1. Background
The incidence of fungal infections caused by Candida spp. is increasing worldwide, especially among immunocompromised patients (1). Prophylactic and therapeutic uses of antifungal agents have given rise to a significant shift to more resistant non-albicans Candida species associated with fungal infections (1-4). Hence, these more resistant fungal infections may become an important cause of both clinical treatment failure and higher mortality rate (5, 6). Previous studies have shown that significant geographical variations exist in species distribution and antifungal drug susceptibility profiles (4, 7). Therefore, species identification and antifungal minimum inhibitory concentration (MIC) determination have become important for the determining the treatment strategies of Candida infections. In addition, performing of antifungal susceptibility testing is also necessary to study the development of antifungal resistance.
Although most Candida species remain susceptible to amphotericin B, there have been new reports about increasing MICs to amphotericin B among C. krusei and C. glabrata isolates (1). The triazoles are commonly used effective drugs for the treatment of fungal infections. The widespread use of these drugs has resulted in a reduced azole susceptibility among Candida species (1). According to results of the ARTEMIS DISK Antifungal Surveillance Program, the incidence of fluconazole resistance among the isolates was as follows: C. tropicalis (4.1%), C. parapsilosis (3.6%), C. kefyr (2.7%) and C. glabrata (15.7%) (8).
Echinocandins inhibit fungal cell wall synthesis by blockage of 1.3-β-D glucan synthase. These drugs have a spectrum of action against most Candida species as well as azole resistant strains (1). The clinical laboratory standard institute (CLSI) revised species-specific breakpoints for Candida isolates (9). These species-specific breakpoints are more sensitive for detecting antifungal resistance in Candida spp. (10). Moreover, the use of these new breakpoints has resulted in detection of higher resistance rates than those obtained from previous studies.
2. Objectives
There is no previous data available about species distribution and antifungal susceptibility patterns of Candida species other than C. albicans in the region of the current study. Therefore, in the current study, the researchers aimed at identifying the distribution and antifungal susceptibility patterns of non-albicans Candida spp. isolated from clinical specimens in Tokat, Turkey.
3. Methods
3.1. Ethics Statement
The non albicans Candida isolates used in this study were obtained from the culture collection of the mycology laboratory of Gaziosmanpasa University Hospital. Yeast isolates are exempted from ethical approval in Turkey.
3.2. Candida Isolates
One hundred and three non-albicans Candida isolates were obtained from the culture collection at the mycology laboratory of Gaziosmanpasa University hospital. Distribution of non-albicans Candida species by specimens is shown in Table 1. These isolates were collected during a five-year period between January 2009 and December 2014. Isolates were identified by the germ tube test, formation of chlamydospore on Cornmeal-Tween 80 agar (11), and with the use of the RapID Yeast Plus System (Remel, USA). Isolates were stored in skimmed milk (Oxoid Limited, UK) at -80°C until use. Each isolate was sub-cultured on Sabouraud Dextrose Agar (Oxoid Limited, UK) before applying susceptibility testing.
Table 1.Distribution of Non-albicans Candida Species by Specimens
| Specimen | C. kefyr | C. tropicalis | C. parapsilosis | C. glabrata | C. famata | Total |
|---|---|---|---|---|---|---|
| Urine | 22 | 20 | 6 | 2 | 2 | 52 |
| Sputum + endotracheal aspirate | 8 | 7 | 4 | - | - | 19 |
| Vaginal swab | 12 | 3 | - | 2 | - | 17 |
| Blood | 1 | 4 | 6 | - | - | 11 |
| Wound | 1 | 2 | 1 | - | - | 4 |
| Total | 44 | 36 | 17 | 4 | 2 | 103 |
3.3. Antifungal Assay
The researchers determined the susceptibility of 103 non-albicans Candida isolates with the E test method. For this purpose, amphotericin B (0.002 - 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy), anidulafungin (0.002 to 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy), caspofungin (0.002 to 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy), fluconazole (0.016 to 256 µg/mL) (LiofilChem Diagnostic Ltd, Italy), ketoconazole (0.002 to 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy), itraconazole (0.002 to 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy), voriconazole (0.002 to 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy), and posaconazole (0.002 to 32 µg/mL) (LiofilChem Diagnostic Ltd, Italy) E test strips were used.
