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The frequency, antifungal susceptibility and enzymatic profiles of Candida species in cases of onychomycosis infection
Microbial Pathogenesis 116 (2018) 257–262
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
The frequency, antifungal susceptibility and enzymatic profiles of Candida species in cases of onychomycosis infection
T
Hafize Sava,∗, Ayse Barisb, Deniz Turanc, Rabiye Altinbasd, Sümeyye Sene a
Division of Mycology, Kayseri Education and Research Hospital, Kayseri, Turkey Division of Mycology, Sisli Etfal Hamidiye Education and Research Hospital, Istanbul, Turkey c Division of Mycology, Fatih Sultan Mehmet Education and Research Hospital, Istanbul, Turkey d Division of Mycology, Kayseri Yunus Emre Hospital, Eskişehir, Turkey e Istanbul University, Cerrahpasa Medical Faculty, Department of Microbiology, Istanbul, Turkey b
A R T I C L E I N F O
A B S T R A C T
Keywords: Candida Onychomycosis Antifungal susceptibility method Virulence Broth microdilution
Although the frequency of candidal onychomycosis is increasing daily, there is little information in literature about the epidemiology, pathogenesis, and antifungal susceptibility of this dermatological disease. This study aimed to provide information about the epidemiology, pathogenesis, and azole susceptibility of Candida species isolated from patients living in a region with continental climate. After identification of the isolated strains using conventional methods, proteinase and phospholipase activities were determined by a plate method and biofilmforming ability was determined using the microplate method. Susceptibility of the same species to fluconazole (FLU), voriconazole (VRC), miconazole (MNZ), itraconazole (ITZ), and ketoconazole (KTZ) were determined by microdilution method. The 50 Candida isolates included 23 C. parapsilosis (46%), 13 C. albicans (26%), 4 C. guilliermondii(8%), 4 C.tropicalis (8%), 2 C.krusei(2%), 1 C.lusitaniae (2%), 1 C. sake (2%), and 1 C. kefyr (2%) isolates. The geometric mean (GM) of the minimum inhibitory concentration (MIC) for FLU, KTZ, VRC, MNZ, and ITZ was 0.4 μg/mL, 0.08 μg/mL, 0.08 μg/mL, 0.2 μg/mL, and 0.6 μg/mL, respectively. Proteinase, phospholipase, and biofilmforming ability were detected in 18%(9/50), 20%(10/50), and 6%(3/50) of the Candida isolates, respectively. We found that the most frequently isolated species is C.parapsilosis. On the basis of the GM values, the most effective azoles are ketoconazole and voriconazole. The isolated Candida species exhibited low phospholipase, proteinase, and biofilm formation activities.
1. Introduction Onychomycosis constitutes 30% of superficial fungal infections and 50% of nail infections [1]. The most frequently isolated species are dermatophytes, yeasts, and non-dermatophyte moulds. Previously, only dermatophytes were considered as the most common causative agents; however, Candida species have recently been reported among the important pathogens causing onychomycosis infections [2,3]. To understand the pathogenesis of onychomycosis caused by Candida species, the host factors and characteristics of Candida species need to be examined. Certain factors, such as the age and occupation of patients and the localization of infection are important host factors. In addition to host factors, the virulence factors that microorganism uses to defend itself against the host are also important. These include biofilm formation by the microorganism and production of hydrolytic enzymes (proteinase, phospholipase, and haemolysin). Fungal cells in
∗
the extracellular matrix form a thick fungal biomass, leading to difficulties in eradication of the fungal nail infection [4].Phospholipases, a class of hydrolytic enzymes, hydrolyse one or more ester linkages in glycerophospholipids, whereas proteinases break down proteins such as albumin, haemoglobin, and keratin [5]. Due to these enzymes, Candida species adhere to the host cell and form an infection by forming lysis. High-cost and long-term treatments, ineffective management of infection, uncooperative patients, and hepatotoxic and gastrointestinal side effects of drugs result in adverse outcomes in treatment. Therefore, treatment protocols are being developed by international committees [6].Topical and systemic antifungals are used for onychomycosis infection treatment.Fluconazole,itraconazole, imidazoles (clotrimazole, ketoconazole, and miconazole), and allylamines (terbinafine) are agents that are effective against onychomycosis and have a safe profile for human application. When dermatophytes are isolated as agents, terbinafine shows good results whereas azoles, especially itraconazoles,
Corresponding author. Tel.: +905053887684. E-mail address: savhafi[email protected] (H. Sav).
https://doi.org/10.1016/j.micpath.2018.01.036 Received 9 October 2017; Received in revised form 4 December 2017; Accepted 22 January 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
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are effective in yeast isolates [7,8]. In this study, proteinase, phospholipase, and biofilm formation characteristics of isolated Candida species were investigated to understand the pathogenesis of onychomycosis caused by Candida spp. In addition, the sensitivity of these species to various azoles was determined by a reference microdilution method to guide clinicians regarding the treatment protocols that can be used. 2. Materials and methods 2.1. Material intake and identification of isolated Candida species Nail samples were obtained from 1810 patients suspected of onychomycosis in the mycology laboratory of the Kayseri Educational and Research Hospital. First, the nail area was cleaned with alcohol to remove contaminants. Sampling was performed particularly from the proximal and lateral corners of the nail in patients with possible onychomycosis. Nail specimens were then treated with 30% KOH and examined directly under a microscope.The fingernail and toenail specimens were incubated with Sabouraud dextrose agar (SDA, Acumedia, Baltimore, Maryland, USA) supplemented with 50 μg/ml gentamycin (Sigma,Steinheim, Germany) and 100 μg/mL cycloheximide (Sigma, Steinheim,Germany). After inoculation, one of the tubes was placed at 25 °C and the other at 37 °C, and the tubes were assessed weekly. Germ tube test, morphological appearance on corn meal agar with Tween 80, and the API ID 32C (Bio Merieux, France) kit were used to identify the growing Candida species.
