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The echinocandin class of antifungal agents is considered to be the first-line treatment of bloodstream infections (BSI) due to Candida glabrata. Recent reports of BSI due to strains of C. glabrata resistant to both fluconazole and the echinocandins are of concern and prompted us to review the experience of two large surveillance programs, the SENTRY Antimicrobial Surveillance Program for the years 2006 through 2010 and the Centers for Disease Control and Prevention population-based surveillance conducted in 2008 to 2010. The in vitro susceptibilities of 1,669 BSI isolates of C. glabrata to fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin were determined by CLSI broth microdilution methods. Fluconazole MICs of ≥64 μg/ml were considered resistant. Strains for which anidulafungin and caspofungin MICs were ≥0.5 μg/ml and for which micafungin MICs were ≥0.25 μg/ml were considered resistant. A total of 162 isolates (9.7%) were resistant to fluconazole, of which 98.8% were nonsusceptible to voriconazole (MIC > 0.5 μg/ml) and 9.3%, 9.3%, and 8.0% were resistant to anidulafungin, caspofungin, and micafungin, respectively. There were 18 fluconazole-resistant isolates that were resistant to one or more of the echinocandins (11.1% of all fluconazole-resistant isolates), all of which contained an acquired mutation in fks1 or fks2. By comparison, there were no echinocandin-resistant strains detected among 110 fluconazole-resistant isolates of C. glabrata tested in 2001 to 2004. These data document the broad emergence of coresistance over time to both azoles and echinocandins in clinical isolates of C. glabrata.
Resistance to the echinocandin antifungal agents, anidulafungin, caspofungin, and micafungin, among the broad population of Candida spp. causing invasive candidiasis (IC), is distinctly uncommon (1, 18, 39, 46, 48, 55–58, 62). Among 15,269 clinical isolates of Candida spp. tested at the University of Iowa (Iowa City), only 25 (0.2%) showed resistance to one or more echinocandins using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method and interpretive criteria (39, 44). As such, the echinocandin class of antifungal agents is recommended as first-line therapy for candidemia and IC, especially in patients with severe sepsis, those previously exposed to azoles, and/or those infected with Candida glabrata (35).
Ever since the introduction of fluconazole in 1990 for the treatment of candidiasis, including IC, empirical antifungal therapy has been driven by fear of C. glabrata (56, 57). Decreased susceptibility of C. glabrata to fluconazole and cross-resistance to other azoles are well known (21, 22, 32, 35, 41, 43, 45, 56, 57). In the United States, C. glabrata has increased as a cause of IC from 18% of all bloodstream infection (BSI) isolates in the time period of 1992 to 2001 to 25% in 2001 to 2007, with a concomitant increase in fluconazole resistance from 9% to 14% (45). Given the distinct differences between the mechanisms of action and resistance for the azoles and the echinocandins (37, 38), the documented lack of cross-resistance between the two classes (27, 31, 42) is not surprising, and thus the recommendation that the echinocandins be used for treatment of IC in patients with prior exposure to azoles and/or infection with C. glabrata is well founded (24, 35, 56, 57). Global surveys have documented the exquisite in vitro susceptibility of C. glabrata to each of the echinocandins (MIC90, 0.015 μg/ml [micafungin], 0.06 μg/ml [caspofungin], or 0.12 μg/ml [anidulafungin]) (39). Furthermore, the clinical experience with the use of echinocandins to treat IC due to C. glabrata is quite favorable (2, 20, 28, 36, 51, 55).
