FKS Mutant Candida glabrata: Risk Factors and Outcomes in Patients With Candidemia

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1 Clinical Infectious Diseases Advance Access published July 9, 2014 MAJOR ARTICLE FKS Mutant Candida glabrata: Risk Factors and Outcomes in Patients With Candidemia Nicholas D. Beyda, 1 Julie John, 1 Abdullah Kilic, 1 Mohammad J. Alam, 1 Todd M. Lasco, 2 and Kevin W. Garey 1 1 Department of Clinical Sciences & Administration, University of Houston College of Pharmacy, and 2 Department of Microbiology, Baylor St. Luke s Medical Center, Houston, Texas Background. Echinocandins are recommended for Candia glabrata candidemia. Mutations in the FKS1 and FKS2 genes are associated with echinocandin resistance. Few studies have assessed risk factors for FKS mutant isolates and outcomes in patients receiving micafungin treatment. Methods. Patients with C. glabrata bloodstream infection admitted to a large, tertiary care hospital between 2009 and 2012 were included in this study. For each isolate, FKS1 and FKS2 genes were sequenced to identify mutations. Risk factors for FKS mutations and treatment outcomes in patients receiving an echinocandin were assessed using multivariate logistic regression. Results. Seventy-two patients were included in the study of which 13 (18%) had an FKS mutant isolate. The only significant predictor for FKS mutations was prior echinocandin exposure (odds ratio [OR], 19.9; 95% confidence interval [CI], ; P.01). Treatment failure occurred in 17 (30%) of 57 patients who received an echinocandin and was more common in patients with FKS mutants (6 of 10; 60%) compared with non-fks mutants (11 of 47; 23%). Underlying gastrointestinal disorder (OR, 4.7; 95% CI, ; P =.04) and prior echinocandin exposure (OR, 8.3; 95% CI, ; P.01) were independent predictors of echinocandin treatment failure. Treatment response and echinocandin minimum inhibitory concentrations varied among specific FKS mutations. Conclusions. FKS mutations were identified in 18% of 72 patients with C. glabrata candidemia. Common risk factors for FKS mutant isolates included previous echinocandin exposure, which also influenced response rates. Keywords. antifungal resistance; Candida glabrata; echinocandins; micafungin; systemic candidiasis. Invasive candidiasis is associated with significant mortality, morbidity, and increased healthcare costs. The incidence of systemic candidiasis caused by non Candida albicans species, such as C. glabrata, has increased and is likely attributable to the increasing population of immunosuppressed and elderly patients and widespread use of azoles [1 3]. Candida glabrata and C. krusei are more likely to be nonsusceptible to azole antifungals; therefore, echinocandins are increasingly being used as empiric and targeted antifungal therapy. However, an increasing number of reports of Candida spp. isolates with elevated echinocandin minimum inhibitory Received 9 December 2013; accepted 21 May Correspondence: Nicholas D. Beyda, PharmD, BCPS, 1441 Moursund Street, Houston, TX (ndbeyda@uh.edu). Clinical Infectious Diseases The Author Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please journals.permissions@oup.com. DOI: /cid/ciu407 concentrations (MICs) are being reported [4]. The echinocandins work by inhibiting the β-1,3-glucan synthase enzyme (encoded by FKS1, FKS2, and FKS3 genes in C. glabrata), which synthesizes a major component of the fungal cell wall. Mutations in FKS1 or FKS2 have been associated with elevated echinocandin MICs, reduced glucan synthase enzyme sensitivity, and outcomes in animal models, with significant differences observed between specific mutations [5, 6]. However, few studies have defined the clinical risk factors and outcomes associated with FKS mutations in patients with C. glabrata candidemia. The purpose of this study was to assess the risk factors and outcomes of FKS mutant C. glabrata candidemia. METHODS This study was conducted at a university-affiliated tertiary care hospital in the Texas Medical Center, Houston, as part of an ongoing surveillance study of patients Outcomes in FKS Mutant Candida glabrata CID 1