The E test method was performed according to the supplier’s recommendation. The RPMI-1640 medium (Sigma, USA) supplemented with 1.5% agar and 2% glucose and buffered to a pH of 7.0 with 0.165 molL-1 MOPS (3-[N-morpholino] propanesulfonic acid) (Sigma, USA) in 130-mm diameter plates were used for application of E test strips. The final yeast inoculum was adjusted to 0.5 McFarland in a sterile saline solution by CrystalSpec (Becton Dickinson, USA). The final inoculum was then spread on the agar plates by a sterile cotton swab. E test strips were placed on the agar surface after the plates were dried in the safety cabinet for 15 minutes. The MIC was read after incubation in ambient air at 35°C for 48 hours. The MIC was determined as 80% inhibition for azoles and echinocandins and 100% inhibition for amphotericin B. Candida albicans ATCC 90028 and C. krusei ATCC 6258 were used as quality strains.
Interpretation of susceptibility was carried out using species-specific breakpoints suggested by the CLSI M27-S4 document (9). Because species-specific breakpoints for C. kefyr have not been proposed in the CLSI document, the researchers did not calculate sensitivity rates for C. kefyr isolates. The CLSI has not determined breakpoints for amphotericin B, therefore for amphotericin B, MIC breakpoints suggested by Park et al. were used (12) (Table 2). The One-way Analysis of Variance (ANOVA) test was used to compare resistance rates for each species and P < 0.005 was considered as statistically significant.
Table 2.Clinical Breakpoints for Candida Species (µg/mL)
| Organism/Antifungal | Susceptible | Susceptible Dose Dependent | Intermediate | Resistant |
|---|---|---|---|---|
| C. tropicalis | ||||
| Amphotericin Ba | ≤ 1 | - | - | ≥ 1 |
| Anidulafunginb | ≤ 0.25 | - | 0.5 | ≥ 1 |
| Caspofunginb | ≤ 0.25 | - | 0.5 | ≥ 1 |
| Fluconazoleb | ≤ 2 | 4 | - | ≥ 8 |
| Itraconazoleb | ≤ 0.12 | 0.25 - 0.5 | - | ≥ 1 |
| Voriconazoleb | ≤ 0.12 | 0.25 - 0.5 | ≥ 1 | |
| C. parapsilosis | ||||
| Amphotericin Ba | ≤ 1 | - | - | ≥ 1 |
| Anidulafunginb | ≤ 2 | - | 4 | ≥ 8 |
| Caspofunginb | ≤ 2 | - | 4 | ≥ 8 |
| Fluconazoleb | ≤ 2 | 4 | - | ≥ 8 |
| Itraconazoleb | ≤ 0.12 | 0.25 - 0.5 | - | ≥ 1 |
| Voriconazoleb | ≤ 0.12 | - | 0.25 - 0.5 | ≥ 1 |
| C. glabrata | ||||
| Amphotericin Ba | ≤ 1 | - | - | ≥ 1 |
| Anidulafunginb | ≤ 0.12 | - | 0.25 | ≥ 0.5 |
| Caspofunginb | ≤ 0.12 | - | 0.25 | ≥ 0.5 |
| Fluconazoleb | - | ≤ 32 | - | ≥ 64 |
| Itraconazoleb | ≤ 0.12 | 0.25 - 0.5 | - | ≥ 1 |
| Voriconazoleb | - | - | - | - |
4. Results
The most frequently isolated non-albicans Candida species were C. kefyr (44 isolates, 42.8%) followed by C. tropicalis (36 isolates, 35%), C. parapsilosis (17 isolates, 16.5%), C. glabrata (four isolates, 3.8%), and C. famata (two isolates, 1.9%). The in vitro activities of amphotericin B, anidulafungin, caspofungin, fluconazole, ketoconazole, itraconazole, voriconazole, and posaconazole against C. kefyr, C. tropicalis and C. parapsilosis are represented in Table 3. For C. kefyr isolates, the Geometric Mean (GM) MIC values of caspofungin and anidulafungin were significantly lower than those of amphotericin B, fluconazole, itraconazole, and posaconazole (P < 0.001). Resistance to caspofungin and anidulafungin were not detected in C. tropicalis, C. parapsilosis, and C. glabrata isolates. Only two of the 36 C. tropicalis isolates were categorized as intermediate resistant to anidulafungin, according to the new CLSI criteria.
