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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.
ABSTRACT
Objective To establish an athymic (nu/nu)BALB/c mouse model with chronic subcutaneous infection due to Phialophora verrucosa (P.verrucosa),and to explore the role of T lymphocytes in defensing against invasive infection due to P.verrucosa.Methods Six immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of P.verrucosa hyphae suspensions at a concentration of 5.0 × 108 colony-forming unit (CFU)/ml into one footpad,while another 6 immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of 5.0 × 108 CFU/ml P.verrucosa conidium suspensions into one footpad.The degree of footpad swelling was measured with a vernier caliper every 3 days.Histopathological characteristics of infected footpads were further analyzed.Biopsy tissues were also subjected to fungal culture to analyze the growth of P.verrucosa in infection foci in mice.Results After the treatment with hyphae or conidium suspensions,the BALB/c mice experienced transient footpad swelling within 12 days,and basically recovered after 50 days.However,the nu/nu BALB/c mice experienced persistent footpad swelling with recurrent ulceration and crusting.As pathological examination revealed,all the inoculation loci in BALB/c mice experienced local infection,and the morphology of P.verrucosa in the infected foci was not changed over time.However,invasive infections due to P.verrucosa hyphae alone or a mixture of P.verrucosa hyphae and sclerotic cells were observed in all the inoculation loci in nu/nu BALB/c mice.The fungal culture showed that P.verrucosa could not grow in the footpads of BALB/c mice after 21 days,while P.verrucosa could persistently grow in the footpads of nu/nu BALB/c mice.Conclusion An experimental model with subcutaneous phaeohyphomycosis due to P.verrucosa has been successfully established by using nu/nu BALB/c mice,and the nu/nu BALB/c mice are more susceptible to P.verrucosa infection compared with the immunocompetent BALB/c mice.
ABSTRACT
Objective To establish an athymic (nu/nu)BALB/c mouse model with chronic subcutaneous infection due to Phialophora verrucosa (P.verrucosa),and to explore the role of T lymphocytes in defensing against invasive infection due to P.verrucosa.Methods Six immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of P.verrucosa hyphae suspensions at a concentration of 5.0 × 108 colony-forming unit (CFU)/ml into one footpad,while another 6 immunocompetent BALB/c mice and 6 nu/nu BALB/c mice were subcutaneously inoculated with 100 μl of 5.0 × 108 CFU/ml P.verrucosa conidium suspensions into one footpad.The degree of footpad swelling was measured with a vernier caliper every 3 days.Histopathological characteristics of infected footpads were further analyzed.Biopsy tissues were also subjected to fungal culture to analyze the growth of P.verrucosa in infection foci in mice.Results After the treatment with hyphae or conidium suspensions,the BALB/c mice experienced transient footpad swelling within 12 days,and basically recovered after 50 days.However,the nu/nu BALB/c mice experienced persistent footpad swelling with recurrent ulceration and crusting.As pathological examination revealed,all the inoculation loci in BALB/c mice experienced local infection,and the morphology of P.verrucosa in the infected foci was not changed over time.However,invasive infections due to P.verrucosa hyphae alone or a mixture of P.verrucosa hyphae and sclerotic cells were observed in all the inoculation loci in nu/nu BALB/c mice.The fungal culture showed that P.verrucosa could not grow in the footpads of BALB/c mice after 21 days,while P.verrucosa could persistently grow in the footpads of nu/nu BALB/c mice.Conclusion An experimental model with subcutaneous phaeohyphomycosis due to P.verrucosa has been successfully established by using nu/nu BALB/c mice,and the nu/nu BALB/c mice are more susceptible to P.verrucosa infection compared with the immunocompetent BALB/c mice.
ABSTRACT
A 19-year-old man was admitted to the hospital for erythema and nodules on the face and postauricular region for 6 years. Microscopic examination of lesion scrapings revealed brown septate hyphae. A restricted, velvety and black colony grew on Sabouraud's dextrose agar. Slide culture on potato dextrose agar plate showed flask-shaped phialides at the apex of or around the hyphae with clear collarettes and flaring apex,mucilage-encapsuled, round to oval, semi-endogenous phialosporae accumulating at the apex of the phialides,giving a flower-like appearance. Anti-fungal susceptibility test showed that the fungus was sensitive to itraconazole, terbinafine and amphotericin B, but resistant to fluconazole. Sequence analysis of the ITS1-ITS4 region revealed a 98% consistency with the reference sequence of ITS1-ITS4 of Phialophora verrucosa. On the basis of above findings, the patient was diagnosed with cutaneous phaeohyphomycosis. Clinical improvement was seen after treatment with oral itraconazole (400 mg/d).