Fig. 1. Proteinase activity.
phospholipase activity (Pz value) of the strains was evaluated by measuring the apparent ring-shaped precipitation zone around the colony and the Pz value was obtained by calculating the ratio of the colony diameter (a) to the precipitation diameter. The results were evaluated as follows:Pz = 1, negative (-); Pz values between 0.9 and 1.00, (+), those between 0.8 and 0.89, (2+), those between 0.7 and 0.79, (3+), and those < 0.69, (4+) [14].C. albicans SC 5314 strain was used as a positive control (Fig. 2).
2.2. Antifungal susceptibility test 2.5. Biofilm formation Fluconazole [Pfizer Pharmaceuticals Group, New York, NY, USA,] voriconazole, itraconazole, miconazole, and ketoconazole [SigmaAldrich, St. Louis, Louis, Missouri, U.S.A.] stock solutions were prepared and administered according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 reference method [9]. The minimum inhibitory concentration (MIC) values of the antifungals were evaluated according to the CLSIM27-S4 reference method [10].RPMI 1640 (L glutamine, without bicarbonate) (Sigma Chemical Co., St Louis, Mo., USA) was used as the medium for the microdilution method. The antifungal concentration at which growth was significantly reduced (about 50%) for the azoles was considered as the antifungal MIC value. C.krusei ATCC 6258 and C. parapsilosis ATCC 22019 isolates were used as control isolates.
A modified microplate method (using a plate prepared with brain heart infusion broth (BHIB) containing 0.25% glucose; MMP-BHIB) was used in the experiment [15].The strains were incubated for 24 h at 35 °C in BHIB containing 0.25% glucose and diluted 120 with prewarmed 0.25% glucose containing BHIB. From these suspensions, 200 μL was transferred to sterile 96-well polystyrene microplates (3 wells were used for each strain). Following 48 h of incubation at 37 °C, the wells were washed with PBS, inverted, and stained with 1% crystal violet for 15 min. After washing, 200 μLof ethanol-acetone (80:20, v/v) was added to each of the tablets.The plates were read at 590 nm to determine the optical densities (OD), and the strains were considered negative for biofilm formation in case the OD < 1, weakly positive (+) if 1 < OD < 2; moderate positive (2+) if 2 < OD < 3, and strong positive (3+) if OD > 3. All experiments were repeated twice. Enterococcus faecalis strain ATCC 29212 was used as the control.
2.3. Proteinase activity For detection of proteinase activity, bovine serum albumin-agar (SSAA) was used, as described previously [11].For this test, yeast colonies were inoculated into yeast peptone dextrose (YPD) medium following 24–48 h of incubation at 37 °C in SDA, and then incubated for 4 h at 30 °C.The cultures were adjusted to 0.5 McFarland turbidity standard after incubation. The suspension (10 μL) was then mixed with SSAA medium and incubated for 6 days at 30 °C. Strains without the melting zone were evaluated as (-) in terms of acid proteinase activity, strains that spread in a maximum area of 1–2 mm in transparent zone were considered as moderate (+), whereas strains that spread in a 3–5 mm area of the transparent zone were considered to be strong (2+) [12].C.albicans ATCC 10231 strains was used as positive control (Fig. 1). 2.4. Phospholipase activity An egg yolk agar (YSA) plaque method was used to detect the phospholipase activity of the isolated strains [13].In this test, 10 μLof egg-wrapped agar was inoculated witha yeast suspension of 0.5 McFarland turbidity and incubated at 37 °C for 4days. The
Fig. 2. Phospholipase activity.
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Fig. 3. Total candidal onychomycosis.
3. Results From amongst patients with suspected onychomycosis, candidal onychomycosis was diagnosed in fifty patients (Fig. 3, Fig. 4 and 5). These patients included 32 (64%) females and 18 (36%) males, and their mean age was 45.68 years. The frequency of cases depending on the site of infection were as follows:28 cases of (56%) fingernail infections and 22 (44%) of toenail infections. The sites of isolation on the nail were as follows: proximal part, 32 (64%); lateral part, 8 (16%); distal part, 8 (16%); and complete nail, 2 (4%). The 50 isolated Candida spp isolates identified by conventional methods were: C.parapsilosis, 23 (46%);C. albicans, 13 (26%);C.guilliermondii, 4 (8%);C.tropicalis, 4 (8%),C.krusei,1 (2%); C.lusitaniae, 1 (2%),C. sake, 1 (2%); and C.kefyr, 1 (2%). In our study, proteinase activity was observed in nine Candida isolates, phospholipase activity was found in10 Candida isolates, and biofilm formation activity was observed in only three Candida isolates.The phospholipase activity of C. albicans was higher than that of C. parapsilosis species and all isolates (100%) were detected as strong phospholipase producers. The proteinase, phospholipase, biofilm formation, and antifungal susceptibility characteristics of the 50 Candida spp. isolates are summarized in Table 1. The geometric mean values of antifungal drugs were evaluated for fluconazole (0.4 μg/mL), miconazole (0.2 μg/mL), itraconazole (0.6 μg/mL), ketoconazole (0.08 μg/mL), and voriconazole (0.08 μg/mL). According to the results of the antifungal susceptibility test, all isolates were found to be susceptible to voriconazole. Evaluation of fluconazole showed that most isolates were
Fig. 5. Paronychia.
