Antifungal Susceptibility Testing
Antifungal susceptibility testing measures the in vitro activity of an antifungal drug against an isolate, as the lowest concentration that inhibits growth (the minimal inhibitory concentration, MIC). The result is interpreted against clinical breakpoints, where these exist, to predict the likelihood of therapeutic success.
It identifies acquired resistance, guides the choice and dose of drug in invasive infection, supports surveillance and infection control, and defines the wild type distribution of a species. Testing is less well established than antibacterial testing, because breakpoints exist for few species and drugs, the correlation between MIC and outcome is weaker, and host factors and the site of infection have a large effect.
| Method | Principle and use |
|---|---|
| Reference broth microdilution (yeasts) | Serial dilutions of the drug in RPMI 1640 medium buffered with morpholinepropanesulphonic acid (MOPS) at pH 7.0 in 96 well plates, inoculated with 0.5 to 2.5 times 10 to the 3 colony forming units per mL, and read after 24 hours (Candida) or 48 to 72 hours (Cryptococcus). The two internationally used reference methods differ in glucose content (2 percent or 0.2 percent), inoculum size, plate shape, and the reading (visual or spectrophotometric), so results differ slightly and breakpoints are tied to the method |
| Reference broth microdilution (moulds) | Conidial inoculum of 0.4 to 5 times 10 to the 4 colony forming units per mL, incubated at 35 degrees Celsius for 24 hours (Mucorales) or 48 hours (Aspergillus, most moulds), with 72 to 96 hours or longer for slow growing species |
| Gradient strip (Etest) | A strip with a gradient of drug on agar; easy to use for yeasts and moulds, with agreement of about 90 percent within one dilution for most drugs against Candida, and more variable results for amphotericin B and moulds; RPMI agar with glucose 2 percent |
| Commercial colourimetric broth microdilution (Sensititre YeastOne) | A plate with an oxidation reduction indicator (Alamar blue); the colour change gives the MIC; widely used in clinical laboratories |
| Automated systems (Vitek 2 yeast cards) | Rapid results for common Candida species; good for fluconazole and echinocandins, but less reliable for amphotericin B, voriconazole, and some species |
| Disk diffusion | Standardised for Candida (fluconazole, voriconazole, and echinocandins in some systems) and, with limited use, for Cryptococcus; a simple screening method |
| Azole screening agar | Agar plates with fixed concentrations of itraconazole (4 mg per L), voriconazole (1 mg per L), and posaconazole (0.5 mg per L) for screening Aspergillus fumigatus for azole resistance, and a four well plate format |
| Molecular detection of resistance | Sequencing or polymerase chain reaction for cyp51A mutations (TR34/L98H, TR46/Y121F/T289A) in Aspergillus fumigatus, FKS1 and FKS2 hot spot mutations in Candida, and ERG11 mutations (Candida auris, Candida albicans); faster, and can be done on clinical specimens, but misses new mechanisms |
| Time kill and synergy testing (checkerboard) | Research tools, with no routine clinical use |
| Susceptibility testing of dermatophytes | Reference method available for research (terbinafine resistance is emerging in Trichophyton indotineae), and sequencing of SQLE mutations |
| Endpoint | Application |
|---|---|
| Inhibition of 50 percent of growth (compared to the drug free control) | Azoles, echinocandins against yeasts, and flucytosine; trailing growth (reduced but persistent growth over a range of concentrations) is common with azoles and is read at 24 hours |
| Complete inhibition of visible growth (100 percent) | Amphotericin B (yeasts and moulds), and azoles against moulds |
| Minimal effective concentration (MEC) | Echinocandins against moulds (the lowest concentration producing short, abnormal, branched hyphae); a different endpoint from the MIC |
| Paradoxical (Eagle) effect | Regrowth at high echinocandin concentrations, mainly with caspofungin and Candida and Aspergillus; its clinical importance is uncertain |
| Trailing | Persistent reduced growth in azole susceptible Candida albicans and Candida tropicalis after 48 hours, so 24 hour reading is used |
| Concept | Meaning |
|---|---|
| Clinical breakpoint | An MIC value that separates susceptible, intermediate (or susceptible dose dependent), and resistant categories, based on microbiological data, pharmacokinetics and pharmacodynamics, and clinical outcome |
| Susceptible dose dependent | Fluconazole against Candida glabrata and the susceptible category with a high dose (800 mg or 12 mg per kg per day) |
| Epidemiological cutoff value (ECV, ECOFF) | The upper limit of the wild type MIC distribution of a species; isolates above it carry acquired resistance, but the value does not predict clinical outcome |
| Wild type and non wild type | Isolates with and without acquired resistance mechanisms |
| Intrinsic resistance | A property of the species (Candida krusei to fluconazole, Aspergillus terreus to amphotericin B, Mucorales to voriconazole, Cryptococcus to echinocandins, Fusarium and Scedosporium to multiple drugs) and testing is not needed to establish it |
| 90 to 60 rule | Infections caused by susceptible isolates respond in about 90 percent of cases, and infections caused by resistant isolates respond in about 60 percent, because the host and site also affect outcome |
Breakpoints for Candida (examples of clinical breakpoints in micrograms per mL, reference broth microdilution; the values differ between methods and are revised periodically):
| Species and drug | Susceptible | Intermediate or dose dependent | Resistant |
|---|---|---|---|
| Candida albicans, fluconazole | 2 or less | 4 | 8 or more |
| Candida albicans, voriconazole | 0.12 or less | 0.25 to 0.5 | 1 or more |
| Candida albicans, caspofungin, micafungin, anidulafungin | 0.25 or less | 0.5 | 1 or more |
| Candida glabrata, fluconazole | None (dose dependent at 32 or less) | 32 or less (dose dependent) | 64 or more |
| Candida glabrata, caspofungin | 0.12 or less | 0.25 | 0.5 or more |
| Candida glabrata, micafungin | 0.06 or less | 0.12 | 0.25 or more |
| Candida parapsilosis, fluconazole | 2 or less | 4 | 8 or more |
| Candida parapsilosis, echinocandins | 2 or less | 4 | 8 or more |
| Candida tropicalis, fluconazole | 2 or less | 4 | 8 or more |
| Candida krusei, fluconazole | Intrinsically resistant | ||
| Candida krusei, echinocandins | 0.25 or less | 0.5 | 1 or more |
| Amphotericin B | No clinical breakpoints; an MIC above 1 microgram per mL suggests resistance |
Candida auris has no species specific breakpoints in the reference methods. Tentative resistance thresholds from public health agencies are fluconazole 32 micrograms per mL or more, amphotericin B 2 micrograms per mL or more, and echinocandins (anidulafungin and micafungin 4 or more, caspofungin 2 or more). The majority of isolates are fluconazole resistant, and a minority are multidrug resistant.
