Oral Prevalence of Candida Species in Patients Undergoing Systemic Glucocorticoid Therapy and the Antifungal Sensitivity of the Isolates

Introduction

Candida species are common asymptomatic colonizers of the oral cavity that may cause opportunistic infections in debilitation host. Symptoms of candidiasis include loss of taste, bleeding, a sense of burning or soreness, and stomatitis; these may result in profound difficulties with eating and/or swallowing. Candidiasis may also lead to invasive infections associated with spread into the systemic circulation; this may result in esophageal, gastrointestinal, respiratory, and urinary tract infections.¹ Oral candidiasis is diagnosed frequently in children, the elderly, and patients undergoing systemic corticoid (SC) or immunosuppressive therapy due to eg, organ transplantation.² Earlier studies revealed that the use of inhaled corticosteroids (ICS) by adults and children with bronchial asthma could have a significant impact on the diversity of yeast species detected as well as on the incidence of candidiasis.³ In females, systemic intake of corticoids increases not only the incidence of vulvovaginal candidiasis (VVC) but also the frequency of infections with non-Candida albicans (NCA) species that are less susceptible to commonly used antifungal drugs.⁴ Topical or inhaled corticosteroids were both factors that promote an increased risk of oral candidiasis; however, the impact of systemic glucocorticoids (SGCs) therapy on the nature of the fungal species that comprise the normal oral yeast flora has not been determined.⁵ The present study aimed to compare the diversity and drug susceptibility of Candida species in the oral cavity among individuals undergoing treatment with SGCs compared with non-users as controls.

Methods

Patients’ Recruitment

The study was conducted at the First Affiliated Hospital, College of Medicine, Zhejiang University and Zhejiang Hospital, Hangzhou, China, during the period from February 1 to September 30, 2019. Study participants included patients with oral complaints who were under treatment with SGCs for unrelated underlying diseases for at least 4 weeks before enrollment. Patients who were undergoing treatment with other immunosuppressants or who were under antifungal prophylaxis, as well as those diagnosed with diabetes or undergoing treatment with mineralocorticoids, were excluded from the study. Sex-matched patients of similar age who were not undergoing SGCs treatment were recruited as the control group. Use of the oral rinse samples was approved by the Ethical Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (reference number 2019–40-1). Written informed consent was obtained from all participants. This research study is in full compliance with the principles of the Declaration of Helsinki.

Collection and Identification of Samples

Samples were collected using a concentrated oral rinse method as previously described with several small and necessary changes.^6,7 Briefly, 10 mL of sterile saline was used to rinse the oral cavity; the samples were sent to the microbiological laboratory within 30 min where they were subjected to centrifugation (2300g for 20 min). The supernatants were removed, and the pellets were resuspended in 1 mL saline. A sterile cotton swab was used to sample the suspension, which was spread evenly on Sabouraud glucose agar (SGA) supplemented with chloramphenicol (0.5 g/L). SGA plates were then cultured at 37°C for 4 days; isolates were subsequently inoculated onto CHROMagar Candida (CHROMagar, Paris, France) for detection of mixed cultures. The API 20C system (bioMérieux, Marcy l’Etoile, France) was used for phenotypic identification. Samples that could not be identified by phenotypic analysis were evaluated with molecular identification via sequencing of the conserved ribosomal internal transcribed spacer (ITS) region using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as described in a previous study.⁸ Methods for extraction and quantification of genomic DNA, as well as for amplification and sequencing were as previously described.⁹ Amplification by polymerase chain reaction (PCR) was conducted as described using the following cycling parameters: initial at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s, and then a final extension step at 72 °C for 10 min. Sequence data were analyzed using SeqMan Pro (DNAStar, Madison, WI, USA) and species identification was verified by BLAST searches in GenBank (NCBI, Bethesda, MD, USA). Selected isolates were incubated on SGA slants at 37°C for 24–48 h and then maintained in distilled water at 4°C until use.

Antifungal Susceptibility Testing

In vitro susceptibilities for nine common antifungal drugs, including anidulafungin, amphotericin B, micafungin, caspofungin, 5-flucytosine, posaconazole, voriconazole, itraconazole, and fluconazole, were determined using the Sensititre YeastOne^TM YO10 panel (Thermo Fisher Scientific, Waltham, USA) according to manufacturer’s recommendations and the Clinical and Laboratory Standards Institute (CLSI) M27-A3 protocol.¹⁰ Candida strains were cultured on SGA plates for 24 h. Several colonies of >1 mm diameter were selected and suspended in sterile water and adjusted to the 0.5 McFarland standard at the 530 nm wavelength; this ensures that the concentration in the final inoculum for the 96-well plates was between 1.5× 10³ to 8× 10³ colony-forming units per milliliter as recommended by the manufacturer. After transfer, the plates were placed in a non-CO₂ incubator at 35°C for 24 h. Candida parapsilosis ATCC 22,019 and Candida krusei ATCC 6258 were used as quality controls. Tests were conducted in triplicate on three different days. The concentration in the first blue-colored well was recorded as the minimum inhibitory concentration (MIC). Antifungal susceptibility of Candida species-specific was interpreted by clinical breakpoints (CBPs) based on the CLSI M60;¹¹ when CBPs were not available, epidemiological cutoff values (ECVs) that were used to differentiate wild-type (WT) from non-WT isolates based on the CLSI M59 and the previous study were applied.^12,13

