A rise in incidence of infection caused by yeasts, especially those caused by Candida Species has been noted in the recent times. The spectrum of infections caused by Candida species ranges from superficial mucosal infections to widespread disseminated blood stream infections. Invasive Candidiasis is the leading cause of morbidity and mortality in both immunocompetent as well as immunocompromised individuals (40% and 20% respectively)[1] Prolonged use of broad spectrum antibiotics, increased length of hospital stays, use of immunosuppressive agents post organ transplantation, increase in absolute number of immunocompromised patients on cancer chemotherapy agents can be attributed to increase in incidence of  invasive Candidiasis[2].

Among the Candida species, Candida, albicans is the most common species implicated however infections caused by Non albicans Candida species are also becoming common. Non albicans Candida infection account for >50 % of infection[3,2]. The commonly isolated Non albicans Candida species include  C.tropicalis, C.parapsilosis, C.glabrata, C.kefyr, C.krusei, C.pelliculosa, C.guillermondii. In recent times, outbreaks due to MDR C.auris were also witnessed.

Some of the Non-albicans Candida is known to be inherently resistant to   the commonly used antifungal agents’ .For eg. Candida krusei C.hemulonii, C.auris are  known to be resistant to Fluconazole, C.lusitanie is known to be resistant to Amphotericin B [4] In addition to this, indiscriminate use of antifungal agents as empirical therapy and for prophylaxis has led to emergence of acquired resistance to the commonly used antifungal agents in some of the previously known susceptible strains. Knowledge about the species distribution and the susceptibility profile helps in making appropriate empirical choice of antifungal agents for therapy. Further, availability of the antifungal susceptibility result of the species aids in making a definitive choice of antifungal agent for therapy and help reduce the emergence of resistance   

Different modalities available for identification include conventional or automated. Antifungal susceptibility testing can be carried using disc diffusion, Etest, Micro broth dilution or automated technique.

Manual methods are cumbersome and time consuming. Whereas automated methods provide an advantage of shorter TAT but at the same time are expensive. In a resource constrained setting, availability of antifungal susceptibility testing facility may be limited, which could either result in an increased dependence on empirical therapy or delay in treatment. In such situation knowledge of the local epidemiology of the strains and the susceptibility profile will prove to  be useful.

This study was carried out to determine the epidemiology of Candida isolated at our center (Private diagnostic Microbiology laboratory) with respect to species and susceptibility profile .

A retrospective study was carried out in a Microbiology department of a referral center of a Private Diagnostic Microbiology laboratory over a period of 18 months from Jan 2018 to June 2020.All isolates of Candida species recovered from different clinically significant specimen during the study period were included in the studyDuplicate isolates obtained from same patient and same site were excluded.

Identification-

The samples received were proceesed on Sabourauds Dextrose Agar supplemented with Chloramphenicol plus Gentamicin and Brain heart infusion agar. They were incubated at 37°C and 25°C  for 72 hours. Blood received for fungal cultures were inoculated in BATCEC MYCO/F bottles. Any growth of yeast obtained was presumptively identified as Candida Species based on Gram stain morphology. The identification was confirmed using Germ tube test, Candida ChromAgar, API ID 32C automated VITEK2 system. The identification by API and VITEK2 was performed as per the manufacturer’s instruction. 

Antifungal susceptibility testing-

Antifungal susceptibility testing was carried by Sensititre Yeastone or VITEK2. The test was performed as per the manufacturer’s instruction. Nine antifungal agents were tested using SensititreYeastone which included Fluconazole(0.12- 256 ug/ml), Posaconazole (0.08-8ug/ml), Itraconazole (0.015-16 ug/ml) ,Voriconazole(0.008-8ug/ml) , AmphotericinB (0.12-8 ug/ml) ,5 flucytocine (0.08-64 ug/ml) , Anidulafungin (0.015-8 ug/ml) , Micafungin (0.008-8 ug/ml), and Capsofungin(0/008-8). Antifungal agents tested by VITEK 2 included Fluconazole (0.25- 16 ug/ml), Voriconazole (0.12-8ug/ml),5 Flucytosine (1-64ug/ml), Micafungin 0.06-8 ug/ml), Capsofungin(0.125-8 ug/ml).

