Prevalence of Candida species and their Susceptibility to Triazoles in Clinical Isolates from a Tertiary Care Hospital

In the recent past, the incidence of Candidiasis has witnessed a concerning upsurge, resulting in a significant healthcare challenge. These infections are further exacerbated by factors like the widespread use of broad-spectrum antimicrobials, chemotherapy-induced neutropenia, and the presence of medical devices. The present study is designed to address the critical need for identifying the Candida species responsible for clinical infections and assessing their susceptibility to key antifungal drugs Fluconazole, Voriconazole, and Itraconazole. Two hundred clinical samples from Rohilkhand Medical College & Hospital, Bareilly were analyzed. Using Vitek-2 Compact (Biomerieux, France), the Candida spp. and the antifungal drug sensitivities were identified for Fluconazole and Voriconazole. E-test was done to identify Itraconazole sensitivity. This study found that C. albicans accounted for 21.5% while Non-albicans Candida (NAC) constituted 78.5%. Prolonged medication was the most common factor making susceptible for Candidiasis (43.5%), followed by indwelling biomedical devices (23%), Diabetes mellitus (16%), surgical causes (5.5%), trauma (5%), pregnancy (5%), and HIV (2%). Antifungal susceptibility testing showed that 68.5%, 72%, and 69.5% of Candida spp. isolates were sensitive to Fluconazole, Voriconazole, and Itraconazole, respectively. In conclusion, non-albicans Candida infections are increasing due to predisposing conditions, and some of these species are inherently resistant to the routinely used antifungal drugs. The study emphasizes the importance of identifying Candida spp. and their susceptibility to antifungals. This can limit the indiscriminate use of antifungal drugs, aid in selecting appropriate treatments, and reduce treatment costs, hospital stays, and patient morbidity and mortality.


INTRODUCTION
The prevalence of Candida infections has reached alarming levels in recent past, leading to significant mortality and morbidity.Candida species, including Candida albicans and Non-albicans Candida spp.such as C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. dubliniensis, and C. auris are major contributors to human fungal infections.Candidiasis, once rare, has become increasingly common among bedridden and critically ill patients, despite improvements in medical care.These fungal infections pose a substantial risk to both healthy individuals and those with compromised immune systems. 1 Candida spp.are also a leading cause of nosocomial infections.Candida overcomes immune defenses, invades tissues, and causes severe infections, often facilitated by medical devices.Factors such as broad-spectrum antibiotics, chemotherapyinduced neutropenia, reduced phagocytic activity, and parenteral nutrition contribute to the growth and invasion of Candida.Among Candida spp., Candida albicans and Non-albicans Candida spp.such as C. tropicalis, C. glabrata, C. parapsilosis, and C. krusei account for as high as 90% of all the infections.However, Candida auris a notoriously multiple drug resistant species, pose a great challenge, particularly in critically ill patients.Another interesting aspect is that the diversity of Candida spp.differ from multicenter studies to single center studies.
Candidiasis, caused by various Candida spp., necessitates a diverse range of antifungal drugs for effective treatment.Polyenes, azoles, echinocandins, nucleoside analogues, and allylamines are commonly used drug classes.Candida spp.exhibit variations in virulence and drug sensitivity patterns.Notably, C. krusei displays intrinsic resistance to fluconazole due to low fluconazole-ERG11 affinity, rendering it ineffective for treatment.Studies have shown high resistance rates among C. krusei and C. glabrata to fluconazole and azoles, respectively.Timely identification of Candida spp. is crucial prior to initiating treatment, as empirical therapy can prolong the disease and lead to increased morbidity and mortality.Determining the sensitivity pattern of Candida spp. to antifungal drugs is vital for selecting appropriate and targeted therapy.Although significant progress has been made in the management of Candidiasis, the increased prevalence of drugresistant Candida strains poses a significant hurdle, impacting treatment outcomes.Despite the introduction of novel antifungal agents, the survival rates of critically ill patients with Candida infections remain largely unchanged, which can be partly attributed to the emergence of drug resistance.
As the Candida spp.differ in the antifungal sensitivity pattern, it is important to identify the species of Candida.
The study aims to find the distribution of Candida spp. in clinical samples and find the susceptibility pattern to triazole antifungal drugs namely Fluconazole, Voriconazole and Itraconazole.

MATERIALS AND METHODS
A cross sectional study was done from the year 2019 to 2022 at Rohilkhand Medical College & Hospital, A Tertiary Care Hospital in Bareilly.A total of 1437 culture positive samples collected from patients in Intensive Care Units (ICU), In-Patients Wards (IP) and Out Patients Departments (OPD).200 isolates of Candida spp.obtained from these samples.The samples included respiratory samples (sputum, Endotracheal (ET) aspirate, bronchoalveolar lavage (BAL), throat swabs), blood, urine, vaginal swabs, CSF, pus and catheter tips.

Sample collection
Enough sample was collected from active lesions under strict aseptic conditions using sterile instruments following the universal safety precautions.All the samples were transported immediately to the laboratory without delay.

Sample processing
The samples (Urine, Respiratory samples, Vaginal swabs, CSF, Pus, Catheter tips) were subjected to direct Gram's Staining and were inoculated into Blood Agar and Mac Conkey Agar and incubated at 37°C for 24-48 hours.If there is no growth after 48 hours of incubation, the sample is considered sterile for aerobic organisms.If there is growth, the colonies were picked up and a Gram's stain is done.In the Gram's stain preparation, the appearance of Gram positive, oval, budding yeast like fungi with pseudohyphae are presumptively identified as Candida spp.Additionally, the samples were inoculated in Saboraud's Dextrose Agar and incubated at 25°C for 48 hours to one week.Blood samples were incubated in BacTAlert3D (Biomerieux, France) automated blood culture system.A positive blood culture is subcultured in Blood Agar and Mac Conkey Agar and processed like explained above for other samples.

