The Spectrum of Pathogenic Yeast Infection in a Tertiary Care Hospital in Assam, India

Context: In view of the growing incidence of pathogenic yeast infection all over the world, this study was undertaken to understand its etiology and epidemiology in Assam. Aims: To characterize and study the antifungal susceptibility pattern of the pathogenic yeasts from the clinical samples. Settings and Design: The study was a hospital-based cross-sectional study. Methods and Material: 150 patients were enrolled in the study and from which clinical samples were collected. A total of 83 samples showing the growth of yeast in culture were included in the study. The yeasts were identified by conventional and BioMerieux ID 32C and VITEK 2TM. Antifungal susceptibility test was done by disk diffusion method as per Clinical and Laboratory Standards Institute (CLSI), M44-A2. Statistical analysis used: Data was analyzed using statistical software Epi-Info 7.1.2.0 (2013; CDC, Atlanta, USA). For comparison of categorical data, the Chi-square test or Fisher exact test was used. A value of p less than 0.05 was considered statistically significant. Results: The most affected population was the age group of ≤10 years (32.5%) with male preponderance (67.5%). Yeasts were mostly isolated bloodstream infections (49.3%). The major risk factor was prolonged antibiotic intake. Predominant yeast isolates were Candida albicans (43.4%) followed by Candida tropicalis (19.3%). Emerging yeasts like Kodaemea ohmeri (4.8%), Pichia anomala (2.4%), and Candida auris (1.2%) were also isolated. Amphotericin B was effective against all yeast isolates. All the isolates of Candida krusei were resistant to all the azoles. Conclusions: The study reflects that there is a growing incidence of emerging yeast infections and efforts are to be made for their identification and antifungal susceptibility testing for the initiation of appropriate therapy.


Introduction
Infections have dramatically increased over the last few decades due to pathogenic yeasts. The increasing risk of opportunistic fungal infections is due to growing numbers of immunocompromised hosts, including AIDS patients, the abuse of broad-spectrum antibiotics, immunosuppressive agents after organ transplantation, and cancer chemotherapy [1,2]. Apart from Candida species, various fungi like Trichosporon, Cryptococcus neoformans, and some emerging yeasts have been widely reported [3,4]. These opportunistic yeasts are ubiquitous and they can be acquired from the normal endogenous flora (Candida) or obtained from host surroundings (Cryptococcus) [5]. The ARTEMIS global program is one of the most comprehensive and long-running fungal surveillance programs. This program generates massive amounts of data that have been externally validated and that can be used to identify temporal and geographic trends in the species distribution of pathogenic yeasts [6]. On the global scale, Candida species accounted for 95-97% of all clinical isolates in each study year from 1997 to 2005 [6]. Candida albicans was the most frequent (overall, 65.6% of all Candida species), followed by Candida glabrata (11.2%), Candida tropicalis (7.0%), and Candida parapsilosis (5.8%). Among the non-candidial yeast, C. neoformans (31.2%) was most frequent, followed by Saccharomyces species (9.6%), Trichosporon species (6.7%), and Pichia anomala (2.5%) [7]. In India also prevalence rate of Candida infection was reported to be 6% in a 5-year study (2001)(2002)(2003)(2004)(2005) [8].There is an increasing drift of disease by non-albicans Candida (NAC) [8,9]. C. neoformans is a saprophytic encapsulated haploid yeast that is distributed worldwide in association with avian excreta, mostly pigeons [10]. There is a rising trend of cryptococcosis in India which is posing a serious threat. As per different Indian studies, Cryptococcal infection was seen in the range of 2.09-53.1% [10][11][12]. Fluconazole resistance among NAC ranges from 14.5% to 41.6% [10,11]. Candida auris has caused increased clinical attention due to its multidrug resistance [13][14][15]. To manage patients suffering from yeast infections, antifungal susceptibility has become important for initiating treatment. Antifungal susceptibility testing using a disk diffusion method is a simple, rapid, and cost-effective method for screening the susceptibility pattern of yeast [16]. Although the epidemiology of pathogenic yeast infection has been reported from various parts of India, there is a paucity of data on pathogenic yeast infection in Assam [17]. Also, there is a lack of data concerning the in-vitro susceptibility of pathogenic yeasts and yeast-like fungi from Assam. Considering the lack of availability of local epidemiological data, this study was undertaken to characterize and study the antifungal susceptibility pattern of the pathogenic yeasts from the clinical samples.

Materials And Methods
The study was conducted in the Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, for a period of one year from June 2017 to May 2018. It was a cross-sectional study. One hundred and fifty patients were enrolled in the study and from whom clinical samples were collected. The samples were collected from the patients attending OPD and indoors during the study period. Eighty-three samples showing growth of yeast in culture were further processed for their identification and antifungal susceptibility testing. Approval from the Institutional Ethical Committee (IEC) was obtained before the commencement of the study. The clinical specimens (blood, CSF, other body fluids, and sputum) were collected under aseptic conditions. The samples were processed as per standard protocol [18]. Direct microscopy (Gram Stain, Indian Ink) was done. Isolation was done from the culture in Sabouraud's dextrose agar with chloramphenicol (0.05 mg/ml). Identification of different Candida species was done by Germ tube test, Corn Meal Agar morphology (Dalmau Technique), HiChrom Candida Agar, sugar assimilation and fermentation tests and using Biomerieux ID 32C, and VITEK 2 TM with YST card for yeast identification system. For confirmation of Cryptococcus species, urease hydrolysis tests and phenol oxidase tests were done. Antifungal disk diffusion susceptibility test was done as per the Clinical and Laboratory Standards Institute (CLSI) protocol (M44-A2, CLSI, USA) to find the susceptibility pattern of the fungal growths (