Candida parapsilosis ATCC 22059 were used as quality control strains for susceptibility testing.The results were interpretated as per   CLSI M27-S4, CLSI M-59 and M-60.

A total of 273 Candida species were isolated during the study period.Of the 273 strain isolated, 200 strains were identified by API ID 30C and 73 were identified using VITEK2.169 isolates were tested for susceptibility using Sensititre Yeast One and 104 by VITEK2.148 (54%) isolates were recovered from males and 125(46%) were from females.

Table 1-Age wise distribution of the isolates

44% isolates were recovered from elderly population >60 years of age. 7% of isolates were from neonates.68% isolates were obtained from the Inpatient department and 32% were from Outpatient department. 

Table 2: Species distribution of Candida species

  C.albicans constituted 42% of the total isolates . 58% of the isolates were  Non albicans. Non albicans species included C.tropicalis (27%), C.sake, (9.3%) C.parapsilosis (8%). C.glabrata (4.3%), C.pelliculosa (3%), C.auris (3%) C.kruseii, C.kefyr, C.rugosa, C.famata ,C.colliculosa and C.duoboshaemulonii were isolated in frequency of less than 1%

Table 3- Specimen wise species distribution of Candida species

114(42%) isolates were from urine followed by  blood 70 (26%) . 14.5% , 8% and 20% were obtained from Sputum , BAL and ET Secretions. 14 (5% ) of isolates were recovered from vaginal swab and 11( 4%)  isolates from pus/wound swab/tissue. Around 1% each of isolates were from site likes ICD fluid, CSF, Stool, Oral swab.

Table 4a- Antifungal Susceptibility Profile of Candida Species

Table 4b- Antifungal Susceptibility Profile  of Candida Species contd.

Table 4c: - Antifungal Susceptibility Profile  of Candida Species contd.

Among the C.albicans, 19 % strains were resistant to Fluconazole, 15% were resistant to Voriconzole and  3% strains were resistant to Amphotericin B. 1% resistance was noted to Capsofungin and Micafungin. All isolates were susceptible to anidulafungin. 6% isolates had MIC value >=16 for Itraconazole and  7.6 % had MIC value >=8 for Posaconazole .

Among C.tropicalis 7% and 8.5 % isolates showed resistance to Fluconazole and Voriconazole..  Resistance to Micafungin and Capsofungin was 1% and no resistance was noted to Anidulafungin. 6.5 % and 4.3% of isolate showed resistance to Itraconazole and Posaconazole.

Resistance to Fluconazole, Voriconazole, Amphotericin was 45% , 10% and 5% respectively  for C.parapsilosis and all isolates were sensitive to Echinocandins .

58% of C.glabrata were Intermediate to Fluconazole and 17% were resistant to Capsofungin and Micafungin. All isolates were susceptible to Anidulafungin and Amphotericin B.

One out of two isolates of C.krusei showed MIC value >=16 for Amphotercin B.. All isolates were sensitive to Voriconazole, and Eichocandins.

Table-5 Susceptibility profile of other Candida species

NA- antifungal agent were not tested against that particular isolate.

All isolates of C.guillermondii were susceptible to all the antifungal agents tested except one which showed resistance to Fluconazole.

 C.rugosa exhibited a high MIC values Fluconazole (>=32µg/ml ) and Voriconazole (>=1µg/ml).

MIC value of C.famata to Fluconazole was 4 μg/ml. For all other Antifungals the MIC values ranged 0.06µg/ml to 0.25µg/ml.

C.duoboshaemulonii showed high MIC value to Fluconazole (32µg/ml), AmphotericinB (8-16µg/ml) and 5FC (>=64µg/ml.)

All isolates of C.auris were resistant to Fluconazole. 17% were resistant to Voriconazole. 64% resistance was noted to 5-Flucytosine. 100 % susceptibility was noted to Capsofungin, Micafungin, Anidulafungin and Amphotericn B.

