The purpose of the study was to evaluate the interlaboratory agreement of broth dilution susceptibility methods for five species of conidium-forming (size range, 2 to 7 m) filamentous fungi. The methods used included both macro-and microdilution methods that were adaptations of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (m27-P). The MICs of amphotericin B, fluconazole, itraconazole, miconazole, and ketoconazole were determined in six centers by both macro-and microdilution tests for 25 isolates of Aspergillus flavus, Aspergillus fumigatus, Pseudallescheria boydii, Rhizopus arrhizus, and Sporothrix schenckii. All isolates produced clearly detectable growth within 1 to 4 days at 35؇C in the RPMI 1640 medium. Colony counts of 0.4 ؋ 10 6 to 3.3 ؋ 10 6 CFU/ml (mean, 1.4 ؋ 10 6 CFU/ml) were demonstrated in 90% of the 148 inoculum preparations. Overall, good intralaboratory agreement was demonstrated with amphotericin B, fluconazole, and ketoconazole MICs (90 to 97%). The agreement was lower with itraconazole MICs (59 to 79% median). Interlaboratory reproducibility demonstrated similar results: 90 to 100% agreement with amphotericin B, fluconazole, miconazole, and ketoconazole MICs and 59 to 91% with itraconazole MICs. Among the species tested, the MICs for S. schenckii showed the highest variability. The results of the study imply that it may be possible to develop a reference method for antifungal susceptibility testing of filamentous fungi.Despite the lower volume of serious infections caused by filamentous fungi compared with that of serious infections caused by yeasts, the performance of antifungal susceptibility testing for these opportunistic pathogens is important in the clinical laboratory (8). Progress has been made in developing guidelines for the antifungal susceptibility testing of yeasts to deal with the standardization of different testing parameters such as inoculum preparation, medium composition and pH, length of incubation, and endpoint criteria (3,5,6,(9)(10)(11)(12). The standardization of these antifungal susceptibility testing steps has led to increased interlaboratory reproducibility which has opened the possibility of developing standards by adopting and tailoring these steps for antifungal susceptibility tests for the filamentous fungi. The first priority of the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests was to develop a standard for the preparation of inoculum suspensions. Among four procedures evaluated, a spectrophotometric method was recommended as the procedure that gives the least variable results for the preparation of inoculum suspensions of yeast cells (11). This recommendation has been substantiated by ensuing collaborative studies of the subcommittee and other studies (2,3,5,6,12).The spectrophotometric method has been evaluated further in a single laboratory for the preparation of conidial suspensions of selected medically important filamentous fungi (1). In t...

x 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 g/ml for C. albicans and C. tropicalis, 0.031 to 0.5 g/ml for C. glabrata, and 0.063 to 1 g/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.

We describe a patient with recurrent episodes of oropharyngeal candidiasis who required progressively higher doses of fluconazole to control and infection. The patient was treated for 14 infections over a 2-year period with doses of fluconazole that ranged from 100 to 800 mg per day. Clinical response, two methods of in vitro susceptibility testing, and molecular epidemiologic techniques were evaluated for 12 of the 14 episodes. Ultimately, the patient became unresponsive clinically to a dose of 800 mg of fluconazole per day. In vitro susceptibility testing of isolates obtained during these successive episodes of infection revealed the development of resistance to fluconazole, and molecular epidemiologic techniques confirmed the persistence of the same Candida albicans strain throughout all 12 episodes.

Several members of the order Mucorales (subphylum Mucoromycotina) are important agents of severe human infections. The identification of these fungi by using standard mycologic methods is often difficult and time consuming. Frequently, the etiological agent in clinical cases is reported either as a Mucor sp., which is not the most frequent genus of zygomycetes, or only as a member of the Mucorales. For this reason, the actual spectrum of species of zygomycetes and their incidences in the clinical setting is not well known. The goals of this study were to compare the results of the molecular identification of an important set of clinical isolates, received in a mycological reference center from different regions of the United States, with those obtained by using the traditional morphological methods and to determine the spectrum of species involved. We tested 190 isolates morphologically identified as zygomycetes by using sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. Molecular identification revealed that Rhizopus oryzae represented approximately half (44.7%) of these isolates. The remainder was identified as Rhizopus microsporus (22.1%), Mucor circinelloides (9.5%), Mycocladus corymbifer (formerly Absidia corymbifera) (5.3%), Rhizomucor pusillus (3.7%), Cunninghamella bertholletiae (3.2%), Mucor indicus (2.6%), Cunninghamella echinulata (1%), and Apophysomyces elegans (0.5%). The most common anatomic sites for clinically significant zygomycetes, as determined by isolates sent to the Fungus Testing Laboratory for identification and/or susceptibility testing and included in this study, were the sinuses, lungs, and various cutaneous locations, at 25.8%, 26.8%, and 28%, respectively. These sites represented approximately 80% of the isolates evaluated. A high level of correlation (92.6%) between morphological and molecular identifications was found.