ABSTRACT
Chromoblastomycosis is a chronic fungal disease of the skin and subcutaneous tissues caused by a group of dematiaceous (black) fungi. The most common etiologic agents are Fonsecaea pedrosoi and Cladophialophora carrionii, both of which can be isolated from plant debris. The infection usually follows traumatic inoculation by a penetrating thorn or splinter wound. Several months after the injury, painless papules or nodules appear on the affected area; these papules then progress to scaly and verrucose plaques. We report a case of chromoblastomycosis caused by Phialophora richardsiae, which has been rarely associated with chromoblastomycosis. The case involved a 43-year-old male, who for the past 2 months had noted an erythematous, pustulous plaque that was somewhat dark brown in color on his right shin; the plaque also had intermittent purulent discharge and crust formation. On histopathological examination, chronic granulomatous inflammation and sclerotic cells were seen. The tissue fungus culture grew out the typical black fungi of P. richardsiae, which was confirmed by polymerase chain reaction. The patient has been treated with a combination of terbinafine and itraconazole for 3 months with a good clinical response.
Subject(s)
Adult , Humans , Male , Chromoblastomycosis , Fungi , Inflammation , Itraconazole , Naphthalenes , Phialophora , Plants , Polymerase Chain Reaction , Skin , Subcutaneous TissueABSTRACT
Objective To rapidly detect and identify pathogenic fungi of some deep fungal infections by PCR.Methods The suspensions of22pathogenic fungi(23strains)were amplified by PCR with fungal universal primers ITS86and ITS4which were labeled by FAM.The precise length of amplified fragments was determined by ABI PRISM TM 377Sequencer and Genescan analysis software,then compared with that of am-plicons of corresponding fungal DNA which were previously extracted.Results(1)Amplification of17pathogenic fungi with ITS4,ITS86resulted in a unique fragment length(except for A.nidulans and A.niger,C.albicans and C.stellatoidea,F.pedrosoi and E.dermatitidis).(2)No significant difference of the length of am-plicons was found between the fungal suspension and control organisms,based on the results of Genescan analysis.(3)The whole process took only6h to complete the detection.Conclusion The combination of fun-gal suspension PCR with ITS fungal universal primers and Genescan analysis might provide an accurate,spe-cific,sensitive,and rapid approach to detect and identify22pathogenic fungi causing deep fungal infections,and hold promise to be applied for the diagnosis of deep fungal infection.
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Objective To report a case of chromomycosis caused by Phialophora verrucosa and explore the laboratory features of the pathogen. Methods Skin lesion was examined by histopathology and fungus culture. The morphology of the isolate was observed by microscopy and scanning electron microscopy. The coenzyme Q system of this isolate was analyzed by HPLC assay. The DNA sequences of LSU rDNA D1/D2 region of this isolate and a standard fungus strain were compared. Results The initial lesion was an erythematous papule that subsequently developed into one or multiple coalescing warty papules or plaques slowly. The bronze-colored spores could were observed in the dermis or macrophages. The isolate grew very slowly, requiring 4 weeks of incubation. Microscopically, no characteristic structures were found on Sabourand′s dextrose agar, while there were vase-like structures, which were referred to as phialides on potato dextrose agar (PDA) and corn meal agar I (CMA-I). The phialides on PDA mostly grew at the top of hypha, but on CMA-I they mostly grew on the side of hypha. The isolate contained coenzyme Q-10, and its DNA sequence of LSU rDNA D1/D2 region completely consistent with those of the standard strain. Conclusion Chromomycosis caused by Phialophora verrucosa is rare in China. It can be diagnosed by fungus culture and histopathological examination. Coenzyme Q system analysis and DNA sequencing can exclude the interference from different phenotypes.
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Um paciente procedente da Amazônia do Brasil, seringueiro, portador de uma branqueada de haseníase, tratado com sulfonas, tendo sequelas lesões disfróficas das extremidades e manchas hipocrômicas, com pertubações da sensibilidade, apresentou no hipocôndrio esquerdo uma lesão verrucosa extensa, com evolução de mais de 10 anos, rebeldes aos tratamentos feitos. Foram feitas biopsias para esclarecimento histopatologico da doença, tendo-se concluido trata-se de dermatite verrucosa cromomicótica, com presença dos fungos bem caracterizados pelas colorações de rotina, histoquíomicas. A cultura do material obtido da lesão em meio ágar-Sabouraud deu crescimento a colônia gigante com as caracteristicas típicas do genêro Philophora: hifasseptadas e conidioforos com morfologia de taca. O tratamento, na falta de 5-fluorocitosina, foi feito com infiltrações de anfotericina B, associada a iodureto de sódio, endovenosamente, estando a lesão em plena regressão. Os exames histopatológicos das lesões discrômicas mostraram apenas infiltrado linfoplasmocitário e ausência de bacilos álcool-ácido resistentes íntegros (AU).