Clinical features of patients with infections caused by Candida

J Antimicrob Chemother 2013; 68: 2632 – 2635 doi:10.1093/jac/dkt214 Advance Access publication 13 June 2013

Clinical features of patients with infections caused by Candida guilliermondii and Candida fermentati and antifungal susceptibility of the isolates at a medical centre in Taiwan, 2001–10 Chien-Yuan Chen1,2, Shang-Yi Huang1, Jih-Luh Tang1, Woei Tsay1, Ming Yao1, Bo-Sheng Ko1, Wen-Chien Chou1,3, Hwei-Fang Tien1 and Po-Ren Hsueh1,3* 2

1 Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan; Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan; 3Department of Laboratory Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan

Received 2 April 2013; returned 22 April 2013; revised 3 May 2013; accepted 7 May 2013 Objectives: This study was intended to analyse the clinical characteristics and outcomes of patients with infections due to Candida guilliermondii complex and evaluate in vitro susceptibilities of the isolates. Methods: We searched the Mycology Database of the National Taiwan University Hospital and identified patients with infections due to C. guilliermondii complex from 2001 to 2010. Isolates were identified to species level by two yeast identification systems and restriction fragment length polymorphism of the riboflavin synthetase gene. MICs of nine antifungal agents were determined using the Sensititre YeastOne system (Trek Diagnostic Systems) and were interpreted by breakpoints (BPs) for three echinocandins and epidemiological cut-off values (ECVs) for the other agents. Results: Fifty-two patients with infections due to C. guilliermondii complex were evaluated. The majority (90%, n ¼47) of the isolates were C. guilliermondii, followed by Candida fermentati (10%, n ¼5). Among them, 42 (81%) were isolated from blood cultures. Among the 52 patients, 27 (52%) had underlying malignancy and 15 (29%) had undergone abdominal surgery. The 30 day mortality rates among patients with C. guilliermondii and C. fermentati infections were 45% and 60%, respectively. Among C. guilliermondii isolates, 98%, 100% and 98% were susceptible to caspofungin, micafungin and anidulafungin, respectively, by BPs. Nearly all (96% –100%) C. guilliermondii isolates belonged to wild-type for the other agents by ECVs. All five C. fermentati were susceptible to three echinocandins and belonged to wild-type for the other agents. Conclusions: The currently used antifungal agents exhibited good in vitro activities against C. guilliermondii complex isolates. Keywords: candidaemia, malignancy, in vitro antifungal susceptibility, drug resistance, outcomes

Introduction The incidence of candidaemia has increased over the past 20 years in the USA and Europe.1,2 Bloodstream infections due to Candida species cause significant morbidity and mortality, with mortality rates ranging from 30% to 60%.3,4 Candida guilliermondii is widely distributed in the environment, is frequently isolated from soil, plants, insects, seawater and the atmosphere, and forms part of the saprophytic microflora on human skin and mucosal surfaces. The incidence of candidaemia due to C. guilliermondii ranges from 1% to 3%, depending on the geographic region.5,6 Although its incidence is not high, C. guilliermondii has been shown to be more resistant to antifungal agents than other Candida species.1,5,7 In addition to candidaemia, C. guilliermondii can also cause

osteomyelitis, septic arthritis, candiduria, endocarditis and skin lesions.8 – 11 C. guilliermondii complex is a genetically heterogeneous complex comprising several phenotypically indistinguishable taxa, including C. guilliermondii, Candida fermentati, Candida carpophila and Candida xestobii. Recent advances in molecular identification methods have improved the accuracy of differentiating between closely related species of fungi in Candida complexes.12 In this study, we retrospectively analysed the clinical characteristics and outcomes of 52 patients with infections due to C. guilliermondii complex who were treated at a medical centre during the period 2001 –10 and evaluated the in vitro susceptibilities of the isolates from those patients to a battery of antifungal agents.

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*Corresponding author. Tel: +886-2-23123456, ext. 65355; Fax: +886-2-23955072; E-mail: hsporen@ ntu.edu.tw

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Candida guilliermondii and Candida fermentati infection

Methods

Results

Hospital setting and patients

Identification of Candida species

We retrospectively analysed the clinical manifestations, laboratory and microbiological data, and outcome of all HIV-negative patients whose cultures yielded C. guilliermondii during the period January 2001 to December 2010 at the National Taiwan University Hospital, a 2500 bed tertiary care teaching hospital in northern Taiwan. The overall 30 day mortality rate was also assessed. This study was approved by the National Taiwan University Hospital Institutional Review Board (NTUH-IRB-201205039).

