High prevalence of itraconazole resistance among Candida parapsilosis isolated from Iran

Background and Purpose: Candida parapsilosis isolates usually have a low minimum inhibitory concentration (MIC) against azoles. Although Candida parapsilosis isolates usually have low MICs against azoles, recent studies candida invasive infections due to azole resistant-C. parapsilosis isolates . Regarding this, the main aim of this study was to determine the susceptibility pattern of Iranian clinical C. parapsilosis against available azole antifungal drugs. Materials and Methods: This study was conducted on 105 previously-identified isolates of C. parapsilosis sensu stricto. For the purpose of the study, the isolates were subjected to antifungal susceptibility testing against fluconazole (FLZ), itraconazole (ITZ), voriconazole (VRZ), and two new azole drugs, namely luliconazole (LUZU) and lanoconazole (LZN). The broth microdilution reference method adopted in this study was according to the Clinical & Laboratory Standards Institute M27-A3 and M27-S4 documents. Results: According to the results, 89% (n=94) of C. parapsilosis isolates showed a MIC of ≥ 1 µg/ml, indicating resistance against ITZ. Multi-azole resistance was observed in 3.8% of the isolates. In addition, LUZU and LZN demonstrated the highest efficacy with the MIC50 values of 0.5 and 1 µg/ml, respectively. Conclusion: The majority of the isolates showed high MIC values against ITZ. This may have been associated with the long-term ITZ prophylaxis/therapy in patients infected with candidiasis. Hence, the adoption of an appropriate antifungal agent is a crucial step for starting the treatment.


Introduction
ncidence of invasive fungal infections due to non-albicans Candida species has increased, especially in immunocompromised or hospitalized patients with serious underlying diseases. The most common Candida species isolated from blood samples are C. albicans (42.1%), C. glabrata (26.7%), and C. parapsilosis, respectively [1][2][3]. An epidemiological study on 3,648 patients in North America showed that the proportion of candidemia caused by non-albicans Candida species (57.9%) was higher than that caused by C. albicans (42.1%). Similarly, C. parapsilosis has been recognized as the third common cause (34.4%) of candidemia in Iran [4].
Candia parapsilosis is a normal human commensal agent that can also live freely in environmental niches and transmit horizontally via the hands of healthcare workers and medical devices [5]. In neonates, C. parapsilosis is recognized as the most frequent nonalbicans Candida species that causes invasive candidiasis [6,7]. Among antifungal drugs, azoles and amphotericin B have been used as the main choice for the treatment of invasive candidiasis. However, new antifungal agents, such as echinocandins, have been applied as an alternative therapy in neonates [8,9]. Candida parapsilosis isolates are usually reported to be susceptible to azoles. Nonetheless, the results of a recent study were indicative of the incidence of invasive Candida infections as a result of azolesresistant C. parapsilosis isolates [10,11]. Itraconazole (ITZ), a triazole antifungal agent, is a water-soluble orally active compound with a wide spectrum of antifungal activities. Recently, this agent has been used for the prophylaxis of opportunistic fungal infections, especially in patients at the risk of candidiasis, such as patients with chronic recurrent vaginal candidiasis, chronic mucocutaneous candidiasis, and AIDS, as well as those receiving immunosuppressant drugs [12]. New representatives of this class of antifungal agents (e.g., voriconazole [VRZ], posaconazole [POS], luliconazole [LUZU], and lanoconazole [LZN]) are extensively active against Candida species [5,13,14].
In the present study, a large number of C. parapsilosis sensu stricto isolates were subjected to antifungal susceptibility testing against several azole antifungals, such as fluconazole (FLZ), ITZ, VRZ, LZN, and LUZU. The aim of this study was to evaluate the susceptibility pattern of a large number of C. parapsilosis isolates against a comprehensive collection of available azoles. This study also involved the examination of the susceptibility pattern of C. parapsilosis isolates against two new antifungals, namely LUZU and LZN.

Strains and Antifungal agents
This study was conducted on a total of 105 C. parapsilosis sensu stricto strains. These species had been isolated from the different body parts of the patients infected with various clinical forms of candidiasis during 2014-2017 ( Figure 1). These parts included the nails (74), hands (6), skin (2), vagina (2), urine (1), interdigital space (6), sputum (2), ear (2), and other cutaneous parts [10]. All the studied isolates were C. parapsilosis sensu stricto which had been previously screened by polymerase chain reaction (PCR) amplification of the secondary alcohol dehydrogenase-encoding gene (SADH), followed by digestion with the restriction enzyme BanI. In the mentioned investigation, C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139, and C. metapsilosis ATCC 96144 were used as controls.
Stock cultures in this study were maintained in the reference culture collection of the Invasive Fungi Research Center (IFRC, Sari, Iran). They were cultured on the 2% malt extract agar (MEA, Difco, USA) and incubated at 24°C for 2 days. The antifungal drugs (i.e., FLZ, ITZ, VRZ, LCZ, and LUZU) were in form of standard powders by their manufactures (Pfizer, Central Research, Sandwich, Kent, and the UK, respectively). The FLZ and ITZ/VRZ were resolved in sterile distilled water and dimethyl sulfide, respectively. Stoke solution of each drug was stored at -80°C.

