Introduction
Candida is yeast like fungi consisting of more than 150 species which can cause a range of infections from superficial infections to deep invasive infections. The incidence of urinary tract infections caused by Candida species has increased substantially in the recent times.1 Although community acquired urinary candidiasis is rare, nosocomial UTIs are more common. 2 Candida species can account for 9-40% of all nosocomial urinary tract infections. 3 Various factors like female gender, pregnancy, diabetes mellitus, urinary catherization, prolonged use of broad spectrum antibiotics, corticosteroid therapy and immunocompromised states have been associated with the risk of urinary candidiasis. 4
Among the pathogenic Candida species, C.albicans is the most commonly isolated species from urinary tract infections. However in the recent past many Candida non-albicans species such as C.tropicalis, C.glabrata, C.parapsilosis and C.krusei are frequently more isolated 5
Azole group of drugs such as fluconazole are commonly used in the treatment of urinary candidiasis. Unfortunately many Candida non-albicans species are increasingly becoming resistant to fluconazole. 6 Few Candida species like C. krusei are also known to be intrinsically resistant to fluconazole. 7 In view of this, routine species identification and antifungal susceptibility testing of Candida species is crucial for successful clinical outcomes.
This study was undertaken to analyse the distribution and risk factors associated with urinary candidiasis and also to determine the resistance patterns of different Candida species to various antifungal agents using phenotypic methods.
Materials and Methods
This is an observational study conducted over a period of 15 months from September 2016 to December 2017 in the department of microbiology of a rural tertiary health care centre in Puducherry. All urine samples from patients with UTI attending the hospital were included in the study. All non-repeat urine samples yielding pure growth of Candida species with more than 105 colony forming units (CFU) were included in the study. Samples which did not yield a pure growth of yeast, samples with colony count equal to or less than 104 CFU/ml and samples with growth other than Candida were excluded from the study. The institute ethics committee approval (No: SMVMCH-EC/DDO/AL/225/2016) was obtained prior to commencement of the study.
Candida isolation and identification
The urine samples submitted were inoculated on to Cysteine Lactose Electrolyte Deficient agar (CLED) and incubated at 37⁰C for 24-48 hours. White creamy colonies on CLED confirmed to be yeasts by Gram stain were subcultured on to Sabouraud’s dextrose agar (SDA) plates and were incubated at 37°C for 48 hours. Presumptive identification of the Candida species was done by urease test and germ tube test. Final identification of the Candida species was derived based on the growth character on Chrom agar Candida. The isolates were inoculated on HiCrome Candida Differential Agar (Himedia labs, Mumbai) and the species were identified by characteristic growth and colour after 24 to 48 hours of incubation at 37⁰C. Light green smooth colonies- C.albicans, metallic blue colonies- C.tropicalis, cream to white smooth colonies-C.glabrata, purple fuzzy colonies- C.krusei, and white creamy colonies- C.paprapsilosis.
Antifungal susceptibility testing
Disk diffusion method was employed for antifungal susceptibility testing following the Clinical Laboratory Standards Institute (CLSI) guidelines (M44 A-2 document). 8 The media used for testing was the Muller Hinton agar with 2% glucose and 0.5 mcg/ml methylene blue. The media and the antifungal disks were procured from HiMedia Laboratories, Mumbai. The colonies from 24 hour old cultures of Candida species on SDA were suspended in 5ml of 0.85% saline and the turbidity was matched to 0.5 McFarland standards. A lawn culture of the suspension was made on the test medium using sterile cotton swab. Antifungal disks with 25 mcg fluconazole, 1 mcg voriconazole, 10 mcg ketoconazole and 100 U amphotericin B were placed on the lawn cultures, and the plates were incubated for 24 hours at 37°C. After 24 hours the zones of inhibition around each disk were measured. The interpretation of zone diameters for fluconazole and voriconazole were done using the CLSI M44-S3 document guidelines. 9 The interpretation of zone diameters for ketoconazole and amphotericin B were done using manufacturer instructions as there are no established interpretive criteria. For fluconazole, more than or equal to 19 mm was interpreted as Susceptible (S), 15 to 18mm as Susceptible-Dose Dependent (SDD), and less than or equal to 14 mm as Resistant (R). For voriconazole, more than or equal to 17 mm was interpreted as S, 14 to 16 mm as SDD and less than or equal to 13 mm as R. For ketoconazole, more than or equal to 32 mm was interpreted as S and less than or equal to 20 mm as R. For amphotericin B, more than or equal to 15 mm was interpreted as S and less than or equal to 10 mm as R. C.albicans (ATCC 90028) was used as the quality control strain for the antifungal susceptibility testing.