Although documentation of acquired resistance to the echinocandins remains sporadic (3, 18, 37, 44, 48, 58, 62), several recent reports of acquired resistance in clinical isolates of C. glabrata focus concern on this species (5, 7, 11–15, 18, 19, 48, 58, 59, 62). Data from global surveys demonstrate that the frequency of echinocandin resistance among clinical isolates of C. glabrata ranges from 1 to 3% and is higher among isolates from North America (3%) than among those from Europe (1%), Latin America (0.0%), or the Asia-Pacific region (0.0%) (39, 47). It is now clear that clinical isolates of C. glabrata with decreased susceptibility to one or more echinocandins harbor mutations in fks1 and/or fks2 and are also resistant clinically (5, 15, 37, 48, 59, 62). Chapeland-Leclerc et al. (5) reported a case of IC due to C. glabrata in which the infecting strain acquired resistance to flucytosine, fluconazole, voriconazole, and caspofungin through successive independent events following prolonged exposure to each class of antifungal agent. The recovery of different isolates exhibiting clonality for microsatellite markers but genetic diversity for antifungal resistance markers (three unique resistance mechanisms) demonstrates the high propensity of C. glabrata to readily mutate in vivo in a single patient (5). Additional reports from medical centers in the United States and Denmark provide further documentation of multidrug-resistant (MDR) (resistant to two or more classes of antifungal agents) strains of C. glabrata (13, 18, 19, 48, 62). One potential explanation for the emergence of MDR in C. glabrata is that the haploid nature of the organism makes it particularly adept at acquiring and expressing resistance mutations in response to drug pressure (5, 37, 48, 62).
The generally excellent wild-type (WT) susceptibility of C. glabrata to the echinocandins coupled with broadening azole resistance has driven the use of echinocandins for treatment of infections due to C. glabrata and at the same time has generated selection pressure for resistant organisms (4, 5, 11, 18, 24, 33–35, 48, 56, 57, 62). The fact that echinocandins are recommended for use in the setting of prior azole exposure and specifically for the treatment of IC due to C. glabrata raises the concern that acquired resistance to the echinocandins may emerge independently in fluconazole-resistant strains due to mutations in fks (5, 24, 48, 62). In an effort to further examine this issue, we have addressed the frequency of decreased susceptibility to the echinocandin class of antifungal agents among fluconazole-resistant strains of C. glabrata BSI isolates from two large antifungal surveys, the SENTRY Global Surveillance Program for the years 2006 through 2010 and the Centers for Disease Control and Prevention (CDC) population-based surveillance conducted in the Atlanta, GA, and Baltimore, MD, metropolitan areas between 2008 and 2010. Those isolates with decreased susceptibility (either intermediate [I] or resistant [R]) to one or more of the echinocandins were further examined for mutations in fks1 and fks2.
A total of 1,669 isolates of C. glabrata from blood or other normally sterile sites were obtained from diverse medical centers worldwide (SENTRY; 847 isolates) or from those in the Atlanta and Baltimore metropolitan areas (CDC; 822 isolates). All isolates represented incident episodes of IC (first positive blood culture). Isolates were identified by standard methods and stored in water at ambient room temperature (SENTRY) or in glycerol at −70°C (CDC) until used in the study. Before testing, each isolate was passaged on Sabouraud dextrose agar (Remel, Lenexa, KS) and CHROMagar (Becton, Dickinson, Sparks, MD) to ensure purity and viability.
A total of 21 fluconazole-resistant isolates of C. glabrata for which MICs for one or more echinocandin were I or R were further characterized for the presence or absence of mutations in the hot spot (HS) regions of fks1 and fks2 as described previously (3, 62).
All isolates were tested for in vitro susceptibilities to anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole using CLSI BMD methods (8, 9). MIC results for all agents were read visually following 24 h of incubation as the lowest concentration of drug that caused a significant diminution (≥50% inhibition) of growth compared with control levels (8, 9). The recently revised CLSI clinical breakpoints were used to identify strains of C. glabrata that were either I or R to the echinocandins and R or nonsusceptible (NS) to the azoles (40, 41, 44): anidulafungin and caspofungin MIC values of 0.25 μg/ml were considered I, and MIC values of ≥0.5 μg/ml were considered R; micafungin MIC values of 0.12 μg/ml were considered I, and MIC values of ≥0.25 μg/ml were considered R; fluconazole MIC values of ≥64 μg/ml were considered R, and voriconazole MIC values of >0.5 μg/ml were considered NS. Quality control was performed by testing the CLSI-recommended strains Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 (8, 9).
