Evaluation of Epidemiological Pattern of Candida Species Associated with Candidemia from A Tertiary Care Facility in South India

Candidemia ranks the 4 th most prevalence cause of bloodstream infections and stands out as the primary cause of invasive fungal infections among hospitalized patients. Its incidence varies globally from 0.33 to 6.51 episodes per 1000 admissions, representing a major public health burden due to its increasing incidence and high mortality rates. The present research work has been conducted to identify the distribution of Candida species among septicemic patients and to determine the patterns of antifungal susceptibility of Candida species isolates from them in a tertiary care center in South India. Among the 88 Candida isolates, 13 (14.8%) were speciated and identified as C. albicans and 75 (85.2%) were Candida non-albicans . Of them, C. tropicalis (42%) ranks more prevalent. The distribution of virulence factors among 88 Candida isolates revealed that 49 isolates (55.7%) exhibited phospholipase activity, hemolysin production was detected in 68.2% of isolates, biofilm production was demonstrated in 73.9% isolates and coagulase activity was observed in 46.7% isolates. In the present study, Candida species were most sensitive to Amphotericin B (94.3%), which is followed by Caspofungin (93.2%), Voriconazole (92%), Micafungin (90.9%), and the least was observed with Flucytosine (78.4%) and Fluconazole (71.5%). Thus, in order to improve treatment responses, the insights acquired from this research will aid in clinical management and the development of antifungal stewardship recommendations.


INTRODUCTION
Candidemia remains the most prevalent invasive nosocomial fungal infection worldwide.Its incidence varies globally from 0.33 to 6.51 episodes per 1000 admissions, representing a major public health burden due to its increasing incidence and high mortality rates. 1 Frequently administering antibiotics, employing central venous catheters, as well as other invasive medical devices, along with conditions including stays in the intensive care units (ICUs) and could make patients at risk of obtaining a Candida infection. 2lobal reports have indicated rising incidence of candidemia brought on by Candida krusei, Candida tropicalis, Candida glabrata, Candida parapsilosis, despite the fact that Candida albicans remains the most prevalent species being responsible for more than 50% of cases in some series. 3 a n d i d a s p p . a r e c o m m e n s a l organisms present in the gut and skin of 50-70% of healthy individuals in low numbers due to competition within the microbiome. 4][6] The evolution of Candida from a commensal organism to a formidable pathogen is facilitated by a number of host variables as well as the virulence of the infecting species.Patients who are critically sick yet are immunocompetent can also frequently have candidiasis as Candida formerly thought to play a passive part in the overall process of infection initiation and development. 7Candida has a variety of virulence factors that determine its capacity for penetration of host tissues.The virulence factors of Candida are multifaceted and include the formation of biofilms as well as the release of extracellular hydrolytic enzymes that damage tissue, such as phospholipases, hemolysin, and proteinases.The capacity for it to adhere to medical equipment and host tissues, as well as the development of pseudohyphae makes it pathogenic. 8Comprehension and evaluation of these virulence characteristics in newly developing drug-resistant yeast infections will aid in the creation of innovative target-specific medications in the future. 9 growing number of strains of Candida species are emerging that are resistant to azoles, mostly in the form of prophylaxis and to a lesser extent with echinocandins have been associated with the occurrence of breakthrough infections with resistant Candida species. 10his is an extremely disturbing trend, possibly associated with increased use of azoles as prophylaxis, especially in surgical units and intensive care units. 11Therefore, there is a need for continuous surveillance to monitor trends in incidence, species distribution and antifungal drug susceptibility profiles of Candida BSI. 12 Hence, the present research work has been conducted to identify the distribution of Candida species among septicemic patients and to determine the patterns of antifungal susceptibility of Candida species isolates from them in a tertiary care center in South India.

