Journal de Mycologie Médicale (2014) 24, e43—e50
Available online at
ScienceDirect www.sciencedirect.com
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Species distribution and antifungal susceptibility of Candida spp. isolated from superficial candidiasis in outpatients in Iran ` ces et sensibilite ´ aux antifongiques de Distribution des espe ´ de candidose superficielle chez les malades en Candida spp. isole consultation externe en Iran M. Razzaghi-Abyaneh a,*, G. Sadeghi a, E. Zeinali a, M. Alirezaee a, M. Shams-Ghahfarokhi b, A. Amani c, R. Mirahmadi d, R. Tolouei a a
Department of Medical Mycology, Pasteur Institute of Iran, 13164 Tehran, Iran Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, 14115-331 Tehran, Iran c Medicinal Chemistry Laboratory, Pasteur Institute of Iran, 13164 Tehran, Iran d Laboratory of Arboviruses and Viral Hemorrhagic Fevers, Pasteur Institute of Iran, 13164 Tehran, Iran b
Received 21 April 2013; received in revised form 4 December 2013; accepted 7 January 2014 Available online 21 April 2014
KEYWORDS Candidiasis; Candida species; Antifungal drugs; Broth microdilution; CHROMagar Candida; Disk diffusion
Summary Objective. — Candidiasis is the most prevalent fungal infection affecting human and animals all over the world. This study represents the epidemiological aspects of superficial candidiasis in outpatients and in vitro antifungal susceptibility of etiologic Candida species. Patients and methods. — Clinical samples were taken from 173 patients including skin and nail scrapings (107; 61.8%), vaginal discharge (28; 16.2%), sputum (20; 11.6%), oral swabs (7; 4.0%), bronchoalveolar lavage (6; 3.5%) and 1 specimen (0.6%) of each eye tumor, gastric juice, urine, biopsy and urinary catheter and confirmed as candidiasis by direct microscopy, culture and histopathology. Susceptibility patterns of the isolated Candida species were determined using the disk diffusion and broth microdilution methods. Results. — Among 173 Candida isolates, C. albicans (72.3%) was the most prevalent species followed by C. parapsilosis (11.5%). Other identified species were C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. intermedia and C. sake. Majority of the Candida isolates were susceptible to fluconazole (95.4%) followed by 5-flucytosine (89.6%), voriconazole (78.6%)
* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Razzaghi-Abyaneh). 1156-5233/$ — see front matter # 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2014.01.004
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M. Razzaghi-Abyaneh et al. itraconazole (48.0%) and ketoconazole (42.8%). Caspofungin was the most potent antifungal drug against C. albicans (MICs; 0.062—1 mg/mL), ketoconazole for C. parapsilosis and C. tropicalis (MICs; 0.031—0.25 mg/mL) and itraconazole for C. krusei, C. glabrata and C. guilliermondii (MICs; 0.031—1 mg/mL). Conclusion. — This study reinforces the significance of superficial candidiasis as an important fungal infection with multiple clinical presentations. Our results further indicate that susceptibility testing to commonly used antifungals is crucial in order to select the appropriate therapeutic strategies which minimize complications while improving patients’ life. # 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Candidose ; Espèces de Candida ; Antifongiques ; Microdilution ; CHROMagar Candida ; Diffusion des disques
Re ´sume ´ Objectif. — La candidose est l’infection fongique la plus courante chez les animaux et l’homme partout dans le monde. Cette étude présente les aspects épidémiologiques de la candidose superficielle chez les patients et la sensibilité antifongique in vitro des espèces de Candida isolées. Patients et me ´thodes. — Les prélèvements cliniques ont été effectués sur 173 patients, il s’agit de la peau et des ongles (107 ; 61,8 %), des secrétions vaginales (28 ; 16,2 %), de l’expectoration (20 ; 11,6 %), de prélèvements oraux (7 ; 4,0 %), de lavage broncho-alvéolaire (6 ; 3,5 %) et d’un échantillon (0,6 %) de tumeur de l’œil, de suc gastrique, d’urine, de biopsie et d’un cathéter urinaire. Ils ont été confirmés par l’examen direct, la culture et l’histopathologie comme candidose. La sensibilité des souches de Candida isolées a été déterminée par la méthode des disques et la méthode de microdilution. Re ´sultats. — Parmi 173 isolats de Candida, l’espèce la plus fréquente a été C. albicans (72,3 %), suivie par C. parapsilosis (11,5 %). Les autres espèces identifiées ont été C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. intermedia et C. sake. La majorité des isolats de Candida s’est montrée sensible au fluconazole (95,4 %), suivie par la 5-flucytosine (89,6 %), le voriconazole (78,6 %), l’itraconazole (48,0 %) et le kétoconazole (42,8 %). La caspofungine a été le médicament le plus efficace contre C. albicans (CMI ; 0,062—1 mg/mL), le kétoconazole pour C. parapsilosis et C. tropicalis (CMI ; 0,031—0,25 mg/mL) et l’itraconazole pour C. krusei, C. glabrata et C. guilliermondii (CMI ; 0,031—1 mg/mL). Conclusion. — Cette étude montre l’importance de la candidose superficielle ayant des présentations cliniques différentes. Les résultats obtenus par cette étude montrent la nécessité de faire le test de sensibilité aux médicaments couramment utilisés pour choisir les meilleures stratégies de traitement en vue de diminuer les complications de la maladie et améliorer la qualité de vie des patients. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Worldwide occurrence of fungal infections has dramatically been increased in recent years due to a continuous augment in immunosuppressive conditions like AIDS, and other predisposing factors including organ transplantation, leukemia, broadspectrum antibiotics, indwelling catheters, diabetes and intravenous drug misuse. Superficial mycoses caused by species of the genus Candida has been reported from all over the world. They include some of the most common mucosal infections, such as thrush, vaginal candidiasis, cutaneous candidiasis, onychomycosis and chronic mucocutaneous candidiasis [34]. It has been estimated that an approximate of 20 to 30% of women have vaginal Candida colonization. The prevalence of oral cavity colonization by yeasts in normal individuals varies, from 20 to 40% in the general population. In the medical community, oral candidiasis and vaginitis caused by Candida account for a significant number of clinical complaints brought to colleagues of different specialties. The prevalence of carriage is greater in hospitalized patients and in those with conditions that predispose to candidiasis. Among Candida species, C. albicans is responsible for the majority cases of superficial candidiasis. However, increasing
numbers of other species such as C. tropicalis, C. parapsilosis, C. glabrata, C. guilliermondii have been reported as common etiologic agents [10,34]. Different methods with a wide range of successfulness have been used for identification of Candida species isolated from clinical specimens. Aside from conventional morphological and biochemical methods, a vast array of novel techniques including molecular assays, MALDI-TOF mass spectrometry, specific antibody-based agglutination techniques and omics analyses have been successfully used for identification of Candida species and clinical candidiasis [11,25,29]. A combination of such methods increases considerably the chance of reliable identification of Candida at the genus and species level [20]. Besides the necessity for identification of Candida isolates at species level, dramatic emergence of multidrug resistance urged the scientists to determine susceptibility patterns of this commensal pathogen of human and animals to commonly used antifungal agents. In this way, isolation and identification of causative Candida species and determination of their susceptibility to antifungal drugs is important for ensuring the efficacy of therapeutic strategies. Like as many other parts of the world, fungal infections are major concerns in Iran with an increasing numbers of new
Distribution and antifungal susceptibility of Candida species reports of superficial to deep infections every year. Although Candida species has been a subject of several studies in Iran [1,2,4,13,27,40], there is no comprehensive data gathering of all aspects of Candida infections from clinic to the laboratory. In our previous 4-year study on the prevalence and distribution of superficial fungal infections, candidiasis comprised about 16.0% of total dermatomycoses in Tehran [35]. The present study was designed to investigate: the epidemiological trends of candidiasis among patients who referred to the medical mycology department of Pasteur Institute of Iran; to determine species distribution and antifungal susceptibility patterns of Candida isolates against commonly used antifungal drugs from major classes i.e. polyenes, azoles and echinocandins with therapeutic values for different types of candidiasis.
Patients and methods Sample preparation and Candida identification During a two-year period from January 2010 to December 2011, 173 clinical specimens from outpatients suspected to candidiasis referred to the department of medical mycology, Pasteur Institute of Iran (a reference center for fungal identification and antifungal susceptibility testing) by Imam Khomeini hospital as a major center of infectious diseases in Tehran were examined. Candidiasis was proven for all the specimens by a combination of clinical features, direct microscopy, culture and histopathology. The specimens were placed directly onto a microscope slide, covered with 20% potassium hydroxide (KOH) and examined under a light microscope for fungal elements. A portion of the specimens were inoculated on Sabouraud dextrose agar (SDA; E. Merck, Germany) supplemented with 0.005% chloramphenicol and incubated for 2 weeks at 28 8C. By visual inspection, colonies suspected to Candida were isolated for further confirmation and species identification by a combination of germ tube test (GTT), chlamydospore formation (CF) on cornmeal agar plus tween 80 (Becton Dickinson, France), chromogenic assay on CHROMagar Candida (CHROMagar Candida, France), carbohydrate assimilation in ID32C system (bioMeriéux, France) and specific monoclonal antibody-based agglutination assays (Fumouze diagnostics, France). All identified Candida species are now available at Pathogenic Fungi Culture Collection of the Pasteur Institute of Iran (www.http://en.pasteur.ac.ir/pages.aspx?id=586).
