In Vitro Antifungal Susceptibility of Candida Species Isolated from Iranian Patients with Denture Stomatitis

Background Candida-associated denture stomatitis (CADS) is a common fungal infection in people who wear dentures. The main objective of this study was to make molecular identification of causative agents of CADS and in vitro antifungal susceptibility testing (AFST) in the Iranian patients with denture stomatitis. Methods A total of 134 Candida spp. were obtained from patients with denture stomatitis. The Candida spp. were identified using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) involving the universal internal transcribed spacer (ITS1 and ITS4) primers, which were subjected to digestion with MspI and BlnI restriction enzymes. The in vitro antifungal susceptibility of Candida spp. to fluconazole (FLC), terbinafine (TRB), itraconazole (ITC), voriconazole (VRC), posaconazole (POS), ketoconazole (KET), amphotericin B (AMB), and caspofungin (CAS) was evaluated using the Clinical and Laboratory Standards Institute M27-A3 and M27-S4 guidelines. Results Overall, C. albicans was the most commonly isolated species (n = 84; 62.6%), followed by C. glabrata (n = 23; 17.2%), C. tropicalis (n = 16; 12%), and C. parapsilosis (n = 11; 8.2%). Posaconazole had the lowest geometric mean minimum inhibitory concentration (MIC) (0.03 μg/ml), followed by AMB (0.05 μg/ml), ITC (0.08 μg/ml), VRC (0.11 μg/ml), CAS (0.12 μg/ml), KET (0.15 μg/ml), and FLC (0.26 μg/ml). Discussion Our study showed that C. albicans was most prevalent in Iranian patients with CADS and was susceptible to both azoles and amphotericin B. In addition, POS could be an appropriate alternative to the current antifungal agents used for the treatment of CADS, as well as in the treatment of recurrent candidiasis.


Introduction
Candida-associated denture stomatitis (CADS) is a chronic atrophic complication of the oral cavity that mainly affects people who wear removable dentures [1]. Several evidencebased studies have shown that Candida albicans is the main etiological agent of denture stomatitis (DS), followed by C. tropicalis, C. parapsilosis, and C. glabrata [2][3][4]. The early diagnosis of pathogenic fungal agents and the determination of their susceptibility to antifungal drugs are critical to the treatment of the infection and to establish preventive healthcare-associated strategies [5,6].
In recent years, non-albicans Candida infections and antifungal resistant isolates have increased; thus, developing a reliable diagnostic method is essential for the management of candidiasis [7,8].
A molecular-based method, such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), is a promising technique that is used in the identification of pathogenic Candida spp. [9].
The management of CADS depends on a wide-ranging treatment strategy [10], which includes detecting and eradicating possible significant risk factors, preventing a systemic Candida infection, and reducing any associated discomfort [11,12]. The use of oral formulations of antimicrobial 2 BioMed Research International agents, such as amphotericin B (AMB), nystatin (NYS), and miconazole (MIC), and systemic drugs, such as fluconazole (FLC), voriconazole (VRC), posaconazole (POS), itraconazole (ITC), and ketoconazole (KET), has been shown to be effective in the treatment of CADS [13][14][15][16]. Echinocandins, such as caspofungin (CAS), are a class of antifungal drugs that appear to be highly effective against all Candida spp., including those that are less sensitive or are resistant to FLC and/or ITC [15]. However, previous studies have described the recurrence and clinical relapse of CADS after treatment [1,17,18]. Having sufficient information about the antifungal susceptibility testing (AFST) of the Candida spp. involved in CADS may help in the selection of alternative antifungal treatments for recurrent oral candidiasis. In the current study, we evaluated the in vitro AFST of a collection of molecularly identified Candida spp. isolated from Iranian patients with DS.