2 with candidemia [7, 8]. In this institutional review board (IRB) approved study, Candida isolates are collected from the clinical microbiology laboratory and clinical information is collected from the medical chart. The hospital clinical microbiology laboratory routinely performed caspofungin susceptibility testing for the entire time period of the study using broth microdilution (BMD) (Sensititre YeastOne; Trek Diagnostic Systems, Cleveland, OH) according to Clinical and Laboratory Standards Institute (CLSI) M27-A3. A waiver of informed consent was approved for this study by the University of Houston and the hospital IRB. This analysis included patients admitted between 2009 and 2012 with at least 1 blood culture positive for C. glabrata. Patients were excluded if their medical record could not be accessed or if their isolate was unavailable for testing. Micafungin was the formulary echinocandin during the time period of this study. All patients received micafungin 100 mg intravenously every 24 hours during their course of treatment. Genomic DNA was extracted from cells grown twice on Sabouraud dextrose agar (Hardy Diagnostics, Santa Maria, CA) at 35 C for 24 hours using the Ultraclean Microbial DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA). Hot spots 1and 2 of FKS1 and FKS2 were amplified by polymerase chain reaction (PCR) as previously described [9]. Standard Sanger sequencing of purified PCR amplicons was performed using a 3730 DNA analyzer (Applied Biosystems, Carlsbad, CA). DNA Dynamo software (Blue Tractor Software, North Wales, UK) was used to analyze DNA sequences. A new episode of candidemia was defined as the isolation of C. glabrata from the blood more than 30 days from the initial positive culture. In patients with more than 1 episode, only the most recent episode was included. For the FKS mutations risk factor analysis, all patients with C. glabrata candidemia were included. For the echinocandin treatment outcomes analysis, only patients receiving an echinocandin for 3 or more days for empiric or definitive treatment were included. Treatment failure was assessed at day 14 using consensus definitions of outcomes for antifungal therapy or as a breakthrough infection while receiving an echinocandin for 3 or more days as empiric therapy [10]. The following variables were assessed as risk factors for FKS mutant C. glabrata or echinocandin treatment failure: age, gender, race, solid organ transplant, gastrointestinal disorders (including those that compromise gastrointestinal membrane integrity), renal dysfunction, hemodialysis, diabetes, receipt of corticosteroids, gastrointestinal surgery within the previous 30 days, total parenteral nutrition within the previous 30 days, admission to the intensive care unit at time of candidemia, presence of a central venous catheter, central venous catheter removal within 72 hours of positive culture, prior episode of C. glabrata candidemia, mean days of echinocandin exposure prior to candidemia, echinocandin exposure prior to candidemia, fluconazole exposure prior to candidemia, and breakthrough infection. Statistical analysis was performed using STATA version 11 software (StataCorp LP, College Station, TX). Risk factors associated with FKS mutant C. glabrata and echinocandin treatment failure were assessed by comparing hospitalization and host risk factors between patients with FKS mutant C. glabrata candidemia with those of patients without FKS mutant C. glabrata candidemia. Univariate statistics were used as appropriate (Pearson χ 2 or Fisher exact test for categorical variables and Student t test for continuous variables). Independent predictors of FKS mutant C. glabrata and echinocandin treatment failure were assessed using stepwise multivariate logistic regression, including all variables with a P value <.2 in univariate analysis. All statistical tests were 2 tailed, and a P value <.05 was considered significant. RESULTS In Vitro Caspofungin Susceptibility Testing and FKS Mutations A total of 72 patients with C. glabrata candidemia were included. Six patients (8%) developed breakthrough candidemia while receiving an echinocandin and 6 (11%) while receiving fluconazole. Among the 72 C. glabrata isolates, the caspofungin MICs obtained using YeastOne BMD ranged from 0.03 to 8 µg/ml; the MIC 50 and MIC 90 were 0.12 and 0.5 µg/ml, respectively (Figure 1). Applying the revised CLSI clinical breakpoints for caspofungin, 64% (46/72) of isolates were classified as susceptible (MIC 0.12 µg/ml), 25% (18/72) intermediate (MIC = 0.25 µg/ ml), and 11% (8/72) resistant (MIC 0.5 µg/ml). FKS mutations were detected in 18% (13/72) of isolates. Five FKS mutations were identified, occurring in hot spot 1 in both FKS1 and FKS2. The most common mutations in FKS1 were I634V (n = 4), F625C (n = 1), and S629P (n = 1). The most common mutations in FKS2 were S663P (n = 6) and S663F (n = 1). Caspofungin MICs ranged from 0.12 to 8 µg/ml among the FKS mutant isolates. Risk Factors for FKS Mutant C. glabrata Candidemia