Among 1,669 BSI isolates of C. glabrata collected during the course of the two surveillance programs, 162 (9.7%) were resistant to fluconazole, including 62 of 847 (7.3%) isolates from the SENTRY Program (years 2006 to 2010) and 100 of 822 (12.2%) isolates from the CDC population-based surveillance (years 2008 to 2010). Among the 162 fluconazole-resistant isolates, 160 (98.8%) were NS to voriconazole (MIC > 0.5 μg/ml) (40).
Among the 162 fluconazole-resistant isolates of C. glabrata, there were 21 isolates (13.0%) that were either I or R to one or more of the echinocandins (Table 1): 3.7% and 9.3% were I or R, respectively, to anidulafungin, 3.7% and 9.3% to caspofungin, and 4.9% and 8.0% to micafungin. Mutations in either fks1 or fks2 were detected in 19 of the 21 (90.5%) echinocandin I or R isolates (Table 1). There were 18 fluconazole-resistant isolates (11.1% of all fluconazole-resistant isolates) that were R to one or more of the echinocandins (MIC ≥ 0.25 μg/ml [micafungin] or ≥ 0.5 μg/ml [anidulafungin and caspofungin]), all (100.0%) of which demonstrated an acquired mutation in fks1 or fks2.
Table 2 provides a comparison of the frequency of I or R among 110 fluconazole-resistant strains of C. glabrata collected from diverse medical centers worldwide between 2001 and 2004 (27, 42) and that of the present collection, representing the time period 2006 to 2010. Results from the two time periods were obtained with CLSI reference methods in different laboratories; however, quality control procedures were rigorously performed during all studies, suggesting that testing conditions did not influence the differences noted.
Whereas there were no echinocandin-resistant strains among 110 fluconazole-resistant isolates of C. glabrata tested between 2001 and 2004, a period where only caspofungin was available, there were 18 echinocandin-resistant strains (11.1%) among the 162 fluconazole-resistant isolates representing the latter time period. All of these had mutations in fks (Tables 1 and and22).
These data document for the first time the broad emergence of coresistance over time to both azoles and echinocandins in clinical isolates of C. glabrata. Whereas resistance to azoles in isolates of C. glabrata has been documented to be due to the overexpression of CDR efflux pumps (52, 53), there is little or no evidence that efflux pumps are involved in resistance to the echinocandins (31, 50). The documentation of fks mutations in isolates of C. glabrata showing in vitro resistance to both azoles and echinocandins suggests the sequential accumulation of acquired resistance mechanisms as demonstrated by Chapeland-Leclerc (5). The lack of coresistance in C. glabrata in the 2001-to-2004 time period is not surprising given that among the echinocandins only caspofungin was available, having been just approved in the United States and Europe in 2001. Since that time, the overall use of echinocandins in the United States has increased significantly, from 7.7 ± 5.3 days of therapy (DOT) per 1,000 patient-days in 2004 to 13.1 ± 8.6 DOT per 1,000 patient-days in 2008 (mean ± standard deviation [SD]) (P < 0.001) (33). During the same time period (2004 to 2008), the use of azoles increased as well, from 67.6 ± 29 to 72 ± 33 DOT per 1,000 patient-days, but this change was not significant (P = 0.1570) (33).
Whereas a shift to non-albicans species of Candida, particularly C. glabrata, has been a cause for alarm among clinicians caring for profoundly ill patients in the hospital (23, 26, 61), others have suggested that this is not a concern in the current “echinocandin era” (16, 56, 57). Prior to the availability of the echinocandins, concern over the reduced azole susceptibility of C. glabrata was a major issue for clinicians (10, 22, 29, 32, 54). Both in vitro and clinical studies have documented excellent activities of the echinocandins against C. glabrata and other potentially azole-resistant species, and the echinocandins have been rapidly replacing the azoles as first-line therapy for IC (4, 17, 18, 24, 30, 33, 35, 55, 58). Thus, in addition to the continued high use of azoles, the significant increase in the use of echinocandins places considerable selection pressure on C. glabrata, resulting in the emergence of MDR strains. It must be noted that thus far, Candida isolates demonstrating reduced echinocandin susceptibility appear following prolonged exposure to the drug, and these infections are manifested as recurrent episodes of candidemia in severely immunocompromised patients (48, 58).