Antifungal susceptibility testing
Antifungal susceptibility testing was performed according to the guidelines of the Clinical and Laboratory Standards (CLSI), M27-A3 and M27-S4 (4 th edition) [15]. The antifungal agents were diluted in a standard RPMI 1640 medium (Sigma Chemical Co.), and then buffered to pH 7.0 with 0.165 3-(N-Morpholino) propanesulfonic acid (MOPS, sigma chemical Co.) with l-glutamine without bicarbonate to yield two times their concentration. Subsequently, they were distributed into 96-well microdilution trays (Nunc, UK) with a final concentration of 0.016-16 µg/ml for ITZ, VRZ, LCZ, and LUZU. Regarding FLZ, this concentration was considered as 0.063-64µg/ml.
Conidial suspensions were prepared from the isolates grown for 24 h. They were then suspended in a sterile saline solution and adjusted by spectrophotometric measurements at 530 nm wavelengths to a percent transmittance range of 75-77. A working suspension was made by a 1:10 dilution, followed by a 1:100 dilution of the stock suspension with RPMI 1640 medium, which resulted in 2.5-5×10 3 CFU/ml. A 100-μl volume of yeast inoculum and an equal volume of antifungal agents were added to each well. Drug-free and yeast-free wells were included as positive and negative controls, respectively. The MICs were reported as the lowest drug concentration that inhibits 50% of the growth, compared to positive controls. The microdilution plates were incubated at 35°C and examined visually after 24 h. Candida krusei (ATCC6258) and C. parapsilosis (ATCC 22019) were used as quality controls. Based on the interpretative guidelines, the MIC values of ≤ 2, ≤ 4, and ≥ 8µg/ml were the breakpoints for: susceptible, susceptible dose-dependent (SDD), and resistant FLZ, respectively. Regarding ITZ and VRZS, these values were considered as ≤ 0.12, 0.25-0.5, and ≥ 1 µg/ml, for the aforementioned features, respectively. However, no breakpoints have been reported for LUZU and LNZ yet.

Results and discussion
Different origins of isolates are indicated in Figure  1. Table 1 summarizes the obtained results for antifungal susceptibility testing of all 105 C. parapsilosis isolates. The majority of the isolates showed high MIC values against ITZ. As the results indicated, 89% (n=94) of C. parapsilosis isolates showed a MIC of ≥ 1 µg/ml that were ITZ-resistant according to the CLSI guideline. Sixty-seven C. parapsilosis isolates that were resistant to ITZ were isolated from the nails obtained from Esfahan (n=45), Mazandaran (n=25), and Tehran (n=24). The most active antifungal agent against C. parapsilosis isolates was VRZ, followed by LULZ, LCZ, and FLZ.
The ITZ is mainly used for the treatment of mucosal and nosocomial infections of children [16]. Clinical studies have shown that ITZ is equally efficient for the treatment of vaginal and oropharyngeal candidiasis as well [17,18]. The growing prevalence of C. parapsilosis isolated from blood in neonates is associated with different environmental sources [19,20]. Presently, the azole class of antifungals has significant advantages rendering a broad spectrum of antifungal activity and fewer side effects [21]. The widespread use of FLZ for prophylaxis, as well as empirical therapy, has been interpreted as the cause of a shift in the epidemiology of Candida infections. This has led to the use of other azoles for the therapy of systemic Candida infections [22].
Recent studies have shown that ITZ could be a useful alternative for FLZ resistance Candida species [22]. Most of the clinical C. parapsilosis isolates are susceptible to azoles; however, some studies have reported an increase in the incidence of invasive infections due to azole-resistance isolates [23,24].
In this study, reduced susceptibility to ITZ was notable. Previous studies indicated that ITZ is highly active against C. parapsilosis. In this regard, in a study, only 3 out of 120 C. parapsilosis isolates showed a MIC value of ≥ 1µg/ml (2.5%) against ITZ [25]. In an investigation performed on 124 medical centers worldwide, Pfaller et al. found no evidence of increasing azole resistance among C. parapsilosis isolates [26]. However, high MICs against ITZ (62%) were reported for Iranian Candida species isolated from the vagina [27].
In the present study, out of 105 C. parapsilosis isolates, 94 (89.5%) strains showed a MIC of ≥ 1µg/ml against ITZ. These results clearly indicate the high prevalence of ITZ resistance in Iranian C. parapsilosis isolates. The major origin of resistant isolates was the nail which could have been caused by the nonpenetration of drugs into the nail plate [17]. It is also possible that the nail plate provides a better environment for higher biofilm formation rate. On the other hand, in recent years, ITZ has been used as an alternative drug against FLZ-resistant Candida isolates. Hence, long-term prophylaxis in high-risk patients can be caused by reduced ITZ susceptibility [28].
In conclusion, the high rate of resistance against ITZ, which is extensively used in Iran, seems to be an issue. In addition to echinocandins, awareness of the fact that C. parapsilosis is likely to have high MICs against ITZ seems to be a considerable issue to be addressed. It is suggested that the susceptibility pattern for isolates collected from deep candidiasis be evaluated in future studies. Moreover, further research needs to be carried out on the mechanisms of resistance. Financial disclosure