NJBMS - Volume 11, Issue 2, October - December 2020

Pages: 201-210

Date of Publication: 06-Oct-2020

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IDENTIFICATION, SPECIATION AND ANTIFUNGAL SUSCEPTIBILITY PATTERN ON URINARY ISOLATES OF CANDIDA SPECIES ISOLATED FROM HOSPITALISED PATIENTS IN A TERTIARY CARE HOSPITAL - A PROSPECTIVE STUDY.

Author: Arasi Samyuktha1, Ramya R 2, Shajahan O. M 3, AnakhaKaladharan4

Category: Microbiology

Abstract:

Aim: The aim is to speciate Candida isolated from the urine samples of hospitalized patients, to correlate risk factors associated and to find out the Antifungal susceptibility of the Candida species isolated. Methods: This is a single centre, cross – sectional study with study group of 100 hospitalized patients with candiduria having a colony count of more than 10 4/ml of urine. Patients satisfying the inclusion criteria were interviewed by structured questionnaire and their hospital records were used to know about the history, risk factors, duration of Candiduria and treatment details. Results: A total of 100 patients were included in the study, Males constituted 54% and females 46% of the total population, with asymptomatic presentation 46%, symptomatic 25% and unconsciousness (patients unable to voice their symptoms), 29%. Fever (17%) and dysuria (13%) were the common symptoms in patients with candiduria. C.albicans constituted 14% and non-albicans Candida spp. constituted 86% of the total isolates obtained. Study showed Hi-Chrom agar was the best method of speciation, compared to Sugar Assimilation Test (SAT) using Rapid identification kit. Hi-chrom agar showed 100% sensitivity while SAT showed only 86% of sensitivity. Resistance to fluconazole was seen in 34% of isolates, with C.tropicalis being the most resistant (14%).72% isolates were susceptible to itraconazole. 90% of the isolates were susceptible to amphotericin B. Conclusion: The increase in rates of resistance particularly among the non-albicans Candida spp. emphasizes the need for speciation and antifungal susceptibility testing. Hence the present study was conducted to speciate Candida isolated from the urine samples of hospitalized patients, to correlate risk factors associated and to find out the Antifungal susceptibility of the Candida species isolated.

Keywords: Candiduria, Speciation, Hi-Chromagar, SAT, Risk factors, Antifungal susceptibility

DOI URL: https://dx.doi.org/10.31975/NJBMS.2020.11204

Full Text:

INTRODUCTION:
Candida species can cause wide spectrum of clinical diseases ranging from superficial infections of skin, nails and mucosal surfaces to deep seated infections involving various internal organs and disseminated disease causing significant morbidity and mortality. It can cause lower urinary tract infections and renal infection in hospitalised patients commonly. Epidemiological surveillance indicates that Candida species are the most common pathogens causing nosocomial blood stream and urinary tract infection1.
Candiduria is defined as the presence of Candida spp. in urine and represents colonization or infection. It should never be ignored, as it may be one of the early indications of disseminated and invasive candidiasis, especially in critically ill patients2,3.The common risk factors for candiduria include indwelling urinary tract devices, prior surgical procedures, recent use of antibiotics, advanced age, female sex, Diabetes mellitus, immunosuppressive therapy and prolonged hospital stay4.
Over the last decade, however, there has been an increase in the incidence of candidiasis caused by other Candida species, such as C. glabrata, C. krusei, C. tropicalis and C.parapsilosis5.Candida albicans accounts for 40-60% yeasts isolated in developed countries, whereas Indian reports show an increased predominance of non-albicans Candida spp.
C.glabratais less susceptible and Candida kruseiis intrinsically resistant to Fluconazole. C. tropicalis has the highest adherence rate to inanimate materials such as urinary and vascular catheters, and is often involved in biofilm formation, that is more resistant to antifungal agents. Resistance to azoles in C. Tropicalis and C.albicans has also been increasingly reported6,7,8.
So it is important to know the Candida species causing UTI before initiating the treatment as non- albicans Candida species are on the rise in the hospital environment and majority are inherently resistant to treatment with Fluconazole9.
As the conventional identification of Candida take several days, employing chromogenic media may help to reduce the time of isolation and identification by 48 -72 hrs. This will help the clinician in optimizing the selection of antifungal agents and provide a more rational and customized therapy10.
