Mycoses once very uncommon are increasingly reported in recent years, primarily due to surge in number of immunocompromised populations, increase use of broad-spectrum antimicrobial agents and immunosuppressive drugs, high incidence of hospitalization, aggressive surgical and medical interventions.^{1, 2, 3}

Among various fungal pathogens, Candida spp. is probably the only fungi that is able to cause a wide spectrum of clinical manifestation that ranges from a mere mucosal overgrowth to serious fatal invasive infections like candidemia.⁴ Although traditionally, most of infections are attributed to C. albicans, recent studies document emergence and upsurge of non albicans Candida (NAC) spp.⁵

The emergence of NAC spp. is of great concern as these ‘cryptic’ fungal pathogens are often treatment resistance as they are either innately resistant or may develop resistance during course of therapy to commonly prescribed antifungal drugs.⁶

Therefore the present study was planned at a tertiary care academic hospital with an aim to identify clinical isolates of Candida isolates and to evaluate their antifungal susceptibility profile.

The present descriptive cross-sectional study was conducted in the Department of Microbiology for a period of three years (January 2022 to December 2024). The study included clinical isolates of Candida spp. Candida isolates were identified upto species level by the standard mycological protocol for identification of yeast and yeast like fungi including germ tube production, carbohydrate assimilation test and colony morphology and color on Hichrom Candida agar (Himedia Laboratories, Pvt Ltd., Mumbai).7 Antifungal susceptibility testing of isolates was performed by the disc diffusion method as outlined in the Clinical and Laboratory Standards Institute’s (CLSI) M44-A guidelines. As suggested by the CLSI, disc diffusion test was performed by Kirby-Bauer disc diffusion method on Mueller Hinton agar supplemented with 0.2% glucose and 0.5μg/mL methylene blue agar (MH-GMB).8 Clinical isolates of Candida were tested for susceptibility against antifungal drugs like amphotericin B (20 μg), fluconazole (25 μg), itraconazole (10 μg) and ketoconazole (10 μg). All of these antifungal discs were procured from Himedia Laboratories, Pvt Ltd. Mumbai. Statistical analysis was done using statistical packages SPSS 19.0 (SPSS Inc. Chicago, IL). A P value <0.05 was considered to be significant. Pictorial presentation of the key findings was done by using appropriate statistical graph.

During the study period, a total of 5853 clinical specimens were received in the Department of Microbiology. Out of these, 2432 (41.6%) were culture positive. Of these, 2432 culture positive specimens, bacteria were isolated from 2034 (83.6%) specimens whereas 398 (16.4%) were fungal pathogens.  The rates of isolation of bacteria and fungi from various clinical specimens in this study were 83.6% and 16.4%.  

Among a total of 398 fungal pathogens, a total of 247 (62.1%) isolates were Candida spp.  Isolation of Candida spp. was significantly high among various fungal pathogens (Fisher Exact test P value <0.05).

The maximum number of Candida isolates were isolated from urine samples (39.7%) followed by vaginal swabs (21.5%). A total 51 (20.6%) Candida spp. were isolated from blood culture.

Figure 1: The clinical specimen wise distribution of Candida spp.

Female sex, geriatric age, diabetes mellitus, use of broad spectrum antibacterial drugs and indwelling Foley’s catheter were important risk factors associated with increased isolation of Candida spp. from urine specimens whereas pregnancy, HIV infection and antibacterial therapy were major risk factors identified for vulvovaginal candidiasis (VVC). In case of orophrayngeal candidiasis (OPC) HIV infection and diabetes mellitus were the only risk factors noted. ICU stay, presence of ventilator support, indwelling Foley’s catheter and central venous catheterization (CVC) were risk factors for candidemia.

In the present study, a total of 62 (25.1%) isolates were identified as C. albicans whereas 185 (74.9%) isolates belonged to NAC spp. Isolation of NAC spp. was significantly high (Fisher Exact test P value <0.05).

The species wise distribution of Candida isolates is shown table 1. C. tropicalis (38.5%) was the predominant isolate in the present study. C. krusei was isolated from 35 (14.2%) clinical specimens.

Table 1: The species wise distribution of Candida isolates.

Antifungal susceptibility profile of clinical isolates of Candida spp. by disc diffusion method is shown in table 2. Fluconazole resistance was observed in 29.1% of Candida isolates. Fluconazole resistance was noted in C. tropicalis (33.7%) and C. glabrata (23.8%)

Itraconazole resistance was noted in 29 (11.7%) isolates.  It was noted in C. tropicalis (23.2%) and C. krusei (20%).  Ketoconazole resistance was observed in 20.6% of Candida isolates. In this study, no clinical isolate of Candida spp. were resistant to Amphotericin B.  

