In vitro activity of four triazole antifungal drugs against clinically common and uncommon yeast species

Background and Purpose: Incidence of fungal infections caused by opportunistic fungal pathogens, such as yeasts and yeast-like species, has undergone an increase in otherwise healthy individuals. These pathogens account for high mortality and show reduced susceptibility to the routine antifungal drugs. Accordingly, antifungal susceptibility testing is an urgent need in the determination of the susceptibility spectrum of antifungals and selection of appropriate antifungal agents for the management of patients with fungal infection. Materials and Methods: The present study was conducted on 110 yeast strains belonging to 15 species recovered from clinical specimens. Susceptibility of the isolates to four antifungal drugs (i.e., fluconazole, itraconazole, voriconazole, and posaconazole) was tested according to the Clinical and Laboratory Standards Institute guidelines M27-A3 and M27-S4. Results: Fluconazole exhibited no activity against 4.3% (n=2) of C. albicans isolates, whereas the remaining 44 isolates had a minimum inhibitory concentration (MIC) range of 0.125-4 μg/ml. Voriconazole had the lowest geometric mean MIC (0.03 µg/ml) against all isolated yeast species, followed by posaconazole (0.07 µg/ml), itraconazole (0.10 µg/ml), and fluconazole (0.60 µg/ml). Overall, all of the isolates had reduced voriconazole MICs with a MIC range of 0.016-0.5 μg/ml, except for one isolate of C. albicans that had a MIC of 1 μg/ml. Candida haemulonii as a multidrug-resistant fungus showed a fluconazole MIC of > 64 μg/ml. Conclusion: The current study provides insight into the antifungal susceptibility profiles of clinically common and uncommon yeast species to four triazole antifungal agents. According to our findings, voriconazole was the most active agent. Awareness about antifungal susceptibility patterns is highly helpful in the selection of appropriate antifungal drugs and identification of the efficiency of the currently used agents.


Introduction
ver the last few years, the incidence of fungal infections caused by opportunistic fungal pathogens, such as yeasts and yeast-like species, has witnessed a dramatic increase. The most important yeasts isolated from clinical specimens are Candida species. These species infect hospitalized patients, especially those admitted to intensive care units or oncology wards. According to the statistics, invasive Candida infection is associated with mortality having a range of 40-70% [1][2][3].
While Candida albicans continues to be a major cause of candidiasis, however, the evidence is indicative of the emergence of other Candida and uncommon yeast species with high mortality and reduced susceptibility to the currently administered antifungal drugs. Some of these species isolated from different clinical sources include C. parapsilosis, Kluyveromyces marxianus (C. kefyr), Meyerozyma guilliermondii (C. guilliermondii), C. intermedia, C. lusitaniae, C. haemulonii, C. auris, and atypical forms of Candida albicans (i.e., Candida africana, Candida dubliniensis, and Candida stellatoidea) [4][5][6][7]. The routine antifungal agents for candidiasis treatment are still restricted to polyenes, azoles, and the recently developed echinocandins [8,9]. Toxic effects of amphotericin B as an efficient polyene antifungal agent have limited the application of this medicine for humans [8]. Minimal side effects and high therapeutic index of azole compounds have made them as first-line therapy for the treatment of Candida infections (for many years), antifungal prophylaxis, and empirical or pre-emptive treatment [10]. Nevertheless, the number of Candida species with variable susceptibilities or acquired resistance to these antifungal agents has been on a growing trend over the past decade [11].
Determination of the antifungal susceptibility patterns of yeast species isolated from clinical sources and the selection of appropriate antifungal agents can be useful for the management of fungal infection. Regarding this, the current investigation was conducted to evaluate the in vitro antifungal susceptibility of a large number of yeast strains isolated from different clinical sources to four triazole antifungal agents, namely fluconazole, voriconazole, itraconazole, and posaconazole, using microdilution broth method.
Fluconazole (Sigma-Aldrich, USA) was dissolved in deionized-distilled water. Furthermore, itraconazole (Sigma-Aldrich, USA), voriconazole (Sigma-Aldrich, USA), and posaconazole (Sigma-Aldrich, USA) were dissolved in dimethyl sulfoxide (Sigma). Fluconazole was prepared at a final concentration of 0.063-64 μg/ml, while a concentration of 0.016-16 µg/ml was considered for itraconazole, voriconazole, and posaconazole. For the purpose of the study, RPMI 1640 medium containing L-glutamine without bicarbonate (Gibco, UK) buffered to pH 7 with 0.165 mol/l 3-Nmorpholinepropanesulfonic acid (MOPS, Sigma) was used. Drug-free and yeast-free controls were also included in the study for comparative purposes.
Plates were stored at -70°C until they were used. Briefly, all isolates were grown on potato dextrose agar (PDA, Difco, Leeuwarden, the Netherlands) plates at 35°C for up to 3 days. Inoculum suspensions were prepared in a sterile saline solution and then adjusted spectrophotometrically at a wavelength of 530 nm and a percent transmission range of 75-77%. The microdilution plates were incubated at 35°C and read visually after 24 h to determine the MIC values of the antifungal agents. The P. kudriavzevii (C. krusei) ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains, and analysis of these strains was performed with every new batch of MIC plates. The MIC endpoints for all antifungals were defined as the lowest drug concentration causing 50% growth inhibition, compared with the growth of a drugfree control.

