Identification and Antifungal Susceptibility of Medically Important Fungi
These descriptions have by necessity been kept brief and many have been based on descriptions by other authors. For further information regarding any of the mycoses or pathogenic fungi mentioned, the reader is referred to the citations provided. For the precise definitions of the mycological terminology used, the reader is referred to Ainsworth and Bisby’s Dictionary of the Fungi (Kirk et al. 2008).
Note: Nomenclature changes in mycology are ongoing. To search for current accepted fungal names go to Index Fungorum (www.indexfungorum.org) and Mycobank (www.mycobank.org).
The antifungal susceptibility data presented is from the National Mycology Reference Centre at SA Pathology using the CLSI M27-A and M38-A protocols for yeasts and moulds respectively. Data is from clinical isolates collected over the last 10 years. Published data from many rare or uncommon species have also been listed although it must be stressed that in many cases insufficient numbers have been tested for an accurate assessment of MIC data.
Select from one of the following groups:
- Dimorphic Pathogens
- Hyphomycetes (hyaline moulds)
- Hyphomycetes (dematiaceous moulds)
- Prototheca (achlorophyllous algae)
Key Morphological Characters
• Surface texture [glabrous, suede-like, powdery, granular, fluffy, downy, cottony]
• Surface topography [flat, raised, heaped, folded, domed, radial grooved]
• Surface pigmentation [white, cream, yellow, brown, pink, grey, black etc]
• Reverse pigmentation [none, yellow, brown, red, black, etc]
• Growth rate [colony diameter <5 cm in 14 days or >5 cm in 15 days]
• Growth at 37C, 40C, 45C.
Zygomycota. Sporangia characteristics:
• Arrangement of sporangiospores [multispored, sporangiola, merosporangium]
• Arrangement of sporangiophores [unbranched often in groups or frequently branched]
• Sporangium shape [pyriform, spherical, flask-shaped etc]
• Sporangium size [<100 μm diam. or >100 μm diam.]
• Columella [Present or Absent]
• Apophyses [Present or Absent]
• Sporangiophore height [<0.8 mm or >1 mm]
• Rhizoids [Present or Absent] (look in the agar)
• Sporangiospore size [<6 μm or >6 μm]
Hyphomycetes - Conidial Moulds
1. Conidial characteristics:
• Septation [one-celled, two-celled, multicelled with transverse septa only, or multicelled with both transverse and longitudinal septa]
• Shape [spherical, sub-spherical, pyriform, clavate, ellipsoidal, etc]
• Size [need a graduated eyepiece, length <10 μm or >10 μm]
• Colour [hyaline or darkly pigmented]
• Wall texture [smooth, rough, verrucose, echinulate]
• How many conidial types present? [i.e. micro and macro]
2. Arrangement of conidia as they are borne on the conidiogenous cells:
• Solitary [single or in balls]
• Catenulate (in chains) [acropetal (youngest conidium at the tip) or basipetal (youngest conidium at the base]
3. Growth of the conidiogenous cell:
• Determinant (no growth of the conidiophore after the formation of conidia)
• Sympodial (a mode of conidiogenous cell growth which results in the development of conidia on a geniculate or zig-zag rachis)
4. Type of conidiogenous cell present:
• Phialide (specialised conidiogenous cells that produces conidia in basipetal succession without increasing in length)
• Annellide (specialised conidiogenous cell producing conidia in basipetal succession by a series of short percurrent proliferations (annellations). The tip of an annellide increases in length and becomes narrower as each subsequent conidium is formed)
5. Any additional features present:
• Hyphal structures [clamps, spirals, nodular organs, etc]
• Synnemata, Sporodochia, Chlamydoconidia, Pycnidia
• Confirmatory tests for dermatophytes
Molecular and/or MALDI-TOF MS Identification:
The use of PCR-based assays, DNA sequencing, and other molecular methods, including those incorporating proteomic approaches such as matrix assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results to aid in accurate species identification of fungal cultures. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended (Halliday et al. 2015). Molecular-based fungal identification is particularly helpful for fungi that lack distinguishing morphological features, e.g. Apophysomyces elegans, or to distinguish between species of the Aspergillus fumigatus complex. Comparative sequence analysis is now the ‘gold standard’ for identification of fungi. Methods are referenced where available and in many instances are recommended for more definitive identifications.
Schematic diagram of the fungal rDNA gene cluster (adapted from CLSI MM18-A and Halliday et al. 2015). The 18S, 5.8S and 28S rDNA genes are separated by the two internal transcribed spacers. The 28S and 5S rDNA genes are separated by the intergenic spacer 1 (IGS1). The intergenic spacer 2 (IGS2) separates the rDNA repeat units from each other.
Regardless of the genetic locus selected, accurate sequence-based identification is dependent upon database accuracy and adequate species representation. GenBank is well known to contain numerous errors in sequences and the species names attributed to the sequences, which are rarely corrected. Therefore caution must be used when interpreting sequencing comparisons against this database, and the use of multiple sequence databases is encouraged. Well-curated databases that are helpful for species identification include:
1. International Society for Human and Animal Mycoses (ISHAM) ITS database (http://its.mycologylab.org/).
2. CBS-KNAW Fungal Biodiversity Centre database (http://www.cbs.knaw.nl).
Frequently used molecular targets for species identification are outlined below:
For many species, antifungal susceptibility data has also been provided. This has been derived from both the literature and data from Australian clinical isolates generated by using the CLSI M27-A Standard for yeasts and the CLSI M38-A Standard for moulds. This composite data is provided as a guide only. In many cases the clinical relevance of in vitro antifungal susceptibility results remains difficult to interpret, and expert advice from a consulting microbiologist or infectious disease specialist may be required.
CLSI M27-S4 clinical breakpoints are marked where available (green for susceptible, yellow for susceptible dose dependant or intermediate, red for resistant).
Abbreviations: Amphotericin B (AmB), Fluconazole (FLU), Itraconazole (ITRA), Posaconazole (POSA), Voriconazole (VORI), Anidulafungin (ANID), Caspofungin (CAS), Micafungin (MICA), 5-Fluorocytosine (5FC), Terbinafine (TERB).
Risk group (RG) recommendations
These are based on published data and on current definitions in accordance with the Australian/New Zealand Standard AS/NZS 2243.3:2010. Safety in laboratories Part 3: Microbiological safety and containment. Note: International biosafety guidelines vary in their RG ratings of fungal species.