Considering the above facts, the present study was conducted to speciate Candida isolated from the urine samples of hospitalized patients, to correlate risk factors associated and to find out the
Antifungal susceptibility of the Candida species isolated.
MATERIALS AND METHODS:
This is a single centre, cross – sectional study, carried out over a period of 1 year after the approval of Institutional Ethical Committee. The Study group included 100 hospitalized patients with candiduria having a colony count of more than 10 4/ml of urine.
Inclusion Criteria:
Hospitalized Patients with urine colony count of any Candida species more than or equal to 10 4/ml.
Patients older than 12 yrs of age.
Both males and females were included.
Exclusion Criteria:
Candiduria with colony counts less than 10 4/ml of urine.
Patients less than 12 yrs of age.
Outpatients.
Patients satisfying the inclusion criteria were included in the study and after getting informed consent, they were assigned serial numbers. They were interviewed by structured questionnaire and their hospital records were used to know about the history, risk factors, duration of Candiduria and treatment details.
Specimen Collection:
Specimens collected were mid-stream urine specimens, and catheter collections. They were immediately transported to laboratory without any delay, as urine is an excellent culture medium for other microorganisms to grow. Delay of more than 1-2 hr in transportation if unavoidable, it should be stored in refrigerator at 4°C or transported in a container with 1.8% boric acid.
Specimen Processing:
Direct Microscopical Examination:
Microscopic visualization of uncentrifuged urine was done. One to three Candida per high power field was found to be equivalent to colony count of approximately 15000/ml with 80% accuracy. A wet mount of uncentrifuged urine was done. A small volume of urine was applied to a glass microscope slide, allowed to air dry, stained with Gram’s stain, and examined microscopically for presence of grampositive budding yeast cells with or without pseudohyphae.
Culture:
The uncentrifuged mid-stream urine was cultured on blood agar, Mac Conkeyagar, Cystine lactose electrolyte deficient (CLED) agar and Sabouraud’s Dextrose Agar (SDA) for primary isolation of Candida spp.
Examination of Culture: On SDA:
Colonies were cream colored, pasty and smooth. A wet mount, lacto phenol cotton blue (LPCB) mount and Gram’s stain were done from the culture isolates to confirm it as Candida species.
Species Identification:
The various Candida spp. were identified based on the following tests
Germ Tube Test:
Isolates producing germ tubes were presumptively identified as C.albicans or C.dubliniensis.
Growth at 45°C:
Isolates of C.albicans grow, while C.dubliniensisdo not grow at 45°C.
Candida Hi - Chrom Agar:
The various species of Candida were identified by their colony colour, size, texture, and presence of color diffusion into the surrounding agar presumptively in 48hrs.
Sugar Assimilation Test:
Sugar assimilation test (SAT) was done using rapid Hi-Candida identification kit (KB006) from Hi-media Mumbai. KB006 kit is a standardized colorimetric identification system utilizing twelve conventional biochemical tests.
Table 1: Various species of Candida on Chrom Agar:
Candida species
Colour on HI-CHROM agar
C. albicans
Light green colour colonies
C. parapsilosis
Cream coloured colonies.
C. tropicalis
Steel blue colonies with a pink halo
C. krusei
Pale pink, dry rough colonies with spreading, pale edges.
C. glabrata
Pink to Purple colonies.
Antifungal Susceptibility Testing:
Antifungal susceptibility testing for Candida isolates was done by, Disc diffusion method, as per CLSI Guidelines on Antifungal Susceptibility testing in M-44A document
RESULTS:
Basic Demographic Data:
In about 100 patients, Males contributed 54% and females contributed 46% with majority of the study population above 60years age group (28%), followed by (21%) patients in 20-29 & 40-49 years age group. Table 2 showed that females were predominated in the age group of 20-29 yrs (28.2%), where as males predominated after 60 yrs (31.4%).