susceptible, but two C. parapsilosis and two C. tropicalis isolates were susceptible depending on the dosage. One of two C. krusei isolates was completely resistant to fluconazole.The MIC intervals, MIC50, and MIC90 values for voriconazole, fluconazole, miconazole, itraconazole, and ketoconazole are shown in Table 2. 4. Discussion Fifty Candida isolates obtained from nails were investigated in this study. Conventional methods were used to identify the species and the proteinase, phospholipase, and biofilm formation characteristics of these isolates were investigated to understand their pathogenesis in the clinical setting. The sensitivity of these isolates to azoles was evaluated using a reference microdilution method.In our study, C. parapsilosis was the most commonly isolated species. No significant resistance pattern was observed against any of the azole antifungal agents.The proteinase, phospholipase, and biofilm formation characteristics of the isolates may play a minor role in the pathogenesis of candidal onycomycosis. C.parapsilosis species can cause onychomycosis in nails subjected to trauma since such nails are found in the soil and in animals [16].Segal et al. [17] reported C. parapsilosis isolated from toenail as the most common cause of candidal onychomycosis in Israel. However, in Denmark and Iran, C.albicans species were reported to be most commonly isolated from fingernails [18,19].Ilkit et al. [20] reported C. tropicalis as the most frequently isolated onychomycosis causative agent in Adana province. In our study, we found C. parapsilosisas the most frequently isolated species in onychomycosis infections, followed by C. albicans, C. guilliermondii, C. tropicalis, C. krusei,C. glabrata,C.lusitaniae,C.sake, and C.kefyr. Epidemiological studies show that the causative agents of
Fig. 4. Proximal candidal onychomycosis.
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Table 1 The proteinase, phospholipase, biofilm formation and antifungal susceptibility characteristics of the Candida isolates. no
species
region
Disease zone
age
flu
mika
ıtra
keto
vori
proteinase
phospolipase
biofilm
gender
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C glabrata C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C sake C tropicalis C tropicalis C tropicalis C tropicalis C lusitania C kurusei C kurusei C kefyr C. guilliermondii C. guilliermondii C. guilliermondii C. guilliermondii
hand hand hand hand hand hand hand hand hand hand hand hand hand food hand hand hand hand hand hand hand hand hand hand hand hand hand hand food food food food food food food food food food hand food food food food food food food food food food food
distal distal distal distal distal lateral distal proximal distal lateral distal lateral lateral lateral proximal total total proximal proximal proximal proximal proximal lateral proximal proximal proximal proximal proximal proximal lateral proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal lateral proximal proximal proximal proximal proximal proximal
70 31 22 33 28 88 58 50 50 42 51 32 25 45 43 25 51 56 45 68 58 42 27 29 59 56 58 42 54 52 27 45 57 35 60 30 34 27 65 55 40 22 30 62 28 34 54 66 47 76
0,5 0,25 0,25 0,125 0,125 0,125 0,125 0,125 0,125 0,06 0,06 0,06 0,06 2 16 16 8 8 4 4 1 1 0,5 0,5 0,5 0,5 0,5 1 0,25 0,25 0,25 0,125 0,125 0,125 0,06 0,06 0,06 0,25 0,125 16 16 0,125 0,25 2 > 64 0,25 2 0,06 0,06 0,125
0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 4 2 2 1 1 1 1 1 0,5 0,5 0,5 0,25 0,25 0,5 0,25 0,25 0,25 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 4 4 0,06 0,06 0,5 4 0,06 1 0,06 1 0,06
1 1 0,5 0,5 0,5 0,5 0,5 0,25 0,25 0,25 0,125 0,06 0,06 4 4 4 4 2 2 2 2 2 2 1 1 1 0,5 2 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,06 0,06 0,25 0,06 4 4 0,5 0,25 0,25 4 1 2 0,06 2 0,25
0,25 0,25 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,125 0,6 0,25 0,25 0,25 0,25 0,125 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,25 0,25 0,06 0,06 0,125 0,25 0,06 0,06 0,06 0,25 0,06
0,125 0,125 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,6 0,25 0,25 0,25 0,125 0125 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,125 0,25 0,06 0,06 0125 0,25 0,06 0,06 0,06 0,25 0,06
–
++++
– – – – ++ – – ++
++++ – – ++++ – – ++++ ++++
– – – – – – – ++ – – – ++ – – – – – – ++ – – – – – – ++ ++ ++ – – – – – – ++ – – – –
++++ – – – – ++++ – – ++++ – ++++ ++++ – – – – – – – –
– – – – + – – – – – – – – – – – – – + – – – – – – – – – – – – – – – – – – – – – – +++ – – – – – – – –
W M M M W W W W W W M W M W M M W W W W M M W W M W M M W W W M W W W M W W M W W W W W W W W M M M
– – – – – – – – – – – – – – – – – –
Flu; fluconazole mika; miconazole ıtra; ıtraconazole keto; ketoconazole vori;voriconazole.