Seventy-two patients with C. glabrata candidemia were included in this analysis. On univariate analysis, patients with FKS mutant C. glabrata candidemia were more likely to have had a breakthrough infection while receiving an echinocandin, had echinocandin exposure prior to initial candidemia, an underlying gastrointestinal disorder, or a prior episode of C. glabrata candidemia (Table 1). Using multivariate logistic regression analysis, prior echinocandin exposure was the only independent predictor of FKS mutant C. glabrata candidemia (odds ratio [OR], % confidence interval [CI], ; P.01). Caspofungin MICs were 0.25 µg/ml for 2 (22%) and 4 (100%) of the FKS mutant isolates with and without documented echinocandin exposure prior to infection, respectively. 2 CID Beyda et al

3 Figure 1. Caspofungin MIC distribution and FKS mutations among 72 Candida glabrata isolates. Abbreviation: MIC, minimum inhibitory concentrations. Risk Factors for Echinocandin Treatment Failure Fifty-seven patients received 3 or more days of echinocandin treatment and were evaluable at day 14. Seventeen (30%) patients failed echinocandin treatment (9 had repeat positive blood cultures after receiving more than 5 days of echinocandin treatment; 6 had breakthrough infection while receiving echinocandin empirically for 3 or more days; 2 expired after receiving 3 or more days of an echinocandin). Presence of an FKS mutation (P =.02), prior echinocandin exposure (P.01), caspofungin MIC 0.25 µg/ml (P =.04), and underlying gastrointestinal disorder (P.01) were significantly associated with echinocandin treatment failure in univariate analysis (Table 2). On multivariate analysis, underlying gastrointestinal disorder (OR, 4.72; 95% CI, ; P =.04) and prior echinocandin exposure (OR, 8.27; 95% CI, ; P.01) were found to be independent predictors of echinocandin treatment failure. Among the 31 patients with underlying gastrointestinal disorder, pancreatitis (21%), small bowel obstruction (12%), gastric outlet obstruction (9%), and ileus (9%) were the most common. The performance of 2 caspofungin breakpoints ( 0.25 µg/ml and 0.5 µg/ml) and presence of an FKS mutation in predicting echinocandin treatment failure were assessed (Table 3). The positive predictive value was highest for a breakpoint of 0.5 µg/ml (100% [6/6]), followed by presence of an FKS mutation (60% [6/10]) and a breakpoint of 0.25 µg/ml (47% [9/19]). Among the 10 episodes involving an FKS mutant, treatment failure was 100% (5/5) if the caspofungin MIC was 0.5 µg/ml compared with 20% (1/5) with a caspofungin MIC 0.25 µg/ml (Figure 2). DISCUSSION The incidence of FKS mutations in C. glabrata varies among institutions, with reports ranging from 3% to 18% [11 13]. In this study 18% of C. glabrata isolates had an FKS mutation. Prior echinocandin exposure was the only significant predictor for Outcomes in FKS Mutant Candida glabrata CID 3

4 Table 1. Risk Factors for FKS Mutant Candida glabrata Candidemia Variable a FKS Mutation (n = 13) No FKS Mutation (n = 59) Univariate OR, 95% CI, Multivariate OR, 95% CI, Mean age (range) 56 (24 74) 62 (28 97) N/A, N/A,.11 Male gender 11 (85%) 32 (54%) 4.64, ,.06 Race b Caucasian 4 (31%) 29 (49%) 0.46, ,.35 African American 6 (46%) 14 (24%) 2.75, ,.10 Hispanic 3 (23%) 12 (20%) 1.18, ,.83 Underlying conditions (%) Solid organ transplant 1 (8%) 5 (9%) 0.9, ,.93 Gastrointestinal disorder 11 (85%) 30 (51%) 5.31, ,.03 Renal dysfunction 6 (46%) 38 (64%) 0.47, ,.22 Hemodialysis 2 (15%) 26 (45%) 0.23, ,.07 Diabetes 3 (23%) 31 (53%) 0.27, ,.07 Corticosteroids 2 (15%) 22 (35%) 0.35, ,.32 GI surgery previous 30 d (%) 7 (54%) 26 (44%) 1.48, ,.52 TPN previous 30 d (%) 9 (69%) 27 (46%) 2.67, ,.22 ICU at time of candidemia (%) 7 (54%) 42 (71%) 0.47, ,.22 Presence of CVC (%) 12 (92%) 53 (90%) 1.36, ,.79 CVC removed within 72 h (%) c 8 (67%) 34 (64%) 1.12, ,.87 Prior episode of CG candidemia (%) 3 (23%) 1 (2%) 17.6, ,.02 Mean days of echinocandin exposure (range) 13 (0 32) 1 (0 14) N/A, N/A, <.01 Echinocandin exposure previous 60 d 9 (69%) 6 (10%) 19.88, , < , , <.01 Fluconazole exposure previous 60 d 5 (38%) 21 (36%) 1.13, ,.85 Breakthrough infection (%) Fluconazole 1 (8%) 7 (12%) 0.62, ,.66 Echinocandin 4 (31%) 2 (3%) 12.67, ,.01 Abbreviations: CG, Candida glabrata; CI, confidence interval; CVC, central venous catheter in place at time of initial positive culture; GI, gastrointestinal; ICU, intensive care unit; N/A, not applicable; OR, odds ratio; TPN, total parenteral nutrition. a Other variables which were non-significant on univariate analysis: malignancy, liver