Retrospective analysis on distribution and antifungal susceptibility profile of Candida in clinical samples: a study from Southern India

Introduction Candida is one of the rising primary causes of infections connected with health care. However, their distribution and susceptibility patterns vary widely amongst different regions. Method The study was carried out to retrospectively analyze the distribution of Candida in various clinical samples, their species types and susceptibility, in a tertiary care hospital, in India for 4 years using the Vitek-2 database. Results Candida infection was identified in 751 clinical samples, and the major source of infection was found to be urine samples accounting for about 58.32%. A total of 18 different Candida species were recorded. Non-albicans Candida (NAC) 73.64% (n = 553) predominated Candida albicans 26.36% (n = 198). Candida tropicalis was found to be identified at a higher frequency followed by C. albicans, Candida glabrata and Candida parapsilosis. Candida tropicalis was the only species which were recovered from bile; Candida pelliculosa was recorded merely from blood and Candida lipolytica from urine and blood and not in any other samples. In vaginal swabs, C. albicans accounted for 63.64% (n = 14) compared to NAC 36.36% (n = 8). The susceptibility test revealed that 75.44% (n = 559) isolates were susceptible and 24.56% (n = 182) were resistant to one or more drugs tested. Major resistance was exhibited to flucytosine by C. tropicalis 77.46% (n = 55) compared to C. albicans 11.27% (n = 8). Apart from C. albicans, NAC-C. tropicalis, C. glabrata and Candida krusei showed resistance to echinocandins, and Candida haemulonii to amphotericin-B. Conclusion The knowledge of the incidence, resistance and emergence of different species might guide clinicians to select an appropriate antifungal therapy and plan effective strategies to control invasive and systemic Candida infections.


Introduction
Candida has been documented as the most common fungi associated with nosocomial infections due to their invasive interventions and indwelling nature in prosthetic devices (1). Also, they are the most common opportunistic infections in patients with immunocompromised conditions and those with impaired physiological and cellular barriers (2). Their dimorphic nature, adherence to host tissues/medical devices, invasive character, biofilm formation and secretion of  extracellular hydrolytic enzymes have swapped the commensal character to pathogenic. Additionally, the factors such as profuse usage of widespectrum antibiotics, and corticosteroids, expanded use of antifungal drugs, and geographical difference in the epidemiology have increased their occurrence rate, distribution, and diversity frequency (3)(4)(5)(6)(7)(8).
Among Candida species, Candida albicans is recorded as the most prevalent species, but its prevalence varies considerably depending on geographical regions; and a shift to non-albicans Candida (NAC) has been observed globally (9,10). NAC have emerged as clinically interested pathogens and are exhibiting a high degree of resistance to the azole group of antifungals compared to C. albicans (11)(12)(13).
The rise of superbug Candida infections auris with increased morbidity and mortality in different parts of the continent is challenging the diagnosis methods and treatments (14). Candida is known to cause infections in cutaneous, mucosal, and deep-seated organs. Various species have been isolated from multiple clinical sites. The increased incidence of candidiasis, the surge of NAC, and the change in susceptibility patterns have emphasized the need to monitor laboratory data and select the appropriate antifungal for therapy (14)(15)(16)(17). The knowledge of epidemiological surveillance on species distribution and their emerging resistance to antifungal agents can be the guide for effective management and antifungal stewardship.
The study aimed to retrospectively analyse the data on Candida species diversity, their distribution in different clinical samples and susceptibility profile in a tertiary care hospital, in Karnataka, India. Though individual institution data does not represent the total population, surveillance on species distribution and antifungal susceptibility test (AST) of a particular geographical area improve clinical decisions.

Materials and methods
A retrospective analysis was carried out to understand the distribution of Candida species in different clinical samples and their drug susceptibility pattern from the Department of Microbiology database of an 1800-bed tertiary care hospital in Mysuru, Karnataka, India, for 4 years. The database included patient identifiers, hospital      Distribution of Candida albicans and non albicans Candida species year wise. Candida species (recovered >1%) from different clinical specimens.
wards/outpatient services/departments, sample collection date, specimen type, body site of isolation and results of speciation and AST pattern. The samples were processed as per the standard microbiological procedures. The isolates were identified to the species level, and AST patterns were determined using the Vitek 2 system (BioMerieux, France). The AST panel included the antifungals such as amphotericin B, 5 flucytosine, caspofungin, micafungin, fluconazole, and voriconazole. The susceptibility results were concordant with CLSI and EUCAST Methodologies and were interpreted as sensitive (S), intermediate (I) and resistant (R) (18, 19).
The clinical samples received by the laboratory for routine analysis were processed as per the standard protocols. Therefore, the study did not require oversight by the institutional ethics committee because of its descriptive nature.

Statistical analysis
Descriptive analysis was carried out to analyse the distribution of C. albicans and NAC, in different clinical samples and susceptibility to antifungal drugs year-wise. The Mann-Whitney U-test and Pearson's Chi-square Test were used to compare the differences and find the association between variables. The Mann-Whitney U-test with a 5% significance level and Pearson's Chi-square Test with a p-value <0.05 was significant. Statistical analysis was performed using SPSS 20.0 program.