rates, the proteinase activity of C.parapsilosis species was higher, but a statistical evaluation could not be performed owing to the small number of species obtained. Phospholipase enzyme breaks down phospholipids present in the host cell membrane and alters the cell surface properties (such as perforation or adhesion).Thus,phospholipase production can be used as a parameter to distinguish non-invasive colonized species from invasive species. In their study, Pakshir K et al. [24]evaluated the phospholipase, esterase, and haemolytic activity properties of Candida species obtained from lichen planus and onychomycosis patients and reported that fewer C.parapsilosis isolates from onychomycosis patients secreted these enzyme types.DeepaKet al. [25]found 52.6% of phospholipase activity and 86.8% of proteinase activity when evaluating phospholipase, proteinase,haemolysin, and esterase activities in Candida spp isolated from the oral cavity. They found phospholipase and proteinase activity to be most frequently positive in C.albicans species. In our study,phospholipase activity was detected in C. albicans(6) and C. parapsilosis(4) species. Phospholipase activity was detected most frequently in
onychomycosis differ according to the countries and regions. In our study, the frequency of onychomycosis caused by Candida spp. was higher in female patients (32 of 50 patients, 64%) compared to male patients (18 of 50 patients, 36%)). Candida species causing onychomycosis were more commonly isolated from the fingernail (28 of 50 patients, 56%) compared to the toenail. The higher frequency of Candida species in female patients and in the fingernails may be associated with the higher exposure of women to aquatic and mechanical traumas, or it may be due to cosmetic reasons. The proteinase enzyme found in Candida species damages mucosal membranes and allows colonization and invasion of the host cell and provides nitrogen for fungal growth [21]. The activities and properties of the proteinase enzymes secreted by different species are different. The proteinase activities of C. parapsilosis species isolated from the vagina and epithelium are reported to be higher [22,23].In our study,C. parapsilosis (4), C. albicans (2) C. tropicalis (1), C. sake (1), and C. kefyr (1) were detected as proteinase producing isolates. Proteinase activity was detected most frequently in C.parapsilosis. Based on these positivity 260
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0,06 0,06 0,06 0,06
MIC (50)
0,06 0,06 0,06 0,06
MIC (50)
0,06 0,5
MIC (90)
MIC range 1 - 0,06 4 - 0,06 4 4 - 0,25 0,25 4 - 0,06 0,25 1 2 - 0,06
Ketoconazole
MIC (90)
MIC range 0,25–0,06 0,6–0,06 0,125 0,25–0,125 0,06 0,25–0,06 0,06 0,06 0,25–0,06
Voriconazole
MIC (90)
MIC range 0,125–0,06 0,6–0,06 0,06 0,25–0,125 0,06 0,25–0,06 0,06 0,06 0,25–0,06
C.parapsilosis among the non-albicans species. All C. albicans and C. parapsilosis species showed strong enzymatic activities (Pz values ≤ 0.69). When considered in a wide spectrum, the effect of biofilm productionby fungi on onychomycosis treatment is controversial [26]. In our study, only three isolates of Candida species were positive for biofilm production. The low biofilm positivity rate in isolated Candida species suggests that its impact on the pathogenesis of onychomycosis may be a minor factor. Although not approved by the FDA, fluconazole may be used as an alternative therapy, because of the side effects of terbinafine and itraconazole in onychomycosis treatment [27].Miconazole and ketoconazole are the first azole derivatives used in onychomycosis treatment and have serious side effects and drug interactions. Various studies have reported invitro susceptibility results against miconazole and ketoconazole [28,29]. Voriconazole, on the other hand, is not used for treatment because of its high cost. In our study, low MICs of fluconazole were obtained for all Candida isolates, but only two C.parapsilosis and two C.tropicalis species were found as dose-dependent sensitive isolates.Voriconazole showed excellent activity against Candida species isolated from onychomycosis in this study. The CLSI standardized broth microdilution method remains a reference for antifungal susceptibility testing but the certain breakpoint MICs of itraconazole have not yet been established for onychomycosis therapy.Examination of itraconazole showed that the MIC values of itraconazole against C.parapsilosis species were high. The geometric mean values ofmiconazole and ketoconazole were 0.2 μg/mL and 0.08 μg/mL, respectively. According to our study ketoconazole was more effective than miconazole in Candida onychomycosis.
MIC (50) MIC (90)
0,06 0,06
MIC (50)
0,06 0,25
The epidemiology of onychomycosis caused by Candida species may vary. In our study, the most frequently isolated agent of candidal onychomycosis in our region was C.parapsilosis.Besides, examination of enzymatic and non-enzymatic properties showed no interesting association between the candidal onychomycosis isolates and their characteristics of phospholipase and proteinase activities and biofilm formation. Examination of the susceptibility of these species to fluconazole, voriconazole, miconazole, itraconazole, and ketoconazole using the reference method showed no significant resistance pattern. In this case, it was concluded that clinicians should take into account drug interactions, side effects and the costs they incur while establishing treatment protocols.
MIC range 0,06–0,06 4 - 0,06 0,06 4 - 0,5 0,06 4 - 0,06 0,06 0,06 1 - 0,06
0,5 1
Itraconazole Miconazole
5. Conclusion
0,5–0,06 16 - 0,06 2 16–2 0,25 16 - 0,125 0,25 0,25 2 - 0,06
The authors declare no conflict of interest. Acknowledgements This study was made from patient samples analyzed in Kayseri Educational Research Hospital and was not financed by any organization.
MIC;Minimum Inhibitor Concentration MIC;μg/ml.
0,06 0,06 0,125 0,5 26 46 2 4 2 8 2 2 8 albicans (13) parapsilosis (23) glabrata (1) krusei (2) sake (1) tropicalis (4) lusitania (1) kefyr (1) guilliermondii(4) C. C. C. C. C. C. C. C. C.
MIC range MIC (90) MIC (50) Candida spp(n)
%
Fluconazole Antifungal (ug/ml)
Table 2 In vitro susceptibilities of the Candida isolates to antifungal agents.