Table 2: Age distribution of the study population
Age (years)
Total number of cases n=100 no (%)
Male
n=54 no(%)
Female
n=46 no(%)
<20
4(4%)
3(5.5%)
1(2.1%)
20-29
21(21%)
8(14.8%)
13(28.2%)
30-39
7(7%)
2(3.7%)
5(10.8%)
40-49
21(21%)
11(20.37%)
10(2.1%)
50-59
19(19%)
13(24%)
6(13.04%)
>60
28(28%)
17(31.4%)
11(23.91%)
TOTAL
100(100%)
54(100%)
46(100%)
Based on Symptoms:
Most of the patients had asymptomatic candiduria (46%), followed by symptomatic patients (25%) and unconscious patients who were unable to tell the symptoms (29%). Symptomatic and unconscious patients were common in the age group of > 60yrs and asymptomatic patients were common in the age group of 40-49yrs and 50-59yrs.Among the patients with candiduria, fever (17%) and dysuria (13%) were the predominant symptoms. Symptoms of frequency, urgency and lower abdominal pain were present in 9%, 8% and 5% of patients respectively. Dysuria, increased frequency and lower abdominal pain were predominant in males than in females (Table 3).
Table 3: Various symptoms in patients with candiduria
Symptoms
Male n=54
PERCENT
Female n=46
No (%)
TOTAL n=100
No (%)
Fever
13
(24.07%)
4
8.6%
17
17%
Dysuria
10
(18.51%)
3
6.5%
13
13%
Frequency
7
(12.96%)
2
4.3%
9
9%
Urgency
6
((11.11%)
2
4.3%
8
8%
Lower abdominal pain
3
(5.5%)
2
4.3%
5
5%
Incontinence
4
(7.4%)
1
2.1%
5
5%
Urine Sample:
73% of urine samples were catheterized urine samples followed by 27% of midstream urine samples. 74.07% of samples from males and 71.1% of samples from females were catheterized urine samples.
Patient Distribution:
Patients from medical ward had Diabetes mellitus as a major risk factor and patients from urology and nephrology had CKD and other diseases of urinary tract as the major risk factors. ICU, Surgery, Orthopaedics and neurosurgery patients had antibiotic use and catheterization as major risk factor. In addition, immune suppressives were a major risk factor for neurosurgery patients. (Table 4)
Table4: Ward distribution in patients with candiduria
Ward
Number of patients(n)
Risk factors associated & no (%)
Medicine
36
Diabetes mellitus-32(88.8%)
Antibiotics-26(72.22%)
Catheterization-23(63.88%)
Unconsciousness-14(38.88%)
Imunosuppresives-10(27.7%)
Nephrology
19
CKD and other diseases of urinary tract-19(100%)
Hemodialysis-10(52.6%)
Antibiotics-14(73.68%)
Transplant-4(21.05%)
Immunosuppresives-4(21.05%)
Urology
14
CKD and other diseases of urinary tract-14(100%)
Catheterization-14(100%)
Antibiotics-12(85.71%)
ICU(Intensive Care Unit)
12
Antibiotics-12(100%)
Catheterization-12(100%)
Unconsciousness-12(100%)
Diabetes mellitus-6(50%)
Surgery
9
Antibiotics-8(88.88%)
Catheterization-8(88.88%)
Diabetes mellitus-2(22.22%)
Orthopaedics
6
Catherization-6(100%)
Antibiotics-6(100%)
Diabetes mellitus-2(33.33%)
Neurosurgery
4
Catheterization-4(100%)
Antibiotics-4(100%)
Immunosuppresives-3(75%)
Unconsciousness-3(75%)
Diabetes mellitus-2(50%)
Risk Factor Assessment:
Major risk factors included antibiotics use in 82%, catheterization of urinary tract in 67%, followed by Diabetes in 44% and Chronic Kidney Disease in 33% of the patients with candiduria (Table 5). Non-albicans Candida spp was more common in catheterized patients (70.9%) and unconscious patients (30.23%).
Table 5: Risk factors in candiduria due to C.albicans and non albicans candida species.
Species Identification:
Of the 100 Candida isolates, 62 were C.tropicalis, 14 isolates were C.albicans, followed by 10 isolates of C.glabrata. 9 isolates of C.krusei and 5 isolates of C.parapsilosis. C.tropicalis 62% was the predominant isolate among the non-albicans Candida species (Table 6).
C.albicans was more common in midstream urine (37%) sample than from catheterized urine samples. C.tropicalis was more common in catheterized urine samples (72.6%) than from midstream urine samples (33.33%).
