1. Background
Candiduria is one of the most common symptoms of urinary tract infections caused by several species of Candida, which is a normal flora of human body. Candida albicans has played an important role in candiduria (1, 2), however during the last decades non-albicans, such as C. glabrata, and C. tropicalis, have gradually increased in the incidence of nosocomial infections (1, 3-6). Old age, long stayin hospital, using broad spectrum antibiotics, and renal defects are the most important predisposing factors for candiduria (1, 5, 7, 8). Several reports have indicated that candiduria is a very common infection in hospitalized patients and its incidence is linked to antibiotic usage, long stay in hospitals, old age etc (2, 6, 9).
There are several valuable antifungals, such as amphotericine B, itraconazole, fluconazole, ketoconazole, econazole and nystatin, that are effective against Candida species. Some of these agents (fluconazole, amphotericine B, ketoconazole, econazole, itraconazole) are systemically used to treat urinary tract infections (UTI) (4, 10). Several reports have demonstrated that antifungal fluconazole has been effective for short-term eradication of candiduria (1, 11). On the other hand, some researches have found that the susceptibility degree of Candida species vary towards the used antifungal drugs (6, 7). For example, C. krusei and C. glabrata are resistant and less susceptible to fluconazole, respectively (7, 8, 12). Recent reports from different countries and hospitals have indicated that there has been an association between non-albicans and the rate of fluconazole resistance (12-14).
The susceptibility degrees of Candida species towards the used antifungal drugs vary and due to the growing use of these antifungals ,resistance to these agents has increased during the last decades (14).
2. Objectives
The present study aimed to determine the susceptibilities to antifungal drugs of Candida species isolated from candiduria in hospitalized patients of educational hospitals in Ahvaz.
3. Materials and Methods
3.1. Tested Yeasts and Identification
In the present study, 92 Candida spp. isolates ,and one Geotrichum spp. isolate were used for susceptibility tests. All species had been previously isolated from urine samples of hospitalized patients in the two educational hospitals in Ahvaz and identified by routine methods. Tested Candida isolates were included, C. albicans (58, 62.3%), C. glabrata (25, 26.8%), C. tropicalis (4, 4.3%), C. krusei (1, 1.1%), and Candida spp. (4, 4.3%). In addition, one isolate (1.1%) of Geotrichum spp. was also used for susceptibility tests. All strains were preserved in sterile distilled water at refrigerator temperature. The isolates were first subcultured on CHROMagar Candida (CHROMagar Candida®, France) plates and incubated at 37°C for 24h, aerobically to check for purity. All isolates were re-identified based on standard mycological methods, morphology on CHROMagar Candida, morphology on cornmeal agar, germ tube production and growth at 45°C. Then a suspension of each isolate of overnight cultures was prepared in 1ml of sterile PBS and adjusted to 0.5 McFarland turbidity standards.
3.2. Susceptibility Method
two sterile swabs were dipped into the suspension and rolled separately on the surface of two series of plates containing Sabouraud dextrose agar SDA (Merck, Germany) as lawn (17). The inoculated plates were dried in laminar hood at ambient temperature for 15mins. Paper disks of antifungals were placed on plates (three antifungal disks for each plate) by forceps and incubated at 37°C for 24h, aerobically. Antifungal disks were nystatin (100U), amphotericine B (20µg), fluconazole (100µg), ketoconazole (10µg), itraconazole (50µg) and econazole (10µg). All antifungal disks were purchased from Liofilchem Bacteriology Products (Italy). After 24h, the zone diameter around each antifungal disk was manually measured by ruler and recorded.
4. Results
4.1. Interpretive Criteria for Susceptibility of Antifungals
The interpretive criteria for the fluconazole, nystatin, amphotericine B, ketoconazole, itraconazole and econazole disks were indicated in Table 1 (15, 16).
| Zone Diameter in mm | |||
|---|---|---|---|
| Sensitive | Dose Dependent | Resistance | |
| Amphotericine B | > 15 | 10-14 | < 9 |
| Nystatin | ≥ 25 | 17-24 | < 16 |
| Fluconazole | ≥ 19 | 15-18 | ≤ 14 |
| Itraconazole | > 16 | 10-15 | < 9 |
| Ketoconazole | ≥ 30 | 29-23 | ≤ 22 |
| Econazole | > 16 | 10-15 | < 9 |
Interpretive Criteria of Susceptibility and Resistance of Used Antifungal Disks
4.2. Susceptibility to Amphotericine B
In the present study 54.8% and 44.1% were dose dependent and sensitive to amphotericine B, respectively (Table 2). 44.8% of C. albicans isolates were dose dependent and the rest of them (55.2) were sensitive to amphotericine B. One isolate (4%) of C. glabrata was resistant to amphotericine B, whereas 15(60%) and 9(36%) were dose dependent and sensitive to amphotericine B, respectively.
