Original article / research
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Speciation and Antifungal Susceptibility Testing of Candida Isolated from Various Clinical Specimens at a Tertiary Care Centre: A Cross-sectional Study |
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Pramod N Sambrani, Uma Chikkaraddi, A Divya, KS Neema 1. Associate Professor, Department of Microbiology, Karnataka Medical College and Research Institute, Hubballi, Karnataka, India. 2. Senior Resident and Infection Control Officer, Department of Microbiology, Karnataka Medical College and Research Institute, Hubballi, Karnataka, India. 3. Assistant Professor, Department of Microbiology, ESIC Medical College and PGIMSR and Model Hospital, Bengaluru, Karnataka, India. 4. Postgraduate Student, Department of Microbiology, Karnataka Medical College and Research Institute, Hubballi, Karnataka, India. |
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| Correspondence
Address : Dr. KS Neema, Postgraduate Student, Department of Microbiology, Karnataka Medical College and Research Institute, Vidyanagar, P.B. Road, Hubballi-580021, Karnataka, India. E-mail: neemaksmtm@gmail.com |
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| ABSTRACT |  | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| : Candida species are a normal commensal flora of the human body, colonising the skin, mucous membranes and gastrointestinal tract. However, they may also be associated with superficial and deep-seated fungal infections. In recent years, Non albicans Candida (NAC) species have emerged as significant pathogens causing severe infections in humans. Commonly used antifungal drugs show significant variation in susceptibility patterns among different Candida species. The incidence of drug resistance has been increasing over the last few decades due to the random use of antifungal agents. Therefore, the introduction of newer antifungal agents and changes in drug susceptibility patterns of Candida species have made in-vitro susceptibility testing of antifungal agents increasingly relevant for selecting sensitive drugs. Aim: To identify species of Candida isolated from various clinical samples and to perform Antifungal Susceptibility Testing (AFST) using the disc diffusion method. Materials and Methods: This study was a single-centre cross-sectional investigation involving 100 consecutive, non duplicate Candida isolates from various clinical samples obtained from patients at the diagnostic Microbiology laboratory of Karnataka Medical College and Research Institute, Hubballi, Karnataka, India from January 2023 to December 2023. One hundred consecutive clinical specimens (blood, urine, stool, sputum, oropharyngeal swabs, vaginal swabs, wound swabs, pus, Cerebrospinal Fluid (CSF), skin and nail samples, and other body fluids) that yielded Candida species were included in the study. Candida species were identified by the type and colour of colonies on HiCrome Candida differential agar as per the manufacturer’s instructions. All isolates were subjected to AFST and interpretation was performed using the disc diffusion method. The data collected were entered into MS Excel and analysis was conducted using statistical software called Statistical Package for the Social Sciences (SPSS) version 28.0. Pearson’s Chi-square test was used to compare differences and to find associations between variables. Results: Out of 100 Candida isolates, 38 (38%) were identified as Candida albicans, while 62 (62%) were NAC species. The most affected age group was from 1 to 10 years, with 27 (27%) cases. The male to female ratio was 1:1.08. Intensive Care Units (ICUs) reported Candida species more frequently, with 51 (51%) cases compared to other areas in the hospital. The predominant source of infection was found to be urine samples, accounting for about 60 (60%). A total of five different Candida species were recorded. Among these, Candida albicans was the most predominant species, with 38 (38%), followed by Candida tropicalis (32, 32%), Candida krusei (16, 16%), Candida parapsilosis (13, 13%), and Candida glabrata (1, 1%). Major resistance was observed to fluconazole (25, 25%) and clotrimazole (6, 6%), while major susceptibility was reported for nystatin (97, 97%). Conclusion: The emergence of different Candida species and the data regarding their resistance patterns may assist clinicians in selecting appropriate antifungal therapy to treat invasive and systemic Candida infections. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Keywords : Candida albicans, Fungal drug resistance, Non albicans Candida | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| INTRODUCTION | | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Candida is an omnipresent human commensal yeast-like fungus. When the host’s resistance is lowered, either locally or systemically, Candida can become a pathogen and cause infections (1). Candida species can cause varied clinical manifestations ranging from acute, subacute and chronic to episodic forms. Involvement may be localised or systemic, as seen in endocarditis, septicaemia and meningitis (2),(3). Historically, Candida albicans (C. albicans) has been the predominant cause of candidiasis. In the 1980s, C. albicans accounted for a greater