Isolation, Speciation and antifungal drug susceptibility of Candida species from clinically suspected infections in a tertiary care hospital

Introduction: The incidence of fungal infections has increased dramatically over the last two to three decades. Study was done on, Clinical samples from suspected patients of candidiasis. Study period was one and a half year from January 2013 to June 2014. Out of 915 clinically suspected samples of candidiaisis processed in the Microbiology Laboratory, 156 (17.04%) yielded growth of Candida species. Methods: Candida was identified from Clinical samples by wet mount, Gram stain and Culture on SDA. The organisms were further speciated by germ tube test, Cornmeal agar Morphology, Sugar assimilation and Fermentation tests and CHROM agar. Antifungal susceptibility testing was done by disc diffusion method with Fluconazole (25 μg), Amphotericin-B(100U), Itraconazole(10 μg), Nystatin(50 μg), Clotrimazole(10 μg) and Voriconazole(1 μg). Results: C.tropicalis (46.79%) was the most common species isolated followed by C.albicans (37.17%) and C.parapsilosis (9.61%). Non albicans Candida species(NAC) (62.82%) was more than C.albicans (37.17%). All species isolated were susceptible to Amphotericin-B(100U), Nystatin(50 μg) and Voriconazole(1μg). Resistance to commonly used antifungal agents was Fluconazole-20.40%, Clotrimazole-18.26% and Itraconazole17.34%. Conclusion: Species identification is important for treatment of Candida infections, as NAC species continue to be increasingly documented and not all species respond to same treatment.


Introduction
The incidence of fungal infections has increased dramatically over the last two to three decades [1,2]. Extensive and inappropriate use of antimicrobial agents as well as use of immunosuppressant drugs in various diseases has contributed to increased propensity for opportunistic fungal infections [3].
Candida species is the fifth most common nosocomial urinary pathogen in India [4]. Candidiasis is the fourth common cause of nosocomial bloodstream infections worldwide, accounting for 9% of all such infections in the United State [5]. As per CDC Guidelines for Sexually transmitted diseases an estimated 75% of women will have at least one episode of VVC, and 40%-45% will have two or more episodes [6]. Predisposing  immunodeficiency, malignancy, increasing number of critically ill patients, surgical procedures, cytotoxic therapies with prolonged neutropenia, other immunosuppressive therapies, prolonged use of broadspectrum antibiotics, indwelling invasive medical devices and intensive care supports [7].
Although Candida albicans is the most prevalent species involved in both mucocutaneous and disseminated infections, the incidence of candidiasis due to non-albicans Candida (NAC) spp. is increasing. The clinical manifestations of infections caused by different members of NAC spp. are usually indistinguishable, but several NAC spp. are inherently resistant or acquire resistance, or both, to commonly used antifungal drugs [8]. Continuous monitoring of trends of species distribution and antifungal resistance pattern is essential to control and optimize therapy of Candida infection [9].

Materials and methods
Present study was carried out in the Department of Microbiology, Ruxmaniben Deepchand Gardi Medical college, Ujjain, MP. This is Hospital based cross sectional study for a period of one and a half year from January 2013 to June 2014. The study was approved by Institutional Ethics Committee. The samples (915) of the present study were received in Microbiology laboratory. Details of the patients were recorded.

Inclusion criteria
i) Clinically suspected patients of Candidiasis were included in study. ii) Patient given consent for participation in study Exclusion Criteria i) Patients with history of receiving any antifungal drug 30 days prior to study. ii) Those patients who were not willing to give the consent All samples were collected under aseptic precautions from Clinically suspected patients of Candidiasis. Clinical samples where urine, blood, vaginal swab , oral swab, sputum, stool, lip scraping, pus, CSF, central line tip, eye swab and umbilical swab. Each sample was processed immediately on receipt in laboratory as per standard microbiological procedures [10]. All the samples were processed in the following manner. Defining criteria of candidiasis-Smear showing budding yeast cells, pseudohyphae and pus cells denoted infection by Candida species. But in case of C.glabrata presence of pus cells and yeast cells were indicative of infection. Isolation of the same yeast in significant numbers from multiple specimens from the same body site was used to prove yeast as a pathogen [11].

B) Isolation of Candida in culture and species identification
Culture-All specimens were inoculated onto Sabouraud's dextrose agar (SDA) with chloramphenicol (0.05gms/litre) and blood agar (HiMedia, Mumbai) and incubated at 37˚C for 24-48 hrs. Colonies appeared within 1-3 days as creamy white,smooth pasty with a yeasty odour. Candida isolates were identified by Gram staining.