Candida species are an important cause of invasive fungal infections. They are the fourth most common cause of bloodstream infection worldwide[3]. Though Candida albicans was the most common species isolated till recently, other non albicans Candida species are becoming more common. Injudicious use of antifungal agents has resulted in development of resistance in the previously known susceptible strains. Some of the Candida species are known to be inherently resistant to some of commonly used antifungal agents. Mostly recently isolated C.auris is known to be  multidrug resistant and  has emerged as threat to public health [2] Eichnocandins are first line agents for empirical Antifungal therapy for management of invasive Candidiasis in neutropenic patients  [4].

Due to the changing trend in the species profile of Candida  and diverse  pattern of resistance noted it is essential to identify and perform the susceptibility of all clinically relevant isolates of [5-6] 

 A total of 273 Candida species were isolated in our study during a period of 18 months.  54% of the isolates were recovered from male and 46% from female. Studies carried out by Dharwad et al.,, Kandhar et al.,, Prasadini guru have observed female predominance which is contrast to our study.

In our study, 44% of the isolates were recovered from patients belonging to age group >60 years, 7.3% from neonates and 5.4% from children below 10 years of age . The frequency of isolation of Candida species in elderly age group was higher in our study as compared to those from other studies, where the infection was more common in 18 to 45 years of age[5,7].Higher isolation rate of Candida in the elderly in our study could be because of high risk factors and co morbidities associated with increasing age which serves as predisposing factor for development of  fungal infection[8]. 

Percentage of specimens obtained from Inpatient department were 68% vs 32%from Outpatient, indicating the serious natures of infections from which the isolates were recovered.

In our study, 42% of the total Candida species were C.albicans and 58% were Non albicans Candida. This finding is  similar to those observed in other studies where a predominance of Non albicans Candida has been  seen [5-9].  

In our study, among the non albicans Candida spp,  C.tropicalis (26%) was  most common followed by C.sake (8.5%) and C.parasilosis(7.3%). Other less commonly isolated species included C.glabrata (4.5%), C.auris 2.5% and C.pelliculosa (2.9%). C.guillermondii, C,krusei, C.kefry, C.rugosa, C.famata, C.colliculosa, C.duoboshaemulonii were isolated in frequency of 0-1%

Candida albicans was the most common isolate from IPD as well as  OPD. C.glabrata was isolated in increased number from OPD. All the C.glabrata obtained from the OPD were  from urine

All the C.sake isolated in our study were identified using APID 32C.  It has been well established that some of the automated platform for fungal identification misidentified C.auris Identification of C.sake, Rhodotorula  glutinis, Candida species not identified, by API 32 C required further work up.  Since the identification of C.sake isolates in our study were not confirmed using molecular methods, it is likely that these isolates were misidentified(Diseases ZI.).

Variation in species distribution has been observed in various geographical areas as shown in table. 

Table 6- Species distribution Candida species in various studies

In our study Fluconazole resistance in C.albicans was noted in 19% of the isolates. Studies carried out across India have demonstrated resistance ranging from 5 % to 67% [8,10]. Voriconazole resistance  against C.albicans noted in our study was 15%, which was higher as compared to other studies carried out in various parts of India where the resistance noted is around 10% [7,11,12]. Studies carried out in other parts of the world have demonstrated 100% susceptibility to Voriconazole against C.albicans[6,10].

Fluconazole and Voriconazole resistance against C.tropicalis observed in the present study was 7% and 8.5% respectively. Azole resistance to C.tropicalis in various studies carried out in Indian setting ranges from 2% to 53% [7,11-14]. High variation to Azoles has also been noted in various parts of the world and overall an increasing trend in resistance to azole has been noted for C.tropicalis. The resistance ranges from 4 to 9% in United States and 0 to 0.5% in Japan and Turkey [10,15-17].

In studies, carried out by Dharwad et al., and S Jangala et al., no resistance to Azoles was seen in C.parapsilosis. In contrast to this, in the present study 45% of C.parapsilosis were resistant to Fluconazole and 10 % were resistant to [7,11]. Similar findings (28.5% resistance) was observed [5] 

Eichnocandins retained 100% activity against C.parapsilosis in our study. This is in contrast to other studies were high MIC values were observed. Deorukhkar et al., in their study noted that C.parapsilosis showed high MIC values to all three echinocandins. Similar finding were observed [18-19].