| Amphotericine B | C. albicans | C. glabrata | Candida sp. | C. tropicalis | C. krusei | Geotrichum | Total |
|---|---|---|---|---|---|---|---|
| Resistance | 0 (0.0%) | 1 (1.1%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 1 (1.1%) |
| Dose dependent | 26 (28.0%) | 15 (16.1%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 51 (54.8%) |
| Sensitive | 32 (34.4%) | 9 (9.7%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0(0.0%) | 41 (44.1%) |
| Total | 58 (62.4%) | 25 (26.9%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 93 (100%) |
| Itraconazole | |||||||
| Resistance | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 1 (1.1%) | 0 (0.0%) | 1 (1.1%) |
| Dose dependent | 52 (55.9%) | 24 (25.8%) | 4 (4.3%) | 4 (4.3%) | 0 (0.0%) | 1(1.1%) | 85 (91.4%) |
| Sensitive | 6 (6.5%) | 1 (1.1%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 7 (7.5%) |
| Total | 58 (62.4%) | 25 (26.9%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 93 (100%) |
| Nystatin | |||||||
| Resistance | 0 (0.0%) | 1 (1.1%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 1 (1.1%) |
| Dose dependent | 23 (24.7%) | 20 (21.5%) | 4 (4.3%) | 2 (2.2%) | 1 (1.1%) | 1 (1.1%) | 51 (54.8%) |
| Sensitive | 35 (37.6%) | 4 (4.3%) | 0 (0.0%) | 2 (2.2%) | 0 (0.0%) | 0 (0.0%) | 41 (44.1%) |
| Total | 58 (62.4%) | 25 (26.9%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 93 (100%) |
| Econazole | |||||||
| Resistance | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
| Dose dependent | 4 (4.3%) | 2 (2.2%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 6 (6.5%) |
| Sensitive | 54 (58.1%) | 23 (24.7%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 33 (93.5%) |
| Total | 58 (62.4%) | 25 (26.9%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 93 (100%) |
| Fluconazole | |||||||
| Resistance | 32 (34.4%) | 7 (7.5%) | 2 (2.2%) | 2 (2.2%) | 1 (1.1%) | 1 (1.1%) | 45 (48.4%) |
| Dose dependent | 17 (18.3%) | 16 (17.2%) | 1 (1.1%) | 2 (2.2%) | 0 (0.0%) | 0 (0.0%) | 36 (38.7%) |
| Sensitive | 9 (9.7%) | 2 (2.2%) | 1 (1.1%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 12 (12.9%) |
| Total | 58 (62.4%) | 25 (26.9%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 93 (100%) |
| Ketoconazole | |||||||
| Resistance | 19 (20.4%) | 4 (4.3%) | 2 (2.2%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) | 25 (26.9%) |
| Dose dependent | 31 (33.3%) | 14 (15.1%) | 0 (0.0%) | 4 (4.3%) | 1 (1.1%) | 0 (0.0%) | 50 (53.8%) |
| Sensitive | 8 (8.6%) | 7 (7.5%) | 2 (2.2%) | 0 (0.0%) | 0 (0.0%) | 1 (1.1%) | 18 (19.4%) |
| Total | 58 (62.4%) | 25 (26.9%) | 4 (4.3%) | 4 (4.3%) | 1 (1.1%) | 1 (1.1%) | 93 (100%) |
Susceptibility of Candida spp. Isolates to Antifungal Drugs
4.3. Susceptibility to Itraconazole
Susceptibility of tested isolates indicated that only one isolate of C. krusei was resistant to itraconazole. 91.4% of isolates were dose dependent and 7.5% were sensitive to itraconazole (Table 2). Overall, it was evident that 89.7% and 96.0% of C. albicans and C. glabrata were respectively exhibited dose dependent and the rest of them were sensitive.