Antifungal susceptibility testing Disk diffusion assay Candida isolates were evaluated for antifungal susceptibility to commonly used antifungal disks (6.0 mm Dia., MAST Diagnostics), i.e. amphotericin B (AmB; 20 mg), fluconazole (FCZ; 25 mg) and 5-flucytosine (5-FC; 1 mg) by the disk diffusion method on Mueller Hinton agar (MHA) supplemented with 2% glucose and 0.5 mg/mL methylene blue. Agar plates were swab inoculated with a suspension of yeast cells adjusted to the turbidity of a 0.5 McFarland standard. The plates were
e45 allowed to dry for 5 to 15 minutes and antifungal disks were placed on the agar surface. The plates were incubated for 24 h at 35 8C. The interpretive criteria for the fluconazole disk test were based on zone diameter of growth inhibition (dz) reported by Barry et al. [5] and CLSI document M44-A [8] and reported as susceptible (S) for dz 19 mm; intermediate susceptible (I) for 15 < dz < 18 mm, and resistance (R) for dz 14 mm. The response to 5-FC and AmB was interpreted according to the manufacturer’ instructions and those published by Kabli [20] as susceptible (S) for dz 23 mm (5-FC) and dz 11 mm (AmB), intermediate susceptible (IS) for 18 < dz < 22 mm (5-FC), and resistant (R) for dz 17 mm (5-FC) and dz 10 mm (AmB).
Broth microdilution testing (CLSI method) Reference antifungal susceptibility testing of Candida isolates was performed using the broth microdilution method described in the Clinical Laboratory Standard Institute (CLSI) document M27-A using sterile disposable 96-well U shaped microplates [9]. Reference powders (Sigma-Aldrich, USA) of fluconazole (FCZ), itraconazole (ITR), ketoconazole (KCZ), voriconazole (VOR), amphotericin B (AmB), nystatin (NYS), 5-flucytosine (5-FC) and caspofungin (CAS) were used. Stock antifungal drug solutions were prepared in dimethyl sulfoxide for all drugs except nystatin and caspofungin which were prepared in distilled water. They were diluted with RPMI 1640 with L-glutamine, without bicarbonate (PAA, Austria) supplemented with 2% dextrose and buffered to pH 7.0 with 0.165M N- morpholinopropansulfonic acid (MOPS) (SigmaAldrich, USA) to obtain 2-fold of the final concentrations. The final concentrations of the antifungal drugs in microplates were 0.031—64 mg/mL for FCZ, 5-FC and CAS, and 0.031—16 mg/mL for the other antifungals. The drugcontaining microplates were inoculated with a suspension of yeast cells adjusted to the turbidity of a 0.5 McFarland standard and incubated for 48 h at 35 8C. MIC end points were read at the end of incubation period. Drug-free and yeastfree controls were included in the tests. Following incubation, the MICs for the azoles and 5-FC were defined as the lowest concentrations that produced a prominent decrease in turbidity compared with that of the drug-free controls. The MICs for AmB were determined as the complete inhibition of the fungal growth. The interpretative criteria for azoles and 5-FC were of those appeared in the documents of CLSI [9]. The MICs for CAS were defined as the lowest drug concentrations that produced a 50% reduction in fungal growth compared with that of the drug-free controls by visual inspection [21]. For visual reading of the MICs for NYS, the least concentration of the drug which produced an optically clear well was recorded [3]. The endpoints for the tested drugs which considered for the classification of the isolates as susceptible (S) or resistant (R) followed the values stated by CLSI are summarized in Table 1.
Results Epidemiological trends and species distribution Clinical observations let to the presumptive identification of candidiasis in outpatients were discolored deformed nails,
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M. Razzaghi-Abyaneh et al.
Table 1 Interpretive guideline for in vitro susceptibility testing of Candida species. ´ tation des tests de sensibilite ´ in vitro des espe ` ces de Candida. Guide d’interpre Antifungal agent
Susceptible (S) MIC (mg/mL)
Susceptible dose-dependent (SDD) MIC (mg/mL)
Intermediate (I) MIC (mg/mL)
Resistant (R) MIC (mg/mL)
Fluconazole Ketoconazole Itraconazole Voriconazole Flucytosine
8 0.0625 0.125 0.5 4
16—32 0.125—0.5 0.25—0.5 1—2 —
— — — — 8—16
64 1 1 4 32
MIC: minimum inhibitory concentration.
paronychia and red-crusted skin lesions in cutaneous infections, excessive sputum, dyspnea and cough in respiratory tract infections, excessive purulent discharge and local inflammation in vaginitis, thrush and cheilitis in oral involvement and polyuria in urinary tract involvement. These observations were confirmed by observation of fungal elements (yeast cells and hyphae) in direct microscopy, isolation of Candida from culture and histopathological demonstration of the fungus in affected tissue specimens. Distribution of candidiasis according to the type of specimens, involved species, gender and age groups is shown in Table 2. Totally, 173 cases, 68 men (39.3%) and 105 women (60.7%) were identified as candidiasis. The mean age of patients was 45 years (range: 1 to 89 years). Candidiasis was predominantly found in the 30—59 years age group (85 cases, 49.1%). Nail scrapings including fingernail (63 cases, 85.1%) and toenail (11 cases, 14.9%), comprised the majority of specimens (74 cases, 42.8%) followed by skin scrapings (33 cases, 19.2%) and vaginal discharge (28 cases, 16.2%). Candidiasis were distributed as skin and nail infections (107 cases; 61.8%), vaginitis (28 cases; 16.2%), respiratory infections (26 cases; 15.0%), thrush (7 cases; 4.0%), urinary tract infection (2 cases; 1.2%), eye tumor (1 case; 0.6%), gastric involvement (1 case; 0.6%), and subcutaneous infection (1 case; 0.6%).
As indicated in Table 2, 72.3% of Candida isolates were identified as C. albicans according to positive results for CF and GTT tests as well as identical criteria on CHROMagar Candida and ID 32C assimilation test. Other identified species, which comprised totally 27.7% of the isolates. The most prevalent species in fingernail infection was C. albicans (41 cases, 65.1%), followed by C. parapsilosis (11 cases, 17.5%), C. tropicalis (3 cases, 4.8%), C. krusei (3 cases, 4.8%), C. guilliermondii (3 cases, 4.8%), C. intermedia (1 case, 1.5%) and C. glabrata (1 case, 1.5%). For toenail specimens, C. parapsilosis (6 cases, 54.5%) was the most prevalent species followed by C. albicans (2 cases, 18.2%), C. glabrata (1 case, 9.1%), C. guilliermondii (1 case, 9.1%) and C. krusei (1 case, 9.1%). C. sake was isolated from urinary catheter.
Antifungal susceptibility patterns The in vitro susceptibility of identified Candida species to AmB, FCZ and 5-FC by disk diffusion is shown in Table 3. The majority of Candida species were susceptible to AmB (96.0%), 5-FC (85.0%) and FLZ (76.3%). All C. krusei isolates and 62.5% of C. glabrata isolates were resistant to FCZ. The MIC ranges and means of Candida species for the eight antifungal drugs are shown in Table 4. FCZ and CAS showed
Table 2 Distribution of Candida species among clinical specimens. ´ partition des espe ` ces de Candida dans les ´echantillons cliniques. Re Specimen
n
Candida species C. albicans (125)
C. parapsilosis (20)
C. glabrata (8)
C. krusei (8)
C. tropicalis (5)
C. guilliermondii (5)
C. sake (1)
C. intermedia (1)
Skin & nail scrapings Vaginal discharge Sputum Oral swabs Bronchoalveolar lavage Eye tumor Gastric juice Urine Tissue biopsy Urinary catheter
107 28 20 7 6 1 1 1 1 1
70 25 16 5 6 0 1 1 1 0
20 0 0 0 0 0 0 0 0 0
3 3 0 2 0 0 0 0 0 0
4 0 3 0 0 1 0 0 0 0
4 0 1 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 1
1 0 0 0 0 0 0 0 0 0
Total
173
125
20
8
8
5
5
1
1
Distribution and antifungal susceptibility of Candida species
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Table 3 Antifungal susceptibility of Candida isolates by disk diffusion (DD). ´ aux antifongiques de Candida par diffusion de disques (DD). Sensibilite Candida species (no. of isolates)