Criteria used to differentiate between infection and colonization for Candida species in various samples.
1) Urine-Candiduria was diagnosed by urine culture. The urine samples were spread by calibrated loop (0.01 ml) as per standard protocol for urine culture. Plates were incubated aerobically at 37 0 C and read at 24 hours. In case of urine samples, colony counts are important to differentiate between colonization and infection. Quantitative culture with colony count of >10 5 cfu/ml of urine is associated with infection in patients without indwelling catheters and >10 3 cfu/ml for catheterized patients. Pyuria usually supports diagnosis of Candida infection. Low colony counts in presence of pyuria were considered significant. Repeat isolation in same patient was also considered significant. [8,12 &13].
2) Sputum-Specimen was considered as acceptable when 25 or more polymorphonuclear leukocytes were seen per low power (10x) field with few (<10) squamous epithelial cells. [14] 3) Blood-Candidemia was defined as the presence of at least one positive blood culture containing pure growth of Candida species with supportive clinical features [13].

Research Article
Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in 185 | P a g e The direct demonstration of pseudohyphae along with yeast cells is an important diagnostic feature. [11].

6) Central venous catheter tip
Greater than 15 colony-forming units (CFU) on roll-plate culture was considered diagnostic of CRBSI.

C) Tests for identification of Candida species
i) Germ tube test [13] ii) Cornmeal agar [11] iii) Sugar fermentation tests [8,13 &15] iv) Sugar assimilation tests [10] v) Chrom agar [10] vi) Tetrazolium reduction media [10] 1) Pagano Levin base (PBL) agar was prepared according to the manufacturer's instructions HiMedia, Mumbai. After autoclaving, PBL media was allowed to cool to 50˚C and aseptically 5ml of 1% 2, 3, 5-triphenyltetrazolium chloride (TTC) solution was added and mixed well and then 5ml of rehydrated contents of 1vial of neomycin suspension was added and mixed well and poured into flat bottomed sterile petri-dishes on a level, approximately 4mm.

Research Article
Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in 187 | P a g e C.tropicalis (46.79%) was the most common species isolated followed by C.albicans (37.17%) and C.parapsilosis (9.61%).  The highest number of isolates was from Uine samples constituting 67 (42.94%). Non albicans Candida species was the common isolate in all clinical samples except vaginal and oral swab.   All isolates were sensitive to Amphotericin-B.

Discussion
Among 915 suspected clinical samples received in Microbiology laboratory, 156 samples were positive for Candida species, so prevalence rate was 17.04%. The prevalence rate by Rizvi et al, [19] Amar CS et al [15]and Feglo et al [20] study was 10.6%, 6.1%, and 12.7% respectively. The prevalence rate was higher in present study (17.04%) in comparison to Rizvi et al [19]. Amar CS et al [15]and Feglo et al [20]. However it was comparable to Roy et al [21] (22.6%).
In the present study it was observed that Candidiasis can occur in all age group and in both sexes. In the present study, youngest study subject was a 1-day old while the oldest was 85 years old man. In the study by Kashid et al [22] the age range was from 1-day to 90 years.
The present study had a slight female preponderance with an overall male :female ratio 0.92 :1. In north-east Indian study by Roy et al [21] it was observed that, the frequency of Candidiasis is more among females (51/113) as compared to males (41/113), but statistically by Chi square test, it has been seen that the incidence of Candida species distribution is independent on sex. Our study results matched with Roy et al [21].
In a study by Dalal PJ and Kelkar S, it was observed that vulvovaginitis was common in the sexually active age group of 21-40 years [23]. In the present study, vulvovaginitis was commonly seen in the same age group.  [26] and 2014 found C. albicans as a common isolate, which does not correlate with the present study.
The rise in frequency of infection of non-albicans Candida species has been also observed in tertiary care centres in India, with the isolation rate ranging from 50% to 96%. [21].
In this study the results of Chrom agar were exactly paralleled to that of Conventional method. No resistance detected in the isolates to amphotericin-B, nystatin and voriconazole in the present study. Rizvi et al [19] also reported same finding, no resistance to amphotericin-B, nystatin and voriconazole. Our findings correlated well with those in the studies by Kashid et al [22], Vijaya D et al [25] and Roy et al [21]. . In these studies, C.albicans and NCA showed 100% sensitivity to amphotericin B while azole group of drugs were used as second choice. In our study Resistance was more in NAC as compared to C. albicans species. Similar finding was noted by Sachin et al [9]. In the present study the Non albicans Candida spp showed 20.40 % resistance to azole group of drugs and C. albicans isolates showed 13.79% resistance to azole group of drugs. Also the finding was correlated with those of a study done by Shivanand et al [26] in which NAC shows 25% resistance to fluconazole. In our study resistance for fluconazole by C. krusei was 100%, This was also confirmed b Amar C S et al [15] and Roy et al [21] who reported that all isolates of Candida krusei tested were resistance to fluconazole.

Association of species and underlying predisposing conditions-
It is well established that Candida krusei is intrinsically resistant to fluconazole. In the current study, amphotericin B was the most effective antifungal agent. Western data have shown that Candida species are reliably susceptible to polyenes, azole and echinocandins. In India, amphotericin B is the drug of choice for invasive Candidiasis with low or no resistance reports.