Candidemia ranks among the top five causes of nosocomial bloodstream infections in the United States of America, while Indian studies have shown Candida as sixth among isolatesfrom bloodstream infections.^1-3 Apart from an attributable mortality that varies between 5-71% in various studies, candidemia adds to morbidity, prolongs hospital stay and increases the cost of healthcare.⁴ As mortality directly correlates with delay in initiation of empiric therapy, knowledge regarding species distribution and extent of fluconazole resistance in an institution or country is important in deciding empiric therapy.⁵Candida non-albicans (CNA), especially C. tropicalis, predominate in India in virtually all recent reports but emergent species such as C. haemulonii complex have been recently described.^6-8 In this report we describe the species distribution and fluconazole susceptibility of candidemia episodes between May 2010 and December 2015.

A cross-sectional study of candidemia episodes between May 2010 and December 2015 was carried out at a 600-bed tertiary care referral hospital in Chennai, South India. Positive blood cultures from a patient were considered as being part of a single candidemia episode if they belonged to the same species and occurred less than two weeks apart. Blood culture was done using the Bact/ALERT system, species identification was performed by VITEK 2 (bioMérieux, Marcy-l'Étoile, France). From January 2014 species confirmation was also performed by VITEK MS MALDITOF, bioMérieux. Results were interpreted as per the Clinical Laboratory Standards Institute guidelines. These lab methods were standard lab procedure for identification of the isolate. Antifungal susceptibility testing for fluconazole (FLU), voriconazole (VRC), amphotericin B (AMB), caspofungin (CAS), micafungin (MFG), and flucytosine was performed using the AST-YS07 card of the VITEK2 until 2011 and later by the AST-YS08 card. The minimum inhibitory concentration (MIC) determination for azoles and echinocandins were based on CLSI document M27-S4. MIC determination of amphotericin B was based on CLSI document M27-A3 and EUCAST 2015 guidelines.^9-11 Inoculum suspensions for Vitek-2 cards were obtained from the overnight cultures, with the turbidity being adjusted to a 1.8-2.2 McFarland standard according to the manufacturer's recommendations.A standardized inoculum suspension was placed into a VITEK-2 cassette along with a sterile polystyrene test tube and an antifungal susceptibility test card for each organism. The loaded cassettes were placed into the VITEK-2 instrument, and the respective inoculum suspensions were diluted appropriately by the instrument, after which the cards were filled, incubated, and read automatically.

A total of 306 blood culture samples grew Candidaduring the study period. Of these 306 isolates, 258 (84.3%) were CNA and 48 (15.7%) were C. albicans. The most common species was C. tropicalis103 (33%), followed by C. albicans 48 (15.7%), C. parapsilosis sensu lato 42 (13.7%), C. haemuloniicomplex 38 (12.4%) and C. glabrata 36 (11.8%). C. krusei, C. famata, C. guilliermondii, C. pelliculosa, C. rugosa, C. utilis, C. lusitaniae make up the remainder. The distribution of species isolated is shown in Fig. 1.

Figure 1. Species distribution in the candidemia study from South India

Fluconazole susceptibility was determined for all isolates. Out of 306 Candida isolates, 237 (77.5%) were susceptible to fluconazole. All C. albicans isolates (100%) were invariably susceptible to fluconazole. Most of C. tropicalis (98%), C. parapsilosis sensu lato (95.2%), C. famata(87.5%) and C. guilliermondii (83.3%) isolates were susceptible to fluconazole. Among C. glabrata isolates only 72.3% were susceptible and only 16.7% of C. krusei isolates were susceptible to fluconazole. All the C. haemulonii complex isolates recovered in this study were resistant to fluconazole and all except one (37 out of 38 isolates) were resistant to amphotericin B; all C. haemulonii complex isolates were susceptible to caspofungin and voriconazole. The fluconazole susceptibility of the remaining isolates is shown in Table 1.