Antifungal susceptibility test by disk diffusion method Fluconazole S
C. C. C. C. C. C. C. C.
albicans (125) parapsilosis (20) glabrata (8) krusei (8) tropicalis (5) guilliermondii (5) sake (1) intermedia (1)
Total (173)
107 13 3 0 3 4 1 1
5-flucytosine I
(85.6) (65.0) (37.5) (0) (60.0) (80.0) (100) (100)
132 (76.3)
13 4 0 0 1 1 0 0
R (10.4) (20.0) (0) (0) (20.0) (20.0) (0) (0)
19 (11.0)
5 3 5 8 1 0 0 0
S (4.0) (15.0) (62.5) (100) (20.0) (0) (0) (0)
22 (12.7)
113 18 3 4 3 4 1 1
Amphotericin B
I (90.4) (90.0) (37.5) (50.0) (60.0) (80.0) (100) (100)
147 (85.0)
R 0 0 5 3 2 1 0 0
(0) (0) (62.5) (37.5) (40.0) (20.0) (0) (0)
11 (6.4)
12 2 0 1 0 0 0 0
S (9.6) (10.0) (0) (12.5) (0) (0) (0) (0)
15 (8.6)
121 19 8 6 5 5 1 1
R (96.8) (95.0) (100) (75.0) (100) (100) (100) (100)
166 (96.0)
4 1 0 2 0 0 0 0
(3.2) (5.0) (0) (25.0) (0) (0) (0) (0)
7 (4.0)
Values are given as n (%). S: susceptible; I: intermediate susceptible; R: resistant.
excellent antifungal activities against C. albicans, with MICs of 2 mg/mL for all the isolates. All 125 C. albicans isolates were susceptible to FCZ, while the susceptibility to the other tested antifungals for this species was recorded in the range of 40 to 95%. FCZ was also effective against C. parapsilosis, C. tropicalis, C. sake and C. intermedia. All C. parapsilosis isolates were susceptible to KTZ, FCZ and 5-FC. All C. tropicalis isolates were susceptible to FCZ. Two azoles i.e. VOR and ITZ affected C. albicans at a higher percentage than that of the other Candida species. Antifungal susceptibility of the Candida species to tested antifungals for which CLSI breakpoints are available (Azoles and 5-FC) is summarized in Table 5. According to the obtained susceptibility rates in comparison with CLSI break points mentioned in Table 1, majority of the isolates were susceptible to FCZ (95.4%) followed by 5-FC (89.6%), VOR (78.6%), ITR (48.0%) and KCZ (42.8%). The highest resistance rate (34.0%) among the tested antifungals was reported for KCZ. Resistance to FCZ was reported only for C. krusei, to VOR for C. albicans and C. krusei, to ITR and KCZ for C. albicans, C. krusei, C. glabrata and C. guilliermondii and to 5-FC for only C. albicans. Among 125 C. albicans isolates, two were FCZ-susceptible VOR-resistant which both were isolated from sputum samples. Two C. krusei isolates from sputum and eye tumor and two C. glabrata isolates from oral swab and vaginal discharge were reported to be resistant to caspofungin.
Discussion In the present study, a total number of 173 cases of candidiasis were reported in outpatients referred to our laboratory during January 2010 to December 2011 by involving eight species of Candida as the etiologic agents. The prevalence of candidiasis was found to be different in various gender and age groups. Women were more affected than men. The most frequent site of candidiasis in both genders was nail, followed by skin and vagina. Candidal onychomycosis were frequently found in the age group of 30 to 59 years. Fingernail
infections were more prevalent in women probably due to the more humidity at home, while, the higher occurrence of toenail candidiasis in men may be a consequence of exposure to outdoors with greater physical activity. These results were in accordance with Aghamirian and Ghiasian [2], and in contrast to a published report from Turkey [17]. Our results showed that C. albicans was the main cause in nail and skin specimens, followed by C. parapsilosis. Similar results have been reported in separate studies in Iran [2] and Slovakia [18]. In contrast, C. parapsilosis reported as the most prevalent species from Mexico [26]. Differences in epidemiological trends of candidiasis may be attributed to various factors, of which lifestyle is an important component. Although the incidence of non-albicans Candida species at different types of candidiasis is dramatically increased in recent years, our results showed that C. albicans was the most prevalent species (72.3%), followed by C. parapsilosis (11.5%) as etiologic agents of superficial candidiasis. C. albicans has been reported as the most predominant species of superficial candidiasis in Iran, Turkey and India [1,7,13,27,33,35,36,38]. In the present study, the second commonest agent in skin and nail infections was C. parapsilosis (18.7%) and in vaginal discharge, C. glabrata (10.7%). This is in agreement with the results of separate studies in Tehran-Iran during 2004 to 2008 [24,39]. In a report in the year 2005, C. tropicalis was identified as the most important causative agent of candidiasis in India [33]. In Turkey and India, C. glabrata was reported as the major cause of vulvovaginal candidiasis [15,16,19]. In Ghana as an African country, next to C. albicans, C. glabrata and C. tropicalis were reported as the predominant species [14]. In a recent study from the United States, non-albicans species, particularly C. glabrata were reported as the most prevalent cause of the candidiasis. We did not isolate any C. dubliniensis in the examined specimens. However, Badiee et al. reported C. dubliniensis (13.3% of the total isolate) in mucosal specimens from Shiraz-Iran [4]. Susceptibility patterns of Candida species to tested antifungals revealed that FCZ was the most potent antifungal
Candida species
n (%)
MIC ranges (means) (mg/mL) AmB
C. C. C. C. C. C. C. C.
albicans parapsilosis krusei glabrata tropicalis guilliermondii sake intermedia
125 20 8 8 5 5 1 1
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Table 4 The MIC ranges and means of Candida species for the eight antifungal agents determined by broth microdilution test (Clinical Laboratory Standard Institute, CLSI). ´ termine ´ es par le test de microdilution (Clinical Laboratory Standard Institute, CLSI). Intervalles et moyennes de la CMI de Candida pour les huit agents antifongiques de
(72.3) (11.5) (4.6) (4.6) (2.9) (2.9) (0.6) (0.6)
FCZ
2—4 4—8 2—4 2—4 4—8 0.25—4 4 16
(3.6) (4.4) (3.0) (3.0) (6.4) (1.7) (4.0) (16.0)
ITR
0.125—2 0.5—2 8—64 2—32 0.125—1 2—16 0.5 2
(0.4) (1.2) (36.) (9.5) (0.6) (5.2) (0.5) (2.0)
KCZ
0.031—4 0.031—0.5 0.031—1 0.25—1 0.031—1 0.25—1 0.031 8
(0.3) (0.3) (0.7) (0.4) (0.4) (0.7) (0.03) (8.0)
5-FC
0.031—8 0.031—0.062 0.062—1 0.031—4 0.062—0.25 0.031—4 0.031 0.031
(4.0) (0.05) (0.7) (0.6) (0.1) (0.8) (0.03) (0.03)
VOR
0.031—32 1—4 0.25—8 2—16 0.031—16 0.062—8 1 1
(0.7) (2.1) (3.6) (6.0) (3.4) (3.6) (1.0) (1.0)
0.125—8 1—2 0.25—8 0.5—2 0.5—2 0.062—2 1 1
NYS (0.5) (1.5) (2.9) (0.7) (1.0) (1.2) (1.0) (1.0)
2—8 2—4 2—8 4 2—4 2—4 4 16
CAS (3.2) (3.0) (4.0) (4.0) (3.2) (2.8) (4.0) (16.0)
0.062—1 0.25—4 0.062—8 1—16 0.25—1 0.062—4 1 1
(0.2) (1.8) (2.2) (5.0) (0.5) (1.6) (1.0) (1.0)
MIC: minimum inhibitory concentration; AmB: amphotericin B; FCZ: fluconazole; ITR: itraconazole; KCZ: ketoconazole; 5-FC: 5-flucytosine; VOR: voriconazole; NYS: nystatin; CAS: caspofungin.
Table 5 In vitro susceptibility of 173 Candida isolates to fluconazole (FCZ), voriconazole (VOR), itraconazole (ITR), ketoconazole (KCZ) and 5-flucytosine (5-FC) according to Clinical Laboratory Standard Institute (CLSI) breakpoints. ´ in vitro de 173 isolats de Candida au fluconazole (FCZ), le voriconazole (VOR), l’itraconazole (ITR), le ke ´ toconazole (KCZ) et 5-flucytosine (5-FC) en fonction des Sensibilite concentrations critiques du Clinical Laboratory Standard Institute (CLSI). Candida species (no.)