disease, immunosuppression, and non-gi surgery within previous 30 days. b Four patients with no FKS mutations were deemed Other race. c Only patients with a central venous catheter in place at the time of initial positive culture were analyzed. FKS mutations in multivariate analysis. Our results corroborate the findings from 2 previous studies that demonstrated similar risk factors for FKS mutations. Shields et al reported that in patients with invasive candidiasis due to C. glabrata, infection with an isolate harboring an FKS mutation was significantly associated with any prior echinocandin exposure [13]. Alexander et al also found that any prior echinocandin therapy was a significant predictor of FKS mutant C. glabrata in patients with candidemia (OR, 19.65; 95% CI, ) [11]. In our study there were 4 episodes involving an FKS mutant isolate with no documented prior echinocandin exposure, which is in contrast to previous studies reporting prior echinocandin exposure in all cases of FKS mutant C. glabrata. Of these 4 episodes, all were caused by an isolate with an FKS1 I634V mutation and had a caspofungin MIC of 0.25 µg/ml. While it is possible that prior exposure may have occurred at an outside hospital and was not captured in our records, these findings highlight the need for continued surveillance of FKS mutations to understand their epidemiologic and clinical significance. Fifty-seven patients with C. glabrata candidemia received echinocandin treatment, including 10 (18%) who had an FKS mutant isolate. Risk factors for echinocandin treatment failure in univariate analysis included underlying gastrointestinal disorder, prior echinocandin therapy, and the presence of an FKS mutation. In multivariate analysis, underlying gastrointestinal disorder and prior echinocandin therapy were identified as independent predictors of treatment failure. This is in contrast to a study by Shields et al who found the presence of an FKS mutation to be the sole predictor of treatment failure in patients with invasive candidiasis due to C. glabrata (OR, 41.7; 95% CI, ) [13]. Echinocandin treatment failure among patients with FKS mutant isolates occurred in 6 of 10 episodes (60%) in our study compared with 6 of 7 episodes (86%) in the study by Shields et al [13]. This could possibly 4 CID Beyda et al

5 Table 2. Risk Factors for Echinocandin Treatment Failure Variable a Success (n = 40) Failure (n = 17) Univariate OR, 95% CI, Median age (range) 61 (24 87) 55 (42 68) N/A, N/A,.14 Male gender 27 (89%) 8 (47%) 0.4, ,.15 Underlying conditions (%) Multivariate OR, 95% CI, Gastrointestinal disorder 17 (43%) 14 (82%) 6.31, , < , ,.04 Renal dysfunction 28 (70%) 7 (42%) 0.3, ,.04 Liver disease 7 (18%) 6 (35%) 2.57, ,.14 Presence of an FKS mutation 4 (10%) 6 (35%) 4.91, ,.02 GI surgery previous 30 d (%) 15 (38%) 10 (59%) 2.38,.65 9,.14 TPN previous 30 d (%) 20 (50%) 13 (77%) 3.25, ,.08 ICU at time of candidemia (%) 29 (73%) 11 (65%) 0.7, ,.56 Presence of CVC (%) 37 (93%) 16 (94%) 1.3, ,.93 CVC removed within 72 h (%) b 21 (57%) 11 (69%) 1.68, ,.41 Echinocandin exposure previous 60 d 3 (8%) 8 (47%) 10.96, , < , , <.01 Fluconazole exposure previous 60 d 13 (33%) 8 (47%) 1.85, ,.3 Caspofungin MIC (µg/ml) (25%) 9 (53%) 3.38, ,.04 Abbreviations: CI, confidence interval; CVC, central venous catheter at time of initial positive culture; GI, gastrointestinal; ICU, intensive care unit; MIC, minimum inhibitory concentrations; N/A, not applicable; OR, odds ratio; TPN, total parenteral nutrition. a Other variables which were non-significant on univariate analysis: malignancy, prior episode of C. glabrata candidemia, solid organ transplant, immunosuppression, and non-gi surgery within previous 30 days. b Only patients with a central venous catheter in place at time of initial positive culture were analyzed. Table 3. Performance of YeastOne Caspofungin Minimum Inhibitory Concentrations at Two Breakpoints and the Presence of an FKS Mutation in Predicting Clinical Failure Variable No. of Successes (n = 40) No. of Failures (n = 17) % PPV % NPV % Sensitivity % Specificity Presence of FKS mutation % (6/10) 77% (36/47) 35% (6/17) 90% (36/40) Caspofungin MIC 0.25 µg/ml % (9/19) 79% (30/38) 53% (9/17) 75% (30/40) Caspofungin MIC 0.5 µg/ml % (6/6) 78% (40/51) 35% (6/17) 100% (40/40) Abbreviations: MIC, minimum inhibitory concentrations; NPV, negative predictive value; PPV, positive predictive value. Figure 2. Micafungin treatment outcomes in patients with FKS mutant Candida glabrata. Abbreviation: MIC, minimum inhibitory concentrations. Outcomes in FKS Mutant Candida glabrata CID 5