All of the 52 isolates were identified by the API32C and Vitek II systems as being members of the C. guilliermondii complex. Restriction fragment length polymorphism (RFLP) analysis of the riboflavin synthetase gene revealed that 47 isolates were C. guilliermondii and that 5 isolates were C. fermentati.

Identification of C. guilliermondii and C. fermentati

Antifungal susceptibility testing Antifungal susceptibility testing of the preserved isolates of C. guilliermondii complex to nine antifungal agents was performed using a commercial broth microdilution method, the Sensititre YeastOne system (Trek Diagnostic Systems, Ltd, East Grinstead, UK) in accordance with the manufacturer’s instructions.14,15 Plates containing serial 2-fold dilutions of the antifungal agents across 12 dilutions were inoculated using a prepared suspension of the organism, incubated at 358C, and read at 24 h as recommended by the CLSI.16 For all drugs, the MIC was defined as the lowest concentration of antifungal agent that prevented the development of a red colour (the first blue well). Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as control strains. The MIC results of C. guilliermondii isolates were interpreted by recently assigned breakpoints (BPs) for the echinocandins and epidemiological cut-off values (ECVs) for the other agents.17 Because there are no interpretive criteria for C. fermentati isolates, the BPs and ECVs used for C. guilliermondii isolates were applied to C. fermentati isolates.

Definitions Candidaemia was diagnosed in patients who presented with at least one blood culture that was positive for Candida species and who presented with clinical symptoms of bloodstream infection such as fever and hypotension. Mortality attributable to candidaemia was defined as death within 30 days after the first blood culture that could not be attributed to any other cause of death. Persistent candidaemia was defined in patients from whom the same Candida species was isolated from blood cultures obtained from peripheral veins ≥7 days after the initiation of administration of antifungal agents.

Statistical analysis Categorical variables were compared using the x2 test. Analysis of 30 day survival was conducted with the Kaplan–Meier method and the log rank test. All statistical analyses were performed using the statistical package SPSS for Windows (Version 18, SPSS, Chicago, IL, USA).

No. of patients Clinical characteristics

C. guilliermondii (n¼47)

C. fermentati (n¼5)

Age (years) ≥60 ,60

24 23

1 4

Gender male female

27 20

3 2

Cancer yes no

25 22

2 3

Transplantation yes no

3 44

1 4

Steroid use yes no

9 38

2 3

Neutropenia yes no

8 39

2 3

Central venous catheter-related infection yes 36 no 11

3 2

Total parenteral nutrition yes no

22 25

2 3

Bloodstream isolates yes no

40 7

2 3

Abdominal surgery yes no

8 39

0 5

Shock yes no

14 33

2 3

30 day death yes no

18 29

3 2


All isolates were initially identified as C. guilliermondii by standard laboratory procedures, including morphological identification and two commercial yeast identification systems: API ID32C (bioMe´rieux Vitek, St Louis, MO, USA) and Vitek 2 Yeast cards (bioMe´rieux). Isolates were subjected to PCR with the primers RIBO-F (5′ -ACAGTTGGTCGAGGTGGTC-3′ ) and RIBO-R (5′ -CCTGGGTTCCCAAGTAGTCA-3′ ) for the riboflavin synthetase gene. The amplified products were cleaved by restriction enzyme HgaI or HincII to differentiate between C. guilliermondii and C. fermentati.13 The following four isolates were used as control strains: C. guilliermondii BCRC22093 (ATCC 46036), C. fermentati BCRC23164 (CBS9966), C. carpophila BCRC22807 (CBS7921) and C. xestobii BCRC22660 (ATCC 24001).

Table 1. Clinical characteristics of adult patients with infections caused by C. guilliermondii and C. fermentati

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Chen et al.

There were no isolates of C. carpophila or C. xestobii in this study.

Patient characteristics and clinical manifestations

Treatment outcomes Of the 41 (79%) patients who received antifungal treatment, 35 were treated with fluconazole, 3 were treated with amphotericin

Antifungal susceptibility Susceptibilities of the 47 C. guilliermondii and 5 C. fermentati isolates to nine antifungal agents are shown in Table 2. Among C. guilliermondii isolates, 98%, 100% and 98% were susceptible to caspofungin, micafungin and anidulafungin, respectively, by BPs. Nearly all (96%– 100%) C. guilliermondii isolates belonged to wild-type for the other agents by ECVs. All C. fermentati were susceptible to three echinocandins by BPs and the MICs of the other agents were all within the respective ECVs.

