Advances in studies of Australian dermatophytes and tinea unguium
Creators: name: Hainsworth, Steven
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310705
subject: Mycology

description: Relatively scant research concerning toenail onychomycosis has been undertaken as to the predominant species and strains causing this condition in Australia. Four regions were selected in Eastern Australia: Far North Queensland, Rural Victoria, Melbourne Metropolitan and Tasmania. From each of these areas, communal nail dust bags from podiatric physicians’ work were collected and analysed. A total of 32 dust bags were collected: 10 from Far North Queensland, 8 from Melbourne Metropolitan, 8 from Rural Victoria and 6 from Tasmania. Dermatophyte test medium was used to isolate dermatophytes from the dust, and the colonies were subcultured to Potato Dextrose Agar. Of the bags collected, in total 69% were positive for dermatophytes: 40% from Far North Queensland, 75% from Melbourne Metropolitan, 88% from Rural Victoria and 83% from Tasmania. A total of 148 dermatophyte strains were identified. The predominant species isolated was Trichophyton interdigitale (125 isolates), which was found in all four regions. These isolates were subdivided into four ITS genotypes: the first two were present in all regions, but the third was found only in Melbourne Metropolitan and the fourth only in Tasmania. Only one strain of Trichophyton rubrum was found and only in Rural Victoria. Eighteen isolates of Arthroderma quadrifidum were cultured from Rural Victoria and Tasmania and were classified into three ITS genotypes. Some isolates rarely reported in clinical material were Paraphyton cookei, Arthroderma tuberculatum and Arthroderma crocatum. A potential new species of Arthroderma was found in Melbourne Metropolitan.
 
Arthroderma dermatophytes encompass species occurring in soil, caves, animal burrows, clinical material and other environments. We collected ex-type, reference and authentic strains of all currently accepted Arthroderma species and generated sequences of three highly variable loci (ITS rDNA, β-tubulin, and translation elongation factor 1-α). The number of accepted species was expanded to 27. One novel species, A. melbournense (ex-type strain CCF 6162T = CBS 145858T), found in our investigation is described. This species was isolated from toenail dust in Melbourne, Australia. Trichophyton terrestre, Chrysosporium magnisporum, and Chrysosporium oceanitis are transferred to Arthroderma. A multi-gene phylogeny and reference sequences provided in this study should serve as a basis for future phylogenetic studies and facilitate species identification in practice.
 
To investigate the microbial community of infected nails, 18 mycotic nails were photographed and analysed using metagenomics for fungi and for bacteria. These nails consisted of four from Bass Coast, six from Melbourne Metropolitan and eight from Shepparton. Two normal nails, one from Melbourne Metropolitan and one from Shepparton, were analysed as controls. Overall of the mycotic nails containing T. interdigitale and T. rubrum, the sequences for these dermatophytes were in very high abundance averaging over 90% except for two nails where T. interdigitale was in lesser proportions in combination with an NDM. In Bass Coast there were three nails containing a high abundance of sequences for T. rubrum and one for T. interdigitale. In the Melbourne area nails with a very high abundance of sequences were three of T. interdigitale, one of Aspergillus versicolor, one of Acremonium sclerotigenum and one with a mixture of Fusarium oxysporum and T. interdigitale. Of the eight mycotic nails from Shepparton those with high abundances of sequences were four T. rubrum, three T. interdigitale and one mixture of T. interdigitale with Fusicolla acetilerea. The two control nails contained chiefly Fusarium sp. and Malassezia slooffiae respectively. For bacteria, Staphylococcus epidermidis was the most abundant species, including the control nails with an overall mean rate of 66.01%. Rothia koreensis, Corynebacterium tuberculostearicum, and Brevibacterium sediminis also featured in the analysis. No correlation between clinical appearance and the infecting agent was observed.

To investigate the microbial contents of podiatric communal toenail dust bags, fungal and bacterial metagenomic analysis was conducted on nine of these bags. There were three bags each from Bass Coast, Melbourne Metropolitan and rural Shepparton. The sequence assemblies from the nine bags averaged 21.30% T. mentagrophytes, 21.25% T. rubrum and 0.16% of E. floccosum. One bag from Bass Coast had 0.23% T. mentagrophytes, no T. rubrum or E. floccosum but 86.15% A. penicillioides sequences. The main bacterial sequences were skin commensals with Staphylococcus pettenkoferi averaging 67.98% over the nine bags. Metagenomic analysis revealed the presence of a large variety of non-dermatophyte moulds, yeasts and bacteria.

Tinea unguium is frequently recalcitrant to treatment. There is a growing awareness of terbinafine resistance, but susceptibility testing is rarely performed as part of diagnostic services, because microdilution susceptibility testing is complex, costly and time-consuming. A screening assay was developed to indicate which clinical strains were likely to be resistant. Four 6 cm Petri dishes were used per isolate. Each set of plates contained solid PDA with 0, 0.05 μg/ml, 0.28 or 0.5 μg/ml TRB. For each isolate, an area of 4 mm2 mycelial growth was scraped from the colony surface, suspended in 1 ml of sterile water and vortexed. From this suspension, 0.15 μl was spread onto each plate and incubated for up to 7 days. Growth on terbinafine-containing plates was considered to indicate a degree of resistance. Seventy-four isolates were tested: 60 isolates of T. interdigitale, 2 of T. rubrum, 10 of Arthroderma quadrifidum, 1 of A. melbournense and 1 of Paraphyton cookei, along with reference strains of T. rubrum and T. interdigitale with known MICs. Four strains (6.7%) of T. interdigitale had good growth on all terbinafine containing plates. Two strains (20%) of A. quadrifidum grew well on the 0.05 μg/ml plates. Antifungal susceptibility testing (AFST) was performed on isolates and reference strains. The MICs were compatible with the screening method. These results indicate that this screening method may be useful in screening for resistance.

This research is ground-breaking in Australia and reveals areas for future investigation. The novelty of the culture method on communal nail dust bags opens up a new field of investigation, with perhaps more new species to be discovered. The development of a new successful screening method for terbinafine resistance, points to a need to screen clinical samples and do further research. The field of metagenomics is very promising, and more research should be performed.