Epidemiological cutoff values for Aspergillus fumigatus (micrograms per mL, reference broth microdilution): amphotericin B 2, itraconazole 1, voriconazole 1, posaconazole 0.5, and isavuconazole 1; isolates above these values are non wild type and may carry cyp51A mutations. Clinical breakpoints exist for Aspergillus fumigatus in one of the systems (voriconazole susceptible at 1 or less and resistant above 2).
Cryptococcal ECVs are available for fluconazole (about 8 micrograms per mL for Cryptococcus neoformans) and amphotericin B (0.5 to 1), but they do not predict outcome.
| Drug class | Index linked to efficacy |
|---|---|
| Azoles | Ratio of the area under the concentration time curve to the MIC (AUC to MIC), with a free drug target of about 25 for fluconazole against Candida (or a total drug target of 25 to 50), and for voriconazole (trough more than 1 to 2 micrograms per mL) |
| Echinocandins | Ratio of peak concentration to MIC (Cmax to MIC) and AUC to MIC, with concentration dependent killing |
| Amphotericin B | Ratio of peak concentration to MIC (above 10 for the best effect), with concentration dependent killing and a prolonged post antifungal effect |
| Flucytosine | Time above the MIC (about 40 percent of the dosing interval or more), a time dependent drug |
| Setting | Recommendation |
|---|---|
| Candida from blood and other sterile sites | Test every isolate (fluconazole and an echinocandin at minimum, and amphotericin B for Candida auris and unusual species) |
| Candida with prior azole or echinocandin exposure, breakthrough infection, or no response | Repeat and extend testing |
| Candida glabrata, Candida auris, Candida krusei, Candida lusitaniae, and rare species | Always, with species identification |
| Aspergillus from invasive or chronic disease | Test azoles and amphotericin B; screen for azole resistance in invasive disease and in cases that do not respond |
| Mucorales | Test amphotericin B and posaconazole and isavuconazole where possible (no clinical breakpoints) |
| Fusarium, Scedosporium, Lomentosporium, and other uncommon moulds | Always, because resistance is common and varies by species |
| Cryptococcus | Not routine; in relapse or failure after fluconazole therapy |
| Dimorphic fungi | Limited role, in refractory disease |
| Dermatophytes | Considered for refractory disease and suspected terbinafine resistance (Trichophyton indotineae) |
| Superficial Candida in mucosa | Not routine; refractory fluconazole resistant oropharyngeal and vulvovaginal disease |
| Surveillance and outbreaks | Isolates tested to monitor trends, with molecular typing in outbreaks |
| Limitation | Details |
|---|---|
| Few clinical breakpoints | Absent for most moulds, for Cryptococcus, for dimorphic fungi, for amphotericin B (all species), for flucytosine in most species, and for many Candida and drug pairs |
| Method dependence | MIC values differ by medium, inoculum, incubation, and reading method, so results from different systems are not interchangeable |
| Inter species variation | Breakpoints are species specific, so species identification is essential |
| Inoculum and trailing effects | Complicate the reading of azole MICs |
| Poor correlation in some cases | Amphotericin B MICs separate poorly, and a low MIC does not guarantee success in neutropenia or at poorly perfused sites |
| Host and site | Immune status, site of infection, biofilm, drug penetration, and source control matter as much as the MIC |
| Slow results | Culture and testing take 2 to 5 days, so empirical therapy precedes the result |
| Heteroresistance and tolerance | Subpopulations with higher MICs, and persistent growth, are missed by the standard test |
The report gives the species, the MIC value, and the interpretation (susceptible, intermediate or susceptible dose dependent, resistant, or non wild type), with a comment on intrinsic resistance and on the lack of breakpoints.
Results are used to select or change therapy:
- A Candida glabrata isolate with a fluconazole MIC above 32 micrograms per mL needs an echinocandin.
- An isolate with an FKS mutation or high echinocandin MIC is treated with liposomal amphotericin B.
- An azole resistant Aspergillus fumigatus (TR34/L98H) is treated with liposomal amphotericin B or an echinocandin combination, and not with voriconazole alone.
Therapeutic drug monitoring (voriconazole trough 1 to 5.5 micrograms per mL, posaconazole above 0.7 for prophylaxis and above 1 for treatment, itraconazole above 0.5 to 1, flucytosine peak 30 to 80) complements susceptibility testing.