Susceptibility (%) FCZ
albicans (125) parapsilosis (20) krusei (8) glabrata (8) tropicalis (5) guilliermondii (5) sake (1) intermedia (1)
Total (173)
ITR
KCZ
5-FC
S
SDD
R
S
SDD
R
S
SDD
R
S
SDD
R
S
I
R
100 100 50 75 100 60 100 100
0 0 25 25 0 40 0 0
0 0 25 0 0 0 0 0
96 0 62.5 62.5 80 40 0 0
2.4 100 0 37.5 20 60 100 100
1.6 0 37.5 0 0 0 0 0
48 65 50 0 60 40 100 0
48 35 25 37.5 40 40 0 0
4 0 25 62.5 0 20 0 100
40 55 25 37.5 60 60 100 100
28 45 25 12.5 40 20 0 0
33 0 50 50 0 20 0 0
92 100 62.5 75 80 60 100 100
4 0 37.5 25 20 40 0 0
4 0 0 0 0 0 0 0
78.6
18.5
2.9
48
43.9
8.1
42.8
28.9
28.3
89.6
7.5
95.4
3.5
1.1
S: susceptible; SDD: susceptible-dose-dependent; I: intermediate; R: resistant.
2.9
M. Razzaghi-Abyaneh et al.
C. C. C. C. C. C. C. C.
VOR
Distribution and antifungal susceptibility of Candida species agent overall. The highest resistance rate (34%) among the tested antifungals was reported for KCZ. Resistance to FCZ and VOR was reported only for C. krusei, to ITR for all species except C. parapsilosis, C. tropicalis and C. sake, to KCZ for C. albicans, C. krusei, C. glabrata and C. guilliermondii and to 5-FC for only C. albicans. Totally, the resistance level of the isolates to 5-FC and FCZ were low (2 to 3%). Similar patterns of antifungal susceptibility has been reported in Slovak Republic and Spain [22,30]. There are several reports about increasing resistance of Candida species to azoles [12,31]. Our results showed that the rate of resistance to commonly used azoles was higher than that of reported in the United States [23] and Portugal [23,37]. Although FCZ was reported as the best antifungal agent in our report affecting the growth of the majority of isolates, however, resistance to this drug was common among isolates of C. krusei. It has been shown that he intrinsic resistance manifested by this species may be underrepresented by the in vitro results. Five of eight C. glabrata isolates were resistant to FCZ in our study, while all of C. glabrata isolates reported from Ghana in 2012 were susceptible to FCZ [14]. In a study conducted during 2006— 2008, all Candida species were reported as sensitive isolates to AmB, KCZ, NYS, VOR and CAS, whereas in some cases resistance to FCZ and ITR was noticed [4]. Zomorodian et al. [40] reported the highest rate of resistance to KCZ for C. glabrata which is in accordance to our results. In the present study, we reported two C. albicans isolates with as fluconazole-susceptible voriconazole-resistant strains, both were isolated from sputum. Although this is an unusual event, some other researchers have reported such strains from other parts of the world. As a good example, Pfaller et al. reported one fluconazole-susceptible voriconazole-resistant and also 14 fluconazole-SDD voriconazoleresistant strains among 7725 C. albicans isolates tested [32]. Further works on the molecular mechanisms leading to resistance to azoles are needed to explain such extraordinary pattern of susceptibility. As an interesting result, two C. krusei isolates from sputum and eye tumor and two C. glabrata isolates from oral swab and vaginal discharge were reported to be resistant to caspofungin. The patients’ history of antifungal treatment is not clear. However, this resistance may be attributed to a previous exposure of the patients to candins. A good correlation was reported between disk diffusion and MIC results in broth microdilution for 5-FC and FCZ but not for AmB for the majority of isolated Candida species in the present study. Disk diffusion method is useful for the determination of antifungal resistance, but is not so reliable for differentiation between susceptible and SDD fungal species. The major feature of disk diffusion as an agar-based test is that it can allow a quick answer concerning Candida resistance to antifungal agents, preventing unnecessary patient’s drug abuse. However, concerning drugs like as amphotericin B, data on such agar-based tests are still scarce and interpretation of results is a matter of controversy. CLSI published a specific standard for disk diffusion testing of fluconazole (M44-A), which considerably increased the possibility of implementing it in all routine laboratories. Further research must be carried out to ensure the standardization of this method for other antifungal agents [28]. Among the more than 200 Candida species, only a few can cause human diseases. C. intermedia and C. sake are rarely
e49 reported as a human pathogen and importance of these species is unknown [6]. In the present study, C. intermedia and C. sake were identified in nail scrapings and urinary catheter, respectively. These species must be further confirmed by complementary methods such as DNA sequencing. Taken together, results of the present study reinforce the importance of superficial candidiasis as a serious debilitating fungal infection with multiple clinical presentations. Our findings further indicate that susceptibility testing to commonly used antifungals is crucial in order to select the appropriate therapeutic strategy, which minimizes complications while improving patients’ life. The regular investigation on antifungal susceptibility in medical centers should be performed in order to promote the effective management of candidiasis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements This work was supported financially by the Pasteur Institute of Iran. Authors gratefully thank Manijeh Yousefi-Behzadi from department of epidemiology of the Pasteur Institute of Iran for kind editing of the French language.
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e50 [10] Colombo AL, Guimaraes T, Aranha Camargo LF, Richtmann R. Brazilian guidelines for the management of candidiasis - a joint meeting report of three medical societies: sociedade brasileira de infectologia, sociedade paulista de infectologia and sociedade brasileira de medicina tropical. Infect Dis 2013;17:283— 312. [11] Cornet M, Sendid B, Fradin C, Gaillardin C. Molecular identification of closely related Candida species using two ribosomal intergenic spacer fingerprinting methods. J Mol Diagn 2011;13:12—22. [12] Ellepola AN, Samaranayake LP. Oral candidal infections and antimycotics. Crit Rev Oral Biol Med 2000;11:172—98. [13] Esmaeilzadeh S, Mahdavi Omran S, Rahmani Z. Frequency and etiology of vulvovaginal candidiasis in women referred to a gynecological center in Babol, Iran. Int J Fertil Steril 2009;3:74—7. [14] Feglo PK, Narkwa P. Prevalence and antifungal susceptibility patterns of yeast isolates at the Komfo Anokye Teaching Hospital (KATH), Kumasi, Ghana. Br Microbiol Res J 2012;2:10—22. [15] Goswami D, Rabha B, Baruah S, Lourembam SD, Sawian CE, Baruah D. Pattern of Candida species isolated from patients with diabetes mellitus and vulvovaginal candidiasis and their response to single dose oral 5-flucytosine therapy. J Infect 2006;52:111—7. [16] Ilkit M. Onychomycosis in Adana, Turkey: a 5-year study. Int J Dermatol 2005;44:851—4. [17] Ilkit M, Hilmioglu S, Tasbakan M, Aydemir S. Evaluation of Albicans ID2 and Biggy agar for the isolation and direct identification of vaginal yeast isolates. Med Microbiol 2007;56: 762—765. [18] Jautova J, Viragova M, Ondrasovic E, Holoda E. Incidence of Candida species isolated from human skin and nails: a survey. Folia Microbiol 2001;46:333—7. [19] Jindal N, Gill P, Aggarwal A. An epidemiological study of vulvovaginal candidiasis in women of childbearing age. Indian J Med Microbiol 2007;25:175—6. [20] Kabli SA. In vitro susceptibilities of clinical yeast isolates to antifungal drugs of polyene, pyrimidine, and azoles, and their effect in yeast adhesion and mycelial formation. Saudi J Biol Sci 2008;15:189—98. [21] Kalkanci A, Berk E, Aykan B, Caglar K, Hizel K. Epidemiology and antifungal susceptibility of Candida species isolated from hospitalized patients. J Mycol Med 2007;17:16—20. [22] Kovacicova G, Krupova Y, Lovaszova M, Roidova A, Trupl J, Liskova A, et al. Antifungal susceptibility of 262 bloodstream yeast isolation from a mixed cancer and non-cancer patient population: is there a correlation between in vitro resistance to 5-flucytosine and the outcome of fungemia? J Infect Chemother 2000;6:216—9. [23] Lyon GM, Karatela S, Sunay S, Adiri Y. Antifungal susceptibility testing of Candida isolates from the Candida surveillance study. J Clin Microbiol 2010;48:1270—5. [24] Mahmoudi Rad M, Zafarghandi AS, Amel Zabihi M, Mirdamadi Y, Rahbarian N, Abbasabadi B, et al. Identification of Candida species associated with vulvovaginal candidiasis by multiple PCR method. Infect Dis Obstet Gynecol 2012 [Article ID 872169, doi:10.1155/2012/872169]. [25] Mandviwala T, Shinde R, Kalra A, Sobel JD, Akins RA. Highthroughput identification and quantification of Candida species using high resolution derivative melt analysis of amplicons. J Mol Diagn 2010;12:91—101.