Ethical Statement
The current study was approved by the Ethics Committee of Fasa University of Medical Sciences ethical code: 93210/D,97,247016), Fasa, Iran, and written informed consent was obtained from the patients. Table 1 summarizes the results of the MIC range, geometric mean MIC, MIC50, and MIC90 of four triazole antifungal drugs against a total of 110 clinically Candida species and uncommon yeasts obtained from 14 different Candida species and two Trichosporon species. However, MIC90 was not measured when fewer than nine isolates were available. Candida albicans complex isolates (C. stellatoidea and C. africana) showed high susceptibility to the tested antifungal agents. As the results indicated, fluconazole had the widest range and highest MICs against the isolates (0.063-64 µg/ml).

Antifungal susceptibility testing
The MIC ranges in all clinical strains against antifungal drugs were as follows, in increasing order: posaconazole and voriconazole (0.016-1 µg/ml), itraconazole (0.016-4 µg/ml), and fluconazole (0.063-64 µg/ml). Basically, voriconazole, posaconazole, and itraconazole had low MIC50 against all tested clinical strains (Table 1). Overall, in terms of GM MICs, voriconazole was found to be the most active agent against all isolates (n=110), followed by posaconazole in comparison with itraconazole and fluconazole.
Furthermore, most of P. kudriavzevii (C. krusei) isolates were resistant to fluconazole but not to voriconazole, posaconazole, or itraconazole. In

Discussion
Frequency of fungal infections caused by opportunistic fungal pathogens, particularly the genus of Candida, has undergone a dramatic increase [15,16]. Epidemiologically, most of the isolates withdrawn from various clinical samples are C. albicans. Nevertheless, the elevation of non-albicans Candida and uncommon yeast species with reduced susceptibility to routine antifungals is a serious problem. This issue is much more complicated when affecting patients with immunodeficiency due to the likelihood of yeast invasion to the deeper tissues, resulting in infection dissemination [17,18].
Therefore, the determination of the antifungal resistance patterns of clinical samples is a vital issue facilitating the selection of appropriate antifungal agents for the treatment of fungal infections and surveillance of resistance to antifungal drugs. Azole compounds are the most frequently used clinical antifungal agents for the treatment of candidiasis. However, with the overuse of these agents, the number of drug-resistant fungal isolates is on a growing trend [19]. Regarding this, the current study was focused on the susceptibilities of various Candida species to commonly used azole antifungal agents. The results of the current research demonstrated that fluconazole had desirable activities against most of the isolates.
In line with the present results, Bhattacharjee showed that 66.7% (n=6) of the C. haemulonii strains were resistant to fluconazole [22]. In the current study, 3 (6.5%) C. albicans, 1 (5.9%) C. parapsilosis, and 1 (7.7%) C. tropicalis isolates were fluconazole-SDD, respectively. Compared with our findings, Eksi et al. detected dose-dependent susceptibility to fluconazole in 11.3% and 5.2% of C. albicans and non-albicans Candida, respectively [21]. It seems that fluconazole remains an effective antifungal agent against yeast species in spite of its widespread application in Iran.
No breakpoint has been mentioned for posaconazole in the CLSI M27-S4 reference [14]. In this study, the highest posaconazole MIC90 value was observed against C. albicans, C. glabrata, P. kudriavzevii (C. krusei), and C. parapsilosis (1 µg/ml). This was reported as 2 µg/ml in other studies [28,37,38]. In a study performed by Wisplinghoff et al., posaconazole MIC50 and MIC90 values against C. glabrata were higher than former reports [24]. Similar to other studies, voriconazole and posaconazole had greater activities against most Candida species in comparison to fluconazole [33,39].

Conclusion
With the growth of resistant yeast species to routine antifungal agents, the selection of the most appropriate antifungal agent and effective treatment is a critical issue in clinical practice. According to the results of the present study, voriconazole with a low resistance rate might be used as the drug of choice for the treatment of the infections occurring as a result of Candida species. Regarding this, it is required to perform further studies in each region to determine the antifungal susceptibility patterns of yeast species for the successful treatment of patients with Candida infection.