Sample Collection Process.
After an examination of the oral cavity, denture samples were obtained by scraping sterile swabs across the inner surface of the denture. In a period of 3 years (2013 to 2016), a total of 134 clinical isolates were collected from 103 patients aged 53-86 years affected with DS. All samples were streaked on the Sabouraud dextrose agar (Merck, Darmstadt, Germany) and incubated at 35 ∘ C for 7 days. All suspected colonies were detected by CHRO-Magar Candida (CHROMagar, Paris, France) and PCR-RFLP methods. Each isolate was preserved in the tryptic soy broth (TSB) (Merck, Darmstadt, Germany) and then stored in the culture collection of the Department of Medical Mycology, Babol University of Medical Sciences, Iran.

Genomic DNA Extraction and PCR-RFLP.
The total genomic DNA from the yeast was removed using the method described by Yamada et al., which involved cell disruption with glass beads followed by extraction with phenol-chloroform and precipitation with ethanol [19].
Oligonucleotide primer sequences including ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCC-GCTTATTGATATGC-3 ) were used in this study [20]. Amplification was performed on a thermal cycler (C1000; Bio-Rad Laboratories, Inc.). The amplified products were electrophoresed on 1.5% agarose gels containing 0.5 mg/ml of ethidium bromide and then analyzed under UV light using a gel-doc system (Bio-Rad, USA). The breakdown of the amplified products involved the restriction enzymes BlnI and/or MspI ( Table 1). The digests of the PCR fragments were electrophoresed on 1.5% agarose gels. In this study, C. albicans ATCC 10231 and C. dubliniensis CBS 2747 were used as quality control strains.   [21,22]. Isolates that responded to ≤1 g/ml MIC for AMB were recognized as susceptible isolates according to the CLSI M27-S3 guideline [23]. The breakpoint was not determined for TRB; however, several studies have reported resistance breakpoints ≥ 8 g/ml [24,25]. The breakpoint values for KET were not defined by the CLSI and, thus, the resistant breakpoint of ≥4 g/ml which was determined by Mulu et al. (2013) was used [26]. Isolates from C. krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used as quality control strains.

Discussion
Dentures in the oral cavity are considered to be a reservoir of Candida spp. and, thus, are a predisposing factor for DS in patients, as well as a potential origin of reinfection [27]. CADS is an infection initiated by the oral colonization of Candida spp.; the most frequently identified species is C. albicans, although C. glabrata, C. guilliermondii, C. parapsilosis, C. krusei, and C. tropicalis are less commonly seen [28,29].
In agreement with other studies, our research found that C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata caused CADS [30][31][32]. The recommended drug of choice to treat CADS in patients without an underlying disease commonly includes a NYS suspension or a clotrimazole tablet. However, a topical application of an azole, such as FLC or ITC, can also be used to prevent persistent or chronic fungal infections in the patients [33,34]. Several studies reported the emergence of antifungal resistance to azoles, which has been associated with multiple episodes of recurrence [16,[35][36][37]. In the current study, 15.5% of C. albicans (13/84) was observed to be resistant to FLC. In contrast with our data, Abaci and Haliki-Uztan (2011) reported that 59.4% of C. albicans were resistant to FLC [24].
AMB, also used in the management of CADS, proved effective against Candida spp. [1]. Besides, the findings obtained in the present study were in agreement with the results by Wingeter et al. (2007) [38] regarding the susceptibility of oral Candida strains to AMB.
Some data are available on the AFST of Candida spp. isolated from denture-associated stomatitis (DAS) to echinocandins [15,47]. In the present study, only 2 isolates (2.3%) of the 84 isolates of C. albicans were resistant to CAS. We also found that 14 isolates (28%) of the non-albicans Candida strains were resistant to CAS.
In the present study, TRB was not found to be effective against Candida spp. Ryder et al. (1998) also reported that TRB was not an active drug against C. glabrata and C. tropicalis [25].
Our results revealed that the tested antifungal showed good activity for most isolates; however, variability observed among some isolates and resistance to drugs highlight the need for AFST as a monitor to management of therapeutic procedure.

Conclusion
In conclusion, all Candida spp. isolated from patients wearing dentures were susceptible to POS and AMB. As an antifungal, POS could be a suitable alternative to the present antifungal agents used for the management of CADS and could be also used in the treatment of recurrent candidiasis.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors of this paper reported no conflicts of interest.