Discussion Only 1% – 3% of all cases of candidaemia are due to C. guilliermondii. 5,6 However, C. guilliermondii has been shown to be more resistant to antifungal agents than other Candida species and studies have shown that resistance to these agents is associated with consumption of antifungal drugs.1,5 – 7 Lockhart

Table 2. In vitro susceptibility of 52 isolates of C. guilliermondii and C. fermentati to nine antifungal agents as interpreted by BPs for three echinocandins and ECVs for the other agents No. (%) of isolates by ECVs

MIC (mg/L) Candida species (no. of isolates)

range

MIC50

MIC90

WT

non-WT

C. guilliermondii (47) fluconazole itraconazole voriconazole posaconazole 5-flucytosine caspofungin micafungin anidulafungin amphotericin B

0.25 –16 0.25 –2 0.03 –2 0.06 –0.5 ≤0.06 to .64 ≤0.06 –8 0.25 –2 0.12 –2 ≤0.12 –1

4 0.5 0.06 0.25 ≤0.06 1 1 1 0.5

4 1 0.25 0.5 ≤0.06 2 1 2 0.5

45 (96) 46 (98) 45 (96) 47 (100) 46 (98)

2 (4) 1 (2) 2 (4) 0 (0) 1 (2)

C. fermentati (5)a fluconazole itraconazole voriconazole posaconazole 5-flucytosine caspofungin micafungin anidulafungin amphotericin B

2–8 0.25 –1 0.03 –0.25 0.06 –0.5 ≤0.06 0.25 –2 0.25 –1 0.12 –2 0.25 –1

47 (100)

0 (0)

5 (100) 5 (100) 5 (100) 5 (100) 5 (100)

0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

5 (100)

WT, wild-type; S, susceptible; S-DD, susceptible, dose-dependent; I, intermediate; R, resistant. The BPs and ECVs used for C. guilliermondii isolates were applied to C. fermentati isolates.

a

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0 (0)

No. (%) of isolates by BPs S

S-DD

I

R

46 (98) 47 (100) 46 (98)

0 (0) 0 (0) 1 (2)

1 (2) 0 (0) 0 (0)

5 (100) 5 (100) 5 (100)

0 (0) 0 (0) 0 (0)

0 (0) 0 (0) 0 (0)


Of the 52 patients with infections caused by C. guilliermondii complex, 30 were male and 22 were female. The patients comprised 45 adults and 7 children, ranging in age from 1 to 93 years (median age, 56 years). Most (42 of 52, 81%) isolates were obtained from blood cultures. The other isolates were obtained from patients with infected wounds (n¼ 4), empyema thoracis (empyema pus, n¼ 2), pneumonia (purulent sputum, n¼ 1), urinary tract infection (n¼ 2) and lymphadenitis (n¼ 1). Of the 52 patients, 20 had underlying solid cancer and 7 had underlying haematological cancer (Table 1). Approximately one-third of the patients (15 of 52, 29%) had received abdominal surgery, 20% (10 of 52, 19%) were receiving long-term parenteral nutrition for short bowel syndrome and 8% (4 of 52, 7.7%) had undergone stem cell or organ transplantation procedures. We found that C. guilliermondii was more commonly isolated from bloodstream infections than C. fermentati. In addition, all isolates from patients with persistent candidaemia (n¼ 9) were C. guilliermondii.

B or amphotericin B liposomal, 2 were treated with micafungin and 1 was treated with anidulafungin. The 30 day mortality rate among the 41 patients who received antifungal treatment was 41% (17 of 41 patients), whereas that among patients who did not receive antifungal treatment was 36% (4 of 11 patients). There was no significant difference in 30 day survival between patients who received antifungal treatment and those who did not receive treatment (P ¼0.289).

Candida guilliermondii and Candida fermentati infection

Funding This study was supported by internal funding.

Transparency declarations None to declare.

