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Objective To evaluate the in vitro effects of photodynamic therapy alone or in combination with antifungal agents on the apoptosis of planktonic and biofilm cells of Exophiala dermatitidis (E.dermatitidis).Methods The planktonic suspensions of E.dermatitidis were prepared,and the biofilms of E.dermatitidis were prepared via a modified 96-well plate-based methods.Planktonic and biofilm cells of E.dermatitidis were separately divided into several groups:antifungal agent groups treated with antifungal agents alone,photodynamic therapy group receiving photodynamic therapy alone,combination groups receiving photodynamic therapy followed by the treatment with antifungal agents,and blank control group receiving no treatment.These antifungal agents included amphotericin B,posaconazole,voriconazole and itraconazole.The concentrations of these antifungal agents were all 1 mg/L,and the treatment with antifungal agents lasted 2 hours.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect the apoptosis of planktonic and biofilm cells of E.dermatitidis in all the groups.Results The antifungal agents and photodynamic therapy both affected the apoptosis of planktonic (both P ＜ 0.001) and biofilm cells (beth P ＜ 0.05) of E.dermatitidis.The apoptosis rates of E.dermatitidis planktonic cells in the control group,amphotericin B group,posaconazole group,voriconazole group and itraconazole group were 11.67％ ± 0.21％,13.30％ ± 1.78％,14.30％ ± 3.61％,14.51％ ± 1.91％and 36.17％ ± 4.00％ respectively.The apoptosis rate of E.dermatitidis planktonic cells was significantly higher in the itraconazole group than in the control group (P ＜ 0.05),but no significant differences were observed between the other 3 antifungal agent groups and control group (all P ＞ 0.05).The photodynamic therapy group also showed a significantly higher apoptosis rate of E.dermatitidis planktonic cells (41.37％ ±7.80％) compared with the control group (P ＜ 0.05).After the treatment with photodynamic therapy combined with amphotericin B,posaconazole,voriconazole or itraconazole,the apoptosis rates of E.dermatitidis planktonic cells were 29.23％ ± 6.71％,37.23％ ± 10.86％,43.57％ ± 6.42％ and 69.87％ ± 3.53％ respectively.Moreover,the photodynamic therapy + voriconazole group and photodynamic therapy + itraconazole group both showed significantly higher apoptosis rates compared with the voriconazole group and itraconazole group respectively (both P ＜ 0.05).The apoptosis rate of E.dermatitidis biofilm cells was significantly higher in the photodynamic therapy group than in the control group (32.00％ ± 0.43％ vs.25.30％ ± 1.31％,P ＜ 0.05),as well as in the photodynamic therapy + amphotericin B than in the amphotericin B group (P ＜ 0.05).Conclusion Photodynamic therapy combined with antifungal agents can markedly promote the apoptosis of planktonic and biofilm cells of E.dermatitidis.
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Objective To evaluate the in vitro antifungal activity of 4 antifungal agents alone or in combination against Exophiala dermatitidis (E.dermatitidis) biofilms.Methods E.dermatitidis biofilms were prepared by using a modified 96-well plate-based method.The in vitro antifungal activity of amphotericin B,voriconazole,itraconazole and caspofungin alone or in combination against E.dermatitidis biofilms were investigated via the broth microdilution checkerboard technique.Results The sessile minimum inhibitory concentration ranges resulting in 50％ (SMICS0) and 80％ inhibition (SMIC80) of E.dermatitidis biofilms were all ＞ 32 mg/L for itraconazole,voriconazole and caspofungin,and the SMIC50 and SMIC80 ranges of amphotericin B were 1-2 mg/L and 4-8 mg/L respectively.The combination of amphotericin B with voriconazole showed synergistic inhibitory effects against E.dermatitidis biofilms,while the combination of amphotericin B with itraconazole or caspofungin,as well as the combination of voriconazole with caspofungin,revealed no synergistic effects.No antagonistic effect was observed in any of the combinations.Conclusion Amphotericin B appears more active against E.dermatitidis biofilms,and the combination with voriconazole can enhance the anti-biofilm effects against E.dermatitidis biofilms.
