SUMMARY Trichosporon spp. are basidiomycetous yeast-like fungi found widely in nature. Clinical isolates are generally related to superficial infections. However, this fungus has been recognized as an opportunistic agent of invasive infections, mostly in cancer patients and those exposed to invasive medical procedures. It is possible that the ability of Trichosporon strains to form biofilms on implanted devices, the presence of glucuronoxylomannan in their cell walls, and the ability to produce proteases and lipases are all factors likely related to the virulence of this genus and therefore may account for the progress of invasive trichosporonosis. Disseminated trichosporonosis has been increasingly reported worldwide and represents a challenge for both diagnosis and species identification. Phenotypic identification methods are useful for Trichosporon sp. screening, but only molecular methods, such as IGS region sequencing, allow the complete identification of Trichosporon isolates at the species level. Methods for the diagnosis of invasive trichosporonosis include PCR-based methods, Luminex xMAP technology, and, more recently, proteomics. Treating patients with trichosporonosis remains a challenge because of limited data on the in vitro and in vivo activities of antifungal drugs against clinically relevant species of the genus. Despite the mentioned limitations, the use of antifungal regimens containing triazoles appears to be the best therapeutic approach.

Current Knowledge of Trichosporon spp. and Trichosporonosis

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive “trailing” of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 μg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of ≤8 μg/ml (susceptible), 16 to 32 μg/ml (susceptible dose-dependent [SDD]), or ≥64 μg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 μg of fluconazole/ml, an 84% reduction after exposure to 4 μg/ml, and 95 and 100% reductions after exposure to 16 and 64 μg of fluconazole/ml, respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 57, 73, and 99% after exposure to 1, 4, 16, and 64 μg of fluconazole/ml, respectively. In contrast, 10 resistant isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in the drug-free control. Of 38 isolates which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM may predict clinical outcome more accurately. The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.

Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans

The in vitro activities of amphotericin B, itraconazole, fluconazole, voriconazole, posaconazole, and ravuconazole against 39 isolates of Trichosporon spp. were determined by the NCCLS M27-A microdilution method. The azoles tested appeared to be more potent than amphotericin B. Low minimal fungicidal concentration/MIC ratios were observed for voriconazole, posaconazole, and ravuconazole, suggesting fungicidal activity.

In Vitro Antifungal Susceptibilities of Trichosporon Species

The inhibitory effect of seven essential oils on the apical growth of hyphae of Aspergillus fumigatus was studied using a bio cell tracer by vapour contact in a sealed vessel. Based on the inhibitory pattern, these essential oils were classified into three groups. The first group, composed of citron, lavender and tea tree oils, stopped the apical growth in a loading dose of 63 micrograms ml-1 air, but allowed the regrowth of the hyphae after removal of the vapour, indicating fungistatic action. The second group, consisting of perilla and lemon-grass oils, stopped the apical growth in a loading dose of 6.3 micrograms ml-1 air, and did not allow the regrowth after gaseous contact at 63 micrograms ml-1 air, indicative of fungicidal action. The third group, consisting of cinnamon bark and thyme oils, retarded the growth in a dose of 6.3 micrograms ml-1 air, stopped it in a dose of 63 micrograms ml-1 air, and incompletely suppressed regrowth of the hyphae. Gas chromatographic analysis revealed that vapours of essential oils were absorbed on fungal mycelia and agar medium most abundantly by the first group, followed by the second and third groups, reflecting the volatility of the respective groups. Suppression of the apical growth by vapour contact was ascribed to the direct deposition of essential oils on fungal mycelia, together with an indirect effect via the agar medium absorbed.

Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus by vapour contact.

Concentrations of essential oils showing high volatility decreased substantially in broth and agar media when incubated under open conditions. The decrease in the half life was from 0.7 to 38 hr in broth medium at 27 C. When evaporation was prevented by sealing, MIC values against Aspergillus fumigatus, Candida albicans and Trichophyton mentagrophytes by broth or agar dilution assay were lowered two to eight-fold, as compared with those obtained under open conditions. Addition of Tween 80 caused a rise of the MICs against A. fumigatus by two to four-fold in broth dilution assay, but little affected the MICs in agar dilution assay.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1348-0421.2001.tb02608.x

Effect of Sealing and Tween 80 on the Antifungal Susceptibility Testing of Essential Oils

Mice treated with cyclophosphamide were infected intravenously with titrated doses of Trichosporon asahii TIMM3140 or TIMM3144. Mortality and survival time correlated with inoculum size and fungal growth was found in the lung, heart, liver, spleen, kidney and brain. Fluconazole and amphotericin B prolonged survival time and reduced the CFU in the kidney, with fluconazole being superior to amphotericin B. The serum level of (1-->3)-beta-D-glucan was reduced by antifungal therapy and correlated well with the CFU/organ in kidney, but not CFU/ml in blood.

Experimental disseminated trichosporonosis in mice: tissue distribution and therapy with antifungal agents.

Standard guidelines for the broth microdilution antifungal susceptibility testing of amphotericin B, flucytosine, fluconazole, miconazole and itraconazole are reported. These are a modification of the method developed by the National Committee for Clinical Laboratory Standards (NCCLS) on the following two points: standardization of the means of endpoint determination and the inclusion of miconazole and itraconazole in the testing. MIC was determined to be when the positive control had a turbidity of 0.2 at the 630 nm wavelength. The endpoint was 80% inhibition for azoles and 100% inhibition for other drugs. The method provided good reproducibility, and a wide range of MIC distribution was observed in all antifungal agents except amphotericin B.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1348-0421.1998.tb01970.x

Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination

Evaluation of antifungal susceptibility testing by the broth microdilution method against Candida species: activities of 5 antifungal agents against Candida species isolated from oral candidiasis or other candidal infectious diseases and correlation of vitro data and clinical outcome of fluconazole therapy.

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Experimental disseminated trichosporonosis in mice: tissue distribution and therapy with antifungal agents.

Development of Reference Procedures for Broth Microdilution Antifungal Susceptibility Testing of Yeasts with Standardized Endpoint Determination