Susceptibility testing of fungi: current status of the standardization process

In a previous review of antifungal susceptibility testing (12), several problems were identified that made clinically relevant in vitro tests an illusive goal. Clinical laboratory microbiologists felt little pressure to develop a test that was infrequently requested or that would have little influence on patient management. The few reference laboratories that offered testing derived their methods from their own local experience. This created a diversity of testing methods which produced correspondingly discrepant results (13). Moreover, since virtually no relationship had been established between tests and patient responses, it was not possible to determine which of the methods would best predict benefit from treatment. The National Committee for Clinical Laboratory Standards (NCCLS) established a subcommittee to develop antifungal testing procedures in 1983, but at that time a standardized methodology did not seem possible until some time in the distant future. During the intervening years, rising numbers of both fungal infections and available antifungal drugs have made both prescribing physicians and clinical pathologists increasingly uncomfortable because of the lack of clinical antifungal tests. Fortunately, with continued support from the NCCLS, the possibilities for meaningful testing have improved considerably. Many people have participated in coordinated efforts to develop standards of potential value to clinical laboratories. Through a series of interlaboratory collaborations, consensus has been developed around a reference testing method for Candida species and Cryptococcus neofornans, and in 1992 it was published as NCCLS document M27-P (18). This minireview provides an analysis of how the standard described in NCCLS document M27-P evolved and what specific features of the standard have proved to be important. In addition, information about this standard method's strengths and limitations in identifying resistance for antifungal drugs will be summarized. Finally, potential uses of the method by the research community or prescribing physicians are considered, as is what further work is needed in the decade ahead. The intent here is not to be comprehensive, and more exhaustive reviews of antifungal susceptibility testing have recently been published elsewhere (14, 24). Development of the NCCLS reference standard. The first activity of the subcommittee for antifungal susceptibility tests was to query NCCLS members regarding their current practices (4). Of those laboratories performing antifungal tests, most were interested in testing Candida species and C. neofornans, and therefore, as its immediate priority, the subcommittee focused on susceptibility tests for yeasts. Similarly, a broth macrodilution procedure was a reasonable choice since it was the most commonly used test format. However, subcommittee members were uncertain as to how the method would eventually be configured, and therefore, an added attraction of the broth macrodilution method was its flexibility, which would permit analytic studies of nearly any test condition. As things have developed, this flexibility has proven to be critical, especially in studies to determine the best endpoint definition for azole antifungal agents such as fluconazole. Once the type of test procedure was agreed upon in a general sense, attention was shifted to analyzing a series of specific details of the test configuration. For some details, consensus could be achieved rather easily. For example, a pipetting scheme, previously adopted by the NCCLS for other standards to minimize systematic errors (17), was incorporated for antifungal testing. For other test conditions, collaborative studies demonstrated the suitability of one or another selection. Thus, RPMI 1640, buffered to a pH of 7.0 with morpholinepropanesulfonic acid (MOPS), was selected as the growth medium, 35°C was selected as a practical incubation temperature, and the starting inoculum was prepared by a spectrophotometric method (21, 22). By 1990, there still remained three important issues that had not yet been resolved. First, whether the incubation time should be 1 or 2 days had not been agreed upon. Second, the definition of the MIC endpoint needed clarification. Third, even though the subcommittee had agreed upon how to prepare the starting inoculum, it had not agreed exactly what the inoculum size should be. In past studies, 104 yeast cells per ml had been used, and with this inoculum size, interlaboratory agreement had not always been acceptable (22). Although lowering of the inoculum size might improve reproducibility, testing of a smaller number of yeast cells could theoretically cause problems with yeasts that are heteroresistant, since such resistance might be missed if the inoculum size is smaller than the frequency of resistant clones. This is especially relevant for the susceptibilities of some strains of yeasts for flucytosine (14). These uncertainties led to the formation of a large collaborative study, the results of which were recently published (11). For that study, 13 sites agreed to test the activities of amphotericin B, flucytosine, and ketoconazole against a total of 100 isolates of yeasts. Of these, 85 strains were consecutive, distinct strains of six species which had been isolated from blood cultures at one university medical center, and the other 15 strains were isolates of C. neoformans supplied from another institution. In parallel studies, four of the study sites also tested fluconazole (9). Studies were performed by using two different starting inoculum sizes (5 x 104 and 1 x 106 yeast cells per ml), and readings were performed on the first and second days of growth. Each tube was examined for turbid-