Table 6: Distribution of candida isolates among catheterised and midstream urine samples
SPECIES
Total no of isolates, n=100
No(%)
No. of isolates in catheterized urine samples, n=73
No(%)
No. of isolates in midstream urine, n=27
No(%)
C.albicans
14
4 (5.4%)
10(37%)
C.tropicalis
62
53 (72.6%)
9(33.33%)
C.glabrata
10
6 (8.2%)
4(14.8%)
C.krusei
9
7 (9.5%)
2(7.4%)
C.parapsilosis
5
3 (4.1%)
2(7.4%)
Total
100 (100%)
73 (100%)
29(100%)
Hi-Chrom Agar Vs Sugar Assimilation Kit:
Hi-Chrom agar was the best method of Candida speciation as it identified all Candidaspecies from the 100 isolated samples with a sensitivity of 100%. The ability of sugar assimilation kit using rapid HiCandida identification kit (KB006) to
Risk factors
No. of patients
N (%)
Candiduria due to C.albicans
N=14
Candiduria due to nonalbicans Candida species N=86
TOTAL
(%)
TOTAL
(%)
Prolonged antibiotics
82(82%)
9
64.28
73
84.88
Catheterization
67(67%)
6
42.9
61
70.9
Diabetes mellitus
44(44%)
6
42.9
38
44.1
Chronic kidney disease and diseases of urinary tract
33(33%)
1
7.14
32
37.20
Unconsciousness
29(29%)
3
21.42
26
30.23
Immunosuppressives
17(17%)
2
14.28
15
17.44
ICU stay
12(12%)
2
14.28
10
11.62
Hemodialysis
10(10%)
3
21.42
7
8.1
Renal transplant
4(4%)
0
7.1
4
4.6
identify the different Candida species from the 100 isolated samples was only 86% and rest was unidentifiable by this method. This shows Hi-Chrom agar is more sensitive for Candida speciation (Table7).
Table 7: Comparison of methods of candida speciation
SPECIES
Total no of isolates n=100
Method used
Hi-Chrom agar
Sugar assimilation
n
%
n
%
C.albicans
14
14
14
12
12
C.tropicalis
62
62
62
58
58
C.glabrata
10
10
10
6
6
C.krusei
9
9
9
6
6
C.parapsilosis
5
5
5
4
4
Total
100
100
100%
86
86%
Antifungal Susceptibility by Disk Diffusion Method Fluconazole:
55(55%) Candida isolates were sensitive and 34(34%) isolates were resistant. The overall susceptibility rate of fluconazole for C.albicans was 71.4% and C.tropicalis was 67.7%. Susceptible dose dependent for C.albicans 21.4% and C.tropicalis 9.6%. Resistance for C.albicans was 7.1% and C.tropicalis 22.5%. Resistance for C.albicans was 7.1% and C.tropicalis 22.5%. In our study C.glabrata and C.krusei showed resistance to fluconazole(Table 8).
Table 8: Antifungal susceptibility to fluconazole
SPECIES
No of isolates
Susceptible n(%)<8μg/ml
Susceptible Dose Dependent n(%)
16-32μg/ml
Resistant n (%)>64μg/ml
n
%
n
%
n
%
C.albicans
14
10
71.4
3
21.4
1
7.1
C.tropicalis
62
42
67.7
6
9.67
14
22.5
C.glabrata
10
-
-
-
-
10
100
C.krusei
9
-
-
-
-
9
100
C.parapsilosis
5
3
60
2
40
-
Total
100
55
55
11
11
34
34
Itraconazole:
72% Candida isolates were sensitive and 18% isolates were resistant. The overall susceptibility rate for itraconazole was, for C.albicans 57.1%, for C.glabrata 60%, for C.tropicalis 77.4% and for C.krusei 55.5%. Susceptible dose dependent for C.albicans was 21.4% and C.glabrata 20%. Resistance for C.albicans was 21.4%, for C.glabrata 20%, for C.tropicalis 16.1% and for C.krusei 44.4% (Table 9).
