Emilia Cantón

Interlaboratory Comparison of Results of Susceptibility Testing with Caspofungin against Candida and Aspergillus Species

ABSTRACT Seventeen laboratories participated in a study of interlaboratory reproducibility with caspofungin microdilution susceptibility testing against panels comprising 30 isolates of Candida spp. and 20 isolates of Aspergillus spp. The laboratories used materials supplied from a single source to determine the influence of growth medium (RPMI 1640 with or without glucose additions and antibiotic medium 3 [AM3]), the same incubation times (24 h and 48 h), and the same end point definition (partial or complete inhibition of growth) for the MIC of caspofungin. All tests were run in duplicate, and end points were determined both spectrophotometrically and visually. The results from almost all of the laboratories for quality control and reference Candida and Aspergillus isolates tested with fluconazole and itraconazole matched the NCCLS published values. However, considerable interlaboratory variability was seen in the results of the caspofungin tests. For Candida spp. the most consistent MIC data were generated with visual “prominent growth reduction” (MIC2) end points measured at 24 h in RPMI 1640, where 73.3% of results for the 30 isolates tested fell within a mode ± one dilution range across all 17 laboratories. MIC2 at 24 h in RPMI 1640 or AM3 also gave the best interlaboratory separation of Candida isolates of known high and low susceptibility to caspofungin. Reproducibility of MIC data was problematic for caspofungin tests with Aspergillus spp. under all conditions, but the minimal effective concentration end point, defined as the lowest caspofungin concentration yielding conspicuously aberrant hyphal growth, gave excellent reproducibility for data from 14 of the 17 participating laboratories.

Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Triazoles and Six Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)

ABSTRACT Clinical breakpoints have not been established for mold testing. Wild-type (WT) MIC distributions (organisms in a species/drug combination with no detectable acquired resistance mechanisms) were defined in order to establish epidemiologic cutoff values (ECVs) for five Aspergillus spp. and itraconazole, posaconazole, and voriconazole. Also, we have expanded prior ECV data for Aspergillus fumigatus. The number of available isolates varied according to the species/triazole combination as follows: 1,684 to 2,815 for A. fumigatus, 323 to 592 for A. flavus, 131 to 143 for A. nidulans, 366 to 520 for A. niger, 330 to 462 for A. terreus, and 45 to 84 for A. versicolor. CLSI broth microdilution MIC data gathered in five independent laboratories in Europe and the United States were aggregated for the analyses. ECVs expressed in μg/ml were as follows (percentages of isolates for which MICs were equal to or less than the ECV are in parentheses): A. fumigatus, itraconazole, 1 (98.8%); posaconazole, 0.5 (99.2%); voriconazole, 1 (97.7%); A. flavus, itraconazole, 1 (99.6%); posaconazole, 0.25 (95%); voriconazole, 1 (98.1%); A. nidulans, itraconazole, 1 (95%); posaconazole, 1 (97.7%); voriconazole, 2 (99.3%); A. niger, itraconazole, 2 (100%); posaconazole, 0.5 (96.9%); voriconazole, 2 (99.4%); A. terreus, itraconazole, 1 (100%); posaconazole, 0.5 (99.7%); voriconazole, 1 (99.1%); A. versicolor, itraconazole, 2 (100%); posaconazole, 1 (not applicable); voriconazole, 2 (97.5%). Although ECVs do not predict therapy outcome as clinical breakpoints do, they may aid in detection of azole resistance (non-WT MIC) due to cyp51A mutations, a resistance mechanism in some Aspergillus spp. These ECVs should be considered for inclusion in the future CLSI M38-A2 document revision.

Cryptococcus neoformans-Cryptococcus gattii Species Complex: An International Study of Wild-Type Susceptibility Endpoint Distributions and Epidemiological Cutoff Values for Fluconazole, Itraconazole, Posaconazole and Voriconazole

ABSTRACT Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 μg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 μg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 μg/ml (VGII); itraconazole, 0.25 μg/ml (VNI), 0.5 μg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 μg/ml (VGIV); posaconazole, 0.25 μg/ml (C. neoformans nontyped and VNI) and 0.5 μg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 μg/ml (VNIV), 0.25 μg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 μg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

Epidemiology and antifungal susceptibility of Candida species isolated from blood: results of a 2-year multicentre study in Spain