Development of cross resistance between Voriconazole and Fluconazole is a known phenomenon. Increased MIC values to Voriconazole can be seen in patients on Fluconazole therapy, in absence of known exposure to Voriconazole .Use of Fluconazole as empirical therapy could probably explain the increased Voriconazole resistance observed in our study. The property of C.parapsilosis to form biofilm can lead to emergence of resistance in these strains[14].

42% isolates of C.glabrata isolated in our study were resistant to Fluconazole. Similar finding were noted in other studies carried out in India [13,20,14].. All, C.glabrata isolates, that were sensitive,  were isolated from High Vaginal swab [6,21]. 

 In the present study, resistance to Echinocandins was less common. Increased resistance to Echinocandins was found in 17% of C.glabrata and 34% of C.krusei. Our findings are in concordance with other studies that have observed resistance to Capsofungin in C.krusei  and C.glabrata (50%)[11,22].  2010-2011 SENTRY surveillance observed that  in Asia pacific region 2% C.glabrata were  resistant to all three Echinocandins and 0.6% of C.albicans were oresistant to Capsofungin [23] ^.

All Candida species in our study remained susceptible to anidulafungin.

Amphotericin B resistance against C.albicans, C.tropicalis and C.parasilosis in our study was 2.7% , 5% and 5.5% . Similar studies carried out showed Amphotericin B  resistance to C.albicans ranging  from 0 to 15% and for C.tropicalis 0-8% [7,11-13,24].

Amongst the C.krusei 33% were resistance to Amphotercin B in our study. Studies carried out by V Tak et al.,, Dharwad et al., noted resistance of 33% and 20% respectively (Dharwad, S., & Saldanha, D. R. 2012; & Tak, V. et al., 2014).   None of the C.glabrata isolated in our study were resistant to Amphotericin. Variable resistance pattern of Amphotericin B against C.glabrata has been observed. The resistance observed in various study ranged from 0-30% [7,11-13,24].Increased resistance to Amphotercin B was noted in C.duoboshaemulonii (100%) and C.pelliculosa( 87.5%).

In the present study 91% of C.sake and 100% of C.auris showed resistance to Fluconazole (>256µg/ml). Our findings are consistent with those reported from other studies where they observed that C.auris was uniformly resistant to Fluconazole and showed variable resistance to Voriconazole. The resistance ranged from 15% to 100% in studies carried out in various parts of the world.  In our study 48% of C.sake and 17% of C.auris was resistant to Voriconazole. Amphotericinand Echinocandins retained its activity against C.auris. 100% susceptibility to Eichocandins was noted in our study. 

39% strains of C.sake showed MIC>2µg/ml to Amphotericin B. Amphotercin B resistance to C.auris  noted in India is around 8% [25-27] . In our study, all isolates of C.auris were susceptible to Amphotercin B.

22% and 17% strains a of C.sake and C.auris respectively were resistant to Itraconazole.. 30 % of C.sake and 57% of C.auris were resistant to 5 FC. In another study 14% strains showed resistance to 5- FC[25].Increased resistance to 5FC was noted in our study.

The main limitation of our study was that the clinical relevance of the isolates recovered could not be determined due to lack of availability of clinical information.  . The isolates of C.sake identified by API ID could not be further worked up. Further work up would have avoided misidentification.

To conclude, resistance to various antifungal agents is emerging. The pattern of resistance varies in different geographical areas. It is essential to establish a local susceptibility profile of the various Candida species isolated in a particular area. The idea about the local antibiogram would help in formulation of empirical antifungal policy and aid stewartship. Emergence of newer Candida species with limited literature available about susceptibility profile warrants antifungal susceptibility testing..

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the SRL Limited, Mumbai Reference Lab, India

1. Jain, N., Mathur, P., Misra, M. C., Behera, B., Xess, I., & Sharma, S. P. (2012). Rapid identification of yeast isolates from clinical specimens in critically ill trauma ICU patients. Journal of Laboratory Physicians, 4(01), 30-34. doi:10.4103/0974-2727.104199.

2. Pappas, P. G., Lionakis, M. S., Arendrup, M. C., Ostrosky-Zeichner, L., & Kullberg, B. J. (2018). Invasive candidiasis. Nature Reviews Disease Primers, 4(1), 1-20. doi:10.1038/nrdp.2018.5.

