Study of Colonisation Pattern and Antifungal Sensitivity Profile of Candida Species in Diabetic Patients
Correspondence Address :
Dr. Rakesh Tank,
Associate Professor, Department of General Medicine, SHKM Government Medical College, Nuh, Haryana, India.
Introduction: In recent times, various studies have pointed out the rise in prevalence of Candida species causing Urinary Tract Infections (UTIs). Candiduria appears to be underdiagnosed entity and thus has been the source of morbidity and mortality. Emergence of drug resistant Candida species has further complicated the patient management.
Aim: To understand the colonisation pattern and antifungal sensitivity profile of Candida species in diabetic patients.
Materials and Methods: This cross-sectional study was conducted for a period of six months (July 2019 to December 2019) at a tertiary care teaching centre of southern Haryana, India. A total of 360 urine samples were collected from patients admitted both out-patient and in-patient. Glycated Haemoglobin (HbA1c) ≥6.5% was taken as having diabetes. The urine samples were collected as per standard guidelines. Urine wet mount examination was performed. Confirmation of identification and antifungal susceptibility testing was done for all the Candida isolates. Data was analysed using statistical software.
Results: The prevalence of candiduria was 65 (18.1%) among study subjects. Out of significant candiduria patients 19 (29.23%) were having Candida albicans, 30 (46.16%) were Candida tropicalis, 10 (15.38%) were Candida krusei, 6 (9.23%) Candida glabrata. Candida albicans was found to be sensitive to amphotericin-B in 100% of cases. Sensitivity to flucytosine, voriconazole and fluconazole was found to be 89.47%, 89.47% and 84.21% respectively in case of Candida albicans.
Conclusion: The prevalence of candiduria is definitely high in this region. Non albicans Candida species are more resistant to antifungal drugs compared to Candida albicans.
Colony, Diabetes mellitus, Susceptibility
Diabetes mellitus is a common and frequently encountered health problem among patients, many of them present with UTIs. In past 2-3 decades, various studies have pointed out the rise in prevalence of Candida species causing UTIs. Candida species is found to be responsible for 10-15% of nosocomial UTIs in Indian scenerio (1). Not all cases of candiduria are diagnosed and reported hence it remains underdiagnosed entity and thus causes morbidity and mortality (2). All Candida species has potential of causing UTIs but as per the literature non albicans Candida species have replaced Candida albicans as the predominant pathogen (3).
Candida species are commensal under normal conditions but turns to pathogenic microorganism as defence system and mechanisms becomes weaker and may cause various kinds of infections in the body of host (4). Environmental conditions in the urinary tract of humans usually favour the growth of Candida species as observed in the literature (5). As per the available literature, higher susceptibility of fungal infections (including Candida species) to the the urinary tract of the host, is attributed to higher levels of urine glucose concentration in the host having diabetes (6). Pathogenic bacteria and fungi grow easily in any environment with raised levels of glucose concentrations.
Over a period of time irrational (prolonged and inappropriate) use of empirical therapy resulted in emergence of drug resistant Candida species that has further complicated the patient management. With this background, the present study was carried out to understand the colonisation pattern and antifunfgal sensitivity profile of Candida species in diabetic patients.
The present cross-sectional study was planned and rolled out under the aegis of Department of General Medicine in close collaboration of Department of Microbiology at a tertiary care teaching medical college situated at a lone aspiration district Nuh of southern Haryana, India. Study was conducted for a period of six months July 2019 to December. 2019. The study was instituted only after obtaining necessary clearance from Institutional Ethics Committee (IEC) of the medical college vide letter number SHKM/CM/2016/901 Dated 23.02.2016.
A total of 360 urine samples were collected from patients admitted both outdoor and indoor. Written informed consent was obtained from all the study subjects.
Inclusion criteria: Diabetic patients of both sexes and all age groups were considered for this study. HbA1c ≥6.5% was taken as having diabetes. Both OPD and IPD patients having urinary tract infections were included. Microbiologically, samples showing pure growth of yeast isolates with significant colony count were also included.
Exclusion criteria: Those urine samples in which Candida species was isolated but was not there, were excluded from this investigation. Similarly, those urine samples in which Candida with colony count was ≤1000 Colony Forming Unit (CFU)/mL and showing mixed growth (polymicrobial growth) were also excluded.
Sample size estimation: The calculation of sample size was done (n=360) considering the prevalence of Candida species causing vaginitis as 64.5% (7), with confidence level of 95% and 5% absolute error by applying the following formula:n = (Z1-a/2)2×p(1-p)/d2; where Z= Standard normal variate, a=Level of significance (0.05), p= Prevalence, d= Absolute Allowable error (5%), n= sample size.
The urine samples were collected as per standard guidelines (8). All samples were collected in containers that were sterile, leak proof and had screw capped lids. Samples were shifted to microbiology laboratory with no delay. Urine wet mount examination was performed. After that, urine samples were used for culture. Urine culture was done as per standard protocol (9). Samples were inoculated on Cysteine Lactose Electrolyte Deficient (CLED) and Mac Conkey agar. Incubation of culture plates was done at 37°C for 24-48 hours. Candida species isolated on culture plates with colony count >10000 CFU/mL were taken as significant (10).
Candida species were identified by inoculating isolates on Chromogenic (CHROM) agar. This was incubated for approximately 48 hours at 30°C. Observations were made as per manufacturers instruction (11). The identification of colonies was done on the basis of chromogenic reaction. Candida albicans was identified with Light green colonies on the CHROM Agar. Similarly Candida tropicalis was identified with blue colonies with pink halo, Candida glabrata was identified with cream to white colonies and Candida krusei was identified with purple fussy colonies on the CHROM Agar (12).