number of nosocomial yeast infections than other species of Candida. Over the last few decades, an increase in the prevalence of non albicans species has been noted (4). NAC species such as Candida tropicalis (C. tropicalis), Candida krusei (C. krusei), Candida glabrata (C. glabrata), and Candida parapsilosis (C. parapsilosis) are less susceptible to azoles, particularly fluconazole (5). Therefore, correct identification of Candida species is essential for early and appropriate antifungal therapy. Debilitated patients, especially those in ICUs, are at risk for nosocomial infections such as bloodstream infections caused by Candida (6). To help prevent severe illness and death from these infections, and to reduce delays in diagnosis and treatment, awareness of the possibility of fungal co-infection is crucial (7). To facilitate this, clinical laboratories need to expand their yeast identification capabilities (8). Conventionally, Candida species are identified using the germ tube test, along with sugar assimilation and fermentation tests. Newer methods that have been developed for the speciation of Candida isolates include CHROM agar, API systems, Vitek 2 ID systems and molecular methods (9). Conventional methods are time-consuming and labour-intensive, while automated and molecular methods are often expensive. Chrom agar is a differential medium that allows for the selective isolation of yeasts and the identification of colonies of C. albicans and other NAC species. It contains chromogenic substrates that react with enzymes secreted by microorganisms, producing colonies with various colours. These enzymes are species-specific, allowing organisms to be identified to the species level by their colour and colony characteristics (4). Hence, the present study was undertaken to speciate Candida isolated from various clinical samples using HiCrome Candida differential agar, which is a novel method, and to perform AFST using the disc diffusion method at a tertiary care hospital. The primary objective was to assess the incidence of different species of Candida in various samples received at the Microbiology laboratory, while the secondary objective was to determine the in-vitro susceptibility pattern of Candida species to six antifungals: fluconazole, voriconazole, itraconazole, clotrimazole, nystatin and amphotericin B. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| MATERIAL AND METHODS | | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| This single-centre cross-sectional study was conducted by the Department of Microbiology for a duration of one year, from January 2023 to December 2023, at Karnataka Medical College and Research Institute, Hubballi, Karnataka, India. A total of 100 consecutive non duplicate isolates from various clinical specimens yielding Candida isolates were considered for the study. The study protocols were approved by the ethics committee of the Institute, bearing IEC number: KIMS:ETHICS COMM: 84:2023-24. Inclusion criteria: 1. Candida isolated in pure culture from blood and sterile fluid. 2. Pure or mixed culture from urine, pus, nasal/oral/vaginal swabs, but not more than two isolates showing pseudohyphae along with budding yeast-like cells in a Direct Gram stain. Exclusion criteria: 1. Stool samples yielding Candida species. 2. Mixed culture from urine, pus, nasal/oral/vaginal swabs involving more than two isolates showing pseudohyphae along with budding yeast-like cells in a Direct Gram stain. Sample size: A non probability sampling technique was used. A total of 100 consecutive non duplicate isolates from various clinical specimens yielding Candida isolates were included in the study. The primary laboratory diagnosis of specimens was performed using wet mounts and Gram stains. All suspected samples were inoculated on Sabouraud’s dextrose agar (HIMEDIA- MH063- 500 G, HiMedia Pvt. Ltd., Mumbai) slope, supplemented with chloramphenicol and aerobically incubated at 37°C for 24-48 hours. Any visible growth on the SDA slope was processed for further identification. From the isolated colony, macroscopic examination and Gram staining were performed (Table/Fig 1). After confirmation of Gram-positive budding yeast-like cells on microscopy, a germ tube test (Table/Fig 2) was performed to differentiate C. albicans and Candida dubliniensis from other Candida species. C. albicans was further differentiated from Candida dubliniensis by its ability to grow at 40°C. Simultaneously, all the Candida isolates were inoculated on HiCrome Candida Differential Agar (HIMEDIA- M1297A- 500G, HiMedia Pvt. Ltd., Mumbai) and incubated aerobically at 37°C for 24-48 hours, allowing for species identification based on the type and colour of the colonies on HiChrom Candida Differential Agar, as per the manufacturer’s instructions (Table/Fig 3), (Table/Fig 4) (10). All isolates were subjected to AFST (Table/Fig 5). The inoculum was prepared by suspending five colonies of growth in 5 mL of sterile saline and comparing the turbidity to the 0.5 McFarland standard. A cotton swab was dipped into the inoculum suspension and evenly streaked onto Mueller-Hinton agar supplemented with 2% glucose. Antifungal discs (fluconazole, voriconazole, itraconazole, nystatin, clotrimazole, and amphotericin B) were placed on the