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6 Pfaller M, Neofytos D, Diekema D et al. Epidemiology and outcomes of candidemia in 3648 patients: data from the Prospective Antifungal Therapy (PATH Alliancew) registry, 2004– 2008. Diagn Microbiol Infect Dis 2012; 74: 323–31. 7 Beyda ND, Lewis RE, Garey KW. Echinocandin resistance in Candida species: mechanisms of reduced susceptibility and therapeutic approaches. Ann Pharmacother 2012; 46: 1086– 96. 8 Girmenia C, Pizzarelli G, Cristini F et al. Candida guilliermondii fungemia in patients with hematologic malignancies. J Clin Microbiol 2006; 44: 2458– 64. 9 Pema´n J, Canto´n E, Linares-Sicilia MJ et al. Epidemiology and antifungal susceptibility of bloodstream fungal isolates in pediatric patients: a Spanish multicenter prospective survey. J Clin Microbiol 2011; 49: 4158– 63. 10 Savini V, Catavitello C, Onofrillo D et al. What do we know about Candida guilliermondii? A voyage throughout past and current literature about this emerging yeast. Mycoses 2011; 54: 434– 41. 11 Savini V, Catavitello C, Di Marzio I et al. Pan-azole-resistant Candida guilliermondii from a leukemia patient’s silent funguria. Mycopathologia 2010; 169: 457–9. 12 Cornet M, Sendid B, Fradin C et al. Molecular identification of closely related Candida species using two ribosomal intergenic spacer fingerprinting methods. J Mol Diagn 2011; 13: 12– 22. 13 Lan L, Xu J. Multiple gene genealogical analyses suggest divergence and recent clonal dispersal in the opportunistic human pathogen Candida guilliermondii. Microbiology 2006; 152: 1539– 49. 14 Cuenca-Estrella M, Gomez-Lopez A, Alastruey-Izquierdo A et al. Comparison of the Vitek 2 antifungal susceptibility system with the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference methods and with the Sensititre YeastOne and Etest techniques for in vitro detection of antifungal resistance in yeast isolates. J Clin Microbiol 2010; 48: 1782– 6. 15 Alexander BD, Byrne TC, Smith KL et al. Comparative evaluation of Etest and Sensititre YeastOne panels against the Clinical and Laboratory Standards Institute M27-A2 reference broth microdilution method for testing Candida susceptibility to seven antifungal agents. J Clin Microbiol 2007; 45: 698–706. 16 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Third Edition: Approved Standard M27-A3. CLSI, Wayne, PA, USA, 2008.

2 Bassetti M, Righi E, Costa A et al. Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect Dis 2006; 6: 21.

17 Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010 to 2012. J Clin Microbiol 2012; 50: 2846– 56.

3 Pappas PG, Rex JH, Lee J et al. A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin Infect Dis 2003; 37: 634–43.

18 Lockhart SR, Messer SA, Pfaller MA et al. Identification and susceptibility profile of Candida fermentati from a worldwide collection of Candida guilliermondii clinical isolates. J Clin Microbiol 2009; 47: 242– 4.

4 Sipsas NV, Lewis RE, Tarrand J et al. Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001– 2007): stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009; 115: 4745– 52.

19 Schmalreck AF, Willinger B, Haase G et al. Species and susceptibility distribution of 1062 clinical yeast isolates to azoles, echinocandins, flucytosine and amphotericin B from a multi-centre study. Mycoses 2012; 55: e124–37.

5 Pfaller MA, Diekema DJ, Mendez M et al. Candida guilliermondii, an opportunistic fungal pathogen with decreased susceptibility to fluconazole: geographic and temporal trends from the ARTEMIS DISK antifungal surveillance program. J Clin Microbiol 2006; 44: 3551–6.

20 Co´rdoba S, Vivot W, Bosco-Borgeat ME et al. Species distribution and susceptibility profile of yeasts isolated from blood cultures: results of a multicenter active laboratory-based surveillance study in Argentina. Rev Argent Microbiol 2011; 43: 176–85.

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et al.18 reported that C. fermentati constituted only 9% (13 of 149) of all species in the C. guilliermondii complex, a finding similar to that in this study (5 of 52, 9.6%). In this study, the clinical manifestations of these two Candida species were similar. The only clinical difference was that C. guilliermondii was more commonly isolated from bloodstream infections than C. fermentati [40 of 47 (85%) versus 2 of 5 (40%), P ¼ 0.043]. Twenty-seven (52%) of 52 patients had underlying cancer in this study. We also found that C. guilliermondii tended to be isolated from patients with cancer (25 of 47 patients, 53%), a finding similar to that reported previously.6,8 Previous studies have shown that isolates of C. guilliermondii are relatively resistant to fluconazole and that they routinely display higher echinocandin MIC values than other Candida species.5,19 Pfaller et al.5 found that susceptibility to fluconazole was dose-dependent in 14% of C. guilliermondii isolates and that 11% of the isolates were resistant to that drug. Co´rdoba et al.,20 on the other hand, found that susceptibility to fluconazole was dose-dependent in 38% of C. fermentati isolates. In this study, 96% of C. guilliermondii isolates and 100% of C. fermentati isolates were susceptible to fluconazole. We also found that 98% of C. guilliermondii isolates were susceptible to caspofungin, all were susceptible to micafungin and 98% were susceptible to anidulafungin. In summary, C. guilliermondii was the main species among C. guilliermondii complex isolates and was frequently associated with candidaemia. The currently used antifungal agents exhibited good in vitro activities against C. guilliermondii complex isolates.

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