Results
Analysis of the database revealed that 751 Candida isolates were recovered from different clinical samples over 4 years. The occurrence level had increased successively over the years except for last year (Figure 1).

Candida species isolated from clinical samples
The distribution frequency of C. albicans over NAC by the Manne Whitney U-test revealed that NAC had the highest mean and sum rank compared to C. albicans. The test showed an asymptotic significance (2-tailed) value of p and concluded that NAC was statistically significantly higher than the C. albicans (p = 0.021 at a 5% significance level).
Out of 58.32% of Urine samples, C. tropicalis accounted to be 210 (46.2%), followed by C. albicans 89 (19.58%), C. glabrata 73 (16.06%), and C. parapsilosis 21 (4.62%). Except for Candida pelliculosa, all 17 Candida were recovered from urine samples. Candida tropicalis was isolated from all specimens except CSF, TS, and GA. It was the only species recovered from bile. Apart from C. lusitaniae, Candida norvegensis, and Candida colliculosa, all other species resulted in candidemia. Candida krusei and C. pelliculosa were frequently isolated from blood in concurrence with C. albicans and C. tropicalis; C. lipolytica was recovered only from urine and blood, whereas

Antifungal susceptibility pattern
Drug susceptibility was carried out for all 751 isolates, but the susceptibility profile was available only for 741 isolates in VITEK with no interpretation for Candida famata (7), Candida lambica (2) and C. colliculosa (1). Among the 741 isolates, 182 (24.56%) showed resistance to one or more drugs tested, and 559 (75.44%) were susceptible to all drugs-  Tables 3, 4.
In vitro susceptibility pattern of Candida species to six antifungals-amphotericin B, caspofungin, flucytosine, fluconazole, micafungin, and voriconazole has been tabulated in Table 4. For   Total  88  11  22  17  3  3  1  1  1  3  No. of drug resistant isolates of Candida albicans and non albicans Candida year wise. FIGURE 5 No. of drug resistant isolates of Candida albicans and non albicans Candida.

Discussion
Nosocomial invasive fungal infections are increasing at a higher frequency (20). The present study infers that NAC predominates C. albicans. C. tropicalis being the most common species reported, revealed a significant epidemiological shift compared to our previous study from 2010 to 2014 (16). The increased ratio of NAC over C. albicans reflected the rise in the incidence of NAC, but the reason for this switchover is unknown. Overall, candiduria was recorded in 58.32%, whereas candidemia was observed in 7.46%. Esmailzadeh  (25) and Ballal et al. (26) revealed the presence of only NAC in bile which is in line with our study. In a study by Baghdadi et al. 2016, C. albicans was the most common species isolated from endotracheal tubes accounting for 35.7%. Among NAC, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei were recorded (27). A retrospective analysis of epidemiology for candidemia has reported that central venous catheters and endotracheal intubation as the most common risk factors associated with mortality and highlights the significance of considering the possibility of invasive Candida infection in patients (28). From vaginal swabs C. albicans 63.64% was the predominant species isolated followed by C. glabrata and C. tropicalis. Studies have shown that elevated levels of estrogen have a direct impact on growth of Candida and its adherence to vaginal epithelium suggests an increased incidence of vaginal candidiasis in women. Studying hormone-sensing pathways help in explaining gender bias-associated infections. C. albicans is recorded as the predominant colonizer in the female reproductive tract and a major cause of genital thrush (29). The shift in the frequency from albicans to NAC such as C. glabrata, C. parapsilosis, C. tropicalis may be due to increased use of the counter drugs, a short-course of antifungal therapy, and long-term use of azoles (30). NAC has not only overtaken the albicans in uncontrolled distribution but has also evolved in exhibiting drug resistance to azole, polyene and echinocandins drugs which are alarming the situation. Data insights show that C. tropicalis parallels C. albicans in demonstrating resistance to azole and flucytosine drugs. Increased flucytosine resistance indicates indiscriminate usage of the drug. Apart from C. albicans, NAC such as C. tropicalis, C. glabrata, and C. krusei have Frontiers in Public Health 07 frontiersin.org evolved in exhibiting resistance toward echinocandins. C. haemulonii has demonstrated resistance to amphotericin B, which is the mainstay of systemic antifungal therapy (31). For micafungin, all isolates were found to be sensitive. The limitations of the present study are no other tests were carried out for species confirmation. Also, the data was from a single institution and cannot be extrapolated to other hospitals.
In summary, it is a study which statistically infers the body sites affected by different species of Candida and their antifungal resistance patterns. Each species varies in susceptibility level to the distinct group of antifungal agents. The emergence of new species, indiscriminate usage of drugs and rising drug resistance have worsened the situation. Therefore, continuous surveillance, epidemiology, and clinical investigation studies performed locally from each hospital can provide a database to monitor Candidemia infections. The antibiogram profile could guide clinicians to choose appropriate drugs for the emerging Candida species and plan effective strategies to control invasive and systemic Candida infections.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethics statement
This is an observational study. JSS Academy of Higher Education & Research Ethics Committee has confirmed that no ethical approval is required.

Author contributions
US and MS contributed to the study's conception and design. Material preparation, data collection and analysis were performed by US and MS. The first draft of the manuscript was written by US. All authors contributed to the article and approved the submitted version.