Conflicts of interest
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The frequency, antifungal susceptibility and enzymatic profiles of Candida species in cases of onychomycosis infection
T
Hafize Sava,∗, Ayse Barisb, Deniz Turanc, Rabiye Altinbasd, Sümeyye Sene a
Division of Mycology, Kayseri Education and Research Hospital, Kayseri, Turkey Division of Mycology, Sisli Etfal Hamidiye Education and Research Hospital, Istanbul, Turkey c Division of Mycology, Fatih Sultan Mehmet Education and Research Hospital, Istanbul, Turkey d Division of Mycology, Kayseri Yunus Emre Hospital, Eskişehir, Turkey e Istanbul University, Cerrahpasa Medical Faculty, Department of Microbiology, Istanbul, Turkey b
A R T I C L E I N F O
A B S T R A C T
Keywords: Candida Onychomycosis Antifungal susceptibility method Virulence Broth microdilution
Although the frequency of candidal onychomycosis is increasing daily, there is little information in literature about the epidemiology, pathogenesis, and antifungal susceptibility of this dermatological disease. This study aimed to provide information about the epidemiology, pathogenesis, and azole susceptibility of Candida species isolated from patients living in a region with continental climate. After identification of the isolated strains using conventional methods, proteinase and phospholipase activities were determined by a plate method and biofilmforming ability was determined using the microplate method. Susceptibility of the same species to fluconazole (FLU), voriconazole (VRC), miconazole (MNZ), itraconazole (ITZ), and ketoconazole (KTZ) were determined by microdilution method. The 50 Candida isolates included 23 C. parapsilosis (46%), 13 C. albicans (26%), 4 C. guilliermondii(8%), 4 C.tropicalis (8%), 2 C.krusei(2%), 1 C.lusitaniae (2%), 1 C. sake (2%), and 1 C. kefyr (2%) isolates. The geometric mean (GM) of the minimum inhibitory concentration (MIC) for FLU, KTZ, VRC, MNZ, and ITZ was 0.4 μg/mL, 0.08 μg/mL, 0.08 μg/mL, 0.2 μg/mL, and 0.6 μg/mL, respectively. Proteinase, phospholipase, and biofilmforming ability were detected in 18%(9/50), 20%(10/50), and 6%(3/50) of the Candida isolates, respectively. We found that the most frequently isolated species is C.parapsilosis. On the basis of the GM values, the most effective azoles are ketoconazole and voriconazole. The isolated Candida species exhibited low phospholipase, proteinase, and biofilm formation activities.
1. Introduction Onychomycosis constitutes 30% of superficial fungal infections and 50% of nail infections [1]. The most frequently isolated species are dermatophytes, yeasts, and non-dermatophyte moulds. Previously, only dermatophytes were considered as the most common causative agents; however, Candida species have recently been reported among the important pathogens causing onychomycosis infections [2,3]. To understand the pathogenesis of onychomycosis caused by Candida species, the host factors and characteristics of Candida species need to be examined. Certain factors, such as the age and occupation of patients and the localization of infection are important host factors. In addition to host factors, the virulence factors that microorganism uses to defend itself against the host are also important. These include biofilm formation by the microorganism and production of hydrolytic enzymes (proteinase, phospholipase, and haemolysin). Fungal cells in
∗
the extracellular matrix form a thick fungal biomass, leading to difficulties in eradication of the fungal nail infection [4].Phospholipases, a class of hydrolytic enzymes, hydrolyse one or more ester linkages in glycerophospholipids, whereas proteinases break down proteins such as albumin, haemoglobin, and keratin [5]. Due to these enzymes, Candida species adhere to the host cell and form an infection by forming lysis. High-cost and long-term treatments, ineffective management of infection, uncooperative patients, and hepatotoxic and gastrointestinal side effects of drugs result in adverse outcomes in treatment. Therefore, treatment protocols are being developed by international committees [6].Topical and systemic antifungals are used for onychomycosis infection treatment.Fluconazole,itraconazole, imidazoles (clotrimazole, ketoconazole, and miconazole), and allylamines (terbinafine) are agents that are effective against onychomycosis and have a safe profile for human application. When dermatophytes are isolated as agents, terbinafine shows good results whereas azoles, especially itraconazoles,
Corresponding author. Tel.: +905053887684. E-mail address: savhafi[email protected] (H. Sav).
https://doi.org/10.1016/j.micpath.2018.01.036 Received 9 October 2017; Received in revised form 4 December 2017; Accepted 22 January 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
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are effective in yeast isolates [7,8]. In this study, proteinase, phospholipase, and biofilm formation characteristics of isolated Candida species were investigated to understand the pathogenesis of onychomycosis caused by Candida spp. In addition, the sensitivity of these species to various azoles was determined by a reference microdilution method to guide clinicians regarding the treatment protocols that can be used. 2. Materials and methods 2.1. Material intake and identification of isolated Candida species Nail samples were obtained from 1810 patients suspected of onychomycosis in the mycology laboratory of the Kayseri Educational and Research Hospital. First, the nail area was cleaned with alcohol to remove contaminants. Sampling was performed particularly from the proximal and lateral corners of the nail in patients with possible onychomycosis. Nail specimens were then treated with 30% KOH and examined directly under a microscope.The fingernail and toenail specimens were incubated with Sabouraud dextrose agar (SDA, Acumedia, Baltimore, Maryland, USA) supplemented with 50 μg/ml gentamycin (Sigma,Steinheim, Germany) and 100 μg/mL cycloheximide (Sigma, Steinheim,Germany). After inoculation, one of the tubes was placed at 25 °C and the other at 37 °C, and the tubes were assessed weekly. Germ tube test, morphological appearance on corn meal agar with Tween 80, and the API ID 32C (Bio Merieux, France) kit were used to identify the growing Candida species.