inoculated media. The zone of inhibition around each disc was measured after incubating the media at 37°C for 24 hours and interpretation was done according to Clinical and Laboratory Standards Institute (CLSI) M44-A document guidelines (11). Quality control was performed for AFST with a C. albicans strain for fluconazole and voriconazole (Table/Fig 6), and interpretation was done according to CLSI M44-A document guidelines (11). The MIC was not performed due to a lack of resources and trained personnel. Statistical Analysis The data collected was entered into MS Excel and analysis was performed using the statistical software IBM SPSS Statistics version 28.0. Pearson’s Chi-square test was used to compare the differences and find the association of susceptibility to antifungal drugs between C. albicans and NAC species. A p-value <0.05 was considered significant. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| RESULTS | | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| During the study period, a total of 100 consecutive samples yielding Candida isolates were considered. The sources of the samples from which Candida species were isolated are represented in (Table/Fig 7). The clinical samples processed included urine, vaginal swabs, blood, Endotracheal (ET) secretions, nail clippings, otomycoses debris, ascitic fluid and pus. Candida albicans was the predominant species isolated from urine, vaginal secretions and blood samples. Candida tropicalis predominated in urine samples, followed by Candida krusei in both urine and blood samples (Table/Fig 8). In the demographic data, all age groups from day 1 to 90 years were included in the study, with the most affected age group being from day 1 to 10 years (27, 27%), followed by the age group of 21 to 30 years (23, 23%). Among the paediatric age group, samples from neonates (0-1 month) yielded the maximum number of Candida species (Table/Fig 9). The male-to-female ratio was 1:1.08, with more Candida species isolated from samples collected from females (52, 52%) than from males (48, 48%). A total of five different types of Candida species were isolated from the 100 samples. C. albicans predominated with 38 (38%). Among NAC, Candida tropicalis was the most frequently isolated species (32, 32%), followed by Candida krusei (16, 16%), Candida parapsilosis (13, 13%), and Candida glabrata (1, 1%). The area wise distribution of Candida species is shown in (Table/Fig 10). Antifungal susceptibility pattern: The in-vitro susceptibility pattern of Candida species to six antifungals-fluconazole, voriconazole, itraconazole, nystatin, clotrimazole and amphotericin B- has been tabulated in (Table/Fig 11)a,b. Drug susceptibility testing was carried out for all 100 Candida isolates. Among the 100 isolates, 97 (75%) were susceptible strains, while 25 (25%) were resistant to one or more drugs. Out of 38 C. albicans isolates, 36 (94.7%) were sensitive to all antifungals tested, 35 (92.1%) were sensitive to clotrimazole, and 2 (5.2%) were resistant to all antifungal drugs. Among the 62 NAC isolates, Candida tropicalis showed 32 (100%) sensitivity to nystatin, while 29-27 (90.6%-84.3%) strains were sensitive to azoles, and 30 (93.7%) were sensitive to amphotericin B. All 16 Candida krusei isolates (100%) were sensitive to voriconazole, itraconazole, nystatin, and amphotericin B, but were intrinsically resistant to fluconazole; 13 (81.2%) were sensitive to clotrimazole. All 13 Candida parapsilosis isolates (100%) were sensitive to voriconazole, 12 (92.3%) were sensitive to amphotericin B and nystatin, 11 (84.6%) were sensitive to Fluconazole and itraconazole, and 9 (69.2%) were sensitive to clotrimazole. Candida glabrata was (100%) sensitive to all antifungal drugs tested. The antifungal resistance pattern was comparatively higher in NAC than in C. albicans. The resistance pattern in the study may be due to the increased use of azoles, over-the-counter use of drugs and short-term courses of antifungal therapy prescribed. On comparison of C. albicans and NAC resistance to all six drugs using the Pearson Chi-square test, the p-value was <0.05 (p=0.0012). Thus, there was a statistical significance between the organism and the drugs (Table/Fig 12). The drugs and organism association revealed a significant difference at the 5% level (p-value <0.001). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| DISCUSSION | | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Candida species significantly vary in their antifungal susceptibility. Therefore, it is very important to identify the specific Candida species isolated in the laboratory from different clinical samples to select the most effective antifungal therapy. Fungal infections caused by Candida species have increased in frequency over the last few decades. In this study, a total of 100 consecutive samples yielding Candida isolates were considered from various clinical specimens. The majority of Candida species were isolated from urine and sputum, covering 90%, which indicates a higher incidence and distribution of Candida species causing urinary tract and respiratory tract infections. In the present study, the prevalence of C. albicans was 38 (38%), and NAC was 62 (62%), which was comparable to studies conducted by Yashavanth R et