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We determined the echinocandin minimum effective concentration (MEC) values for caspofungin, micafungin, and anidulafungin against 288 Aspergillus isolates prospectively collected from transplant patients with proven or probable invasive aspergillosis between 2001 and 2006 as part of the Transplant-Associated Infection Surveillance Network (TRANSNET). We demonstrated that the vast majority of Aspergillus isolates had MEC values at or below the epidemiological cutoff values for caspofungin, micafungin, and anidulafungin, including those from patients who had received caspofungin.
Invasive fungal infections are an important source of morbidity and mortality among immunocompromised patients, particularly transplant recipients. Recent studies demonstrated that invasive aspergillosis (IA) was the most common fungal infection among hematopoietic stem cell transplant (HSCT) recipients and the second most common infection among solid organ transplant (SOT) recipients (9, 12). Echinocandins are currently not recommended for primary treatment of Aspergillus infections. However, caspofungin is indicated in patients with invasive aspergillosis that is refractory to or intolerant of other approved therapies, and there is a recommendation for use of caspofungin or micafungin for salvage therapy of invasive pulmonary aspergillosis (17). Epidemiological cutoff values (ECVs), based on the wild-type distribution of MIC/minimum effective concentration (MEC) values, have been established as plausible precursors in the development of breakpoint values (5, 8, 13). Because there are no breakpoints for any antifungal/mold combinations, we must rely on the available ECVs for determination of whether the MIC/MEC value is outside the wild-type distribution and could represent an isolate with a mutation that renders it resistant. There are also reports of species with elevated echinocandin MEC values, so proper species identification is essential (1, 2, 16).
In this study, we combined molecular species identification with determination of echinocandin minimum effective concentration (MEC) values for caspofungin, micafungin, and anidulafungin against 288 Aspergillus isolates from transplant patients with proven or probable aspergillosis collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET). TRANSNET conducted prospective surveillance for invasive fungal infections in SOT and HSCT patients between 2001 and 2006 and has been described in detail elsewhere (9, 12). Aspergillus isolates from proven or probable cases of invasive aspergillosis, as defined by modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria (4), were sent to the Fungal Reference Unit at the Centers for Disease Control and Prevention, Atlanta, GA, for confirmation of species and antifungal susceptibility testing. One isolate per patient was used in this analysis, except for cases in which a second species was isolated from the same patient (19 cases) or an isolate of the same species was collected more than 60 days after collection of the incident isolate (3 cases). Species identification was confirmed at the CDC using standard morphological and previously published molecular methods (6). Broth microdilution was performed according to the standards in the Clinical and Laboratory Standards Institute (CLSI) guideline M38-A2 (7) using frozen custom microbroth panels manufactured by TREK Diagnostics (Cleveland, OH). MECs were defined as the lowest drug concentration at which small, rounded, and compact hyphal forms were observed. All MECs were read visually at 24 h unless there was insufficient growth, in which case they were read at 48 h.
From the 227 proven and probable cases of IA among SOT recipients and the 425 proven and probable cases among HSCT recipients, there were 288 isolates available for susceptibility testing. Isolates came from the following sources: bronchoalveolar lavage fluids (n = 126), sputa (n = 71), lung biopsy specimens (n = 39), skin lesions (n = 20), tracheal aspirates (n = 8), sinus specimens (n = 7), blood samples (n = 5), and others (n = 12). All isolates were characterized by both microscopic and molecular methods, and they included the following: Aspergillus fumigatus (n = 179), Aspergillus flavus (n = 28), Aspergillus niger (n = 25), Aspergillus terreus (n = 23), Aspergillus calidoustus (n = 7), Aspergillus tubingensis (n = 6), Aspergillus versicolor (n = 5), Aspergillus lentulus (n = 4), Neosartorya udagawae (n = 4), Neosartorya pseudofischeri (n = 3), Aspergillus sydowii (n = 2), Aspergillus nidulans (n = 1), and Emericella quadrilineata (n = 1) (6).
The MEC range for all isolates was as follows: 0.008 to 4 μg/ml for caspofungin, 0.008 to 0.125 μg/ml for anidulafungin, and 0.008 to 0.125 μg/ml for micafungin (Table 1). The 90% MEC (MEC90) values were 0.06 μg/ml for caspofungin, 0.015 μg/ml for anidulafungin, and 0.015 μg/ml for micafungin. For each species with more than 10 isolates, the MEC50 and MEC90 were calculated. The MEC50 values for the individual species A. fumigatus, A. flavus, A. niger, and A. terreus were the same for the three echinocandins, with MEC50 values of 0.03 μg/ml for caspofungin and 0.008 μg/ml for anidulafungin and micafungin. The MEC90 values were more variable depending on the species and the echinocandin used. A. terreus isolates had the highest overall values, with MEC90 values of 0.03 μg/ml for micafungin, 0.06 μg/ml for anidulafungin, and 0.5 μg/ml for caspofungin.