Statistical Analysis

The Statistical Product and Service Solutions (SPSS) v21.0 was used for χ² test to evaluate differences in the proportion of categorized variables. P-values of < 0.05 were considered to be statistically significant.

Results

Two-hundred and forty patients were included in the study; this included 120 patients undergoing treatment with SGCs and 120 non-user controls. There were no differences in mean age or gender composition between the two groups. There were also no statistically significant differences concerning oral complaints and problems, including impacted teeth and residual roots, although the patients undergoing treatment with SGCs were more likely to develop periodontitis and caries (P=0.006 and P=0.026, respectively; Table 1). Patients enrolled in the study had undergone SGCs treatment for at least 4 weeks before evaluation; underlying diagnoses included rheumatoid arthritis, systemic lupus erythematosus, scleroderma, other connective tissue diseases, organ transplantation, inflammatory bowel disease, allergic diseases, cancer, and cutaneous vasculitis, as well as several rare diseases, including adult-onset Still’s disease and immune thrombocytopenia. Of this group, 72 patients were treated with prednisolone oral tablets (2.5–20 mg per day), and 48 patients were treated with methylprednisolone tablets (2–16 mg per day).

Table 1 Demographic Data of Patients Undergoing Treatment with Systemic Glucocorticoids (n=120) and Non-Users (n=120)

Of the 120 patients undergoing treatment with SGCs, 52 (43.33%) were diagnosed with oral candidiasis based on a clinical sign associated with mycotic stomatitis compared with only 14 (11.67%) of the non-users; this difference was statistically significant (P < 0.05); There were no significant differences concerning the symptoms associated with oropharyngeal candidiasis (Table 1). In clinical samples from those undergoing treatment with SGCs, 82 of 120 (68.33%) tested positive for a single species, whereas three samples included two fungi (multi-fungal); a total of 88 strains (73.33%) were identified. In the control group, 46 of 120 samples (38.33%) included a single fungal species, whereas one was multi-fungal, in a total of 48 strains (40%) were identified (P< 0.05). Multi-fungal specimens included those in which Candida albicans was combined with NCA species. NCA species were identified primarily as C. parapsilosis, Candida glabrata, Candida tropicalis, and C. krusei and occasionally as Candida lusitaniae or Candida guilliermondii, which were both identified in samples from the SGC group. The frequencies of detection of C. albicans were 45.45% vs 66.67% in those treated with SGCs compared with non-user controls (P = 0.033). Similarly, NCA species were more prevalent in samples from those undergoing treatment with SGCs than in those from non-user controls (51.14% vs 33.33%, respectively; P = 0.046), although no significant differences were identified for any single NCA species (Table 2). Four strains of Saccharomyces cerevisiae were identified in samples from both groups; four C. lusitaniae, one C. guilliermondii, and four S. cerevisiae isolates were verified by molecular identification methods.

Table 2 Frequency of Yeast Strains Isolated from the Oral Cavities of Patients Undergoing Treatment with Systemic Glucocorticoids (n=88) vs Non-Users (n=48)

In vitro susceptibility testing of nine antifungal agents against 132 Candida strains showed revealed an average 90% MIC₉₀ values for isolates from those undergoing treatment with SGCs vs non-user controls were as follows: for anidulafungin, 0.5 vs 0.125 μg/mL; for amphotericin B, 1 vs 0.5 μg/mL; for micafungin, 0.25 vs 0.125 μg/mL; for caspofungin, 0.5 vs 0.125 μg/mL; for 5-flucytosine, 2 vs 0.25 μg/mL; for posaconazole, 0.5 vs 0.06 μg/mL; for voriconazole, 1 vs 0.25 μg/mL; for itraconazole, 2 vs 1 μg/mL; and for fluconazole, 64 vs 64 μg/mL. The echinocandins, amphotericin B, voriconazole, and posaconazole were all active antifungal agents with low MICs that were effective against 90% of the strains isolated from study participants in both groups; however, C. albicans strains isolated from patients undergoing treatment with SGCs were more resistant to itraconazole (P = 0.004) and fluconazole (P = 0.001) than those from non-users (Table 3). Except for the case of fluconazole, the other eight antifungals exhibited low MIC values (≤1 μg/mL) for the four S. cerevisiae strains; the MIC for fluconazole was no greater than 8 μg/mL.