MATERIALS AND METHODS
The present cross-sectional study has been undertaken at a tertiary care centre, and from the Institute Ethical Committee for Human Studies, ethical permission (2896/IEC/2021) was acquired.A sum of 2114 blood samples from suspected septicaemia cases was collected in blood culture bottles throughout ICUs and various wards between August 2021 and December 2022.In order to detect gram-positive budding yeasts, bottles displaying positive signals were subjected to direct gram staining technique and cultured onto Sabouraud dextrose agar (HiMedia, India) incubated for a duration of 24 to 48 hours at 25°C and 37°C.Candida colonies were detected and speciated in VITEK 2 Compact (Biomerieux) using VITEK®

Analysis of MIC results
To compute the percentage of essential agreement (A) between the MIC values and the species identification, the repeatability and accuracy of the data produced with the VITEK 2 system were compared to the reference methodologies.The critical agreement was determined by comparing differences in MIC endpoints of more than ±2 log2 dilutions, following Pfaller et al. 13 Comparisons were made between the reference broth microdilution (BMD) panels read at 24 and 48 hours and the MIC values from the VITEK 2 yeast susceptibility test.5][16] The essential agreement (EA) was determined by comparing the MIC endpoints of more than two dilutions (two wells) in disagreement.The CLSI guidelines (M27-A2 4 th Edition) were utilised to interpret the MIC breakpoints for Flucytosine, Micafungin, Amphotericin B, Voriconazole, Fluconazole, and Caspofungin.The MICs produced by VITEK 2 and the reference BMD were then compared to calculate the percentage of categorical agreement (CA). 17When the VITEK 2 MIC showed S and the reference MIC indicated R, very substantial errors (VME) were found.When the isolate was categorised as R by VITEK 2 and S by the reference technique, major errors (ME) were found.When the findings of one test technique were S or R and the other was SDD, minor flaws were identified.The C. krusei MIC data were utilised as-is (in micrograms per millilitre) to evaluate EA; however, while calculating CA, the CLSI forced the results into the R category. 17

Assessments of virulence factors Phospholipase Production.
The standard test strain was then inoculated onto agar enriched with egg yolk using a 5 µL aseptically produced inoculum.Following a 48-hour incubation period at 37°C, the Petri plates were given time to dry at room temperature.The colony was encircled with precipitation zone existence was then studied and this showed that the phospholipase enzyme was expressed.C. albicans ATCC 10231 served as the positive control.The phospholipase index (Pz) is obtained by dividing the colony diameter by the precipitation zone ratio.Positive activity is shown by Pz < 1, whereas no phospholipase synthesis is shown by Pz > 1.For every isolate, the test was conducted three times in duplicate to reduce experimental error. 18

Haemolysin production
I t w a s d e m o n s t r a t e d o n S D A supplemented with sheep blood and gentamicin and inoculated with 10 µL of prepared inoculum obtained from the isolates.Following this, C. albicans ATCC 90028 was used as a control and 48 hours of 37°C incubation was required for the aforementioned medium-containing petri plates.Hemolytic activity (Hz) is determined as the ratio of the colony diameter to the visible hemolysis zone (in millimetres). 19

Biofilm production -crystal violet assay
The Crystal Violet test technique described by Kuhn et al. was used to measure the production of biofilms. 20The microtiter plate was added with 0.1% aqueous solution of crystal violet (120 µL) for 15 minutes.Sterilised distilled water was used to wash the wells four times.A 15-minute incubation period at room temperature was then followed by the addition of 125 µL of 95% methanol to each well to de-stain the experiment.Then, using an Enzyme-Linked Immunosorbent Assay reader, the de-stained wells were examined using spectroscopy at 570 nm.Reference strains of C. albicans (ATCC 90028) served as a positive control strain, while C. glabrata (ATCC 90030) was used as a negative control strain. 21,22he cut-off optical density (ODc) was defined as three standard deviations above the mean OD of the negative control, and the strains were classified as follows: OD Values Biofilm Formation ODc < OD strong biofilm producer 2 x ODc < OD ≤ 4 x ODc moderate biofilm producer ODc < OD ≤ 2 x ODc weak biofilm producer OD ≤ ODc no biofilm producer

Coagulase activity
Coagulase production was measured in accordance with the procedure devised by Yigit et al. 23 After adding 500 µL of rabbit plasma as a supplement, 0.1 mL of an overnight inoculum was aseptically added to a tube.The tubes were examined after being incubated at 35°C for 2, 4, 6, and 24 hours.When a coagulase test is positive, clot formation will appear.

Data analysis
SPSS software (version 22, IBM Inc.Chicago) was used for analysis following data collection.P-values <0.05 were significant.

Virulence activity among Candida isolates
The  3).