Fig. 1. Proteinase activity.
phospholipase activity (Pz value) of the strains was evaluated by measuring the apparent ring-shaped precipitation zone around the colony and the Pz value was obtained by calculating the ratio of the colony diameter (a) to the precipitation diameter. The results were evaluated as follows:Pz = 1, negative (-); Pz values between 0.9 and 1.00, (+), those between 0.8 and 0.89, (2+), those between 0.7 and 0.79, (3+), and those < 0.69, (4+) [14].C. albicans SC 5314 strain was used as a positive control (Fig. 2).
2.2. Antifungal susceptibility test 2.5. Biofilm formation Fluconazole [Pfizer Pharmaceuticals Group, New York, NY, USA,] voriconazole, itraconazole, miconazole, and ketoconazole [SigmaAldrich, St. Louis, Louis, Missouri, U.S.A.] stock solutions were prepared and administered according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 reference method [9]. The minimum inhibitory concentration (MIC) values of the antifungals were evaluated according to the CLSIM27-S4 reference method [10].RPMI 1640 (L glutamine, without bicarbonate) (Sigma Chemical Co., St Louis, Mo., USA) was used as the medium for the microdilution method. The antifungal concentration at which growth was significantly reduced (about 50%) for the azoles was considered as the antifungal MIC value. C.krusei ATCC 6258 and C. parapsilosis ATCC 22019 isolates were used as control isolates.
A modified microplate method (using a plate prepared with brain heart infusion broth (BHIB) containing 0.25% glucose; MMP-BHIB) was used in the experiment [15].The strains were incubated for 24 h at 35 °C in BHIB containing 0.25% glucose and diluted 120 with prewarmed 0.25% glucose containing BHIB. From these suspensions, 200 μL was transferred to sterile 96-well polystyrene microplates (3 wells were used for each strain). Following 48 h of incubation at 37 °C, the wells were washed with PBS, inverted, and stained with 1% crystal violet for 15 min. After washing, 200 μLof ethanol-acetone (80:20, v/v) was added to each of the tablets.The plates were read at 590 nm to determine the optical densities (OD), and the strains were considered negative for biofilm formation in case the OD < 1, weakly positive (+) if 1 < OD < 2; moderate positive (2+) if 2 < OD < 3, and strong positive (3+) if OD > 3. All experiments were repeated twice. Enterococcus faecalis strain ATCC 29212 was used as the control.
2.3. Proteinase activity For detection of proteinase activity, bovine serum albumin-agar (SSAA) was used, as described previously [11].For this test, yeast colonies were inoculated into yeast peptone dextrose (YPD) medium following 24–48 h of incubation at 37 °C in SDA, and then incubated for 4 h at 30 °C.The cultures were adjusted to 0.5 McFarland turbidity standard after incubation. The suspension (10 μL) was then mixed with SSAA medium and incubated for 6 days at 30 °C. Strains without the melting zone were evaluated as (-) in terms of acid proteinase activity, strains that spread in a maximum area of 1–2 mm in transparent zone were considered as moderate (+), whereas strains that spread in a 3–5 mm area of the transparent zone were considered to be strong (2+) [12].C.albicans ATCC 10231 strains was used as positive control (Fig. 1). 2.4. Phospholipase activity An egg yolk agar (YSA) plaque method was used to detect the phospholipase activity of the isolated strains [13].In this test, 10 μLof egg-wrapped agar was inoculated witha yeast suspension of 0.5 McFarland turbidity and incubated at 37 °C for 4days. The
Fig. 2. Phospholipase activity.
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Fig. 3. Total candidal onychomycosis.
3. Results From amongst patients with suspected onychomycosis, candidal onychomycosis was diagnosed in fifty patients (Fig. 3, Fig. 4 and 5). These patients included 32 (64%) females and 18 (36%) males, and their mean age was 45.68 years. The frequency of cases depending on the site of infection were as follows:28 cases of (56%) fingernail infections and 22 (44%) of toenail infections. The sites of isolation on the nail were as follows: proximal part, 32 (64%); lateral part, 8 (16%); distal part, 8 (16%); and complete nail, 2 (4%). The 50 isolated Candida spp isolates identified by conventional methods were: C.parapsilosis, 23 (46%);C. albicans, 13 (26%);C.guilliermondii, 4 (8%);C.tropicalis, 4 (8%),C.krusei,1 (2%); C.lusitaniae, 1 (2%),C. sake, 1 (2%); and C.kefyr, 1 (2%). In our study, proteinase activity was observed in nine Candida isolates, phospholipase activity was found in10 Candida isolates, and biofilm formation activity was observed in only three Candida isolates.The phospholipase activity of C. albicans was higher than that of C. parapsilosis species and all isolates (100%) were detected as strong phospholipase producers. The proteinase, phospholipase, biofilm formation, and antifungal susceptibility characteristics of the 50 Candida spp. isolates are summarized in Table 1. The geometric mean values of antifungal drugs were evaluated for fluconazole (0.4 μg/mL), miconazole (0.2 μg/mL), itraconazole (0.6 μg/mL), ketoconazole (0.08 μg/mL), and voriconazole (0.08 μg/mL). According to the results of the antifungal susceptibility test, all isolates were found to be susceptible to voriconazole. Evaluation of fluconazole showed that most isolates were
Fig. 5. Paronychia.