Results
Eighty-three samples showing the growth of yeast in culture were further processed for their identification and antifungal susceptibility testing. Maximum number of the patients (32.5%) were of ≤10 years of age followed by 24.1% in the >60 years age group. This shows that higher incidence of infection with pathogenic yeast was common in the extremes of age. Majority (49.3%) of the patients presented with bloodstream infection (BSI). The second common clinical presentation was bronchopneumonia and pleural effusion (36.1%) followed by meningitis (12%) and tuberculosis (2.4%).
With regard to the risk factors, 88% of the patients in this study had a history of prolonged antibiotic intake (≥7 days) within the last 1 month and it was the leading risk factor in this study. The antibiotics that were used were cephalosporins, carbapenems, and aminoglycosides mostly. Another leading risk factor in this study was ≥7 days stay in the hospital (88%). The second commonest risk factor was indwelling devices like central line, intravenous cannula, and urinary catheter. 92.8% of the patients had history of indwelling devices.
In this study, the maximum number of isolates (

Discussion
This study was done to determine the occurrence of pathogenic yeast infections and their antifungal susceptibility pattern in Assam. Most of the studies showed the growing incidence of pathogenic yeasts in immunocompromised patients. The present study showed a higher incidence of infection with pathogenic yeast was common in the pediatric age group as well as in the geriatric age group as the immunity is low in the extremes of age, which is in concordance with the study by Kim et al. (2016) who reported that 75.8% of the patients were in the age group of >60 years of age [20]. The present study showed a higher incidence in males. However, the finding was not statistically significant. A similar finding was also reported by Shaik et al. (2016) who also found that 62.6% males and 37.3% females had an infection due to pathogenic yeasts [13]. In the present study, the majority (49.3%) of the patients presented with BSI. The second most common clinical presentation was bronchopneumonia and pleural effusion (36.1%) followed by meningitis (12%) and tuberculosis (2.4%). This data was statistically significant. Shaik et al (2016) found medical causes 21 (14%) which included pleural effusion, bronchopneumonia, and tuberculosis associated with the isolation of species [13]. With regard to the risk factors, as consistent with the study by Juyal et al. (2013) found that 61.36% of the patients had a history of prolonged broad-spectrum antibiotic use [21]. The second commonest risk factor was indwelling devices like central line, intravenous cannula, and urinary catheter. 92.8% of the patients had a history of indwelling devices. Juyal et al. (2013) in their study found that 64.39% of the patients had a history of indwelling catheters [21]. The study of Kim et al. (2016) reported a higher incidence of fungal isolates in blood after urine, which was consistent with our study [20]. A study by Shaik et al (2016) found 26.9% of fungal isolates in respiratory samples [13].
In the present study maximum preponderance of isolates was C. albicans and this finding is in concordance with Kim et al., 2016 (48.6%) [20]. Among the NAC, C. tropicalis was the most common isolate. Chakrabarti et al. (2015) also found C. tropicalis as the most common isolate with an isolation rate of 31.2% and 41.6% [22].
The study of Taj-Aldeen et al. (2006) reported K. ohmeri in neonatal candidemia [23]. In the present study, the third most common Candida species was C. parapsilosis (10.8%). This finding corresponds with the finding of Chakrabarti et al. (2015) who also reported C. parapsilosis to be the third most common Candida species (10.9%) [22].
The study showed that P. anomala isolates were isolated from blood in neonates. This shows a higher incidence of infection in neonates which also corresponds with the findings of the study by Pasqualotto et al. (2005) [24]. Shaik et al. found a similar isolation rate of C. krusei (4.7%) [13].
The antifungal susceptibility pattern showed that all the yeast isolates were sensitive to amphotericin B, which correlates with the report of Shaik et al (2016) [13]. Shaik et al. (2016) also reported that 76.6% and 63.3% of C. albicans were sensitive to fluconazole and voriconazole, respectively, which is in concordance with the present study [13]. As fluconazole has been found to be widely resistant so discovery of benzylthioanalogs of fluconazole as a potent antifungal [25].
In the present study, it was not possible to identify K. ohmeri, C. auris, P. anomala by conventional methods, and they were identified by automated culture methods only. Therefore, conventional and automated methods are required for the identification of pathogenic yeast. Variation of species distribution is also dependent on variation in methods adopted in different studies.
The limitations of the study were that the characterization and antimicrobial susceptibility of the organisms are done using conventional methods and automated blood culture system but molecular characterization could not be done. The sample size is also less as the study was done for only a year.

Conclusions
To conclude, the incidence of pathogenic yeast infections is increasing with a gradual increase in infection with some emerging yeasts, many of them showing resistance to commonly used antifungals, making it necessary for their speciation. Hence, identification of the yeasts up to their species level is of utmost importance for the commencement of appropriate antifungal therapy.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Gauhati Medical College Institutional Ethics Committee issued approval 190/2017/pt-1/EC/120. Study was conducted after getting ethical clearance from institutional ethics committee, Gauhati Medical College and Hospital, Guwahati, Assam. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.