Table 3.In Vitro Antifungal Activities of Amphotericin B, Anidulafungin, Caspofungin, Fluconazole, Itraconazole, Ketoconazole, Voriconazole and Posaconazole Against C. kefyr, C. tropicalis and C. parapsilosis Isolates
| Non albicans Candida Species/Antifungal Drugs | MIC Range, µg/mL | MIC50, µg/mL | MIC90, µg/mL | GM, µg/mL | Mean ± SEM MIC, µg/mL | Resistant, % |
|---|---|---|---|---|---|---|
| C. kefyr (n = 44) | ||||||
| Amphotericin B | 0.038-32 | 1 | 2 | 1.15 | 2,49 ± 0.98 | - |
| Anidulafungin | < 0.002 - 0.38 | 0.003 | 0.047 | 0.008 | 0.02 ± 0.009 | - |
| Caspofungin | < 0.002 - 0.38 | 0.032 | 0.19 | 0.01 | 0.06 ± 0.01 | - |
| Fluconazole | 0.032 - > 256 | 0.125 | 0.38 | 0.19 | 11.79 ± 8.12 | - |
| Itraconazole | 0.004 - > 32 | 0.016 | 0.064 | 0.02 | 0.75 ± 0.72 | - |
| Ketoconazole | 0.003 - 2 | 0.006 | 0.012 | 0.007 | 0.08 ± 0.05 | - |
| Voriconazole | 0.002 - > 32 | 0.008 | 0.016 | 0.008 | 0.73 ± 0.72 | - |
| Posaconazole | 0.003 - > 32 | 0.032 | 0.064 | 0.04 | 0.8 ± 0.7 | - |
| C. tropicalis (n = 36) | ||||||
| Amphotericin B | 0.25 - 1.5 | 0.5 | 0.75 | 0.55 | 0.6 ± 0.04 | 0 |
| Anidulafungin | < 0.002 - 0.75 | < 0.002 | < 0.002 | 0.002 | 0.03 ± 0.02 | 0 |
| Caspofungin | < 0.002 - 0.125 | < 0.002 | 0.047 | 0.003 | 0.01 ± 0.004 | 0 |
| Fluconazole | 0.094 - > 256 | 0.25 | 1 | 0.39 | 7.5 ± 7.0 | 2.7 |
| Itraconazole | 0.016 - > 32 | 0.064 | 0.094 | 0.05 | 0.94 ± 0.88 | 2.7 |
| Ketoconazole | < 0.002 - 1 | 0.012 | 0.032 | 0.01 | 0.04 ± 0.02 | - |
| Voriconazole | 0.004 - 0.125 | 0.023 | 0.047 | 0.02 | 0.02 ± 0.004 | 0 |
| Posaconazole | 0.012 - > 32 | 0.032 | 0.064 | 0.03 | 0.92 ± 0.88 | - |
| C. parapsilosis (n = 17) | ||||||
| Amphotericin B | < 0.002 - 0.75 | 0.38 | 0.75 | 0.1 | 0.47 ± 0.11 | 0 |
| Anidulafungin | < 0.002 - 0.002 | < 0.002 | < 0.002 | 0.002 | 0.002 ± 0.0 | 0 |
| Caspofungin | < 0.002 - 0.75 | < 0.002 | 0.38 | 0.009 | 0.1 ± 0.05 | 0 |
| Fluconazole | 0.19 - 3 | 0.25 | 2 | 0.52 | 0.94 ± 0.24 | 0 |
| Itraconazole | 0.012 - 0.094 | 0.016 | 0.064 | 0.02 | 0.03 ± 0.006 | 0 |
| Ketoconazole | 0.006 - 0.064 | 0.008 | 0.047 | 0.01 | 0.02 ± 0.005 | - |
| Voriconazole | 0.002 - 0.032 | 0.016 | 0.032 | 0.01 | 0.01 ± 0.002 | 0 |
| Posaconazole | 0.006 - 0.094 | 0.023 | 0.047 | 0.02 | 0.03 ± 0.006 | - |
Abbreviations: GM, geometric mean; MIC50, MIC for 50% of the isolates; MIC90, MIC for 90% of the isolates, SEM, standard error of mean.
Although no significant differences were observed between the GM MIC values of caspofungin and voriconazole (P > 0.05), the GM MIC values of anidulafungin was significantly lower than that of voriconazole for C. tropicalis isolates (P < 0.01). Anidulafungin was found to be more effective than amphotericin B (P < 0.001), fluconazole (P < 0.001), itraconazole (P < 0.001), voriconazole (P < 0.01) and posaconazole (P < 0.001) for C. tropicalis isolates. Caspofungin was more active than amphotericin B (P < 0.01), fluconazole (P < 0.001), and itraconazole (P < 0.001) for C. tropicalis isolates. Anidulafungin was also found to be more active than amphotericin B (P < 0.001), fluconazole (P < 0.001), itraconazole (P < 0.01), and posaconazole (P < 0.01) against C. parapsilosis isolates. On the other hand, caspofungin was as active as itraconazole, ketoconazole, and voriconazole, and was more active than amphotericin B (P < 0.01) and fluconazole (P < 0.001) against C. parapsilosis isolates.