 

Table 1. Species distribution and fluconazole susceptibility profile of isolates from the candidemia study in South India

 

 

There has been a gradual worldwide shift in the epidemiology of invasive Candida infections from C. albicans to other species over the past two decades, although C. albicans remains the most common species worldwide.¹² CNA is becoming more common and there is a great variability between different parts of the world and among hospitals as well.¹³ Among the CNA, C. glabrata is the most common species in the West, while C. tropicalis predominates in Asia and other countries. A large study performed at 127 study sites over 6.5 years (1997 to 2003) by Pfaller et al. (2005) collected Candida isolates data from countries across the globe; medical centers from North America, Asia, Middle East, Europe, and Latin America participated.¹⁴They found that 5 species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei) accounted for 92% of cases of candidemia. Among the top 5, although there was variability in the occurrence of species, data from this registry showed that there was an increasing trend of isolating C. tropicalis and decreasing trend in C. albicans isolation, although C. albicans still remained the most common, accounting for 62% of cases. Among the CNA, C. glabrata was noted as the most common species.¹⁴ In the review by Guinea J (2014) on global trends in the distribution of Candida spp., the influence of the geographic area, patient risk factors and the influence of local hospital epidemiology were also considered to impact the species distribution and variability.¹³ In the study by Tah et al. (2015), which included data from Asian countries, among the CNA, the highest incidence was recorded for C. tropicalis, followed by C. glabrata and C. parapsilosis, which had almost similar incidence. It was also noted than C. tropicalis was common in tropical countries and isolated frequently in hemato-oncology wards.¹⁵In a literature review on candidemia isolates in Latin America by da Matta et al., although C. albicans remained the most common species, C. parapsilosiswas the most frequently isolated CNA, followed by C. tropicalis and C. glabrata.¹⁶

Current guidelines call for initiating empiric therapy for candidemia with an echinocandin but fluconazole is an acceptable alternative in selected cases, such as patients who are not critically ill and considered unlikely to have an infection with fluconazole-resistant Candida.¹⁷Knowing the species distribution and fluconazole resistance among Candidaspecies in an institution is important in selecting appropriate empiric therapy for candidemia. Our study showed a predominance of CNA (84.3%), similar to other studies.^3,6 In a recent prospective, nationwide, multicentric, observational study conducted by Chakrabarti et al. (2015) at 27 Indian intensive care units (ICU) from April 2011 to September 2012, CNA accounted for 79.1%, which is very similar to our finding.⁸ The most common isolate in our study was C. tropicalis (33.7%), similar to other Indian studies which reported rates ranging between 29.2-42.1%.^3,6-8,18 The next two most common species were C. albicans (15.7%) and C. parapsilosis (13.6%), similar to the study by Chakrabarti et al. (2015).⁸

Overall 77.5% of our Candida isolates were susceptible to fluconazole. This is in contrast to the study conducted by Chakrabarti et al. (2015) where a lower overall fluconazole resistance was reported, specifically 6.2%.⁸ Oberoi et al. (2012) in a casuistic on CNA bloodstream infection over 10 years (1999-2008) noted a high level of fluconazole resistance (31.2%).⁶ High rates of fluconazole resistance are a concern as switch to echinocandins may be required for empiric therapy for candidemia. After commencement of testing for caspofungin susceptibility from September 2012, all our isolates (except for one C. glabrataisolate) were found susceptible to caspofungin.

Species specific susceptibility was looked into. In our study C. albicans, the second most common isolate, was uniformly susceptible to fluconazole (100%); some studies show fluconazole resistance up to 5% among C. albicans species related to candidemia.^8,12 The resistance rates among C. albicans isolates in the multicenter Indian study conducted by Chakrabarti et al. (2015) was noted to be 5.2% and global resistance patterns are around 1.4-5%, which is higher than in our study.^8,19

Among C. tropicalis, fluconazole resistance was 1.9%, which is comparable to the other major Indian study, which reported a rate of 2.6%.⁸ In contrast, several other Indian studies showed higher fluconazole resistance rates among C. tropicalis varying from 9.5% to 19% and 2.2-4.9% globally as in the SENTRY trial by Pfaller et al. (2011).^6,7,12,18 In our study, 4.8% of C. parapsilosis were fluconazole resistant, which is again similar to the study by Chakrabarti et al. (2015) where 4% resistance was noted.⁸ In one study, a very high resistance level of 33.9% among C. parapsilosis was noted and attributed to the widespread use of fluconazole prophylaxis and biofilm formation, which confers relative resistance to antifungals.⁶ Globally, fluconazole resistance rates among C. parapsilosis sensu lato isolates were observed as 4.3 to 6.8 %.¹²