M. Razzaghi-Abyaneh et al. [26] Manzano-Gayosso P, Mendez-Tovar LJ, Arenas R, HernandezHernandez F, Millan-Chio B, Torres-Rodriguez JM. Onychomycosis-causing yeasts in four Mexican dermatology centers and their antifungal susceptibility to azolic compounds. Rev Iberoam Micol 2011;28:32—5. [27] Nazeri M, Mesdaghinia E, Moraveji SAR, Atabakhshiyan R, Soleymani F. Prevalence of vulvovaginal candidiasis and frequency of Candida species in women referred to gynecology obstetrics clinic at Kashan-Iran since 2008 to 2010. J Mazand Univ Med Sci 2012;22:262—9. [28] Negri M, Henriques M, Svidzinski TIE. Correlation between Etest, disk diffusion and microdilution method for antifungal susceptibility testing of Candida species from infection and colonization. J Clin Lab Anal 2009;23:324—30. [29] Olaya-Abril A, Jimenez-Munguia I, Gomez-Gascon L, RodriguezOrtega MJ. Surfomics: shaving live organisms for a fast proteomics identification of surface proteins. J Proteomics 2014;97:164—76. [30] Peman J, Canton E, Quindos G, Eraso E, Alcoba J, Yague G. Epidemiology, species distribution and in vitro antifungal susceptibility of fungaemia in a Spanish multicentre prospective survey. J Antimicrob Chemother 2012;67:1181—7. [31] Pfaller MA, Jones RN, Doern GV, Sader HS, Messer SA, Houston A, et al. Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and Latin America, 1997—1998. Antimicrob Agents Chemother 2000;44:747—51. [32] Pfaller MA, Messer SA, Boyken L, et al. Use of fluconazole as a surrogate marker to predict susceptibility and resistance to voriconazole among 13,338 clinical isolates of Candida spp. tested by clinical and laboratory standards institute-recommended broth microdilution methods. J Clin Microbiol 2007;45:70—5. [33] Rajinder Capoor M, Nair D, Deb M, Kumar Verma P, Srivastava L, Aggarwal P. Emergence of non-albicans Candida species and antifungal resistance in a tertiary care hospital. Jpn J Infect Dis 2005;58:344—8. [34] Roderick JH. The management of superficial candidiasis. J Am Acad Dermatol 1999;40:S35—42. [35] Sadeghi G, Abouei M, Alirezaee M, Tolouei R, Shams-Ghahfarokhi M, Mostafavi M, et al. A 4-year survey of dermatomycoses in Tehran from 2006 to 2009. J Mycol Med 2011;21:260—5. [36] Shokohi T, Bandalizadeh Z, Hedayati MT, Mayahi S. In vitro antifungal susceptibility of Candida species isolated from oropharyngeal lesions of patients with cancer to some antifungal agents. Jundishapur J Microbiol 2011;4:S19—26. [37] Silva AP, Miranda IM, Lisboa C, Pina-Vas C, Rodrigues AG. Prevalence, distribution and antifungal susceptibility profiles of Candida parapsilosis, C. orthopsilosis and C. metapsilosis in a tertiary care hospital. J Clin Microbiol 2009;47:2392—7. [38] Zaini F, Gerami Shoar M, Kordbacheh P, Khedmati E, Safara M, Gharaeian N. Performance of five phenotypical methods for identification of Candida isolates from clinical materials. Iranian J Publ Health 2006;35:25—32. [39] Zaini F, Mahmoudi M, Mehbod ASA, Kordbacheh P, Safara M. Fungal nail infection in Tehran, Iran. Iranian J Publ Health 2009;38:46—53. [40] Zomorodian K, Rahimi MJ, Pakshir K, Motamedi M, Ghiasi MR, Rezashah H. Determination of antifungal susceptibility pattern among the clinical isolates of Candida species. J Global Infect Dis 2011;3:357—60.
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Species distribution and antifungal susceptibility of Candida spp. isolated from superficial candidiasis in outpatients in Iran ` ces et sensibilite ´ aux antifongiques de Distribution des espe ´ de candidose superficielle chez les malades en Candida spp. isole consultation externe en Iran M. Razzaghi-Abyaneh a,*, G. Sadeghi a, E. Zeinali a, M. Alirezaee a, M. Shams-Ghahfarokhi b, A. Amani c, R. Mirahmadi d, R. Tolouei a a
Department of Medical Mycology, Pasteur Institute of Iran, 13164 Tehran, Iran Department of Medical Mycology, Faculty of Medical Sciences, Tarbiat Modares University, 14115-331 Tehran, Iran c Medicinal Chemistry Laboratory, Pasteur Institute of Iran, 13164 Tehran, Iran d Laboratory of Arboviruses and Viral Hemorrhagic Fevers, Pasteur Institute of Iran, 13164 Tehran, Iran b
Received 21 April 2013; received in revised form 4 December 2013; accepted 7 January 2014 Available online 21 April 2014
KEYWORDS Candidiasis; Candida species; Antifungal drugs; Broth microdilution; CHROMagar Candida; Disk diffusion
Summary Objective. — Candidiasis is the most prevalent fungal infection affecting human and animals all over the world. This study represents the epidemiological aspects of superficial candidiasis in outpatients and in vitro antifungal susceptibility of etiologic Candida species. Patients and methods. — Clinical samples were taken from 173 patients including skin and nail scrapings (107; 61.8%), vaginal discharge (28; 16.2%), sputum (20; 11.6%), oral swabs (7; 4.0%), bronchoalveolar lavage (6; 3.5%) and 1 specimen (0.6%) of each eye tumor, gastric juice, urine, biopsy and urinary catheter and confirmed as candidiasis by direct microscopy, culture and histopathology. Susceptibility patterns of the isolated Candida species were determined using the disk diffusion and broth microdilution methods. Results. — Among 173 Candida isolates, C. albicans (72.3%) was the most prevalent species followed by C. parapsilosis (11.5%). Other identified species were C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. intermedia and C. sake. Majority of the Candida isolates were susceptible to fluconazole (95.4%) followed by 5-flucytosine (89.6%), voriconazole (78.6%)
* Corresponding author. E-mail addresses: [email protected], [email protected] (M. Razzaghi-Abyaneh). 1156-5233/$ — see front matter # 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.mycmed.2014.01.004
e44
M. Razzaghi-Abyaneh et al. itraconazole (48.0%) and ketoconazole (42.8%). Caspofungin was the most potent antifungal drug against C. albicans (MICs; 0.062—1 mg/mL), ketoconazole for C. parapsilosis and C. tropicalis (MICs; 0.031—0.25 mg/mL) and itraconazole for C. krusei, C. glabrata and C. guilliermondii (MICs; 0.031—1 mg/mL). Conclusion. — This study reinforces the significance of superficial candidiasis as an important fungal infection with multiple clinical presentations. Our results further indicate that susceptibility testing to commonly used antifungals is crucial in order to select the appropriate therapeutic strategies which minimize complications while improving patients’ life. # 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Candidose ; Espèces de Candida ; Antifongiques ; Microdilution ; CHROMagar Candida ; Diffusion des disques
Re ´sume ´ Objectif. — La candidose est l’infection fongique la plus courante chez les animaux et l’homme partout dans le monde. Cette étude présente les aspects épidémiologiques de la candidose superficielle chez les patients et la sensibilité antifongique in vitro des espèces de Candida isolées. Patients et me ´thodes. — Les prélèvements cliniques ont été effectués sur 173 patients, il s’agit de la peau et des ongles (107 ; 61,8 %), des secrétions vaginales (28 ; 16,2 %), de l’expectoration (20 ; 11,6 %), de prélèvements oraux (7 ; 4,0 %), de lavage broncho-alvéolaire (6 ; 3,5 %) et d’un échantillon (0,6 %) de tumeur de l’œil, de suc gastrique, d’urine, de biopsie et d’un cathéter urinaire. Ils ont été confirmés par l’examen direct, la culture et l’histopathologie comme candidose. La sensibilité des souches de Candida isolées a été déterminée par la méthode des disques et la méthode de microdilution. Re ´sultats. — Parmi 173 isolats de Candida, l’espèce la plus fréquente a été C. albicans (72,3 %), suivie par C. parapsilosis (11,5 %). Les autres espèces identifiées ont été C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. intermedia et C. sake. La majorité des isolats de Candida s’est montrée sensible au fluconazole (95,4 %), suivie par la 5-flucytosine (89,6 %), le voriconazole (78,6 %), l’itraconazole (48,0 %) et le kétoconazole (42,8 %). La caspofungine a été le médicament le plus efficace contre C. albicans (CMI ; 0,062—1 mg/mL), le kétoconazole pour C. parapsilosis et C. tropicalis (CMI ; 0,031—0,25 mg/mL) et l’itraconazole pour C. krusei, C. glabrata et C. guilliermondii (CMI ; 0,031—1 mg/mL). Conclusion. — Cette étude montre l’importance de la candidose superficielle ayant des présentations cliniques différentes. Les résultats obtenus par cette étude montrent la nécessité de faire le test de sensibilité aux médicaments couramment utilisés pour choisir les meilleures stratégies de traitement en vue de diminuer les complications de la maladie et améliorer la qualité de vie des patients. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Worldwide occurrence of fungal infections has dramatically been increased in recent years due to a continuous augment in immunosuppressive conditions like AIDS, and other predisposing factors including organ transplantation, leukemia, broadspectrum antibiotics, indwelling catheters, diabetes and intravenous drug misuse. Superficial mycoses caused by species of the genus Candida has been reported from all over the world. They include some of the most common mucosal infections, such as thrush, vaginal candidiasis, cutaneous candidiasis, onychomycosis and chronic mucocutaneous candidiasis [34]. It has been estimated that an approximate of 20 to 30% of women have vaginal Candida colonization. The prevalence of oral cavity colonization by yeasts in normal individuals varies, from 20 to 40% in the general population. In the medical community, oral candidiasis and vaginitis caused by Candida account for a significant number of clinical complaints brought to colleagues of different specialties. The prevalence of carriage is greater in hospitalized patients and in those with conditions that predispose to candidiasis. Among Candida species, C. albicans is responsible for the majority cases of superficial candidiasis. However, increasing