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Objective To evaluate in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against Exophiala dermatitidis (E.dermatitidis).Methods The minimum inhibitory concentrations (MICs) of itraconazole and terbinafine against 12 strains of E.dermatitidis were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M38-A2 Document).A broth microdilution checkerboard method was used to evaluate the in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against E.dermatitidis.Results The MIC ranges of terbinafine and itraconazole against E.dermatitidis were 0.060-0.125 mg/L and 0.5-1 mg/L,respectively.The combination of tacrolimus with terbinafine showed synergistic inhibitory effects against 5 strains of E.dermatitidis,while the combination of tacrolimus with itraconazole revealed synergistic effects against 10 strains of E.dermatitidis.No antagonism was observed in either of the two combinations.Conclusion In vitro combination of tacrolimus with itraconazole or terbinafine can enhance the antifungal activity of itraconazole or terbinafine against E.dermatitidis.
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Objective To investigate the value of Real-time PCR in the diagnosis of invasive aspergillosis(IA) and to compare it with galactomannan antigen assays.Methods A retrospective study was performed on 110 episodes of hospitalization of 88 patients who were at risk of invasive aspergillosis at Peking University First and Renmin Hospital from May 2008 to December 2010.23 cases with diagnosis and clinical diagnosis IA were classified as infection group and 87 cases with suspected diagnosis and non-IA were classified as non-infeciton group according to the international criterion.Real-time PCR and gaiactomannan antigen detections were performed on 257 serum samples.A receiver operating characteristic curve (ROC)was developed based on the quantitative cycle numbers and an optimal cut off value of quantification cycle (Cq) was determined.The sensitivity (Se),specificity (Sp),positive predictive value (PPV) and negative predictive value (NPV) were calculated under different considerations among which McNemar chi-square tests were used for statistical analyze.Results The area under ROC curve of Real-time PCR for the diagnosis of IA was 0.91 (95％ CI:0.825-0.995) and the optimal cut off value of Cq was 39.45.The Se and Sp of one positive PCR,two positive PCR,one positive GM and two positive GM were 87.0％,79.3％ ; 58.3％,97.8％ ; 78.3％,63.2％ ; and 58.3％,82.6％,respectively.When one positive PCR was considered as the diagnostic criterion of IA,Real-time PCR was able to diagnose 100％ and 84.2％ of proven and probable IA cases,respectively.The Sp of one/two positive PCR were statistically higher than one/two positive GM (P ＜ 0.05),respectively.The Sp of two positive PCR was statistically higher than one positive PCR (P ＜0.05).The Se and Sp were 65.2％,89.7％ and 100％,52.3％ for one positive PCR combined one positive GM and one PCR or GM positive,respectively.Conclusions Real-time PCR assays have better sensitivities and specificities than GM in the diagnosis of invasive aspergillosis.When two PCR positive were considered,better specificity and positive predictive value were achieved.
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A 5-year-old Mongolian girl from Inner Mongolia presented with a painless,round skin lesion on the left cheek for 10 months.Several weeks prior to the development of lesions,her left cheek was seratched by a dog.Subsequently small asymptomatic erythematous papules developed at the scratched site and gradually enlarged.Direct microscopy of scales from the lesions revealed septate,branching hyphae and fungal culture grew Eurotiom amstelodami(perfect stage of aspergillus).She was initially diagnosed as cutaneous aspergillosis and treated with itraconazole 200 mg per day for 2 months,but limited improvement was achieved.Histopathological examination with Wright-Giemsa staining of skin biopsies revealed abundant leishman Donovani hodies intracellulatry and extracellularly in the dermis and subcutaneous tissue; PAS staining of tissue specimens showed no fungal element and fungal culture was negative.She was finally diagnosed with cutaneous leishmaniasis.After treatment with terbinafine 125 mg per day for 2 months,the lesion subsided.