None of the C. parapsilosis isolates were found to be resistant to fluconazole, itraconazole, and voriconazole according to revised CLSI breakpoints. Only one of four C. glabrata isolates was detected as resistant to fluconazole, while the other three isolates were dose-dependent susceptible. One of the 36 C. tropicalis isolates was determined to be resistant to all tested azoles, except voriconazole.
5. Discussion
In this study, C. kefyr was the most prevalent non-albicans Candida species (44 isolates, 42.8%) followed by C. tropicalis (36 isolates, 35%). In a previous study from Turkey, Eksi et al. reported that C. parapsilosis was the most common non-albicans Candida species isolated from blood cultures (13). In a recent study from Turkey, Dagi et al. documented that the majority of non-albicans Candida isolates were C. glabrata (14). These differences in species distribution might be attributed to geographical and local variations.
The current research found that most of the isolates were susceptible to amphotericin B. The MIC values of > 2 µg/mL was observed in only three of the C. kefyr isolates. Similar to the current results, Dagi et al. reported that C. kefyr isolates had MIC values of 2 µg/mL for amphotericin B (14). In another study from Turkey, Eksi et al. reported that the MIC values for amphotericin B were in the range of 0.003 to 1 µg/mL in Candida species (13). Even though several studies from different countries have indicated that amphotericin B has good activity against all Candida spp. (15-19), Bustamante et al. reported the amphotericin B resistance rate as 7% among C. parapsilosis isolates (20). Krogh-Madsen et al. also documented the emergence of amphotericin B-resistant C. glabrata isolates during therapy (21). In a Candida surveillance study from the USA, Lyon et al. reported amphotericin B MICs in the range of 0.5 to ≥ 8 mg/L for C. glabrata isolates (22). The rates of amphotericin B resistance were reported as 10% in C. krusei, 15% in C. glabrata, 22.3% in C. parapsilosis, and 33.3% in C. tropicalis strains isolated from immunocompromised patients in a study from Iran (23).
In this study, resistance to anidulafungin and caspofungin was not observed at any of the non-albicans Candida isolates. Similar results were reported by other researchers (14, 20, 24-26). Lyon et al. reported that echinocandins had significant activity against all Candida spp., except C. parapsilosis (22). Pfaller et al. summarized the results of the Sentry antimicrobial surveillance program between 2010 and 2011 (26). They had not detected any caspofungin or anidulafungin resistant C. tropicalis isolate in North America, Europe, Latin America, and Asia-Pacific Regions (26). They also reported that all strains of C. parapsilosis were susceptible to caspofungin, while 1% and 1.2% of C. parapsilosis isolates from Latin America and North America, respectively, were resistant to anidulafungin (26).
One of the C. tropicalis isolates was found to be resistant to fluconazole and itraconazole. The statistical analysis of MIC results showed that fluconazole was less active than ketoconazole, itraconazole, voriconazole, and posaconazole against C. tropicalis isolates (P < 0.001). Previous studies have documented that voriconazole and posaconazole had greater activity than fluconazole against most Candida species (1). In contrast to the current results, Orasch et al. have reported a higher resistance rate for voriconazole than for fluconazole in C. tropicalis isolates (24). In the current study, fluconazole was found to be a less effective azole drug against C. parapsilosis isolates (P < 0.05). No resistance to azole drugs was detected among the C. parapsilosis isolates. The current findings were in concordance with previous studies (13, 14, 19, 20). On the other hand, the current results were different from Tortorano et al. who documented a higher fluconazole resistance rate in C. tropicalis and C. parapsilosis isolates (25). In spite of the extensive use of fluconazole in Turkey, fluconazole remains effective against C parapsilosis and C. tropicalis isolates.
One of the C. glabrata isolates was detected to be resistant to fluconazole, while others were susceptible, in a dose dependent manner. The decreased susceptibility to fluconazole in C. glabrata isolates was noted in previous studies (8, 27). Pfaller et al. reported that voriconazole resistance was seen in 59.2% of fluconazole resistant C. glabrata isolates (8). Tortorano et al. detected that posaconazole and voriconazole resistance rates were higher than that of fluconazole in C. glabrata isolates (25).
6. Conclusions
Candida kefyr was the most common non-albicans Candida species followed by C. tropicalis and C. parapsilosis. Most of the non-albicans Candida species were found to be susceptible to tested antifungal drugs. Therefore, use of routine antifungal agents like amphotericin B and fluconazole, which are available in this region, are suggested.