Table 9: Antifungal susceptibility to itraconazole
Species
No of isolates n=100
Susceptible
n(%)
0.25μg/ml
Susceptible dose dependent n(%)
0.25-0.50μg/ml
Resistant
n(%)
>1μg/ml
n
%
n
%
n
%
C.albicans
14
8
57.1
3
21.4
3
21.4
C.tropicalis
62
48
77.4
4
6.4
10
16.1
C.glabrata
10
6
60
2
20
2
20
C.krusei
9
5
55.5
-
-
4
44.4
C.parapsilosis
5
5
100
-
-
-
-
Total
100
72
72
9
9
19
19
Amphotericin B: Antifungal susceptibility testing to amphotericin B shows minimal resistance pattern and high susceptibility rate to different Candida species. 90% of the Candida isolates were sensitive. Susceptibility rate for C.albicans was 85.7%, for C.glabrata was 100%, C.tropicalis was 93.4% and for C.krusei was 66.6%. Resistance for C.albicans was 14.2%, for C.glabrata 0%, for C.tropicalis 6.4% and for C.krusei 33.33% (Table 10).
Table 10: Antifungal susceptibility to amphotericin b
Species
Number of isolates n=100
Susceptible
Resistant
N (%)
%
N (%)
%
C.albicans
14
12(85.7)
85.7
2(14.2)
14.2
C.tropicalis
62
58(93.54)
93.54
4(6.4)
6.4
C.glabrata
10
10(100)
100
-
-
C.krusei
9
6(66.6)
66.6
3(33.33)
33.33
C.parapsilosis
5
4(80)
80
1(20)
20
TOTAL
100
90
90
10
10
DISCUSSION:
A significant rise in prevalence of Urinary Tract Infections (UTI) due to Candida species has occurred over the last decade with the upsurge of non albicans Candida species. Clinical importance of species level identification is important as they differ in expression of virulence factors and antifungal susceptibility pattern. The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungal therapy.
Current study was undertaken to speciate the Candida isolated from urine samples of hospitalized patients and to find their antifungal susceptibility pattern. The study also concentrated on the changes observed in species distribution and the surge of non-albicans Candida spp. in our hospital. In this study, males contributed to 54% of the study population. This was similar to the results of study by Arlene O.Cantillep et al11. Although there is an increased risk
in female gender, the other associated risk factors like Diabetes and chronic kidney disease were common in males in our study. There was predominance of patients in the age group of >60 yrs contributing to 28% of total patients, followed by 21% in 40-49 yrs. This correlated well with the study done by S.Krcmery et al, were the mean age was 62.4 yrs 12.
An important observation was that about 46%of patients with candiduria were asymptomatic. It is an important complicating factor in defining candiduria. Many patients on long term urinary catheterization cannot vocalize on symptoms of dysuria or increased frequency.Asymptomatic patients were common in the age groups 40-49 and 50-59 yrs. According to a study done by Mauricio Carvalho et al in 2001, only 13% of patients had symptoms suggesting UTI. Current study showed symptomatic candiduria in 25% of patients. Symptoms of UTI were predominant in the age group of > 60 yrs and were more common in males.In the present study, about 29% of patients were unconscious and on prolonged catheterization. So these patients could not be categorized either as symptomatic or asymptomatic. Fever was the most common presenting symptom in 17% of patients with candiduria followed by dysuria in 13%.This was lesser than the results of the study by Paul A Tambyah et al13, where in fever was present in 17.7% patients and dysuria in 6% of catheterized patients with UTI. Tambyah et al’s study population was that of both bacterial and fungal infections in catheterized patients were as the current study included only patients with candiduria. Catheterization also makes the patient asymptomatic, as the presence of a catheter in the urethra prevents continuous exposure of urethral mucosa to organisms in infected urine thereby preventing infectious urethritis that produces dysuria, urgency in infected non catheterized patients.
Among the type of urine samples obtained, catheterized samples contributed to a total of 73% and midstream urine samples in 27% of patients. This was comparable to the study by Cl´audia CasteloBrancoArtiaga Kobayashi et al14and study by Arlene O.Cantillep et al11where in 84.4% and 89% of patients were catheterized. Catheterization was described by many authors as the most important risk factor for Candiduria. Most of the patients in our study belonged to medical ward, contributing to 36% of patients, followed by nephrology 19%. This was similar to the study by Stephen P storfer15. The risk factors were also found to vary in different wards. Diabetes mellitus was the most common risk factor in patients from medical wards, CKD and other diseases of urinary tract were common in nephrology and urology. ICU and surgery ward patients showed prolonged antibiotic use and catheterization as the common risk factors11.