Pfaller and coworkers (14) established ECVs for caspofungin against five species of Aspergillus. An ECV of ≤0.06 μg/ml was established for caspofungin against A. fumigatus, A. flavus, A. niger, and A. terreus, and an ECV of ≤0.125 μg/ml was established for A. versicolor. Among our isolates, 7 A. fumigatus isolates, 4 A. terreus isolates, and 1 A. niger isolate had MEC values for caspofungin above the ECV (Table 2). Less than 5% of our isolates of each species had MEC values above the ECV. The notable exception was those of A. terreus, in which 17% (4/23) of the isolates had MEC values above the ECV. If the ECVs for anidulafungin and micafungin were established at ≤0.03 μg/ml, a reasonable value based on previous work (15), there would be only 4 A. fumigatus isolates, 3 A. terreus isolates, and 1 A. calidoustus isolate with MEC values above the ECV for anidulafungin and only 2 A. fumigatus isolates and 1 A. calidoustus isolate with MEC values above the ECV for micafungin. Overall, our results are similar to MEC values published in several other studies (3, 11, 14, 15), with the exception of increased MEC values in only two of our A. calidoustus isolates but in none of our A. lentulus isolates (1, 2, 16).
Unfortunately, there are no publications with the MEC values of Aspergillus isolates from patients who either responded to or failed therapy, leaving a gap in our knowledge of predicting which patients may fail to respond to therapy. There is only a single retrospective study with cases of breakthrough IA for which MECs were generated (10). The caspofungin MEC values were comparable to the elevated values that we saw with some of our isolates.
In our study, there were 37 isolates from patients who had received caspofungin as prophylaxis, as empirical therapy, or in the treatment of another invasive fungal infection. Only 2 of the 15 Aspergillus isolates with elevated MEC values came from patients who had received caspofungin; A. terreus isolate IFI-02-0156 came from a patient who had received caspofungin for another infection, and E. quadrilineata isolate IFI-06-0126 came from a patient who had received caspofungin as prophylaxis. Given that the MEC values of isolates from 35 patients who had received caspofungin were not elevated and that 13 isolates with elevated MEC values, including seven A. fumigatus, three A. terreus, one A. niger and two A. calidoustus isolates came from patients who had not received caspofungin, it is not likely that receiving caspofungin was a cause of the elevated echinocandin MEC values in this patient population.
This study, using invasive isolates from transplant patients, corroborates earlier routine surveillance studies which showed that the percentage of Aspergillus isolates with elevated echinocandin MEC values remains low (11, 14, 15). As echinocandins are increasingly used in clinical practice, especially in combination with other agents, it is important that we establish rigorous ECVs for each species/echinocandin combination as a precursor to the establishment of legitimate breakpoints. This includes collecting data on wild-type MEC values, monitoring for increased MEC values in Aspergillus isolates, and exploring the mechanisms of resistance in Aspergillus isolates from patients who have failed echinocandin therapy.
A.J.Z. was supported by the Association of Public Health Laboratories (APHL). TRANSNET was sponsored by Astellas, Pfizer, Merck, and Schering-Plough. This work was funded by a grant from Merck. Potential conflicts of interest are as follows: P.G.P. receives research support and is an ad hoc advisor for Merck, Pfizer, and Astellas; J.W.B. received research grants from Merck, Astellas, and Pfizer and is a consultant/advisor for Merck and Pfizer; J.I.I. received honoraria for speakers' activities from Astellas, Cubist, Merck, and Pfizer and is a consultant for Sigma Tau; D.R.A. has received grants from and is a consultant for Merck, Astellas, and Pfizer; C.A.K. receives research support from Merck and chairs a data adjudication committee for Pfizer; K.A.M. received research grants from Merck, Astellas, and Pfizer and is on the advisory board or is consulting for Astellas, Basilea, Merck, and Pfizer; D.P.K. received research support and honoraria from Schering-Plough, Pfizer, Astellas, Enzon, and Merck; T.J.W. is in consultation with iCo, Vestagen, Trius, and Sigma Tau. All other authors have no potential conflicts.
The findings and conclusions of this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
We thank Carol Bolden and Lauren Smith for their contributions to this work.
Published ahead of print on 13 June 2011.