susceptible, but two C. parapsilosis and two C. tropicalis isolates were susceptible depending on the dosage. One of two C. krusei isolates was completely resistant to fluconazole.The MIC intervals, MIC50, and MIC90 values for voriconazole, fluconazole, miconazole, itraconazole, and ketoconazole are shown in Table 2. 4. Discussion Fifty Candida isolates obtained from nails were investigated in this study. Conventional methods were used to identify the species and the proteinase, phospholipase, and biofilm formation characteristics of these isolates were investigated to understand their pathogenesis in the clinical setting. The sensitivity of these isolates to azoles was evaluated using a reference microdilution method.In our study, C. parapsilosis was the most commonly isolated species. No significant resistance pattern was observed against any of the azole antifungal agents.The proteinase, phospholipase, and biofilm formation characteristics of the isolates may play a minor role in the pathogenesis of candidal onycomycosis. C.parapsilosis species can cause onychomycosis in nails subjected to trauma since such nails are found in the soil and in animals [16].Segal et al. [17] reported C. parapsilosis isolated from toenail as the most common cause of candidal onychomycosis in Israel. However, in Denmark and Iran, C.albicans species were reported to be most commonly isolated from fingernails [18,19].Ilkit et al. [20] reported C. tropicalis as the most frequently isolated onychomycosis causative agent in Adana province. In our study, we found C. parapsilosisas the most frequently isolated species in onychomycosis infections, followed by C. albicans, C. guilliermondii, C. tropicalis, C. krusei,C. glabrata,C.lusitaniae,C.sake, and C.kefyr. Epidemiological studies show that the causative agents of
Fig. 4. Proximal candidal onychomycosis.
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Table 1 The proteinase, phospholipase, biofilm formation and antifungal susceptibility characteristics of the Candida isolates. no
species
region
Disease zone
age
flu
mika
ıtra
keto
vori
proteinase
phospolipase
biofilm
gender
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C albicans C glabrata C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C parapsilosis C sake C tropicalis C tropicalis C tropicalis C tropicalis C lusitania C kurusei C kurusei C kefyr C. guilliermondii C. guilliermondii C. guilliermondii C. guilliermondii
hand hand hand hand hand hand hand hand hand hand hand hand hand food hand hand hand hand hand hand hand hand hand hand hand hand hand hand food food food food food food food food food food hand food food food food food food food food food food food
distal distal distal distal distal lateral distal proximal distal lateral distal lateral lateral lateral proximal total total proximal proximal proximal proximal proximal lateral proximal proximal proximal proximal proximal proximal lateral proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal proximal lateral proximal proximal proximal proximal proximal proximal
70 31 22 33 28 88 58 50 50 42 51 32 25 45 43 25 51 56 45 68 58 42 27 29 59 56 58 42 54 52 27 45 57 35 60 30 34 27 65 55 40 22 30 62 28 34 54 66 47 76
0,5 0,25 0,25 0,125 0,125 0,125 0,125 0,125 0,125 0,06 0,06 0,06 0,06 2 16 16 8 8 4 4 1 1 0,5 0,5 0,5 0,5 0,5 1 0,25 0,25 0,25 0,125 0,125 0,125 0,06 0,06 0,06 0,25 0,125 16 16 0,125 0,25 2 > 64 0,25 2 0,06 0,06 0,125
0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 4 2 2 1 1 1 1 1 0,5 0,5 0,5 0,25 0,25 0,5 0,25 0,25 0,25 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 4 4 0,06 0,06 0,5 4 0,06 1 0,06 1 0,06
1 1 0,5 0,5 0,5 0,5 0,5 0,25 0,25 0,25 0,125 0,06 0,06 4 4 4 4 2 2 2 2 2 2 1 1 1 0,5 2 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,06 0,06 0,25 0,06 4 4 0,5 0,25 0,25 4 1 2 0,06 2 0,25
0,25 0,25 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,125 0,6 0,25 0,25 0,25 0,25 0,125 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,25 0,25 0,06 0,06 0,125 0,25 0,06 0,06 0,06 0,25 0,06
0,125 0,125 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,6 0,25 0,25 0,25 0,125 0125 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,06 0,125 0,25 0,06 0,06 0125 0,25 0,06 0,06 0,06 0,25 0,06
–
++++
– – – – ++ – – ++
++++ – – ++++ – – ++++ ++++
– – – – – – – ++ – – – ++ – – – – – – ++ – – – – – – ++ ++ ++ – – – – – – ++ – – – –
++++ – – – – ++++ – – ++++ – ++++ ++++ – – – – – – – –
– – – – + – – – – – – – – – – – – – + – – – – – – – – – – – – – – – – – – – – – – +++ – – – – – – – –
W M M M W W W W W W M W M W M M W W W W M M W W M W M M W W W M W W W M W W M W W W W W W W W M M M
– – – – – – – – – – – – – – – – – –
Flu; fluconazole mika; miconazole ıtra; ıtraconazole keto; ketoconazole vori;voriconazole.