This study, included in the prospective survey of candidaemia in Europe supported by the European Confederation of Medical Mycology, presents the epidemiological and antifungal susceptibility results of 290 cases of candidaemia (80 in children <15 years old) reported from September 1997 to August 1999 by 19 Spanish hospitals. Presence of an intravenous catheter and previous antibiotic therapy were the most frequent risk factors. The percentages of the four most common species isolated (adults/children) were as follows: Candida albicans (46/36.2), C. parapsilosis (21.9/50), C. tropicalis (12.8/3.75), and C. glabrata (10.1/5). As initial therapy, fluconazole was preferred in adults (54%) and liposomal amphotericin B in children (58%). The 30-day mortality rate was 40.6%, and the species most frequently associated with a fatal outcome was C. krusei (60%). The rates of susceptibility to antifungal agents were as follows: amphotericin B, 91%; flucytosine, 99%; fluconazole, 93.6%; itraconazole, 87.4%; and voriconazole, 92%. These results provide baseline data for future epidemiological and susceptibility studies and for evaluating the impact of new antifungal agents on the distribution of species and the mortality rates associated with candidaemia in Spain.

Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: Should the clinical laboratory Be testing this agent?

ABSTRACT Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 μg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 μg/ml for C. glabrata, and 0.063 to 1 μg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

Epidemiology, species distribution and in vitro antifungal susceptibility of fungaemia in a Spanish multicentre prospective survey

OBJECTIVES To update the knowledge of the epidemiology of fungaemia episodes in Spain, the species implicated and their in vitro antifungal susceptibilities. METHODS Episodes were identified prospectively over 13 months at 44 hospitals. Molecular methods were used to determine the cryptic species inside the Candida parapsilosis and Candida glabrata complexes. Susceptibility to amphotericin B, anidulafungin, caspofungin, fluconazole, flucytosine, itraconazole, micafungin, posaconazole and voriconazole was determined by a microdilution colorimetric method. New species-specific clinical breakpoints (SSCBPs) for echinocandins, fluconazole and voriconazole were applied. RESULTS The incidence of the 1357 fungaemia episodes evaluated was 0.92 per 1000 admissions. The incidence of Candida albicans fungaemia was the highest (0.41 episodes/1000 admissions), followed by Candida parapsilosis sensu stricto (0.22). Candida orthopsilosis was the fifth cause of fungaemia (0.02), outnumbered by Candida glabrata and Candida tropicalis. Interestingly, the incidence of fungaemia by C. parapsilosis was 11 and 74 times higher than that by C. orthopsilosis and Candida metapsilosis, respectively. Neither Candida nivariensis nor Candida bracarensis was isolated. Fungaemia was more common in non-intensive care unit settings (65.2%) and among elderly patients (46.4%), mixed fungaemia being incidental (1.5%). Overall susceptibility rates were 77.6% for itraconazole, 91.9% for fluconazole and 96.5%-99.8% for the other agents. Important resistance rates were only observed in C. glabrata for itraconazole (24.1%) and posaconazole (14.5%), and in Candida krusei for itraconazole (81.5%). CONCLUSIONS Fungaemia is more common in non-critical patients. C. albicans is the most common species, followed by C. parapsilosis and C. glabrata. Nearly 90% of yeasts are susceptible to all antifungal agents tested. Resistance rates change moderately when applying the new SSCBPs.

Prospective Multicenter Study of the Epidemiology, Molecular Identification, and Antifungal Susceptibility of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis Isolated from Patients with Candidemia

ABSTRACT A 13-month prospective multicenter study including 44 hospitals was carried out to evaluate the epidemiology of Candida parapsilosis complex candidemia in Spain. Susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin was tested by the microdilution colorimetric method. A total of 364 C. parapsilosis complex isolates were identified by molecular methods: C. parapsilosis (90.7%), Candida orthopsilosis (8.2%), and Candida metapsilosis (1.1%). Most candidemias (C. parapsilosis, 76.4%; C. orthopsilosis, 70.0%; C. metapsilosis, 100%) were observed in adults. No C. orthopsilosis or C. metapsilosis candidemias occurred in neonates. C. parapsilosis was most frequent in adult intensive care unit (28.8%), surgery (20.9%), and internal medicine (19.7%) departments; and C. orthopsilosis was most frequent in hematology (28.6%), pediatrics (12.0%), and neonatology (11.5%) departments. The geographic distribution of C. orthopsilosis and C. metapsilosis was not uniform. According to CLSI clinical breakpoints, all C. orthopsilosis and C. metapsilosis isolates were susceptible to the nine agents tested. Resistance (MICs > 1 mg/liter) was observed only in C. parapsilosis: amphotericin B, posaconazole, itraconazole, and caspofungin (0.3% each), anidulafungin (1.9%), and micafungin (2.5%). Applying the new species-specific fluconazole and echinocandin breakpoints, the rates of resistance to fluconazole for C. parapsilosis and C. orthopsilosis increased to 4.8% and 0.3%, respectively; conversely, for C. parapsilosis they shifted from 1.9 to 0.6% (anidulafungin) and from 2.5 to 0.6% (micafungin). Our study confirms the different prevalence of C. parapsilosis complex candidemia among age groups: neither C. orthopsilosis nor C. metapsilosis was isolated from neonates; interestingly, C. metapsilosis was isolated only from adults and the elderly. The disparity in antifungal susceptibility among species could be important for therapy.

Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species

Minimum fungicidal concentrations (MFCs) of amphotericin B were obtained for 165 bloodstream isolates (104 Candida parapsilosis, 14 C.glabrata, 13 C.tropicalis, 15 C.krusei, and 19 C.albicans) and 36 C.dubliniensis from oropharyngeal infections. Minimum inhibitory concentrations (MICs) were determined by the M27-A microdilution method. MFCs (> or =99.9% killing) were obtained following MIC determination (inoculum size, 10(4) CFU/ml) by seeding the entire volume of all clear wells. The best fungicidal activity was for C. albicans, (MFC90 1 microg/ml) and the lowest for C.parapsilosis, C.tropicalis and C.glabrata (MFC90 16 microg/ml). Although MFCs were > or =16x MIC for some isolates, including C. glabrata, the overall MFCs were > or =2x MICs. However, major differences between MICs and MFCs were observed for C.parapsilosis and C.dubliniensis (3.8% and 8.9%, respectively, were tolerant: MFC > or =32MIC). MFCs for C.tropicalis and C. glabrata were > or =2 microg/ml. By this more stringent method we found substantial differences from those previously reported between amphotericin B MIC and MFCs for Candida spp.

Wild-Type MIC Distributions and Epidemiological Cutoff Values for Amphotericin B, Flucytosine, and Itraconazole and Candida spp. as Determined by CLSI Broth Microdilution

ABSTRACT Clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs) have been established for several Candida spp. and the newer triazoles and echinocandins but are not yet available for older antifungal agents, such as amphotericin B, flucytosine, or itraconazole. We determined species-specific ECVs for amphotericin B (AMB), flucytosine (FC) and itraconazole (ITR) for eight Candida spp. (30,221 strains) using isolates from 16 different laboratories in Brazil, Canada, Europe, and the United States, all tested by the CLSI reference microdilution method. The calculated 24- and 48-h ECVs expressed in μg/ml (and the percentages of isolates that had MICs less than or equal to the ECV) for AMB, FC, and ITR, respectively, were 2 (99.8)/2 (99.2), 0.5 (94.2)/1 (91.4), and 0.12 (95.0)/0.12 (92.9) for C. albicans; 2 (99.6)/2 (98.7), 0.5 (98.0)/0.5 (97.5), and 2 (95.2)/4 (93.5) for C. glabrata; 2 (99.7)/2 (97.3), 0.5 (98.7)/0.5 (97.8), and 05. (99.7)/0.5 (98.5) for C. parapsilosis; 2 (99.8)/2 (99.2), 0.5 (93.0)/1 (90.5), and 0.5 (97.8)/0.5 (93.9) for C. tropicalis; 2 (99.3)/4 (100.0), 32 (99.4)/32 (99.3), and 1 (99.0)/2 (100.0) for C. krusei; 2 (100.0)/4 (100.0), 0.5 (95.3)/1 (92.9), and 0.5 (95.8)/0.5 (98.1) for C. lusitaniae; −/2 (100.0), 0.5 (98.8)/0.5 (97.7), and 0.25 (97.6)/0.25 (96.9) for C. dubliniensis; and 2 (100.0)/2 (100.0), 1 (92.7)/−, and 1 (100.0)/2 (100.0) for C. guilliermondii. In the absence of species-specific CBP values, these wild-type (WT) MIC distributions and ECVs will be useful for monitoring the emergence of reduced susceptibility to these well-established antifungal agents.

Antifungal drug resistance mechanisms

Antifungal resistance is a prominent feature in the management of invasive mycoses, with important implications for morbidity and mortality. Microbiological resistance, the most common cause of refractory infection, is associated with a fungal pathogen for which an antifungal MIC is higher than average or within the range designated as the resistant breakpoint. Four major mechanisms of resistance to azoles have been described in Candida spp.: decreased intracellular drug concentration by activation of efflux systems or reduction of drug penetration, modification of the target site, upregulation of the target enzyme and development of bypass pathways. Conversely, echinocandins are a poor substrate for multidrug efflux transporters, and their mechanisms of resistance are associated with point mutations and/or overexpression of FKS1 and FKS2 genes. Acquired resistance to flucytosine results from defects in its metabolism through enzymatic mutations, whereas resistance to amphotericin B may be mediated by increased catalase activity or defects in ergosterol biosynthesis.