Table 2: Antifungal susceptibility profile of Candida spp. by disc diffusion method.

Of recent, fungal infections especially candidiasis is reported to be increased in terms of both frequency and severity. In consistent to this observation, the present study also documents the similar findings where 16.4% of isolates from different types of clinical specimens were fungi with 62.1% of fungal isolates being identified as Candida spp. Risk factors for candidiasis are almost similar to those for other mycotic infections but variation is noted in the cause and are mostly associated with medical intervention and compromised immune status of the individual. 9 In this study, 39.7% of Candida spp. were isolated from urine samples. As Candida spp., is often a commensal of genitourinary tract and its isolation from urine cultures usually posesas a challenge to both the clinician and the clinical microbiologist as to whether ignore it’s isolation as a mere colonization/contaminant or, consider it as a potent indicator of lower or upper urinary tract infection (UTI) including ascending pyelonephritis and renal candidiasis with sepsis.11 However, researchers also point out at the finding that isolation of Candida spp from urine specimen should not be overlooked as this condition may at times be the only and often the primary indication of disseminated candidiasis. 12 In routine clinical practice, candiduria/ Candida UTI is a relatively rare finding in an healthy individual with structurally normal urinary tract.13 Female gender, advanced age group, diabetes, use of broad-spectrum antibiotics and presence of urinary catheter were important risk factors of candiduria in the present study. Similar findings were reported by other researchers. 12 14, 15 The waning of immune defences in patients with advance age may relate the high incidence of candiduria in patients belonging to older age group. In women, the Candida colonization of vulvo-vestibular area is too frequent and hence they are more prone for developing candiduria due to ascending infection.16 In diabetes mellitus patients, glycosuria, impaired host immune defence, stasis of urine in neurogenic bladder enhance candiduria. As antibacterial agents suppress and alters commensal perineal bacterial flora, it favours both the colonization and the infection of urinary tract by Candida spp.12 In the present study, a total of 51 Candida spp. were isolated from blood cultures. In the United States, Candida spp. is reported as the 3rd among common causes of hospital associated blood stream infections. Candidemia is associated with high morbidity and mortality in both immunosuppressed and terminally ill immunocompetent patients.17, 18 The disseminated candidiasis also significantly increases the duration of hospital stay and requirement of mechanical ventilation. In this study, predominance of NAC spp. over C. albicans was noted. Recent research studies have revealed emergence of NAC spp. Although various factors like increased empirical use of azoles are cited for emergence of NAC spp., improvement in diagnostic mycological methods like use of chromogenic media with an ability to differentiate Candida spp. as well as introduction of molecular diagnostic techniques appears to be more important. 19, 20 C. tropicalis was the predominant species. Similar to our observation, the predominance of C. tropicalis among various Candida isolates was also reported by Pahwa et al. (2014)21 and Deorukhkar et al (2016).22 C. tropicalis alone, or in association with other Candida spp, is frequently implicated in infections.23It is more frequently isolated in ICU patients, especially those requiring prolonged catheterization, those treated with broad-spectrum antibacterial or with malignancies.21 C. krusei was isolated from 35 (14.2%) clinical specimens. It is considered as an emerging NAC spp. This NAC spp., is highly uncommon outside clinical settings involving exposure to azoles and immunocompromised status.24 In the present study, antifungal susceptibility profile of Candida spp. was studied by disc diffusion method. 25Compared to the Clinical and Laboratory Standard Institute (CLSI) standardized microbroth dilution method, disc diffusion susceptibility testing is convenient and economical.26 In this study, fluconazole resistance was observed in 29.1% of Candida isolates. Among antifungal agents, resistance to fluconazole is of concern as it is the most widely used as first line antifungal agent for treatment and prophylaxis of all forms of candidiasis.27 All isolates of C. krusei were resistant to fluconazole. Various national and international studies have reported total fluconazole resistance in C. krusei isolates. In the present study. no resistance was observed to amphotericin B among the candida isolates. Amphotericin B is generally regarded to have the widest spectrum of antifungal activity and used in serious and life-threatening invasive infections.28 Although, no resistance was observed for amphotericin B, high cost and associated toxicity restricts its wide-spread use.28

       To conclude, non albicans Candida species are important and emerging cause of infections. C. tropicalis, C. krusei and C. glabrata are important NAC spp. As the susceptibility of Candida isolates to antifungal drugs varies as per the species, antifungal susceptibility testing is often necessary to guide clinicians for selecting most appropriate antifungal therapeutic agent.

       Conflict of Interest: Nil.

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