Objective To investigate the role of the ERG11 gene in the drug resistance of Trichosporon asahii (T.asahii), and to explore the relationship between the gene expression and drug concentrations. Methods Stable fluconazole-resistant strains of T.asahii were induced in vitro following exposure to a series of concentrations of fluconazole. Fluconazole-sensitive and-resistant strains of T.asahii were separately cultured in the medium containing fluconazole at concentrations of 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 μg/ml. Real-time quantitative PCR was performed to determine the mRNA expression of ERG11 gene. Results In fluconazole-free medium, the fluconazole-resistant strain of T.asahii showed significantly increased mRNA expression of the ERG11 gene compared with the fluconazole-sensitive strain (7.542 ± 5.311 vs. 1.014 ± 0.012, t=3.002, P=0.03). Additionally, the mRNA expression of ERG11 gene was also significantly higher in the fluconazole-resistant strains than the fluconazole-sensitive strains in the culture medium containing fluconazole at different concentrations of 0.25 (9.183 ± 3.226 vs. 3.281 ± 2.068), 0.5(13.657 ± 5.428 vs. 3.459 ± 1.923), 1(15.292 ± 7.007 vs. 3.242 ± 2.530), 2(13.720 ± 8.550 vs. 3.651 ± 0.728), 4(13.949 ± 2.960 vs. 3.969 ± 1.924)and 8(13.123 ± 6.429 vs. 3.824 ± 1.875)μg/ml(all P<0.05). However, no significant correlation was observed between the mRNA expression of ERG11 gene and fluconazole concentrations(fluconazole-resistant strains: rs = 0.229, P = 0.096; fluconazole-sensitive strains:rs=0.166, P=0.357). Conclusion Overexpression of ERG11 gene is associated with fluconazole resistance in T.asahii, but there is no correlation between the mRNA expression of ERG11 gene and fluconazole concentrations.

Objective To induce fluconazole resistance in T.asahii by culture in medium containing increasing concentrations of fluconazole,and to evaluate the stability of the induced resistance.Methods Two T.asahii strains with a highest sensitivity to fluoconazole,including a clinical isolate CBS2479 (minumum inhibitory concentration (MIC) =0.25 μg/ml) and an environmental isolate CBS8904 (MIC =1.5 μg/ml),were selected from 11 T.asahii strains stored in the laboratory of the Department of Dermatology,General Hospital of Beijing Military Region.Both strains were respectively and serially subcultured in potato dextrose agar (PDA) medium containing growing concentrations of fluconazole (from 0.5 MIC to 256 μg/ml).E-test was performed to evaluate the susceptibility of T.asahii to fluconazole after each passage.To evaluate the stability of fluconazole resistance,the T.asahii isolates with induced resistance (MIC ＞ 256 μg/ml) were serially subcultured in drug-free PDA medium,and drug susceptibility assay was performed after each subculture.Results After serial culture in PDA medium containing fluconazole,high level of fluconazole resistance (MIC ＞ 256 μg/ml) developed in both of the fluconazole-susceptible T.asahii strains CBS2479 and CBS8904.The MIC value of fluconazole remained unchanged in the fluconazole-resistant strain CBS2479R,but gradually decreased to 64 μg/ml in the other resistant strain CBS8904R after 18-day culture in fluconazole-free PDA medium.Conclusions Fluconazole resistance can be induced in T.asahii strains from different origins by serial culture in medium containing growing concentrations of fluconazole,and the stability of the induced fluconazole resistance varies between strains of different origins.