Antifungal susceptibility pattern of Candida isolates by VITEK 2
The 88 Candida isolates underwent Antifungal Drug Susceptibility tests using VITEK 2 against Flucytosine, Micafungin, Amphotericin B (100 U), Voriconazole, Fluconazole, and Caspofungin.Based on their MIC values, the isolates were categorized as Sensitive (S) and Resistant (R) to each antifungal drug.In the present study, the most sensitive antifungal agent was Amphotericin B (94.3%), which is followed by Caspofungin (93.2%),Voriconazole (92%), Micafungin (90.9%), and the least was observed with Flucytosine (78.4%) and Fluconazole (71.5%) as shown in Table 4.
Out of 88 Candida isolates, 13 C. albicans were 100% sensitive to Voriconazole, Amphotericin B, and Caspofungin.Whereas, among them, 3 (23%) isolates showed resistance to fluconazole and 1 (7.7%) isolate was resistant to each of Micafungin and flucytosine.Among CNA, C. auris and C. lipolytica demonstrated 100% sensitivity to all the antifungals used.The highest resistance was observed against Fluconazole (29.5%).Nonetheless, due to their intrinsic resistence, C. krusei (100%) displayed total resistance to fluconazole.In turn, it has been noted in this investigation that CNA isolates exhibited greater resistance to antifungal drugs in comparison to C. albicans (Table 4).