Exposure to fluconazole is recognized by some (6, 22, 25, 49, 60) but not all (10, 54) investigators as a risk factor for fluconazole-resistant C. glabrata. Aside from reports of breakthrough fungemias, some involving fks mutations, there is little comparable data for the echinocandins and their influence on species distribution or resistance patterns in IC (58). Recently Lortholary et al. (24) examined the effect of exposure to fluconazole (n = 159) or caspofungin (n = 61) on the proportions of the five major species of Candida. For both drugs, preexposure was associated with a decreased prevalence of C. albicans in favor of less drug-susceptible species (C. glabrata and C. krusei for the former; C. parapilosis, C. glabrata, and C. krusei for the latter; P = 0.001). Not only did the species distribution change in patients with recent exposure to fluconazole and caspofungin, but the overall susceptibilities of the isolates to these drugs decreased. Notably, two C. glabrata isolates recovered from patients previously exposed to caspofungin during incident candidemia had high caspofungin MICs and harbored mutations in fks1 (24). The authors caution that a new episode of sepsis after a recent prescription of antifungals may be due to isolates with decreased susceptibility to the prescribed drugs, including caspofungin (24). In light of this, it should be noted that among the CDC isolates of C. glabrata with fks mutations for which epidemiological data were available, all came from patients with prior exposure to an echinocandin (62).
In summary, we have documented the emergence of coresistance to the echinocandins among BSI isolates of fluconazole-resistant C. glabrata. Among the 18 fluconazole-resistant isolates with coresistance to the echinocandins, all were from the United States, consistent with our previous observations concerning the geographic distribution of echinocandin resistance in C. glabrata, where the highest frequency of resistance was seen in North America (3%) versus Europe (1%), Latin America (0.0%), and the Asia-Pacific region (0.0%) (47). The increased use of both azoles and echinocandins will most certainly bring selection pressure to bear against C. glabrata, a species that appears to be unique in its ability to sequentially acquire and express resistance mutations. Specifically, the targeted use of echinocandins in severely immunocompromised individuals with prior exposure to azoles and/or infection with C. glabrata will ensure that this dual drug pressure is maintained. Although the vast majority of C. glabrata isolates remain highly susceptible to the echinocandin class of antifungal agents, the increase in MDR C. glabrata strains is a serious concern and argues for continued close resistance surveillance and the increased application of standardized antifungal susceptibility testing. Heightened awareness, rather than complacency, concerning the importance of C. glabrata should be the watchword for those caring for patients at risk for IC.
The antifungal global surveillance program which served as the source of data used in the development of the manuscript was supported in part by Pfizer Inc. and by Astellas.
We acknowledge the excellent assistance of S. Benning and P. Clark in the preparation of the manuscript; Tom Chiller, Carol Bolden, and Naureen Iqbal for data collection at the CDC; Monica M. Farley and Betsy Stein at Emory University School of Medicine and the Atlanta VA Medical Center and surveillance officers of the Georgia EIP and the hospitals in Georgia Health District 3 for assistance with data collection; Lee H. Harrison and Rosemary A. Hollick of Johns Hopkins Bloomberg School of Public Health; and the staff of Maryland ABCs and the hospitals in Baltimore city and Baltimore County, MD. We thank all of the surveillance centers that contributed isolates to both surveillance programs. We also thank G. J. Moet and L. N. Woosley.
The findings and conclusions of this article are ours and do not necessarily represent the views of the Centers for Disease Control and Prevention.
Published ahead of print 25 January 2012