numbers of other species such as C. tropicalis, C. parapsilosis, C. glabrata, C. guilliermondii have been reported as common etiologic agents [10,34]. Different methods with a wide range of successfulness have been used for identification of Candida species isolated from clinical specimens. Aside from conventional morphological and biochemical methods, a vast array of novel techniques including molecular assays, MALDI-TOF mass spectrometry, specific antibody-based agglutination techniques and omics analyses have been successfully used for identification of Candida species and clinical candidiasis [11,25,29]. A combination of such methods increases considerably the chance of reliable identification of Candida at the genus and species level [20]. Besides the necessity for identification of Candida isolates at species level, dramatic emergence of multidrug resistance urged the scientists to determine susceptibility patterns of this commensal pathogen of human and animals to commonly used antifungal agents. In this way, isolation and identification of causative Candida species and determination of their susceptibility to antifungal drugs is important for ensuring the efficacy of therapeutic strategies. Like as many other parts of the world, fungal infections are major concerns in Iran with an increasing numbers of new
Distribution and antifungal susceptibility of Candida species reports of superficial to deep infections every year. Although Candida species has been a subject of several studies in Iran [1,2,4,13,27,40], there is no comprehensive data gathering of all aspects of Candida infections from clinic to the laboratory. In our previous 4-year study on the prevalence and distribution of superficial fungal infections, candidiasis comprised about 16.0% of total dermatomycoses in Tehran [35]. The present study was designed to investigate: the epidemiological trends of candidiasis among patients who referred to the medical mycology department of Pasteur Institute of Iran; to determine species distribution and antifungal susceptibility patterns of Candida isolates against commonly used antifungal drugs from major classes i.e. polyenes, azoles and echinocandins with therapeutic values for different types of candidiasis.
Patients and methods Sample preparation and Candida identification During a two-year period from January 2010 to December 2011, 173 clinical specimens from outpatients suspected to candidiasis referred to the department of medical mycology, Pasteur Institute of Iran (a reference center for fungal identification and antifungal susceptibility testing) by Imam Khomeini hospital as a major center of infectious diseases in Tehran were examined. Candidiasis was proven for all the specimens by a combination of clinical features, direct microscopy, culture and histopathology. The specimens were placed directly onto a microscope slide, covered with 20% potassium hydroxide (KOH) and examined under a light microscope for fungal elements. A portion of the specimens were inoculated on Sabouraud dextrose agar (SDA; E. Merck, Germany) supplemented with 0.005% chloramphenicol and incubated for 2 weeks at 28 8C. By visual inspection, colonies suspected to Candida were isolated for further confirmation and species identification by a combination of germ tube test (GTT), chlamydospore formation (CF) on cornmeal agar plus tween 80 (Becton Dickinson, France), chromogenic assay on CHROMagar Candida (CHROMagar Candida, France), carbohydrate assimilation in ID32C system (bioMeriéux, France) and specific monoclonal antibody-based agglutination assays (Fumouze diagnostics, France). All identified Candida species are now available at Pathogenic Fungi Culture Collection of the Pasteur Institute of Iran (www.http://en.pasteur.ac.ir/pages.aspx?id=586).
Antifungal susceptibility testing Disk diffusion assay Candida isolates were evaluated for antifungal susceptibility to commonly used antifungal disks (6.0 mm Dia., MAST Diagnostics), i.e. amphotericin B (AmB; 20 mg), fluconazole (FCZ; 25 mg) and 5-flucytosine (5-FC; 1 mg) by the disk diffusion method on Mueller Hinton agar (MHA) supplemented with 2% glucose and 0.5 mg/mL methylene blue. Agar plates were swab inoculated with a suspension of yeast cells adjusted to the turbidity of a 0.5 McFarland standard. The plates were
e45 allowed to dry for 5 to 15 minutes and antifungal disks were placed on the agar surface. The plates were incubated for 24 h at 35 8C. The interpretive criteria for the fluconazole disk test were based on zone diameter of growth inhibition (dz) reported by Barry et al. [5] and CLSI document M44-A [8] and reported as susceptible (S) for dz 19 mm; intermediate susceptible (I) for 15 < dz < 18 mm, and resistance (R) for dz 14 mm. The response to 5-FC and AmB was interpreted according to the manufacturer’ instructions and those published by Kabli [20] as susceptible (S) for dz 23 mm (5-FC) and dz 11 mm (AmB), intermediate susceptible (IS) for 18 < dz < 22 mm (5-FC), and resistant (R) for dz 17 mm (5-FC) and dz 10 mm (AmB).
Broth microdilution testing (CLSI method) Reference antifungal susceptibility testing of Candida isolates was performed using the broth microdilution method described in the Clinical Laboratory Standard Institute (CLSI) document M27-A using sterile disposable 96-well U shaped microplates [9]. Reference powders (Sigma-Aldrich, USA) of fluconazole (FCZ), itraconazole (ITR), ketoconazole (KCZ), voriconazole (VOR), amphotericin B (AmB), nystatin (NYS), 5-flucytosine (5-FC) and caspofungin (CAS) were used. Stock antifungal drug solutions were prepared in dimethyl sulfoxide for all drugs except nystatin and caspofungin which were prepared in distilled water. They were diluted with RPMI 1640 with L-glutamine, without bicarbonate (PAA, Austria) supplemented with 2% dextrose and buffered to pH 7.0 with 0.165M N- morpholinopropansulfonic acid (MOPS) (SigmaAldrich, USA) to obtain 2-fold of the final concentrations. The final concentrations of the antifungal drugs in microplates were 0.031—64 mg/mL for FCZ, 5-FC and CAS, and 0.031—16 mg/mL for the other antifungals. The drugcontaining microplates were inoculated with a suspension of yeast cells adjusted to the turbidity of a 0.5 McFarland standard and incubated for 48 h at 35 8C. MIC end points were read at the end of incubation period. Drug-free and yeastfree controls were included in the tests. Following incubation, the MICs for the azoles and 5-FC were defined as the lowest concentrations that produced a prominent decrease in turbidity compared with that of the drug-free controls. The MICs for AmB were determined as the complete inhibition of the fungal growth. The interpretative criteria for azoles and 5-FC were of those appeared in the documents of CLSI [9]. The MICs for CAS were defined as the lowest drug concentrations that produced a 50% reduction in fungal growth compared with that of the drug-free controls by visual inspection [21]. For visual reading of the MICs for NYS, the least concentration of the drug which produced an optically clear well was recorded [3]. The endpoints for the tested drugs which considered for the classification of the isolates as susceptible (S) or resistant (R) followed the values stated by CLSI are summarized in Table 1.
Results Epidemiological trends and species distribution Clinical observations let to the presumptive identification of candidiasis in outpatients were discolored deformed nails,
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M. Razzaghi-Abyaneh et al.
