( 23 , 24 ). This study also found that the plb1 gene was detected in 10 isolates (71.4%) at 489pb. Phospholipids are the primary components of cell membranes. Therefore, the production of phospholipases is considered an essential attribute for host epithelial invasion; in addition, the fracture of these molecules encourages instability in host cells, resulting in the lysis of cells ( 23 , 24 ). The study by Bassyouni, Wegdan ( 25 ) revealed that 100% of Candida isolates caused VVC in diabetic women secretion phospholipase, and the gene plb1 was identified in (87.5 %) of 35 isolates. Another study with 29 C. tropicalis isolates isolated from various anatomic sites obtained from hospitalized patients revealed low or no activity for phospholipase in C. tropicalis ( 26 ). While Jiang, Li ( 27 ) utilized 52 strains of C. tropicalis that were isolated from various clinical sources and found phospholipase enzyme activity in 31 isolates.

The hwp1 gene controls another significant adhesin found on the hyphal cell wall, which was present in all isolates (100%) of C. tropicalis in this study. Several studies have shown that C. tropicalis is the second species after Candida albicans in terms of its ability to adhere ( 28 , 29 ). According to specific investigations, hwp1p is found in high concentrations at hyphal cell walls while in low concentrations in blastoconidia and pseudohyphae ( 30 ). Modrzewska and Kurnatowski ( 31 ) have mentioned that als, epa, and hwp1 are considered the most important adhesins found on the cell wall of Candida spp. It is also considered the responsible gene for fungal hyphae formation. Naglik, Fostira ( 30 ) have mentioned that expression of the hwp1 gene is high among strains isolated from patients with candidiasis. Moreover, the research conducted by Wan Harun, Jamil ( 32 ) in Malaysia revealed that adhesin was reported for C. tropicalis and that utilizing mRNA expression, it was possible to prove the existence of the hwp1gene in NCAC species. The constant regulation of hwp1 mRNA transcription for C. tropicalis suggests that this species can express the gene for this adhesin. This contradicts ten Cate, Klis ( 33 ) claim that the hwp1 gene is exclusively detected in C. albicans.

Additionally, this study demonstrates that all isolates can produce the cph1 gene, which is necessary for morphogenesis and the transcription of the relevant downstream proteins at 224pb. de Barros, Rossoni ( 34 ) demonstrated that cph1 has a minor role in the transcriptional control of cell wall genes throughout hyphal and yeast growth. It is well documented that to create a biofilm, pathogenicity, as well as hyphae, are required ( 35 ).In conclusion, this study confirmed that the species Candida tropicalis is no less important than the species Candida albicans in terms of pathogenicity and its ability to cause diseases including oral candidiasis and that by having virulence factors that are closely related to the events of infection also use the molecular strategies in diagnosis this species and detecting the genes encoding some virulence factors are necessary due to its high accuracy and the oral candida infection rate increases with age, also are prone to develop in certain populations as a warning sign and present in potentially malignant oral disorders of significance.

Authors' Contribution

E. A. conceived the presented idea, designed the study, and collected the samples. M. F. and M. A. investigated from the practical aspect and verified the analytical methods. M. A. wrote the original draft. K. H. and M. F. wrote, reviewed, and edited the final manuscript. K. H. supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.

Ethics

The current study protocol was obtained from the college of pharmacy, Al-Ayen University, and agreed upon by the management of the training and human development department/ Thi-Qar Health Office. Also, participants filled out written consent on this study, which was voluntary. All authors have seen and approved the study protocol.

Conflict of Interest

There are no conflicts of interest among authors. We hereby confirm that all the Figures and Tables in the manuscript are mine.

Acknowledgment

The authors would thank the staff of Al-Rifai General Hospital and Mohammad Al-Mousawi Children′s Hospital in Thi-Qar province for their help collecting the samples. The study is not supported financially.

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