rates, the proteinase activity of C.parapsilosis species was higher, but a statistical evaluation could not be performed owing to the small number of species obtained. Phospholipase enzyme breaks down phospholipids present in the host cell membrane and alters the cell surface properties (such as perforation or adhesion).Thus,phospholipase production can be used as a parameter to distinguish non-invasive colonized species from invasive species. In their study, Pakshir K et al. [24]evaluated the phospholipase, esterase, and haemolytic activity properties of Candida species obtained from lichen planus and onychomycosis patients and reported that fewer C.parapsilosis isolates from onychomycosis patients secreted these enzyme types.DeepaKet al. [25]found 52.6% of phospholipase activity and 86.8% of proteinase activity when evaluating phospholipase, proteinase,haemolysin, and esterase activities in Candida spp isolated from the oral cavity. They found phospholipase and proteinase activity to be most frequently positive in C.albicans species. In our study,phospholipase activity was detected in C. albicans(6) and C. parapsilosis(4) species. Phospholipase activity was detected most frequently in
onychomycosis differ according to the countries and regions. In our study, the frequency of onychomycosis caused by Candida spp. was higher in female patients (32 of 50 patients, 64%) compared to male patients (18 of 50 patients, 36%)). Candida species causing onychomycosis were more commonly isolated from the fingernail (28 of 50 patients, 56%) compared to the toenail. The higher frequency of Candida species in female patients and in the fingernails may be associated with the higher exposure of women to aquatic and mechanical traumas, or it may be due to cosmetic reasons. The proteinase enzyme found in Candida species damages mucosal membranes and allows colonization and invasion of the host cell and provides nitrogen for fungal growth [21]. The activities and properties of the proteinase enzymes secreted by different species are different. The proteinase activities of C. parapsilosis species isolated from the vagina and epithelium are reported to be higher [22,23].In our study,C. parapsilosis (4), C. albicans (2) C. tropicalis (1), C. sake (1), and C. kefyr (1) were detected as proteinase producing isolates. Proteinase activity was detected most frequently in C.parapsilosis. Based on these positivity 260
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0,06 0,06 0,06 0,06
MIC (50)
0,06 0,06 0,06 0,06
MIC (50)
0,06 0,5
MIC (90)
MIC range 1 - 0,06 4 - 0,06 4 4 - 0,25 0,25 4 - 0,06 0,25 1 2 - 0,06
Ketoconazole
MIC (90)
MIC range 0,25–0,06 0,6–0,06 0,125 0,25–0,125 0,06 0,25–0,06 0,06 0,06 0,25–0,06
Voriconazole
MIC (90)
MIC range 0,125–0,06 0,6–0,06 0,06 0,25–0,125 0,06 0,25–0,06 0,06 0,06 0,25–0,06
C.parapsilosis among the non-albicans species. All C. albicans and C. parapsilosis species showed strong enzymatic activities (Pz values ≤ 0.69). When considered in a wide spectrum, the effect of biofilm productionby fungi on onychomycosis treatment is controversial [26]. In our study, only three isolates of Candida species were positive for biofilm production. The low biofilm positivity rate in isolated Candida species suggests that its impact on the pathogenesis of onychomycosis may be a minor factor. Although not approved by the FDA, fluconazole may be used as an alternative therapy, because of the side effects of terbinafine and itraconazole in onychomycosis treatment [27].Miconazole and ketoconazole are the first azole derivatives used in onychomycosis treatment and have serious side effects and drug interactions. Various studies have reported invitro susceptibility results against miconazole and ketoconazole [28,29]. Voriconazole, on the other hand, is not used for treatment because of its high cost. In our study, low MICs of fluconazole were obtained for all Candida isolates, but only two C.parapsilosis and two C.tropicalis species were found as dose-dependent sensitive isolates.Voriconazole showed excellent activity against Candida species isolated from onychomycosis in this study. The CLSI standardized broth microdilution method remains a reference for antifungal susceptibility testing but the certain breakpoint MICs of itraconazole have not yet been established for onychomycosis therapy.Examination of itraconazole showed that the MIC values of itraconazole against C.parapsilosis species were high. The geometric mean values ofmiconazole and ketoconazole were 0.2 μg/mL and 0.08 μg/mL, respectively. According to our study ketoconazole was more effective than miconazole in Candida onychomycosis.
MIC (50) MIC (90)
0,06 0,06
MIC (50)
0,06 0,25
The epidemiology of onychomycosis caused by Candida species may vary. In our study, the most frequently isolated agent of candidal onychomycosis in our region was C.parapsilosis.Besides, examination of enzymatic and non-enzymatic properties showed no interesting association between the candidal onychomycosis isolates and their characteristics of phospholipase and proteinase activities and biofilm formation. Examination of the susceptibility of these species to fluconazole, voriconazole, miconazole, itraconazole, and ketoconazole using the reference method showed no significant resistance pattern. In this case, it was concluded that clinicians should take into account drug interactions, side effects and the costs they incur while establishing treatment protocols.
MIC range 0,06–0,06 4 - 0,06 0,06 4 - 0,5 0,06 4 - 0,06 0,06 0,06 1 - 0,06
0,5 1
Itraconazole Miconazole
5. Conclusion
0,5–0,06 16 - 0,06 2 16–2 0,25 16 - 0,125 0,25 0,25 2 - 0,06
The authors declare no conflict of interest. Acknowledgements This study was made from patient samples analyzed in Kayseri Educational Research Hospital and was not financed by any organization.
MIC;Minimum Inhibitor Concentration MIC;μg/ml.
0,06 0,06 0,125 0,5 26 46 2 4 2 8 2 2 8 albicans (13) parapsilosis (23) glabrata (1) krusei (2) sake (1) tropicalis (4) lusitania (1) kefyr (1) guilliermondii(4) C. C. C. C. C. C. C. C. C.
MIC range MIC (90) MIC (50) Candida spp(n)
%
Fluconazole Antifungal (ug/ml)
Table 2 In vitro susceptibilities of the Candida isolates to antifungal agents.
Conflicts of interest
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