Table 1 Interpretive guideline for in vitro susceptibility testing of Candida species. ´ tation des tests de sensibilite ´ in vitro des espe ` ces de Candida. Guide d’interpre Antifungal agent
Susceptible (S) MIC (mg/mL)
Susceptible dose-dependent (SDD) MIC (mg/mL)
Intermediate (I) MIC (mg/mL)
Resistant (R) MIC (mg/mL)
Fluconazole Ketoconazole Itraconazole Voriconazole Flucytosine
8 0.0625 0.125 0.5 4
16—32 0.125—0.5 0.25—0.5 1—2 —
— — — — 8—16
64 1 1 4 32
MIC: minimum inhibitory concentration.
paronychia and red-crusted skin lesions in cutaneous infections, excessive sputum, dyspnea and cough in respiratory tract infections, excessive purulent discharge and local inflammation in vaginitis, thrush and cheilitis in oral involvement and polyuria in urinary tract involvement. These observations were confirmed by observation of fungal elements (yeast cells and hyphae) in direct microscopy, isolation of Candida from culture and histopathological demonstration of the fungus in affected tissue specimens. Distribution of candidiasis according to the type of specimens, involved species, gender and age groups is shown in Table 2. Totally, 173 cases, 68 men (39.3%) and 105 women (60.7%) were identified as candidiasis. The mean age of patients was 45 years (range: 1 to 89 years). Candidiasis was predominantly found in the 30—59 years age group (85 cases, 49.1%). Nail scrapings including fingernail (63 cases, 85.1%) and toenail (11 cases, 14.9%), comprised the majority of specimens (74 cases, 42.8%) followed by skin scrapings (33 cases, 19.2%) and vaginal discharge (28 cases, 16.2%). Candidiasis were distributed as skin and nail infections (107 cases; 61.8%), vaginitis (28 cases; 16.2%), respiratory infections (26 cases; 15.0%), thrush (7 cases; 4.0%), urinary tract infection (2 cases; 1.2%), eye tumor (1 case; 0.6%), gastric involvement (1 case; 0.6%), and subcutaneous infection (1 case; 0.6%).
As indicated in Table 2, 72.3% of Candida isolates were identified as C. albicans according to positive results for CF and GTT tests as well as identical criteria on CHROMagar Candida and ID 32C assimilation test. Other identified species, which comprised totally 27.7% of the isolates. The most prevalent species in fingernail infection was C. albicans (41 cases, 65.1%), followed by C. parapsilosis (11 cases, 17.5%), C. tropicalis (3 cases, 4.8%), C. krusei (3 cases, 4.8%), C. guilliermondii (3 cases, 4.8%), C. intermedia (1 case, 1.5%) and C. glabrata (1 case, 1.5%). For toenail specimens, C. parapsilosis (6 cases, 54.5%) was the most prevalent species followed by C. albicans (2 cases, 18.2%), C. glabrata (1 case, 9.1%), C. guilliermondii (1 case, 9.1%) and C. krusei (1 case, 9.1%). C. sake was isolated from urinary catheter.
Antifungal susceptibility patterns The in vitro susceptibility of identified Candida species to AmB, FCZ and 5-FC by disk diffusion is shown in Table 3. The majority of Candida species were susceptible to AmB (96.0%), 5-FC (85.0%) and FLZ (76.3%). All C. krusei isolates and 62.5% of C. glabrata isolates were resistant to FCZ. The MIC ranges and means of Candida species for the eight antifungal drugs are shown in Table 4. FCZ and CAS showed
Table 2 Distribution of Candida species among clinical specimens. ´ partition des espe ` ces de Candida dans les ´echantillons cliniques. Re Specimen
n
Candida species C. albicans (125)
C. parapsilosis (20)
C. glabrata (8)
C. krusei (8)
C. tropicalis (5)
C. guilliermondii (5)
C. sake (1)
C. intermedia (1)
Skin & nail scrapings Vaginal discharge Sputum Oral swabs Bronchoalveolar lavage Eye tumor Gastric juice Urine Tissue biopsy Urinary catheter
107 28 20 7 6 1 1 1 1 1
70 25 16 5 6 0 1 1 1 0
20 0 0 0 0 0 0 0 0 0
3 3 0 2 0 0 0 0 0 0
4 0 3 0 0 1 0 0 0 0
4 0 1 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 1
1 0 0 0 0 0 0 0 0 0
Total
173
125
20
8
8
5
5
1
1
Distribution and antifungal susceptibility of Candida species
e47
Table 3 Antifungal susceptibility of Candida isolates by disk diffusion (DD). ´ aux antifongiques de Candida par diffusion de disques (DD). Sensibilite Candida species (no. of isolates)
Antifungal susceptibility test by disk diffusion method Fluconazole S
C. C. C. C. C. C. C. C.
albicans (125) parapsilosis (20) glabrata (8) krusei (8) tropicalis (5) guilliermondii (5) sake (1) intermedia (1)
Total (173)
107 13 3 0 3 4 1 1
5-flucytosine I
(85.6) (65.0) (37.5) (0) (60.0) (80.0) (100) (100)
132 (76.3)
13 4 0 0 1 1 0 0
R (10.4) (20.0) (0) (0) (20.0) (20.0) (0) (0)
19 (11.0)
5 3 5 8 1 0 0 0
S (4.0) (15.0) (62.5) (100) (20.0) (0) (0) (0)
22 (12.7)
113 18 3 4 3 4 1 1
Amphotericin B
I (90.4) (90.0) (37.5) (50.0) (60.0) (80.0) (100) (100)
147 (85.0)
R 0 0 5 3 2 1 0 0
(0) (0) (62.5) (37.5) (40.0) (20.0) (0) (0)
11 (6.4)
12 2 0 1 0 0 0 0
S (9.6) (10.0) (0) (12.5) (0) (0) (0) (0)
15 (8.6)
121 19 8 6 5 5 1 1
R (96.8) (95.0) (100) (75.0) (100) (100) (100) (100)
166 (96.0)
4 1 0 2 0 0 0 0
(3.2) (5.0) (0) (25.0) (0) (0) (0) (0)
7 (4.0)
Values are given as n (%). S: susceptible; I: intermediate susceptible; R: resistant.
excellent antifungal activities against C. albicans, with MICs of 2 mg/mL for all the isolates. All 125 C. albicans isolates were susceptible to FCZ, while the susceptibility to the other tested antifungals for this species was recorded in the range of 40 to 95%. FCZ was also effective against C. parapsilosis, C. tropicalis, C. sake and C. intermedia. All C. parapsilosis isolates were susceptible to KTZ, FCZ and 5-FC. All C. tropicalis isolates were susceptible to FCZ. Two azoles i.e. VOR and ITZ affected C. albicans at a higher percentage than that of the other Candida species. Antifungal susceptibility of the Candida species to tested antifungals for which CLSI breakpoints are available (Azoles and 5-FC) is summarized in Table 5. According to the obtained susceptibility rates in comparison with CLSI break points mentioned in Table 1, majority of the isolates were susceptible to FCZ (95.4%) followed by 5-FC (89.6%), VOR (78.6%), ITR (48.0%) and KCZ (42.8%). The highest resistance rate (34.0%) among the tested antifungals was reported for KCZ. Resistance to FCZ was reported only for C. krusei, to VOR for C. albicans and C. krusei, to ITR and KCZ for C. albicans, C. krusei, C. glabrata and C. guilliermondii and to 5-FC for only C. albicans. Among 125 C. albicans isolates, two were FCZ-susceptible VOR-resistant which both were isolated from sputum samples. Two C. krusei isolates from sputum and eye tumor and two C. glabrata isolates from oral swab and vaginal discharge were reported to be resistant to caspofungin.
Discussion In the present study, a total number of 173 cases of candidiasis were reported in outpatients referred to our laboratory during January 2010 to December 2011 by involving eight species of Candida as the etiologic agents. The prevalence of candidiasis was found to be different in various gender and age groups. Women were more affected than men. The most frequent site of candidiasis in both genders was nail, followed by skin and vagina. Candidal onychomycosis were frequently found in the age group of 30 to 59 years. Fingernail
infections were more prevalent in women probably due to the more humidity at home, while, the higher occurrence of toenail candidiasis in men may be a consequence of exposure to outdoors with greater physical activity. These results were in accordance with Aghamirian and Ghiasian [2], and in contrast to a published report from Turkey [17]. Our results showed that C. albicans was the main cause in nail and skin specimens, followed by C. parapsilosis. Similar results have been reported in separate studies in Iran [2] and Slovakia [18]. In contrast, C. parapsilosis reported as the most prevalent species from Mexico [26]. Differences in epidemiological trends of candidiasis may be attributed to various factors, of which lifestyle is an important component. Although the incidence of non-albicans Candida species at different types of candidiasis is dramatically increased in recent years, our results showed that C. albicans was the most prevalent species (72.3%), followed by C. parapsilosis (11.5%) as etiologic agents of superficial candidiasis. C. albicans has been reported as the most predominant species of superficial candidiasis in Iran, Turkey and India [1,7,13,27,33,35,36,38]. In the present study, the second commonest agent in skin and nail infections was C. parapsilosis (18.7%) and in vaginal discharge, C. glabrata (10.7%). This is in agreement with the results of separate studies in Tehran-Iran during 2004 to 2008 [24,39]. In a report in the year 2005, C. tropicalis was identified as the most important causative agent of candidiasis in India [33]. In Turkey and India, C. glabrata was reported as the major cause of vulvovaginal candidiasis [15,16,19]. In Ghana as an African country, next to C. albicans, C. glabrata and C. tropicalis were reported as the predominant species [14]. In a recent study from the United States, non-albicans species, particularly C. glabrata were reported as the most prevalent cause of the candidiasis. We did not isolate any C. dubliniensis in the examined specimens. However, Badiee et al. reported C. dubliniensis (13.3% of the total isolate) in mucosal specimens from Shiraz-Iran [4]. Susceptibility patterns of Candida species to tested antifungals revealed that FCZ was the most potent antifungal
Candida species
n (%)
