In its most general terms, susceptibility testing refers to the idea of mixing the fungus with a drug and seeing what happens. In general, we are interested in determining a minimal concentration of drug that appears likely to inhibit (minimum inhibitory concentration, MIC, µg/ml) or kill (minimum fungicidal concentration, MFC, µg/ml) the fungus. If this level is low enough, then the drug may work against an infection. The approach to testing is different for yeasts and moulds. If you simply want to look at some susceptibility results, please see our searchable N/A(L):susceptibility database or review our summaries of usual susceptibility patterns of the fungi.
Susceptibility testing of yeasts
Beginning in about 1990, the National Committee for Clinical Laboratory Standards began the process of developing standardized methods for antifungal susceptibility testing. Under the leadership of John Galgiani, the Antifungal Susceptibility Testing Subcommittee was formed and undertook a lengthy series of collaborative studies to establish reproducible methods (reviewed in ). The committee focused initially on broth-based testing of yeasts and ultimately produced a document called M27 (M for “Method), entitled “Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standard NCCLS document M27-A” . For details of the NCCLS standard method, please refer to the laboratory procedures page. The M27 method itself is, however, rather cumbersome and not always accurate. Thus, variants on this method are widely used.
Unresolved problems and variants of the standard method for yeasts
The NCCLS method was a major achievement in standardization of this difficult area, but it is (not suprisingly) not yet optimal. Some of the test parameters remain problematic and require modifications:
The standard NCCLS method is a broth microdilution. It is labor-intensive and cumbersome. In an effort to develop an alternative, easier method, other methods have been studied. Among these are colorimetric microdilution [519, 691, 1773], E test (ABBiodisk) [685, 690, 2378], and disk diffusion [193, 1508, 1777]. All of these alternative methods in general correlate well with the standard method. However, each also has its own disadvantages. While colorimetric microdilution method is as cumbersome as the standard method, E test is relatively expensive. Disk diffusion is the most attractive alternative method so far investigated. It is not only easy-to-perform and cheap, but also well suited for routine use in mycology laboratories. Preliminary data obtained for testing fluconazole susceptibility of Candida by disk diffusion method are very promising and well-correlated with the standard method. Quite different approaches such as flow cytometric measurements [897, 1829, 2406] and determination of ergosterol content  are reliable but inappropriate for routine use.
The NCCLS proposed incubation period is 48 hours for Candida and 72 hours for Cryptococcus neoformans. While these conditions are acceptable for many isolates, some isolates trail (show partial growth inhibition) rather badly when incubated this long. For these so-called “trailers,” it has been shown that fluconazole MICs obtained at 24 hours may correlate better with the clinical outcome compared to those at 48 hours in a murine model of invasive candidiasis . This phenomenon is also pH-dependent, and is clarified by testing at pH 4.0 .
Amphotericin B susceptibility testing.
Amphotericin B MICs are distributed in a very narrow range. Thus, the standard method is inefficient in discriminating the amphotericin B-resistant isolates from the
-susceptible ones. Modifications in the test medium have been proposed to solve this problem. However, there is yet no consensus about this issue. While the use of “Antibiotic Medium 3” as the test medium instead of RPMI 1640 has highlighted the resistant isolates in hands of some investigators [1378, 1379, 1905], the results obtained by others have been contradictory . However, all of the results appear highly technique-dependent and difficult to standardize.
The difficulties in generation of wide range amphotericin B MICs result in troubles in determination of MIC breakpoint for amphotericin B. Also, some investigators suggest the use of MFC (minimum fungicidal concentration) measurements instead of MICs . A suitable resolution to these problems has not been reached.
Cryptococcus neoformans susceptibility testing.
The growth of Cryptococcus neoformans in standard test medium is slow and poor. This not only necessitates unacceptably long incubation periods for susceptibility testing, but also results in difficulties in determination of MIC breakpoints and the resistant isolates. Modifications in test medium and shaking of culture media during incubation have been suggested to promote the growth of cryptococci [834, 1945]. However, these modifications are neither widely accepted nor standardized.
Susceptibility testing of moulds
Testing of moulds has been studied intensively using the ideas generated during development of M27-A. Under the leadership of Michael A. Pfaller and Ana Espinel-Ingroff, the M38-P document has recently been developed and released . This document describes the basis for a standardized reference method applicable to moulds. For details of the NCCLS standard method, please refer to the laboratory procedures page.
Several test parameters remain in flux. These include the interpretive breakpoints, ATCC numerical designation of the reference Aspergillus isolates, and most significantly, the clinical relevance of the method . Moreover, similar to that with the yeasts, the method is labor-intensive and cumbersome. Studies to reveal the use of the standard method and the influence of its various modifications, as well as its clinical significance are in progress [119, 511, 573, 578, 2051].
Information available on this web site
- General summaries of usual susceptibility patterns for a variety of fungi.
- A detailed N/A(L):susceptibility database that provides a way to search selected data from many different papers.
- A comprehensive set of laboratory procedures for general laboratory work and with fungi, including susceptibility testing of fungi.
- Information on methods used in histopathology for diagnosis of fungal infections.