Candida species identification
HiCrome™ Candida Differential Media (HiMedia, Mumbai) was used to differentiate Candida species based on the color of the colonies after 48 hours of aerobic incubation at 37°C.As the color is purely subjective, when there is any ambiguity, sugar fermentation, sugar assimilation and microscopic morphology on Corn Meal agar media and Vitek-2™ yeast identification cards were used to correctly identify the species.

Antifungal sensitivity testing
A few colonies from the pure culture of Candida were transferred to 3ml of sterile saline and vortexed until the turbidity of the suspension is in the range of 1.80-2.20 McFarland units.The suspension was inoculated into YS09™ (Biomerieux, France) Yeast Antifungal Susceptibility cards and processed in Vitek-2™( Biomerieux, France) system.Sensitivity for the drugs Fluconazole and Voriconazole were obtained in this method.For the sensitivity of Itraconazole, E-test method was used.CLSI guidelines were followed for testing and interpretation of the antifungal sensitivity results.

RESULTS
A total of 200 Candida spp.were isolated from different clinical samples.Of these, 116 (58%) were from Respiratory samples, 34 (17%) from urine, 23 (11.5%) from Blood, 22 (11%) from vaginal swab, 5 (2.5%) from pus samples.The distribution of different Candida spp.from various samples is summarized in Table 1.84% of the total infections were seen in 21-70 age group.It is also evident that males are more prone to Candidiasis than females with a frequency of 130 (65%) and 70 (35%), respectively.The predisposing factors leading to Candidiasis are summarised in Table 2.The ratio of incidence in Male versus Female is 1.86:1 (M:F).

DISCUSSION
Fungal infections to the hospitalized and the immunocompromised is an emerging problem to be tackled.During the last three decades, infections due to Candida spp., especially by the non-albicans species, increased to a great extent. 2 The reason for this increased incidence can be attributed to both fungal virulence and host susceptibility factors.Extended use of antibacterials which eventually alter the normal flora, use of corticosteroids, surgeries, malnutrition and hormonal imbalance contribute to predisposition to Candidiasis in the immunocompetent individuals. 3Prolonged use of routinely used antifungal drugs alter the prevalence of Candida spp. 4,5n concordance with the previous studies, it is found that the Non-albicans Candida spp.][8][9][10] C. tropicalis was the most isolated species in all samples, followed by C. albicans.2][13][14][15] Furthermore, the Non-albicans Candida spp.were found to be the causative agent for invasive or deep rooted Candidiasis.Whereas, C. albicans caused superficial and mucosal infections mostly in skin, urinary tract and other mucosal areas.
Prolonged Medication, for more than a week was found to be the predominant risk factor in this study.This is in concordance with Chakrabarthi A et al. study which found that patients on antibiotics for more than a week and/ or patients receiving multiple antibiotics, are prone to Candidiasis. 14n this study, many Candida spp.isolation were from respiratory samples.This deviates from the previous studies where majority of the Candida isolates were from Urine sample.As the study was done during the COVID-19 pandemic, there was a natural predilection towards respiratory complications and infections.This respiratory predilection was also confirmed by another study conducted in between 2018-

CONCLUSION
This study showed that Non-albicans Candida spp.prevalence is higher compared to that of C. albicans.As the number of predisposing factors increased, the incidence of Non-albicans Candida infections has also increased.
As the Non-albicans Candida spp.are inherently resistant to routine antifungal drugs like Fluconazole, prompt identification of different Candida spp.and simultaneous testing of their susceptibility to antifungal medications will have a dual impact: it will limit the indiscriminate application of antifungal drugs and significantly shape the available treatment choices for medical professionals, ultimately benefiting the patients.
Prompt speciation and susceptibility pattern of Candida spp.will aid the clinicians in selecting the appropriate antifungal drug, and thus contributing to overall reduction in the cost of treatment & the duration of hospital stay and potentially reduces the patient's morbidity and mortality.
A periodic surveillance to susceptibility to antifungal drugs is essential as it would throw light on the rational use of antibiotics and thus preventing the emergence of drug resistance.

Table 1 .
Distribution of Candida spp. in various samples

Table 2 .
Prevalence of Candida albicans and Non-albicans Candida among various predisposition factors

Table 3 .
17iazole antifungal drug susceptibility pattern to Candida spp.Froidefond et al.16It is also found that 92% of all COVID-19 patients who were given broad spectrum antibiotics as part of COVID-19 management were infected by Candida spp.17In the present study, 68.5%, 72% and 69.5% of all the Candida spp.isolates were susceptible to Fluconazole, Voriconazole and Itraconazole, respectively.A comparison of studies by Jayalakshmi et al.,Wang etal., Sukumaran et al., and Kashid et al. 18-21 also denotes that 30% resistance to Fluconazole is not an outlier but is becoming common.I f s e e n a s C . a l b i c a n s v e r s u s Non-albicans Candida spp., 27.9%, 23.2% and 30.2% of C. albicans were resistant to Fluconazole, Voriconazole and Itraconazole, respectively, and 32.5%, 29.3% and 30.6% of Non-albicans Candida were resistant to Fluconazole, Voriconazole and Itraconazole, respectively.20 isolates of C.tropicalis, 10 C. albicans isolates, 5 isolates of C. dubliniensis, 4 isolates of C. krusei and one each of C. glabrata and C. gulliermondi were resistant to all the 3 azole drugs tested.