After that, confirmation of identification and antifungal susceptibility testing was done for all the Candida isolates using Vitek-2 compact system of biomerieux. The antifungal susceptibility was looked for amphotericin B, flucystosine, voriconazole, and fluconazole.
Any possible personal identifiers were delinked with the data before the analysis. Data collected was entered in Microsoft excel 7, and then data was analysed using Statistical Package for the Social Sciences (SPSS) 22.0 statistical software. Descriptive values were expressed as mean and Standard Deviation (SD). Categorical variables were written as the numbers of cases and percentage value.
A total of 65 Candida species were isolated from 360 urine samples thus the prevalence of candiduria was 18.1% among study subjects. Out of significant candiduria patients, different species showed different pattern (Table/Fig 1).
Considering the age and sex distribution maximum percentage of significant candiduria was seen in age group 31-60 yrs of age (50.76%), with male being 33.84% and females being 16.92%. Followed by age group of greater than 60 years (30.76%), Male were 18.46% and females were 12.30%. In age group 1-30 years total of 18.46% % were having significant Candiduria, Male 9.23% and Females 9.23% (Table/Fig 2).
Candida albicans was found to be sensitive to amphotericin B in 100 % of cases. Sensitivity to flucytosine, voriconazole and fluconazole is shown in (Table/Fig 3).
In clinical isolates of Candida tropicalis sensitivity to amphotericin B, flucytosine, voriconazole and fluconazole is shown in (Table/Fig 3) respectively. Antifungal sensitivity pattern in case of Candida krusei with amphotericin B, flucytosine, voriconazole and fluconazole is shown in (Table/Fig 3) respectively. Antifungal sensitivity pattern of Candida glabrata with amphotericin B, flucytosine, voriconazole and fluconazole is shown in (Table/Fig 3) respectively.
More than 150 varieties of Candida species are currently existing around us as saprophytes. Majority of them are harmless, only a few are pathogenic for human beings. Candida infections are frequently encountered in the patients having diabetes mellitus. Present study observed a total of 65 Candida species isolated from 360 urine samples thus the prevalence of candiduria was 18.1% among study subjects. This was in contrast to the study from Madhya Pradesh that which observed the prevalence of candiduria as 65% among diabetic patients (7). On the other hand, another study from Bihar reported the prevalence of candiduria as 11.25% (13).
Authors observed higher prevalence of candiduria in this investigation. It is really important to note the rising burden of non albicans Candida species. This is a matter of concern that needs to be addressed (14). Certain factors are considered responsible for such rising trend of candiduria, a few of them are prolonged antibiotic therapy, prolonged hospital stay, immunocompromised status of patient, immunosuppressive therapy, catherisation, etc (15). Process of catheterisation involves migration of microbes from outer surface into the urinary bladder thus increase the chances of episodes of UTIs. Emergence of resistance for Candida indiscriminate is attributed to irrationale use of antifungal drugs, especially azole group (16). Immunocompromised state and critically ill patients are at high risk of developing candidemia (17).
Present study observed the isolation rate of non albicans Candida was 70.77% i.e. 46.16% were Candida tropicalis, 15.38% were Candida krusei, 9.23 % Candida glabrata. Various scientific reports have proved the rise in prevalence of non albicans Candida species among candiduria. Another study by Kumari KS et al., was also in concordance with present study observations (18). They observed that, of 500 urine specimens, 66 (13.2%) yielded Candida isolates whereas among the 66 Candida species isolated from urine, 43 were non-albicans Candida species and 23 were Candida albicans.
Regarding antifungal sensitivity profile and pattern of Candida species among study subjects, this study noted that Candida albicans was found to be sensitive to amphotericin B in 100% of cases. Sensitivity to flucytosine, voriconazole and fluconazole was found to be 89.47%, 89.47% and 84.21% respectively in case of Candida albicans. Identification of Candida species is important as non-albicans Candida are more resistant to azoles compared to that of Candida albicans.
Candida krusei is intrinsically resistant to fluconazole. Present study also observed that antifungal sensitivity pattern in case of of Candida krusei, antifungal sensitivity pattern with amphotericin B, flucytosine, voriconazole and fluconazole was 70%, 70%, 50% and 40% respectively. Antifungal sensitivity pattern of Candida glabrata with amphotericin B, flucytosine, voriconazole and fluconazole was 83.33%, 50%, 50% and 66.67% respectively. Antifungal susceptibility shows that Candida isolates were more susceptible to amphotericin B and flucytosine as compared to azole group. Clinicians commonly use azole group to treat candiduria thus increasing to fluconazole is noteworthy and challanging to us. Present study observed that Candida albicans were found to be more susceptibility to azole group compared to non albicans Candida. The result of this study was in agreement with previous study by Pramodhini S et al., (19). Understanding the colonisation pattern and antifungal sensitivity profile of Candida species are important for planning the management of such subjects. This adds to the strength of study investigation.
(Table/Fig 4) summarises the pattern of colonisation and sensitivity pattern as observed by various authors from different parts of India (13),(20),(21),(22),(23).
Due to financial and logistics constraints, authors could not perform comprehensive analysis of urine as samples of polymicrobial growth and pyuria were excluded.
The findings of this study shows that the prevalence of candiduria is definitely high in this region. Various species of Candida are responsible for causing UTI. Information on antifungal susceptibility pattern will help clinicians for better management of patients. Non albicans Candida species are more resistant to antifungal drugs compared to Candida albicans. Better understanding of colonisation pattern and antifunfgal sensitivity profile shall definitely help for better management of candiduria.
Date of Submission: Nov 17, 2021
Date of Peer Review: Jan 03, 2022
Date of Acceptance: Mar 04, 2022
Date of Publishing: Jun 01, 2022
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
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