Objective To determine the effect of interferon-gamma (IFN-γ) on the phagocytosis and killing activity of a murine macrophage cell line RAW264.7 against T.asahii,and to estimate the possibility of treating T.asahii infection with IFN-γγ.Methods T.asahii was cultured with or without the presence of different concentrations (10,100,1000 U/ml) of IFN-γfor 18 hours followed by the incubation with RAW264.7 cells for different durations.After additional culture for 45 minutes,the number of T.asahii cells phagocytosed by RAW264.7 cells was counted under an inverted microscope,and the rate of phagocytosis was calculated.The number of colony forming units of T.asahii per milliliter (cfu/ml) was counted after 4-hour additional culture and the growth inhibition rate was determined.Data were processed by the SPSS 16.0 software,and comparisons of parameters between these groups were done by Bonferroni method and analysis of variance after homogeneity test of variance.Results The number of phagocytosed T.asahiicells was 25.12 ± 1.81,35.88 ± 3.56,52.12 ± 3.23,with the phagocytosis rate being 25.12％,35.88％ and 52.12％,respectively in RAW264.7 cells incubated with IFN-γ of 10,100,1000 U/ml,significantly different from that in untreated RAW264.7 cells (13.62 ± 2.39,13.62％,all P ＜ 0.01).The colony forming units of T.asahii per ml after incubation with untreated RAW264.7 cells differed significantly from those after incubation with IFN-γ (10,100,1000 U/ml)-treated RAW264.7 cells ((68.12 ± 3.39) × 500 vs.(58.62 ± 4.89) × 500,(45.50 ± 3.02) × 500 and (34.62 ± 4.24) × 500,all P＜0.01),with the growth inhibition rate being 25.21％,41.95％ and 55.83％ respectively for RAW264.7 cells incubated with IFN-γ of 10,100,1000 U/ml.Statistical differences were also observed in the killing activity between RAW264.7 cells incubated with different concentrations of IFN-γ (all P ＜ 0.01).Conclusion IFN-γ (10-1000 U/ml) may enhance the phagocytosis and killing activity of RAW264.7 cells against T.asahii in a concentration-dependent manner.

Objective To observe morphological characteristics and activity distribution of T. asahii biofilm. Methods The morphological characteristics of T. asahii biofilm were observed under an inverted microscope and scanning electron microscope, and activity was measured and quantitatively analyzed by 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazo-lium hydroxide (XTT) assay and viable count, respectively. Spatial distribution of dead/vital cells, activity and thickness of biofilm at different layers were assessed under a confocal laser scanning microscope (CLSM) following double staining with FDA/PI. Results T. asahii formed a biofilm in vitro on the surface of polystyrene materials. Under a scanning microscope, the biofilm displayed a complex three-dimensional structure which composed of spores, pseudohy-pha and true hypha. As time prolonged, the activity and quantity of biofilm increased. The results of XTT assay were correlated with those of viable count (r = 0.94, P < 0.01). The activity was of no obvious difference between different layers of the biofilm. The thickness of biofilm varied from 14.3 μm to 31 μm. Conclusions The structure of T. asahii biofilm in vitro is more complex than that of planktonic T. asahii. The activity is of no significant difference between different layers of T. asahii biofilm.

OBJECTIVE To identify if Trichosporon asahii exist cph1 gene homolog of Candida albicans,and analyze its nucleotide sequence.METHODS Nuclear DNA was extracted from the cells of T.asahii by using a simplified protocol,designed 29 pairs of primers according to the cph1 gene sequence of C.albicans and amplify by PCR.The PCR products were cloned and sequenced using the ABI377 nucleotide sequenator,and BLAST analysis.RESULTS An 827 bp gene was amplified successfully,which was homolog with the cph1 gene of C.albicans and their identity was 97.3%.CONCLUSIONS This trial determines the clone cph1 gene in T.asahii for the first time,which makes bases for the role of cph1 gene in the hypha formation.

Objective:To evaluate the efficacy and safety of radiofrequency introduction of L-vitamin C in patients with melasma.Methods:From March to June 2019, 20 patients with melasma were admitted to the Department of Dermatology, the Seventh Medical Center of PLA General Hospital, including 19 females and 1 male, aged 30-60 years, with an average age of 43.5 years. All patients were treated with 22 percent of L-vitamin C once a week, a total of 8 times of treatment and followed up for 12 weeks. Each subject was assessed with standardized clinical photo, skin tests (VISIA skin image analyzer and CK multifunctional skin tester) and patient self-assessment. In addition, the adverse reactions were recorded.Results:Physician evaluation and patient self-evaluation showed that skin symptoms were improved obviously after treatment. 90% of the subjects thought that all of the skin moisture, pores, fine lines, glossiness, and color spots were improved after 12 weeks. The skin texture, ultraviolet stain and the brown spots which were detected with VISIA skin image analyzer were all improved after one week and one month. Difference was statistically significant ( P<0.05). Skin glossiness was significantly improved, skin moisture content increased and melanin decreased, which were detected with CK multifunctional skin tester. The differences were statistically significant ( P<0.05). But there was no significant change in transdermal water loss and red pigment index ( P>0.05). Conclusions:22% L-vitamin C can be used to treat melasma and improve photoaging safely without affecting skin barrier function.