al., Awari A, and Golia S et al., (Table/Fig 13) (12),(13),(14),(15),(16),(17),(18),(19). In studies conducted by Madhumati B et al., and Raj Kumari S et al., a comparatively higher proportion of C. albicans was isolated than in the current study (15),(16). As depicted in the table, studies conducted by Roopa C et al., Kanna BV et al., and Devi S et al., showed that C. albicans predominated compared to NAC (17),(18),(19). Although Candida was isolated more frequently from female patients (52, 52%) than from males (48, 48%), there was not much difference in the male-to-female ratio. Similar findings were reported in the studies by Ekpo IA et al., and Goel R et al., (20),(21). However, a study conducted in Sweden by Lindberg E et al., showed a male preponderance (22). Most of the Candida isolates among female and male patients were cases of Urinary Tract Infections (UTI) and sepsis, respectively. The majority of Candida isolates were obtained from urine samples (60, 60%), which was consistent with the study by Khadka et al., (23). However, in studies conducted by Madhumati B et al., Roopa C and Biradar SK and Devi S and Maheshwari M, the highest incidence of Candida was observed in high vaginal swabs, followed by urine and sputum samples (15),(17),(19). In this study, the paediatric age group (27, 27%) was the most affected, followed by the 21-30 years age group (23, 23%), whereas in other studies, the most affected population was above 60 years (12),(21). Among the paediatric age group, the maximum number of Candida isolates was obtained from neonates through blood cultures, implying that Candida is one of the major aetiologies of neonatal sepsis. The second-highest affected age group was 21-30 years, in which most patients were young females suffering from UTIs. Most cases were from patients admitted to the ICU, similar to the findings of a study by Alfouzan WAM, (24). This is likely attributed to the fact that most patients in the ICU are catheterised, critically-ill, and on broad-spectrum antibiotics. The AFST pattern showed that Candida isolates were more susceptible to Nystatin (97, 97%) and Amphotericin B (95, 95%) than to azoles (Fluconazole: 75, 75%), which was similar to the studies conducted by Yashavanth R et al., Roopa C et al., Khadka S et al., Manikandan C et al., Jayalakshmi L et al., and Adhikary R et al., (Table/Fig 14) (12),(17),(23),(25),(26),(27). Resistance to azoles was higher in the NAC group compared to C. albicans, which aligns with the findings of Yashavanth R et al., and Khadka S et al., (12),(23). In the study conducted by Khan M et al., on vulvovaginal candidiasis, the susceptibility to fluconazole was only 36 (33%), which was not in line with the present study (28). Among the azoles, the highest susceptibility was shown to voriconazole and itraconazole (92, 92%), followed by clotrimazole (78, 78%) and fluconazole (75, 75%). Among the frequently isolated species of Candida, the highest susceptibility was observed in C. albicans compared to C. tropicalis. However, in a study conducted by Vijaya D et al., azoles demonstrated comparatively better in-vitro activity, especially against C. albicans (10). The findings of this study can serve as an effective guide for clinical management and antifungal stewardship, as it includes data on the distribution of Candida species and their emerging resistance to antifungal agents. Analysing the laboratory data on the diversity of Candida species, their distribution in different clinical samples, and their susceptibility pattern in a tertiary care centre would help clinicians initiate appropriate antifungal treatment. Limitation(s) Minimum Inhibitory Concentration (MIC) testing was not performed for antifungal susceptibility due to a lack of resources and trained personnel. Speciation of Candida from chromogenic differential agar was not confirmed with additional tests. Predisposing factors influencing the distribution of Candida species in different areas of the hospital (e.g., hospital wards, ICU settings) were not studied. The sample size of this study was small, and a larger sample size would be needed to strengthen the findings, especially for more specific species like Candida glabrata. This individual institution data does not represent the total population, so it has limitations regarding extrapolation to multiple centres. Quality control for AFST was not performed with all the drugs and species involved in the study due to a lack of quality control strains. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| CONCLUSION | | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| The present study statistically represents the body sites affected by different species of Candida and their antifungal resistance patterns. The susceptibility pattern of antifungal agents varies with each species of Candida. Therefore, continuous surveillance, epidemiological studies and clinical investigations performed locally at a tertiary care centre can provide a database to monitor different Candida infections. The fungal antibiogram profile could guide clinicians in formulating effective strategies to control invasive and systemic Candida infections. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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