3. Xess, I., Jain, N., Hasan, F., Mandal, P., & Banerjee, U. (2007). Epidemiology of candidemia in a tertiary care centre of north India: 5-year study. Infection, 35(4), 256-259. doi:10.1007/s15010-007-6007-2.

4. Pappas, P. G., Kauffman, C. A., Andes, D. R., Clancy, C. J., Marr, K. A., Ostrosky-Zeichner, L., ... & Sobel, J. D. (2016). Clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America. Clinical Infectious Diseases, 62(4), e1-e50. doi:10.1093/cid/civ933.

5. Guru, P., & Raveendran, G. (2016). Characterisation and antifungal susceptibility profile of Candida species isolated from a tertiary care hospital. Journal of The Academy of Clinical Microbiologists, 18(1), 32-36. doi:10.4103/0974-2727.185617.

6. Seyoum, E., Bitew, A., & Mihret, A. (2020). Distribution of Candida albicans and non-albicans Candida species isolated in different clinical samples and their in vitro antifungal susceptibility profile in Ethiopia. BMC Infectious Diseases, 20(1), 1-9. doi:10.1186/s12879-020-04939-3.

7. Dharwad, S., & Saldanha, D. R. (2012). Species identification of Candida isolates in various clinical specimens with their antifungal susceptibility patterns. Journal of Clinical and Diagnostic Research, 5(6), 1177-1181. doi:10.7860/JCDR/2011/2441.2280.

8. Kaur, R., Dhakad, M. S., Goyal, R., & Kumar, R. (2016). Emergence of non-albicans Candida species and antifungal resistance in intensive care unit patients. Asian Pacific Journal of Tropical Biomedicine, 6(5), 455-460. doi:10.1016/j.apjtb.2015.12.019.

9. Badiee, P., & Hashemizadeh, Z. (2014). Opportunistic invasive fungal infections: Diagnosis & clinical management. The Indian Journal of Medical Research, 139(2), 195-204. doi:10.4103/0971-5916.132977.

10. Eksi, F., Gayyurhan, E. D., & Balci, I. (2013). In vitro susceptibility of Candida species to four antifungal agents assessed by the reference broth microdilution method. The Scientific World Journal, 2013, Article ID 431262. doi:10.1155/2013/431262.

11. Jangla, S. M., Naidu, R., & Patel, S. C. (2018). Speciation and antifungal susceptibility testing of Candida isolates in various clinical samples in a tertiary care hospital in Mumbai. International Journal of Biomedical Research, 9(3), 106-111. doi:10.7439/ijbr.v9i3.4313.

12. Bhattacharjee, P. (2016). Epidemiology and antifungal susceptibility of Candida species in a tertiary care hospital, Kolkata, India. Current Medical Mycology, 2(2), 0-0. doi:10.18502/jmmy.v2i2.303.

13. Rajgopal, D. S. K. (2017). Prevalence and antifungal susceptibilities of Candida spp. from a South Indian tertiary care hospital. International Journal of Medical Research & Review, 5(2), 98-104. doi:10.17511/ijmrr.2017.i2.05.

14. Oberoi, J. K., Wattal, C., Goel, N., Raveendran, R., Datta, S., & Prasad, K. (2012). Non-albicans Candida species in bloodstream infections in a tertiary care hospital at New Delhi, India. The Indian Journal of Medical Research, 136(6), 997-1003. doi:10.4103/0971-5916.200204.

15. Fan, X., Xiao, M., Liao, K., Kudinha, T., Wang, H., Zhang, L., ... & Xu, Y. C. (2017). Notable increasing trend in azole non-susceptible Candida tropicalis causing invasive candidiasis in China (August 2009 to July 2014): Molecular epidemiology and clinical azole consumption. Frontiers in Microbiology, 8, Article 135. doi:10.3389/fmicb.2017.00135.

16. Berkow, E. L., & Lockhart, S. R. (2017). Fluconazole resistance in Candida species: A current perspective. Infectious Drug Resistance, 10, 237-245. doi:10.2147/IDR.S118892.