6 be due to a higher incidence of FKS mutant isolates with a weak resistance phenotype in our study. In vitro studies have shown that the level of glucan synthase enzyme inhibition varies depending on the specific FKS gene mutation, resulting in isolates with differing elevations in echinocandin MICs [5]. The most prominent FKS mutations in C. glabrata (FKS1 S629P, F625S, D632G or E and FKS2 S663P and F659V or Y) exhibit highly elevated echinocandin MICs and reduced glucan synthase enzyme sensitivity and are unresponsive to even high doses of echinocandins in murine models [6, 14]. In this study all 5 patients whose isolate harbored 1 of these prominent FKS mutations had a caspofungin MIC 0.5 µg/ ml and failed echinocandin treatment (FKS1 S629P, n = 1; FKS2 S663P, n = 4). This is consistent with both in vitro data as well as numerous reports that implicate these specific FKS mutations in patients failing echinocandin treatment [4]. In contrast, treatment failure occurred in only 1 of 5 patients whose isolate harbored a less prominent FKS mutation, all of which had a caspofungin MIC of 0.25 µg/ml (FKS1 I634V, n=3;fks1 F625C, n = 1; FKS2 S663F, n = 1). Alexander et al reported similar results with 100% (8/8) of episodes involving a FKS mutant isolate with a caspofungin MIC 0.5 µg/ml failing echinocandin treatment vs 0% (0/4) with caspofungin MICs 0.25 µg/ml [11]. These findings highlight that not all FKS mutations are equal, and further investigation into the clinical significance of FKS mutations with a weak resistance phenotype is warranted. There are several limitations to our study that should be acknowledged. As this was a single-center study, the incidence of FKS mutations as well as the frequency of specific mutations at our institution may not reflect the local epidemiology elsewhere. Adequate source control outside of central venous catheter removal was not included in our analysis as limitations in our electronic database prevented a reliable assessment. During the study period, the clinical microbiology laboratory only reported caspofungin susceptibility data obtained using Yeast One. A high level of essential agreement (MICs within 2 doubling dilutions) has been observed between echinocandin MICs generated by commercial susceptibility tests (such as YeastOne) and the CLSI reference method. However, the categorical agreement (classification of isolates as susceptible, intermediate, or resistant using new CLSI clinical breakpoints) is weaker, particularly for caspofungin [15]. While our data show that caspofungin MICs obtained by YeastOne were useful in identifying clinically relevant FKS mutations that are likely to fail treatment with micafungin, whether or not these results would correlate with MICs obtained using the CLSI BMD method require further study. With recent concerns over variability in caspofungin MICs obtained by CLSI BMD as well as limited data regarding the performance of echinocandin MICs obtained by commercial testing methods in predicting treatment outcomes, clinicians need to be aware of the testing methods used by their clinical microbiology laboratory [16]. In conclusion, C. glabrata isolates harboring FKS mutations were found in 18% of candidemia episodes at our institution. Previous echinocandin exposure was significantly associated with the presence of an FKS mutation and underlying gastrointestinal disorder, and previous echinocandin exposure was significantly associated with echinocandin treatment failure. Caspofungin MICs obtained using YeastOne were useful in identifying clinically significant FKS mutations that were likely to fail treatment. Further research regarding the clinical significance of less prominent FKS mutations that exhibit a weak resistance phenotype requires further study. Notes Financial support. This work was supported by an investigatorinitiated grant from Astellas Inc. to K. W. G. Potential conflicts of interest. K. W. G. has received research funding fromastellasinc.n.d.bhasreceivedresearch funding from Astellas Inc. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. References 1. Arendrup MC. Epidemiology of invasive candidiasis. Curr Opin Crit Care 2010; 16: Pfaller MA, Moet GJ, Messer SA, Jones RN, Castanheira M. Candida bloodstream infections: comparison of species distributions and antifungal resistance patterns in community-onset and nosocomial isolates in the SENTRY Antimicrobial Surveillance Program, Antimicrob Agents Chemother 2011; 55: Pfaller MA, Moet GJ, Messer SA, Jones RN, Castanheira M. 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