Antifungal Drug Resistance among Candida albicans and Non-albicans Candida Species Isolates from a Tertiary Care Centre at Allahabad

Objective: The aim of our study was to identify the distribution of Candida species among clinical isolates and their sensitivity pattern for common antifungal drugs. Methods: This descriptive study was carried out in a tertiary care hospital in Allahabad. A total of One hundred and four Candida isolates were included in the study. Identification and speciation of the isolates were carried out by Germ-tube test, Chlamydoconidia production test, colony characterization in chromogenic agar medium, carbohydrate assimilation test and growth at 45°C. Four antifungal drugs such as Fluconazole (25 mcg), Clotrimazole (10 mcg), Nystatin (100 U) and Amphotericin-B (100 U) were tested by disk diffusion method. Descriptive statistics was used which involves the use of simple percentage and bar chart to analyze the data. In addition, Chi-square test was performed and P value was calculated. Results: Out of 104 Candida isolates, 60 (58%) were from Urine, 36 (34.5%) were from Sputum and 8(7.5%) were from blood isolates. Of the total of 104 isolates 64 (61.5%) were Candida albicans, 20(19%) Candida dubliniensis, 10 (9%) Candida tropicalis, 7(6.5%) Candida parapsilosis and 4(4%) Candida glabrata respectively. Resistance rates for commonly use antifungal drugs among the isolates are as followed Fluconazole 76 (73%), Amphotericin B 40 (38.5%), Nystatin 4(4%) and Clotrimazole 60 (58%) respectively. For Candida albicans and Candida dubliniensis most active antifungal drug was Nystatin (93%; 100% respectively). Discussion: Species level identification of Candida and their antifungal sensitivity testing should be performed routinely in all microbiology laboratories to prevent the spread of antifungal resistance.


Introduction
Candida is major human fungal pathogens that cause both mucosal and deep tissue infections. In recent years, the incidence of Candida infection has increased largely because of the rising rate of immune suppressive therapy [1] and also enhances the infection with multidrug resistant bacteria which leads to overuse of higher classes of antibiotic and as a result there is a destruction of commensal bacteria, owing to this there is an increased opportunity of Candida to cause infection in those patients. The treatment of such Candidal infections is a major concern since they are showing a high rate of anti-fungal drug resistance.
For many years Candida albicans is the predominant cause of invasive candidiasis in the majority of clinical settings, nevertheless, the epidemiology of Candida infection has changed in recent years, with longitudinal studies reporting that a considerable proportion of patients are now infected with non-albicans candida (NAC) species.
Thus the potential clinical importance of species-level identification has been recognized as Candida species differ in the expression of putative virulence factors and antifungal susceptibility [2,3].
In India, there is insufficient data regarding the Candida species and their susceptibility pattern [4] hence, the present study was undertaken to identify different Candida species and their sensitivity pattern with the available antifungal agents.

Material and methods
This prospective study was conducted during the period from November 2016 to April 2017 from patients of the tertiary care hospitals in Allahabad after obtaining permission from the institutional ethical committee. One hundred and four strains of Candida species with significant growth were isolated from the specimen such as clean catch midstream urine, blood and sputum using standard sterile procedures from the study population. Those subjects who had received antifungal drugs during the past one month, Urine and sputum samples shows insignificant growth of Candida species and polymicrobial infections were excluded.

Isolation and identification
based on colony morphology, Gram staining and by standard biochemical properties which include:

Germ-tube test
Yeast cells were inoculated into 0.5 mL of pool serum and incubated at 37°C for 2.5 hour. After this period, aliquots were removed for microscopic examination. Germ tube was considered as a slender tube with straight walls, without septum and without constriction at the junction between the cells [5].

Chlamydoconidia production test
Chlamydoconidia production test was performed using corn meal agar medium. The samples previously grown in SDA were seeded as 3 parallel streaks in a rectangular piece of corn meal agar placed between two slides, incubated in the wet chamber at 30°C for 72 h and visualized in an optical microscope (10X and 40X magnification). The formation of rounded spores with double-wall isolates was observed as chlamydoconidia and was indicative of Candida albicans or Candida dubliniensis [6].

Chromogenic agar culture
Each isolate was cultured on SDA at 30°C for 48 hour. After this, they were seeded on HiCrome Candida Differential agar (Hi Media, Mumbai) and incubated at 30°C for 48 hr. The CHROMAgar ™ allows selective yeast isolation, identifying colonies of Candida albicans, Candida dubliniensis, Candida tropicalis, Candida glabrata and Candida krusei on the basis of colour and colony morphology. The strains were identified according to the manufacturer's instructions, which define light green colonies as Candida albicans, purple fuzzy colonies as Candida krusei, steel blue to blue as Candida tropicalis and cream to white as Candida glabrata [7].