MIC ranges (means) (mg/mL) AmB
C. C. C. C. C. C. C. C.
albicans parapsilosis krusei glabrata tropicalis guilliermondii sake intermedia
125 20 8 8 5 5 1 1
e48
Table 4 The MIC ranges and means of Candida species for the eight antifungal agents determined by broth microdilution test (Clinical Laboratory Standard Institute, CLSI). ´ termine ´ es par le test de microdilution (Clinical Laboratory Standard Institute, CLSI). Intervalles et moyennes de la CMI de Candida pour les huit agents antifongiques de
(72.3) (11.5) (4.6) (4.6) (2.9) (2.9) (0.6) (0.6)
FCZ
2—4 4—8 2—4 2—4 4—8 0.25—4 4 16
(3.6) (4.4) (3.0) (3.0) (6.4) (1.7) (4.0) (16.0)
ITR
0.125—2 0.5—2 8—64 2—32 0.125—1 2—16 0.5 2
(0.4) (1.2) (36.) (9.5) (0.6) (5.2) (0.5) (2.0)
KCZ
0.031—4 0.031—0.5 0.031—1 0.25—1 0.031—1 0.25—1 0.031 8
(0.3) (0.3) (0.7) (0.4) (0.4) (0.7) (0.03) (8.0)
5-FC
0.031—8 0.031—0.062 0.062—1 0.031—4 0.062—0.25 0.031—4 0.031 0.031
(4.0) (0.05) (0.7) (0.6) (0.1) (0.8) (0.03) (0.03)
VOR
0.031—32 1—4 0.25—8 2—16 0.031—16 0.062—8 1 1
(0.7) (2.1) (3.6) (6.0) (3.4) (3.6) (1.0) (1.0)
0.125—8 1—2 0.25—8 0.5—2 0.5—2 0.062—2 1 1
NYS (0.5) (1.5) (2.9) (0.7) (1.0) (1.2) (1.0) (1.0)
2—8 2—4 2—8 4 2—4 2—4 4 16
CAS (3.2) (3.0) (4.0) (4.0) (3.2) (2.8) (4.0) (16.0)
0.062—1 0.25—4 0.062—8 1—16 0.25—1 0.062—4 1 1
(0.2) (1.8) (2.2) (5.0) (0.5) (1.6) (1.0) (1.0)
MIC: minimum inhibitory concentration; AmB: amphotericin B; FCZ: fluconazole; ITR: itraconazole; KCZ: ketoconazole; 5-FC: 5-flucytosine; VOR: voriconazole; NYS: nystatin; CAS: caspofungin.
Table 5 In vitro susceptibility of 173 Candida isolates to fluconazole (FCZ), voriconazole (VOR), itraconazole (ITR), ketoconazole (KCZ) and 5-flucytosine (5-FC) according to Clinical Laboratory Standard Institute (CLSI) breakpoints. ´ in vitro de 173 isolats de Candida au fluconazole (FCZ), le voriconazole (VOR), l’itraconazole (ITR), le ke ´ toconazole (KCZ) et 5-flucytosine (5-FC) en fonction des Sensibilite concentrations critiques du Clinical Laboratory Standard Institute (CLSI). Candida species (no.)
Susceptibility (%) FCZ
albicans (125) parapsilosis (20) krusei (8) glabrata (8) tropicalis (5) guilliermondii (5) sake (1) intermedia (1)
Total (173)
ITR
KCZ
5-FC
S
SDD
R
S
SDD
R
S
SDD
R
S
SDD
R
S
I
R
100 100 50 75 100 60 100 100
0 0 25 25 0 40 0 0
0 0 25 0 0 0 0 0
96 0 62.5 62.5 80 40 0 0
2.4 100 0 37.5 20 60 100 100
1.6 0 37.5 0 0 0 0 0
48 65 50 0 60 40 100 0
48 35 25 37.5 40 40 0 0
4 0 25 62.5 0 20 0 100
40 55 25 37.5 60 60 100 100
28 45 25 12.5 40 20 0 0
33 0 50 50 0 20 0 0
92 100 62.5 75 80 60 100 100
4 0 37.5 25 20 40 0 0
4 0 0 0 0 0 0 0
78.6
18.5
2.9
48
43.9
8.1
42.8
28.9
28.3
89.6
7.5
95.4
3.5
1.1
S: susceptible; SDD: susceptible-dose-dependent; I: intermediate; R: resistant.
2.9
M. Razzaghi-Abyaneh et al.
C. C. C. C. C. C. C. C.
VOR
Distribution and antifungal susceptibility of Candida species agent overall. The highest resistance rate (34%) among the tested antifungals was reported for KCZ. Resistance to FCZ and VOR was reported only for C. krusei, to ITR for all species except C. parapsilosis, C. tropicalis and C. sake, to KCZ for C. albicans, C. krusei, C. glabrata and C. guilliermondii and to 5-FC for only C. albicans. Totally, the resistance level of the isolates to 5-FC and FCZ were low (2 to 3%). Similar patterns of antifungal susceptibility has been reported in Slovak Republic and Spain [22,30]. There are several reports about increasing resistance of Candida species to azoles [12,31]. Our results showed that the rate of resistance to commonly used azoles was higher than that of reported in the United States [23] and Portugal [23,37]. Although FCZ was reported as the best antifungal agent in our report affecting the growth of the majority of isolates, however, resistance to this drug was common among isolates of C. krusei. It has been shown that he intrinsic resistance manifested by this species may be underrepresented by the in vitro results. Five of eight C. glabrata isolates were resistant to FCZ in our study, while all of C. glabrata isolates reported from Ghana in 2012 were susceptible to FCZ [14]. In a study conducted during 2006— 2008, all Candida species were reported as sensitive isolates to AmB, KCZ, NYS, VOR and CAS, whereas in some cases resistance to FCZ and ITR was noticed [4]. Zomorodian et al. [40] reported the highest rate of resistance to KCZ for C. glabrata which is in accordance to our results. In the present study, we reported two C. albicans isolates with as fluconazole-susceptible voriconazole-resistant strains, both were isolated from sputum. Although this is an unusual event, some other researchers have reported such strains from other parts of the world. As a good example, Pfaller et al. reported one fluconazole-susceptible voriconazole-resistant and also 14 fluconazole-SDD voriconazoleresistant strains among 7725 C. albicans isolates tested [32]. Further works on the molecular mechanisms leading to resistance to azoles are needed to explain such extraordinary pattern of susceptibility. As an interesting result, two C. krusei isolates from sputum and eye tumor and two C. glabrata isolates from oral swab and vaginal discharge were reported to be resistant to caspofungin. The patients’ history of antifungal treatment is not clear. However, this resistance may be attributed to a previous exposure of the patients to candins. A good correlation was reported between disk diffusion and MIC results in broth microdilution for 5-FC and FCZ but not for AmB for the majority of isolated Candida species in the present study. Disk diffusion method is useful for the determination of antifungal resistance, but is not so reliable for differentiation between susceptible and SDD fungal species. The major feature of disk diffusion as an agar-based test is that it can allow a quick answer concerning Candida resistance to antifungal agents, preventing unnecessary patient’s drug abuse. However, concerning drugs like as amphotericin B, data on such agar-based tests are still scarce and interpretation of results is a matter of controversy. CLSI published a specific standard for disk diffusion testing of fluconazole (M44-A), which considerably increased the possibility of implementing it in all routine laboratories. Further research must be carried out to ensure the standardization of this method for other antifungal agents [28]. Among the more than 200 Candida species, only a few can cause human diseases. C. intermedia and C. sake are rarely
e49 reported as a human pathogen and importance of these species is unknown [6]. In the present study, C. intermedia and C. sake were identified in nail scrapings and urinary catheter, respectively. These species must be further confirmed by complementary methods such as DNA sequencing. Taken together, results of the present study reinforce the importance of superficial candidiasis as a serious debilitating fungal infection with multiple clinical presentations. Our findings further indicate that susceptibility testing to commonly used antifungals is crucial in order to select the appropriate therapeutic strategy, which minimizes complications while improving patients’ life. The regular investigation on antifungal susceptibility in medical centers should be performed in order to promote the effective management of candidiasis.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements This work was supported financially by the Pasteur Institute of Iran. Authors gratefully thank Manijeh Yousefi-Behzadi from department of epidemiology of the Pasteur Institute of Iran for kind editing of the French language.
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