In the present study prolonged antibiotic use (82%) and catheterization (67%) were the most common risk factor associated with candiduria. This was lower than the results by Uma Chaudary et al16, who showed antibiotics as a risk factor in 99.6% of patients, catheterization in 90%, because the study population in her study included critically ill patients with candiduria rather than the hospitalized patients in our study. Antibiotics alter the normal flora of the genito-urinary tract, thus making way for colonization by Candida species. The surface of catheters also help in colonization with non-albicans Candida spp.
Diabetes mellitus (44%) was most common disease associated, followed by CKD (33%). This was slightly higher than the study by Cl´audia Castelo Branco et al14, who showed 26.7% of the patients, had Diabetes probably because; Indians are more prone to Diabetes.India is termed as the “Diabetes capital of the world”. Immuno suppressives like steroids were also important risk factors, as they alter the natural immunity to Candida spp. These drugs were used as a therapy for transplant recipients, neurosurgery patients and for patients with auto immune diseases. Unconsciousness was another risk factor, as most of the patients were on prolonged indwelling catheter and antibiotic therapy. The present study showed predominance of non-albicans Candida spp. contributing to 86% of isolates and C.albicanscontributing only 14% of the isolates. This was comparable to the results obtained by Manisha jain et al17, who showed non-albicans Candida spp. as predominant isolate in 71.4% from urine isolates.
The species distribution was as follows, C.tropicalis 62%, C.albicans 14%, C.glabrata 10% C.krusei 9% and C.parapsilosis 5% which is comparable to the results by Manisha Jain et al17 from north India. Her study showed 52.9% of isolates as C.tropicalis and 29.8% as C.albicans. In contrast, the studies by Elza Helena Da Silva et al18and N.Febre et al19 from Brazil showed C.albicans as predominant species contributing to about 56% and 46.15% respectively. Non-albicans Candida spp were common in both catheterized and mid-stream urine samples. This is an important observation as 26.5% of catheter associated infections are due to fungi20. Biofilm formation
of the C. tropicalis strain on the catheter surface may contribute to the colonization in patients with urinary catheter. Biofilms of C.tropicalis, with an extensive, hexosamine-rich matrix, were poorly penetrated by antifungal agents, whereas biofilms of C.albicans, with a less-extensive glucose rich matrix, were more readily penetrated by drugs21. The exact reason for the increase in non-albicans Candida spp. is incompletely understood. C.albicans was found in 37% of the midstream samples but it contributed to only in 5.4% in catheterized patients.C.albicans was more common in midstream urine samples than catheterized patients.
Hi-Chrom agar was used for Candida speciation in our study. The methods of identification of Candida by corn meal agar and sugar assimilation tests are very time consuming; On CMA it takes around (24hr-72hr) and in case of sugar assimilation test it may take around (72hrs-2weeks). Besides, these procedures are labour intensive and take a longer time to determine the diagnosis. Several chromogenicsubstrate containing culture media has been developed. The advantages are, it should support the growth of yeast but not of bacteria. It should facilitate the recognition of specimen containing mixture of yeast species and exposure of fungi to the different indicator substances should not affect the viabilities for subsequent subculture. CHROM agar is a chromogenic substrate containing culture medium which fulfils this entire requirement. CHROM agar Candida is a differential culture medium being widely used to differentiate Candidaspecies. These chromogenic media yield colonies of different colours secondary to chromogenic substance that react with the enzymes secreted by the organisms22. The major advantage of CHROM agar is the ability to detect mixed cultures of yeast in clinical specimens23.
In the present study, C.albicans was identified as light green colour colonies in CHROM agar and C.glabrataproduced pink to purple colour colonies as compared to studies done by Baradkar et al24. C.parapsilosis produced cream coloured colonies andC.tropicalis produced steel blue colour colonies as mentioned in Hi-media.
Study done by Yucesoy et al26, revealed that all C.krusei isolates produced rough, fuzzy spreading big pink colonies on CHROM agar. Our study showed colonies of C.krusei to be large, fuzzy, rough and pink. Rapid identification of C.krusei with chromogenic media is important because it exhibits innate resistance to flucanazole.