17. Katsuragi, S., Sata, M., Kobayashi, Y., Miyoshi, T., Yamashita, Y., Neki, R., ... & Yoshimatsu, J. (2014). Antifungal susceptibility of Candida isolates at one institution. Japanese Journal of Medical Mycology, 55(1), 1-7. doi:10.3314/jjmm.55.1.

18. Montagna, M. T., Lovero, G., Coretti, C., Martinelli, D., De Giglio, O., Iatta, R., ... & Caggiano, G. (2015). Susceptibility to echinocandins of Candida spp. strains isolated in Italy assessed by European Committee for Antimicrobial Susceptibility Testing and Clinical Laboratory Standards Institute broth microdilution methods. BMC Microbiology, 15(1), 1-6. doi:10.1186/s12866-015-0386-3.

19. Deorukhkar, S. C., & Saini, S. (2016). Echinocandin susceptibility profile of fluconazole-resistant Candida species isolated from bloodstream infections. Infectious Disorders - Drug Targets, 16(1), 63-68. doi:10.2174/1871526516016010063.

20. Sandhu, R., Dahiya, S., Sayal, P., & Budhani, D. (2017). Increased role of non-albicans Candida, potential risk factors, and attributable mortality in hospitalized patients. Journal of Health Research and Reviews, 4(2), 78-82. doi:10.4103/2394-2010.210032.

21. Raminder, Sandhu, S. D., & R. K. S. (2015). Isolation and identification of Candida and non-albicans Candida species using chromogenic medium. International Journal of Biomedical Research, 6(12), 958–962. doi:10.7439/ijbr.v6i12.2525.

22. Axner-Elings, M., Botero-Kleiven, S., Jensen, R. H., & Arendrup, M. C. (2011). Echinocandin susceptibility testing of Candida isolates collected during a 1-year period in Sweden. Journal of Clinical Microbiology, 49(7), 2516–2521. doi:10.1128/JCM.02518-10.

23. Pfaller, M. A., Messer, S. A., Woosley, L. N., Jones, R. N., & Castanheira, M. (2013). Echinocandin and triazole antifungal susceptibility profiles for clinical opportunistic yeast and mold isolates collected from 2010 to 2011: Application of new CLSI clinical breakpoints and epidemiological cutoff values for characterization of geographic and temporal trends of antifungal resistance. Journal of Clinical Microbiology, 51(8), 2571-2581. doi:10.1128/JCM.00592-13.

24. Axner-Elings, M., Botero-Kleiven, S., Jensen, R.H., & Arendrup, M.C. (2011). Echinocandin susceptibility testing of Candida isolates collected during a 1-year period in Sweden. J Clin Microbiol. 49(7), 2516–21. 

25. Pfaller, M. A., Messer, S. A., Woosley, L. N., Jones, R. N., & Castanheira, M. (2013). Echinocandin and triazole antifungal susceptibility profiles for clinical opportunistic yeast and mold isolates collected from 2010 to 2011: application of new CLSI clinical breakpoints and epidemiological cutoff values for characterization of geographic and temporal trends of antifungal resistance. Journal of clinical microbiology, 51(8), 2571-2581.

26. Tak, V., Mathur, P., Varghese, P., Gunjiyal, J., Xess, I., & Misra, M. C. (2014). The epidemiological profile of candidemia at an Indian trauma care center. Journal of laboratory physicians, 6(02), 096-101. 

27. Chowdhary, A., Prakash, A., Sharma, C., Kordalewska, M., Kumar, A., Sarma, S., ... & Meis, J. F. (2018). A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009–17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance. Journal of Antimicrobial Chemotherapy, 73(4), 891-899. 

28. Kordalewska, M., & Perlin, D. S. (2019). Identification of drug resistant Candida auris. Frontiers in microbiology, 10, 1918. (AUG). Available from: www.frontiersin.org

29. Kathuria, S., Singh, P. K., Sharma, C., Prakash, A., Masih, A., Kumar, A., ... & Chowdhary, A. (2015). Multidrug-resistant Candida auris misidentified as Candida haemulonii: characterization by matrix-assisted laser desorption ionization–time of flight mass spectrometry and DNA sequencing and its antifungal susceptibility profile variability by Vitek 2, CLSI broth microdilution, and Etest method. Journal of clinical microbiology, 53(6)