Clinical and Laboratory Standards Institute standard M38—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi describes a method for testing the susceptibility to antifungal agents of filamentous fungi (nondermatophyte and dermatophyte moulds) that cause invasive and/or cutaneous fungal infections. Antifungal agent selection, preparation of antifungal stock solutions and dilutions for testing, test procedure implementation and interpretation, and the purpose and implementation of QC procedures are discussed. A careful examination of manufacturer and user responsibilities in QC is also presented. In addition, a brief discussion regarding newly defined epidemiological cutoff values for certain Aspergillus spp. and species complexes are included.

This standard replaces the previous edition of the approved standard, M38-A2, published in 2008. Several changes were made in this edition, including: 

• General: – Revised document format and organization to reflect the CLSI quality system essential and path of workflow document templates and the updated CLSI style – Updated references to the previous informational supplement (M51-S1) to reflect CLSI document M61, the new supplement for broth dilution and disk diffusion mould susceptibility testing – Added references to epidemiological cutoff values and CLSI documents M57 and M59 

• Subchapter 1.4.2, Definitions: 

– Revised the breakpoint and interpretive category definitions for consistency with other CLSI antimicrobial susceptibility testing documents. 

– Added definitions for “wild-type” and “non-wild-type” 

• Chapter 2, Preparing for Antifungal Susceptibility Testing: 

– Added new indications for testing of filamentous fungi, with a discussion of resistance in Aspergillus fumigatus originating from the natural environment 

• Chapter 3, Antifungal Broth Dilution Susceptibility Testing Process for Filamentous Fungi: 

– Added an antifungal susceptibility testing process flow chart 

– Expanded the list of relevant drug concentrations to be tested for echinocandins 

– Replaced procedural text with step-action tables 

– Established guide for reading and interpreting results for filamentous fungi, including dermatophytes 

– Modified text on reading results in Subchapter 3.4 to include new information on echinocandins and isavuconazole antifungal agents and minimal inhibitory concentration (MIC) and minimal effective concentration (MEC) comparison 

• Subchapter 4.6, Quality Control Frequency: 

– Added a note for the preparation of Candida spp. QC strains 

(Subchapter 4.6.1) 

• Appendixes (Original Tables): – Updated and moved the solvent list table from M38 to the new supplement, CLSI document M61 

– Moved the table providing the recommended MIC or MEC limits for QC and reference strains for broth dilution procedures from M38 to the combined supplement, CLSI document M61 

– Corrected Appendix C (Composition of Roswell Park Memorial Institute 1640 Culture Medium) to provide a single riboflavin concentration (0.0002 g/L), as found in CLSI document M27 

– Harmonized dilution schemes for dermatophyte and nondermatophyte isolates with those in CLSI document M27 and revised to encompass the full dilution ranges recommended 

– Deleted the procedure for preparing a 0.5 McFarland (barium sulfate) standard and added a note referring to CLSI document M27 for Candida spp. QC strains to Subchapter 4.6.1

This standard describes the reference broth microdilution testing method for antifungal susceptibility testing of filamentous fungi (moulds) that cause invasive and/or cutaneous fungal infections. This standard also covers testing conditions, including inoculum preparation and inoculum size, incubation time and temperature, media formulation, and end-point determination criteria. QC reference ranges and limits and specific epidemiological cutoff values (ECVs) are summarized in the current editions of CLSI documents M61 and M59, respectively.

The intended audience includes medical laboratory personnel, clinicians, and microbiologists who routinely perform antifungal susceptibility testing and use antifungal susceptibility testing results to select suitable antifungal therapy, as well as those involved in emerging resistance surveillance. The reference method is also useful for establishing ECVs and developing and validating alternate commercial methods for determining antifungal susceptibility of filamentous fungi. Therefore, the standard is also of interest for both diagnostic and pharmaceutical companies and their regulatory counterparts. 

This method has not been evaluated in studies of the yeast or mould forms of dimorphic fungi, such as Blastomyces dermatitidis, Coccidioides immitis/posadasii, Histoplasma capsulatum, or Talaromyces marneffei (Penicillium marneffei), and has been evaluated only for the mycelial form of Sporothrix schenckii species complex. This method also has not been used in studies of dermatophytes with the echinocandins or nondermatophyte moulds with ciclopirox, griseofulvin, or terbinafine. 

Antifungal susceptibility testing of other filamentous fungi that cause infections may also be tested by this method but have not been standardized and evaluated in collaborative studies. The appropriate testing parameters such as inoculum and incubation time for those fungi are unknown. 

Commercially available susceptibility test systems are out of scope for this standard. It is recommended that users of these systems refer to the manufacturer’s instructions as outlined in the package insert.