In this study, sugar assimilation test was done using Rapid identification kit from Hi-media Mumbai [KB006 HiCandida Identification Kit]. KB006 is a standardized test system that can be used for identification and differentitiation of Candida species. Each KB006 Kit is a standardized colorimetric identification system utilizing twelve (Urease, Melibiose, Lactose, Maltose, Sucrose, Galactose, Cellobiose, Inositol, Xylose, Dulcitol, Raffinose, Trehalose) conventional biochemical tests. The test is based on the principle of pH changes which are indicated by a spontaneous colour change in the media. Candida species isolated by using rapid identification kit was C.albicans 12% (12cases), C.glabrata 6% (6cases), C.tropicalis 58%(58 cases), C.krusei 6% (6cases) and C.parapsilosis 4% (4 cases) with a total sensitivity of 86% which was similar to study done by AnjanaGopi et al25.
In this study we observed that Hi-Chrom agar was the best method of speciation as it identified all the Candida species with 98% sensitivity and 100% specificity, which was similar to studies done by Yucesoy et al which showed 97% sensitivity and 100% specificity and Wilinger et al showed 98.8% sensitivity and 100% specificity for all Candida species as compared to sugar assimilation test using rapid identification kit26,27.In vitro antifungal susceptibility testing is becoming important because of the emergence of new non-albicans Candida species and increased intrinsic and acquired resistance to azoles and amphotericin B.Soagar based antifungal susceptibility testing is an alternative to the microdilution method. It is easy to perform and inexpensive for routine laboratories. CLSI M44-A disc diffusion testing with glucose methylene blue Muller Hinton Agar is a very convenient method for antifungal susceptibility testing 28.In the current study, the overall susceptibility rate of fluconazole for C.albicans was 71.4% and C.tropicalis was 67.7%. Susceptible dose dependent for C.albicans 21.4% and C.tropicalis 9.6%.Resistance for C.albicans was 7.1% and C.tropicalis 22.5%. In our study C.glabrata and C.krusei showed resistance to fluconazole, as similar to study done by Sobel et al29.C.krusei and C.glabrata is known for its intrinsic resistance to fluconazole30. The overall susceptibility rate for itraconazole was, for C.albicans 57.1%, for C.glabrata 60%, for C.tropicalis 77.4% and for C.krusei 55.5%. Susceptible dose dependent for C.albicans was 21.4% and C.glabrata 20%. Resistance for C.albicans was
21.4%, for C.glabrata 20%, for C.tropicalis 16.1% and for C.krusei 44.4%. Antifungal susceptibility testing to amphotericin B shows minimal resistance pattern and high susceptibility rate to different Candida species. Susceptibility rate for C.albicans was 85.7%, for C.glabrata was 100%, C.tropicaliswas 93.4% and for C.krusei was 66.6%. Resistance for C.albicans was 14.2%, for C.glabrata 0%, for C.tropicalis6.4% and for C.krusei 33.33%. Our finding shows an overall susceptibility rate of amphotericin B to be 90% which correlate well with the study done by Saldanha et al and Noake et al were Candida species were more susceptible to amphotericin B (92%)31,32.
CONCLUSION:
Candiduria should never be ignored as it can be the only indication of systemic or invasive candidiasis. Our study showed a predominance of non-albicans Candida spp. of about 86%. C.tropicalis(62%) was the most common isolate obtained followed by C.albicans (14%), C.glabrata (10%), C.krusei (9%) and C.parapsilosis (5%). Indwelling urinary catheter was an important associated risk factor for non-albicans candiduria. Multiple risk factors like long term antibiotic therapy, prolonged catheterization and Diabetes mellitus were present in many patients.
Hi-Chrom agar takes only 48 hrs for species identification, and is a comfortable alternative to conventional methods, that take 96-120 hrs. Hi-Chrom agar is superior to other conventional methods available for rapid detection of Candida species. Further the increasing rates of resistance particularly among the non-albicans Candida spp. emphasizes the need for speciation and antifungal susceptibility testing a routine in all microbiology laboratories due to alarming increase of resistant fungal infections.

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