Fluconazole resistance among C. glabrata in our study was 27.8%, which is comparable to 39.2% in another Indian study.⁶Globally C. glabratafluconazole susceptibility pattern varies. Fluconazole resistance among C. glabrata isolates varies from as low as 1.5% in the study by Chakrabarti et al. (2015) to as high as 45.6% in European countries, as observed in the study by Basetti et al. (2013).^8,20A published report on the epidemiology of invasive candidiasis by Pfaller et al. (2007) from the collective data of discharges from US hospitals, noted that fluconazole resistance rates among C. glabrata was very much similar between 1996-2003; 2003 resistance rate was 16.9%.¹⁹ A few years later, again reported by Pfaller et al. (2011) in an interesting observation from the antimicrobial surveillance program between 2008-2009, they noted that the C. glabrata resistance pattern varied from 3.3-7.7%, and was lower in cases with community onset as compared to nosocomial candidemia.¹²

The fourth most common species in our series was C. haemulonii complex accounting for 12.4% of isolates. Its first isolation was reported by Lavarde et al. (1984), from the blood culture of a patient with renal failure undergoing peritoneal dialysis; the patient died despite receiving therapy with amphotericin B and flucytosine.²¹ C. haemulonii complex has emerged as an opportunistic fungal pathogen that is often resistant to both amphotericin B and fluconazole and was associated with clinical failure in the studies from Korea.²² A recent study from New Delhi showed a greatly increased prevalence of C. haemulonii complex (15.5% out of 1206 isolates of Candida in blood); all the isolates were resistant to fluconazole and itraconazole and only 63.8% and 27.6% were susceptible to voriconazole and amphotericin respectively.⁶

Accurate Candidaspecies identification requires sequencing the internal transcribed spacer (ITS) and D1/D2 regions, as automated systems may misidentify C. haemulonii complex for C. pseudohaemulonii. Recent reports using molecular typing methods suggest that many isolates of C. haemulonii complex may actually be C. auris.²³ As diagnostic laboratories do not undertake molecular identification routinely, misidentification of C. auris as C. haemulonii complex could occur. The best identification at the species level relies on the ITS region and MALDI-TOF MS.^23,24 Cendeja-Bueno et al. (2012) reclassified C. haemulonii sensu lato (or complex) by sequence analysis of the four genes studied (ITS, D1/D2, RPB1 and RPB2) into C. haemulonii sensu stricto (C. haemulonii group I) and C. duobushaemulonii (group II). There are also two species closely related phylogenetically, C. pseudohaemulonii and C. auris.²⁴ In our study, 38 (12.4%) of our isolates were C. haemulonii complex of which 18 isolates were identified only by VITEK2 from 2010 until 2013, as MALDITOF MS was not available at our center during this period. From 2014 onwards, all Candida isolates which were initially identified by VITEK2 were later confirmed by MALDITOF MS to the species level, thus confirming the remaining 20 C. haemulonii complex isolates (from 2014 to 2015). Thus, in the initial period of the study, between 2010 and 2013 we cannot exclude the possibility of misidentification of C. auris as C. haemulonii. In the future, molecular tools should help to identify others species in the C. haemulonii sensu lato complex including C. auris isolates.

All our C. haemulonii complex isolates showed MIC of >2 mcg/mL for fluconazole and 37 out of 38 (97.4%) isolates had a high MIC of >1 mcg/mL for amphotericin B; amphotericin susceptibility is extrapolated from EUCAST and CLSI guidelines from other Candida species.^9-11 However all were susceptible to caspofungin and voriconazole. This has important therapeutic and infection control implications, as inadequate isolation precautions pose the risk of transmission and outbreak in an unit due to drug resistant Candida.

CNA species were the most common cause of candidemia in our study group, with C. tropicalis being the most frequent species, followed by C. albicans and C. parapsilosis sensu lato, C. haemulonii complex and C. glabrata, respectively. Although more than three-fourths of our isolates were overall susceptible to fluconazole, C. haemuloniicomplex was the fourth most common species and is an important emerging fluconazole resistant species. Further studies are warranted to look for emergence of this and other fluconazole resistant species, so as to ensure optimal empiric antifungal treatment in patients with suspected candidemia.