4.4. Susceptibility to Nystatin
The details of susceptibility tested isolates to nystatin were shown in Table 2. As indicated, 54.8% and 44.1% of tested isolates were dose dependent and sensitive to nystatin, respectively. In the present study, only one isolate (1.1%) of C. glabrata was resistant to nystatin. Totally 39.7% and 80.0% of C. albicans and C. glabrata were dose dependent to nystatin, respectively, whereas 60.3% and 16.0% of C. albicans and C. glabrata were sensitive to nystatin, respectively.
4.5. Susceptibility to Econazole
The results of susceptibilities to econazole indicated that most of the tested isolates (93.5%) were sensitive to econazole and the rest of them were dose dependent (Table 2). Besides, 93.1% and 92.0% isolates of C. albicans and C. glabrata were sensitive to econazole and the rest of them were dose dependent.
4.6. Susceptibility to Fluconazole
Table 2 shows the susceptibility details of 93 tested isolates to fluconazole. As indicated, the zones around 48.4% of isolates were resistant to fluconazole, 38.7% dose dependent and 12.9% sensitive. When looking into C. albicans, 32 (55.2%) of isolates were resistant to fluconazole followed by, 17 (29.3%) dose dependent and 9 (15.5%) sensitive. Also, results indicated that 7 (28.0%) of C. glabrata were resistant to drug, followed by 16 (64.0%) dose dependent and 2 (8.0%) sensitive.
4.7. Susceptibility to ketoconazole
The susceptibility details of tested isolates to ketoconazole were shown in Table 2. As shown 26.9%, 53.8% and 19.4% of isolates were respectively resistant, dose dependent and sensitive to ketoconazole. Totally 53.4% and 56% of C. albicans and C. glabrata were dose dependent to ketoconazole, respectively. In the present study 32.8% and 13.8% of C. albicans were respectively resistant and sensitive to ketoconazole compared to 16.0% and 28.8% of C. glabrata.
5. Discussion
Fungal UTI has become an important nosocomial infection over the past decades among hospitalized patients. In addition, the extensive use of antifungals in hospitals may be a risk of emergence of resistant fungal strains (17, 18). For example, fluconazole is an important antifungal drug that is usually used to treat systemic fungal infections caused by Candida species. In addition, prophylaxis against systemic fungal diseases is also more prevalent by fluconazole.
The susceptibility of Candida species to frequently used antifungal drugs has various degrees. It has been reported that non-albicans species, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis and C. lusitaniae have had higher resistance rates against fluconazole than C. albicans (19).
C. krusei is one of the rare isolates of candiduria that is basically resistant to fluconazole (20), however several reports have different results. It is important to note that in the present study C. krusei was dose dependent to amphotericine B, nystatin, and ketoconazole and sensitive to econazole. In addition, this isolate was quite resistant to both itraconazole and fluconazole antifungal drugs. Ozcelik et al. (18) have reported that this isolate is quite sensitive to amphotericine B, in contrast, Pfaller et al. (16) showed that C. krusei is resistant to amphotericine B. In addition, Cheng et al. (13) showed that several strains of C. krusei isolated from candidemia were resistant to amphotericine B. In a study conducted by Yang et al. (14) 70% of C. krusei isolates ,collected from different hospitals of several regions of Taiwan, were resistant to fluconazole. They concluded that different resistance rates to fluconazole associated with different conditions in hospitals of each region.
The resistance rate of C. glabrata to fluconazole has gradually increased during last decades (19). Manzano-Gayosso et al. (4) study revealed that itraconazole, amphotericine B, and ketoconazole had less antifungal activity against C. glabrata isolates. In a study conducted by Laverdiere et al. (17), 4% of the Candida species isolated from different parts of ICUs patients were resistant to fluconazole and/or itraconazole. They believed that extensive use of antifungals in hospitals may be a risk of emergence of resistant fungal strains.
It is suggested by the current study that controlled surveys must be undertaken to optimize antifungal therapy based on characteristics of Candida strains. The current study indicated that 7.5% of C. glabrata isolates were resistant to fluconazole. It should be considered when C. glabrata is commonly isolated , fluconazole is a frequent choice for treatment and prevention of fungal diseases. The highest fluconazole sensitivity rates were recognized among C. albicans with 9.7%, while none of the isolates of C. tropicalis, C. krusei and Geotrichum spp. were susceptible.