Carbohydrate assimilation test
The carbohydrate assimilation test determines the ability of a yeast isolate to use a particular carbohydrate substrate as its sole carbon in a medium. Yeasts were cultured in YPD for 24 h at 30°C. After the cultures were centrifuged for 5 min at 2500 g and the pellets were washed 3 times successively, suspended in sterile saline and adjusted to 5 McFarland standards. A volume of 300 μL of Yeast Nitrogen Base agar (Hi Media, Mumbai, India) and 1.5 mL of Candida sp. suspension was mixed with 30 mL of sterile bacteriological agar in Falcon tubes. The suspension was placed in 15 cm Petri dishes and after solidification of the medium, discs with the 2% carbohydrates (maltose, trehalose, xylose, galactose, lactose, sucrose and glucose) were placed. The suspension was incubated at 30°C for 96 hr, and observed daily. Glucose was used as a positive control since all the species of Candida assimilate this carbohydrate. The carbohydrate assimilation was observed with a presence of a halo of growth around each carbohydrate [6,7].

Growth at 45°C
Growth at 45°C has been considered a useful test for the differentiation of Candida dubliniensis (no growth) from Candida albicans (growth). This test was used in the positive samples for the germ-tube test, for chlamydoconidia production and that developed green colonies in HiCrome agar. The temperature test was performed in SDA (with Antibiotic) medium. All samples were incubated at 45°C, and growth was assessed daily for 10 days. 6
Antifungal susceptibility testing was performed by disk diffusion method using Mueller-Hinton Agar + 2% Glucose and 0.

Results
A total of 104 isolates of Candida species were obtained from different clinical specimens of all age group patients visiting the outpatient's department and in-patient department the period from June 2016 to June 2017. Of these 60 (58%) were from Urine, 36 (34.5%) were from Sputum and 8(7.5%) were from blood isolates.
We also found that a maximum number of blood isolates were from non-Candida albicans species [7(7%)], and Candida albicans were most common among sputum and urine isolates. (Table 1

Discussion
The epidemiology of Candida infection has changed in recent years. For many years Candida albicans were the most dominant species to cause invasive infection in most clinical settings, a considerable percentage of patients are now infected with non-albicans Candida species. Infections with these Candida species also have a direct impact on the choice of empiric antifungal therapy and clinical outcome as they show resistance to multiple antifungal drugs.
In the present study, we have found that among the study population maximum numbers of the isolates were from urine, which indicates that other than bacteria in our population Candida which also causes the substantial number of infection. Several other investigator form India also reported a high prevalence of Candida causing UTI [4]. However, the incidence of invasive isolates was less in our study isolates in compare to other studies where they had found a high prevalence of invasive isolates in their study population [9,10].
Result of our study has shown that a maximum number of the isolates were from male patients and elder population were mostly effected which is with the agreement of the study done by Pahwa et al. [4] We are not the first to report that diabetics were one of the most common predisposing factors for candidiasis other investigator also reported the same [11]. These findings indicate that due to the poor immune response by elderly patients and predisposing factor such as diabetes mellitus may act as an important risk factor for candidiasis.
In the present study we observe that in our population with Candida albicans as well as non-albicans species were also causing infection, several other investigators also reported the same from the different part of the country [4,9]. A study done by Deorukhkar et al [12] showed a higher frequency of NAC (63.3%) in comparison to Candida albicans (36.7%). Yet another study by Bajwa and Kulshrestha have quoted that the isolation rate of NAC documented in India ranges from 52% to 96%, with Candida tropicalis being the predominant species instead of Candida glabrata or Candida parapsilosis in all age groups. [13].
The antifungal susceptibility testing revealed that amphotericin B exhibited a higher sensitivity against Candida albicans, Candida dubliniensis respectively, which points out that this drug can serve as a good candidate for empirical therapy of Candida albicans and Candida dubliniensis infections. This finding is similar to the studies done by Kaur et al. [14] and Mondal et al. [15].
However, non-albicans and non-dubliniensis Candida were showing the high degree of resistance against Amphotericin B. The most active antifungal drugs for all the Candida species were Nystatin.
We also found that Clotrimazole was more active against nonalbicans species (80%) which is similar to the study done by Ajitha et al. [16]. Where they have found around 67% of their non-albicans isolates were sensitive to clotrimazole. However, in the present study, we have found the very high label of Clotrimazole resistance among the Candida albicans isolates.

Conclusion
In conclusion, microbiology laboratories must be able to detect the different species of Candida and their resistance pattern in a timely manner, Microbiological excellence is needed more than ever, and the resistance should be detected accurately. In addition, there should be good communication between the microbiologist and the health care worker to make better patient outcomes, facilitating effective infection control, reducing spread of resistant pathogens and helping hospitals to meet accreditation standards.