DISCUSSION
In the current study, C. tropicalis (n=37; 42%) emerged to be the species with the greatest prevalence detected out of the total.][26] Additionally, C. tropicalis (27.7%) was reported to be the major species causing candidemia in Chile by Ajenjo et al. 27 Conversely, the research findings by Tan et al. demonstrated that the most common isolate from individuals with candidemia was C. albicans (41.3%). 28However, over the past several decades, there was a steady rise in the isolation of CNA isolates from candidemia patients.CNAs are known to be associated with greater death rates because of their enhanced virulence and decreased sensitivity to antifungal medications.The result is significant at p < .05A notable finding in the current research is the identification of C. auris in 3 isolates (3.4%) among all cases of candidemia.The findings did not agree with the results of another recent study in India by Rajni et al. and Shastri et al.In the previous research, the prevalence of C. auris accounted for about 11% and 39.9% of candidemia cases, respectively. 25,29The development of this C. auris is extremely worrying since it causes serious infections in critically sick patients and spreads quickly across the hospital due to its resistance to numerous antifungals.The inability of the widely used phenotypic commercial method to correctly detect this fungus is another significant problem.When creating a treatment strategy, a precise identification of this yeast is crucial.We speculate that the low number of studies from India reporting C. auris may be due to misidentification by traditional methods.However, healthcare professionals must be aware of this multidrug-resistant fungus and ready for any possible breakout. 30 wide range of virulence factors, the transition from yeast to hyphae, involving biofilm formation, the release of tissue-damaging hydrolytic enzymes like phospholipases, proteases and hemolysins), adherence and invasion to target cell surfaces, and immune cell evasion, all contribute to Candida's pathogenicity. 31,32Candida species secrete phospholipases that are involved in tissue invasion, and they hydrolyze phospholipids as substrates, rupturing host cell membranes in the process. 31,33Out of the 88 Candida isolates found in this study, 49 (55.7%)showed evidence of phospholipase activity.These results were compared with the findings of a study by Sachin C et al. (60.9%), with C. tropicalis having the highest prevalence of phospholipase activity (38.8%). 34hese results are comparable to those of the study conducted by Sharma et al., which found that in 30% of patients had C. tropicalis displaying significant phospholipase activity. 35On the other hand, according to a prior work by Saiprom et al. all 26.3% of the C. albicans isolates generated high phospholipase activity, but none of the NAC spp.showed phospholipase activity. 31he ability of Candida to produce hemolysin, which lyses red blood cells to extract iron from haemoglobin and promotes hyphal penetration and yeast spread in the host, is a critical component of the virulence of Candida. 31,36emolysin production was found in 60 (68.2%)Candida isolates in our investigation; the current study's findings were consistent with those of Galan-Ladero et al. study, which revealed that around 77.2% of Candida isolates had hemolysin production. 37In this investigation, hemolysin production was predominantly reported in 25 (41.7%) of C. tropicalis.The results of the current investigation were consistent with those of Selvan et al. study, which indicated that 61.1% of C. tropicalis was a hemolysin producer and the proportion of hemolysin producers was significantly lower than that of our study. 26owever, the findings of the study by Nouraei et al. demonstrated that 100% of Candida albicans generated hemolysin activity. 38Additionally, the findings of the study by Badran et al. revealed that isolates of C. albicans (84.9%) exhibited hemolysin activity the most frequently. 39ne of the most studied virulence variables linked to bloodstream infections caused by Candida is biofilm development, which has also been linked to pathogenicity in candidemia connected to catheter use. 40Furthermore, the production of biofilm makes it easier for Candida to adhere to prosthetic joints, vascular catheters, cardiac devices. 41Due to its well-known strong resistance to antifungal medications, Candida biofilm is a major factor in the infection's fatality. 40 Of 42 (46.6%)Candida isolates that showed coagulase activity among 88 Candida isolates, 13 (31%) were C. albicans, which demonstrated the greatest coagulase activity.These results were quite similar to those reported by Yigit et al., whose work found that coagulase activity was found in Candida albicans isolates, accounting for 50.6% of the isolates. 23andida species has a dynamic resistance pattern that differs across different regions. 44The antibiogram pattern of 88 Candida isolates in this study indicated that the most sensitive antifungal agent was Amphotericin B (94.3%), which was consistent with the findings of studies by Siopi et al. and Giri et al. that showed 100% of the Candida isolates were Amphotericin B sensitive. 45,46The current study demonstrates that the greatest resistance was seen against fluconazole (29.5%), and these results were consistent with research studies by Giri et al. that indicated the greatest percentage of Candia isolates (30.8%) were resistant to fluconazole. 46Because fluconazole is the most commonly used antifungal medication that is empirically administered to all high-risk patients, resistance to it is increasing.This has led to the emergence of resistant strains, including C. glabrata and intrinsically fluconazole-resistant forms of C. krusei. 47,48In contrast, CNA species exhibited higher resistance to flucytosine (16.04%) and fluconazole (14.81%) in research by Gautam et al. than C. albicans. 38In line with other research findings by Bhattacharjee et al. and Gautam et al., a lower prevalence of resistance has been determined for Micafungin (8.57%), Voriconazole (8%), Caspofungin (6.8%), and Amphotericin B (1.43%).A few more isolates also showed resistance to Flucytosine (20%). 44,48n this investigation, all 13 of the C. albicans isolates showed complete sensitivity to caspofungin, amphotericin B, and voriconazole.These results coincide with the findings presented by Solomon et al., which demonstrated that all isolates were susceptible to echinocandins (caspofungin), voriconazole, and amphotericin B. 49 Conversely, the results of the study by Bhattacharjee et al. revealed that all of the C. albicans isolates were 100% susceptible to fluconazole, and that resistance to flucytosine (64.3%), amphotericin B (53.6%), itraconazole (21.4%), and voriconazole (10.7%) was identified. 44ut of all the CNA isolates, C. lipolytica and C. auris showed complete sensitivity to every antifungal that was used.In contrast, C. auris was shown to have a high resistance pattern for fluconazole in research by Solomon et al., suggesting that 9 Candida isolates tested against the drug were sensitive. 49here has been a significant shift in the prevalence of Candida bloodstream infections from C. albicans to CNA species.Consequently, the sensitivity of each species to the antifungal medications now in use varies greatly, early and precise detection of Candida infection is imperative.Aside from assisting clinicians to administer the right antifungal medication in a timely manner, laboratory testing for antifungal susceptibility may also limit the empirical use of existing antifungal drugs. 49

CONCLUSION
The present research concludes by emphasizing the rising prevalence of C. tropicalis as the leading cause of candidaemia while highlighting the increasing incidence of rarer Candida species such as C. auris and C. lipolytica.The emergence of various CNA species as significant pathogens underscores the critical need for routine and precise differentiation in clinical laboratories.Additionally, the alarming rise in Fluconazole resistance further accentuates the necessity for accurate species identification and antibiogram assessments in routine laboratory procedures to ensure effective patient management and treatment strategies.

Table 2 .
Comparison of various virulence factors expressed by Candida albicans and Candida non-albicans isolated from candidemia patients

Table 4 .
Distribution of Antifungal susceptibility and minimum inhibitory concentration (MIC) range of isolated Candida According to our findings, C. tropicalis (44.3%) formed the majority of biofilm, which is consistent with earlier research by Sasani et al. that found C. tropicalis (47%) produced the most biofilm relative to other Candida species. 43Alternatively, C. parapsilosis