1.Sievert DM, Ricks P, Edwards JR, et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010.Infect Control Hosp Epidemiol2013;34:1-14. [Crossref]

2.Tabah A, Koulenti D, Laupland K, et al. Characteristics and determinants of outcome of hospital-acquired bloodstream infections in intensive care units: the EUROBACT International Cohort Study. Intensive Care Med 2012;38:1930-45. [Crossref]

3.Chander J, Singla N, Sidhu SK, Gombar S. Epidemiology of Candida blood stream infections: experience of a tertiary care centre in North India. J Infect Dev Ctries 2013;7:670-5. [Crossref]

4.Falagas ME, Apostolou KE, Pappas VD. Attributable mortality of candidemia: a systematic review of matched cohort and case- control studies. Eur J Clin Microbiol Infect Dis. 2006;25:419-25. [Crossref]

5.Skrobik Y, Laverdiere M. WhyCandidasepsis should matter to ICU physicians. Crit Care Clin 2013;29:853-64. [Crossref]

6.Oberoi JK, Wattal C, Goel N, Raveendran R, Datta S, Prasad K. Non-albicansCandidaspecies in blood stream infections in a tertiary care hospital at New Delhi, India. Indian J Med Res 2012;136:997-1003.

7.Chakrabarti, A, Chatterjee SS, Rao KL, et al. Recent experience with fungaemia: change in species distribution and azole resistance. Scand J Infect Dis 2009;41:275-84. [Crossref]

8.Chakrabarti A, Sood P, Rudramurthy SM, et al. Incidence, characteristics and outcome of ICU-acquired candidemia in India. Intensive Care Med 2015;41:285-95. [Crossref]

9.Clinical and Laboratory Standards Institute.Reference method for broth dilution antifungal susceptibility testing of yeasts; 4^th Informational Supplement. CLSI document M27-S4.Wayne: Clinical and Laboratory Standards Institute; 2012.

10.Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeast; approved standard – third edition. CLSI document M27-A3.Wayne: Clinical and Laboratory Standards Institute; 2008.

11.European Committee on Antimicrobial Susceptibility Testing. Antifungal agents. Breakpoint tables for interpretation of MICs. Version 6, 2013.

12.Pfaller MA, Messer SA, Moet GJ, Jones RN, Castanheira M. Candidabloodstream infections: comparison of species distribution and resistance to echinocandin and azole antifungal agents in intensive care unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009). Int J Antimicrob Agents 2011;38:65-9. [Crossref]

13.Guinea J. Global trends in thedistributionofCandidaspeciescausing candidemia. Clin Microbiol Infect 2014;20 Suppl 6:5-10. [Crossref]

14.Pfaller MA, Diekema DJ, Rinaldi MG, et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study: a 6.5-year analysis of susceptibilities of Candidaand other yeast species to fluconazole and voriconazole by standardized disk diffusion testing. J Clin Microbiol 2005;43:5848-59. [Crossref]

15.Tan BH, Chakrabarti A, Li RY, et al. Incidence andspeciesdistributionof candidaemia inAsia: a laboratory-based surveillance study. Clin Microbiol Infect 2015;21:946-53. [Crossref]

16.da Matta DA, Souza ACR, Colombo AL. Revisiting species distribution and antifungal susceptibility of Candidabloodstream isolates from Latin American medical centers. J Fungi (Basel) 2017;3. pii: E24. [Crossref]

17.Pappas PG, Kauffman CA, Andes DR, et al. Executive summary: clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 2016;62:409-17. [Crossref]

18.Adhikary R, Joshi S. Species distribution and anti-fungal susceptibility of candidaemia at a multi super-specialty center in Southern India. Indian J Med Microbiol 2011;29:309-11. [Crossref]

19.Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007;20:133-63. [Crossref]

20.Bassetti M, Merelli M, Righi E, et al. Epidemiology, species distribution, antifungal susceptibility, and outcome of candidemia across five sites in Italy and Spain. J Clin Microbiol 2013;51:4167-72. [Crossref]

21.Lavarde V, Daniel F, Saez H, Arnold M, Faguer B. Peritonite mycosique a Torulopsis haemulonii. Bul Soc Fr Mycol Med 1984;13:173-6.

22.Kim MN, Shin JH, Sung H, et al. Candida haemulonii and closely related species at 5 university hospitals in Korea: identification, antifungal susceptibility, and clinical features. Clin Infect Dis 2009;48:e57-61. [Crossref]

23.Kathuria S, Singh PK, Sharma C, et al. Multidrug resistantCandida aurismisidentified asC. 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. J Clin Microbiol 2015;53:1823-30. [Crossref]

24.Cendejas-Bueno E, Kolecka A, Alastruey-Izquierdo A, et al. Reclassification of theCandida haemuloniicomplex asCandida haemulonii(C. haemuloniiGroup I),C. duobus haemuloniisp. nov. (C. haemuloniiGroup II), andC. haemuloniivar.vulneravar. nov.: three multi resistant human pathogenic yeasts. J Clin Microbiol 2012;50:3641-51. [Crossref]