Table 3 Antifungal Sensitivity of Candida Spp. Isolated from Patients Undergoing Treatment with Systemic Glucocorticoids (n=85) vs Non-Users (n=47)

Discussion

In the present study, we identify multiple strains of Candida from the oral cavities of adults undergoing treatment with SGCs and from non-user controls; we established antifungal susceptibilities characteristic of the strains from both groups. First, our findings confirmed that SGC therapy resulted in an increased risk for oral candidiasis. We also found that the NCA strains were isolated at greater frequency from those undergoing treatment with SGC than those from non-users (51.14% vs 33.33%). Additionally, the ratios of itraconazole- and fluconazole-resistant C. albicans strains were significantly higher among those undergoing treatment with SGCs than those among the non-user controls.

Oral candidiasis is promoted by numerous disorders and most notably associated with those that impair host immunity. Use of prednisone and/or ICS are known to increase the risk of developing oral candidiasis.^2,3 To the best of our knowledge, no previously published studies have focused on fungal pathogens in the oral cavity and sensitivities to antifungal drugs, specifically among those undergoing SGC treatment. Renal transplant recipients, patients with diabetics, and those with pemphigus vulgaris frequently develop oral lesions; those patients have a higher prevalence of Candida species in their oral cavities than do immunocompetent control subjects.^14–16 The underlying diseases associated with SGC use in our target patient cohort were all associated with immunoreactivity and immunosuppression. Among those, SGC use was necessitated by underlying conditions including autoimmune disorders, organ transplantation, or cancer; those findings may explain in part the high frequencies of Candida detected. Additionally, some diseases, including Sjögren’s syndrome and scleroderma, are themselves associated with an increased risk for oral candidiasis.¹⁷ In the present study, we also found that, compare with non-users, patients treated with SGCs have significantly more problems that are associated with periodontitis and caries; this finding is consistent with those reported the previous studies.^18,19

Candida albicans is usually the most prominent of the fungal colonizers found in the oral cavity; indeed, 30% to 45% of healthy individuals are carriers of this organism.⁵ NCA species, including C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, C. stellatoidea, and C. tropicalis, are also detected frequently in the oral cavity.^20,21 In the present study, the prevalence of NCA was higher among the patients undergoing treatment with SGCs; this may be related in part to SGC-mediated immunosuppression of the host, although similar results were obtained from patients diagnosed with diabetics, pemphigus vulgaris, or vulvovaginal candidiasis.^12–14

The first-line therapy for oral candidiasis is topical antifungal treatment; however, previous studies indicated that one in four patients were provided with systemic antifungal therapy as the first-line treatment after initiation of ICS therapy, which was contrary to current guidelines.^22,23 In the present investigation, we found that most of the commonly used antifungal drugs were effective against Candida species in vitro, except for itraconazole and fluconazole. Only a few of the isolates were resistant to echinocandins; this included a strain of C. guilliermondii isolated from a patient undergoing treatment with SGCs with MICs of 16, 8, and 8 μg/mL for anidulafungin, micafungin, and caspofungin, respectively. Likewise, two strains of C. parapsilosis, one from an SGC-treated patient and the other from a non-user exhibited MICs of 8 μg/mL for both anidulafungin and caspofungin. Similarly, there were no significant differences between the responses to amphotericin B and 5-flucytosine among WT or non-WT strains from both study groups.

Azole resistance among Candida species is currently a critical problem in clinical settings; numerous studies exploring the mechanisms underlying acquired resistance have been published. Our results revealed that C. albicans from those undergoing treatment with SGCs had increased rates of resistance; this may be due to genetic modifications within the fungal cell promoted by corticosteroid-induced stress.²⁴ The NCA strains are of particular concern with respect to azole resistance.²⁵ Although the proportion of NCA isolates clearly increased in the SGC treatment group, the numbers of non-susceptible strains for each species remained low; as such, this was unlikely to have an impact on differential drugs sensitivity when comparing the two groups.

Among the limitations of the present study, this work features a relatively small number of patients that were recruited from a single institutional setting. We also did not conduct subgroup analysis and as such as are unable to associate any of our findings with specific underlying diseases.

In conclusion, SGC therapy may result in an increase in the incidence of oral candidiasis together with an increase in the frequency of NCA strains; treatment with SGCs may also promote itraconazole and/or fluconazole resistance among C. albicans species. Our findings provide evidence suggesting that SGC therapy not only influences colonization and infection with Candida spp., but also affects species diversity and azole sensitivity.