Results
A total of 6930 urine samples were received during the study period and 101 Candida isolates were obtained from these samples. C.tropicalis was the most common species isolated followed by C.albicans (Table 1). Females had more incidence of Candiduria (66.3%) compared to males (33.6%). C.albicans was isolated in 7 males (20.6%) and 15 females (22.4%) whereas Candida non–albicans was isolated in 27 males (79.4%) and 52 females (77.6%). Candiduaria was predominantly seen above 50 years of age (38.6%). (Figure 1) Urinary candidiasis was seen in 87(86.1%) hospitalized patients whereas it was present only in 14(13.8%) patients treated on outpatient basis. Urinary catheterization was the predominant risk factor associated with urinary candidiasis in 44(43.5%) patients. (Figure 2) Among the Candia species 50 (49.5%) isolates were found to be susceptible to fluconazole, 81(80.1%) isolates were found to be susceptible to voriconazole, 68(67.3%) isolates were found to be susceptible to ketoconazole, and 99(98%) isolates were found to be susceptible to amphotericin B. Among azoles, voriconazole showed highest susceptibility both to C.albicans and Candida non-albicans. (Figure 3) Among different Candida species isolated, C.parapsilosis and C.dublinensis showed the least susceptibility to the azole group of drugs.Table 2
Table 1
Various species of Candida isolated from urine samples
|
Candida species |
Number isolated (n=101) |
Percentage (%) |
|
C.tropicalis |
32 |
31.6 % |
|
C.albicans |
22 |
21.7% |
|
C.glabrata |
18 |
17.8% |
|
C.parapsilosis |
12 |
11.8% |
|
C.krusei |
10 |
9.9% |
|
C.dublinensis |
07 |
6.9% |
Table 2
Antifungal susceptibilities of different Candida species
Discussion
Urinary tract infections by Candida species are becoming increasingly common.10 In normal adults, yeasts are found in less than 1 % of clean voided urine samples. In this study 101 urine samples (1.45%) yielded growth of Candida species from a total of 6,930 samples. Similar Candida isolation rates (1.37%) were shown in previous studies conducted in South India.11
The prevalance of urinary candidiasis was higher among females (66.3%) when compared to males (33.6%) in this study. This is similar to the observations of N Jain et al, and N Safdar et al, where 77.4% and 77% females respectively had candiduria in their studies.12, 13 However, a few studies have reported a higher prevalance of candiduria in males compared to females. 14, 15 Urinary candidiasis was found to be more prevalent among patients over 50 years of age (38.6%) in our study. A similar observation has been recorded in previous studies. 16, 17 Urinary candidiasis was observerd in 87 (86.1%) patients who were hospitalized prior to the onset of the disease in this study. This was also observed in many previous studies where hospitalized patients are at a higher risk of contracting urinary candidiasis.18, 19
The most common risk factors for urinary candidiasis observed in this study are urinary catheterization (43.5%), diabetes mellitus (33.6%), surgical procedures (22.7%), use of broad spectrum antibiotics (22.7%), pregnancy (21.7%), use of systemic corticosteroids (6.9%) and malignancy (0.9%). Similar observations where urinary catheterisation is the predominant risk factor for urinary candidiasis is also shown in previous studies by Jain N et al, and Navin Paul et al, where the risks were were 61.8% and 66.6% respectively.12, 20
In our study, out of the 101 Candida species isolated, Candida tropicalis predominated (31.6%) followed by Candida albicans (21.7%), Candida glabrata (17.8%), Candida parapsilosis (11.8%), Candida krusei (9.9%) and Candida dubliniensis (6.9%). Many recent studies on candiduria have also shown a considerable increase in the incidence of non albicans Candida species as compared to Candida albicans.21
Among azole group of drugs, both C.albicans and Candida non-albicans showed highest susceptibility to voriconazole (90.9% and 77.2% respectively) whereas fluconazole showed the least susceptibility (63.6% and 47.8% respectively) in the antifungal susceptibility testing. For amphotericin B, one isolate each of C.albicans and C.parapsilosis was found to be resistant. Among the Candida non albicans, C. parapsilosis and C. dublinensis showed the least susceptibility to the azoles fluconazole, voriconazole and ketoconazole (16.6%, 58.3%, 41.6% and 14.2%, 57.1%, 28.5% respectively). Recent studies on urinary candidiasis reveal that many Candida species exhibit higher rates of resistance to fluconazole whereas they still remain susceptible to voriconazole and amphotericin B.22
Conclusion
Hospitalization and urinary catheterization are the important risk factors for developing urinary candidiasis. The antifungal susceptibility varies among different Candida species and hence, identification of Candida to species level along with antifungal susceptibility testing should be practiced as a routine in all clinical mycology laboratories.
Article DOI
