Chinese Journal of Mycology

Simultaneous identification of Aspergillus and Mucorales species by reverse line blot(RLB) hybridization assay

ZHAO Zuo-tao, LI Li-li, WANG Xiao-yang, WAN Zhe, CHEN Wei, LI Ruo-yu

2011, 6(5): 261-266.

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Objective To Rapidly differentiate Aspergillus and Mucorales species by reverse line blot hybridization(RLB).Methods Totally 98 isolates including five Aspergillus strains(Aspergillus fumigatus,Aspergillus flavus,Aspergillus niger,Aspergillus terreus and Aspergillus nidulans) and seven Mucorales species(Mucor heimalis,Mucor racemosus,Mucor cercinelloidea,Rhizopus arrhizus,Rhizopus microsporus,Rhizomucor pusillus and Absidia corymbifera) were obtained from Research Center for Medical Mycology and Mycoses,Peking University.ITS1 and ITS4 fungal universal primers were chosen for PCR amplification,and the amplified products were used for reverse line blot hybridization with 12 fungal specie-specific probes.RLB data were compared with those by traditional fungal morphology and ITS sequencing methods.Results RLB showed high sensitivity and specificity,with 100% correct identification percentage of all the isolates and no cross hybridization between the species-specific probes.Eight negative control stains(Candida albicans,Fusarium solani,Scedosporium apiospermum,Penicillium marneffei,Exophiala verrucosa,Aspergillus clavatus,Aspergillus japonicus and Cunninghamella elegans) also showed negative by RLB.The analytical sensitivity of RLB was 1.8×10^-3 ng/μL by 10-fold serial dilution of Aspergillus fumigatus genomic DNA.Conclusions The RLB assay provides a rapid and reliable option for laboratory diagnosis and identification of Aspergillus and Mucorales species.

Anti-fungal effect of sertraline on Cryptococcus neoformans in vitro and in vivo

ZHOU Nan, HUANG Chen, PAN Wei-hua, LIAO Wan-qing

2011, 6(5): 267-270.

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Objective To assess the effIcacy of sertraline on Cryptococcus neoformans.Methods Fungal-loaded mice were treated by fluconazole(10 mg/mL),sertraline(10 mg/mL,20 mg/mL),or fluconazole combined with sertraline(10 mg/mL & 10 mg/mL,10 mg/mL & 20 mg/mL).Drug-sensitivity tests were performed in vitro with different concentrations of sertraline and fluconazole above.Results Colony numbers of Cryptococcus neoformans decreased in vitro in sertraline group,while much more evidently in combination group of sertraline and fluconazole.In the early stage,both concentrations of sertraline could significantly decrease the colony numbers of Cryptococcus neoformans in lung and brain tissue.However,in the later stage,10 mg/mL of sertraline showed no anti-fungal effect in brain tissue.Fluconazole was superior to sertraline in lung and brain tissue,and much more efficient in antifungal therapy when combined with sertraline.Conclusions Sertraline is efficient on Cryptococcus neoformans,and may have synergistic action to fluconazole.

Cloning a novel catalase gene of Sporothrix schenckii with degenerate PCR and RACE

WANG Xiao-hui, LIU Wei, LI Ruo-yu

2011, 6(5): 271-275.

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Objective To isolate a novel catalase homologous gene from yeast-form Sporothrix schenckii and to make a designation.Methods Oligonucleotide primers were designed according to the conserved areas of the other 7 fungal catalase genes.Partial Sscat cDNA was amplified by PCR,and special primers were designed by RACE method to amplify the 3′cDNA and 5′cDNA.Results The full-length Sscat cDNA sequence was 1746 bp with an open reading frame of 1500 bp encoding 499 amino acids.The predicted molecular mass of Sscat was 56.07 kDa.The deduced amino acid sequence of Sscat showed 66.3% and 56.6% identity with those of Aspergillus oryzae and A.clavatus.An intron was identified within the 933-1063 bp Sscat genomic DNA sequence of S.schenckii.Conclusions Degenerate PCR combined with RACE is effective in searching and isolating novel genes of S.schenckii.

Dynamic study on the susceptibilities of caspofungin and micafungin to Candida species in vitro

ZENG Rong, LI Min, CHEN Qing, WANG Le, LV Gui-xia, SHEN Yong-nian, CAI qing, LI Cai-xia, TANG Rong-cai, LIU Wei-da

2011, 6(5): 276-280.

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Objective To assess the susceptibilities of Candida species to caspofungin and micafungin.Methods Susceptibilities of Candida isolates to caspofungin,micafungin and fluconazole were determined by microdilution method based on CLSI M27-A2 for consecutive seven days.Results After 48 hours,the medians of MIC₅₀ and MIC₉₀ of C.albicans,C.glabrata,and other Candida spp.to caspofungin were 0.03 and 0.03 μg/mL,0.06 and 0.125 μg/mL,0.125 and 0.5 μg/mL,respectively.The medians of MIC₅₀ and MIC₉₀ of C.albicans,C.glabrata,and other Candida species to micafungin were 0.03 μg/mL and 0.03 μg/mL,0.06 μg/mL and 0.06 μg/mL,0.25 μg/mL and 0.5 μg/mL,respectively.The medians of MIC₈₀ and MIC₁₀₀ of C.albicans,C.glabrata,and other Candida species to fluconazole were 2 μg/mL and 128 μg/mL,64 μg/mL and 128 μg/mL,2 μg/mL and 32 μg/mL,respectively.None of the 85 isolates of Candida species showed cross-resistance to caspofungin,micafungin or fluconazole.To caspofungin,there was no change on MIC₅₀ or MIC₉₀ of C.albicans after 24 hours.No increase on MIC₅₀ and MIC₉₀ of C.glabrata was observed after 72 hours and 120 hours.Neither did increase on MIC₅₀ and MIC₉₀ of other Candida species after 168 hours and 96 hours.To micafungin,there was no change on MIC₅₀ and MIC₉₀ of C.albicans and C.glabrata after 24 hours and no increase on MIC₅₀ and MIC₉₀ of other Candida species after 72 hours.Conclusions Caspofungin and micafungin may have great antifungal activities to all candida species,especially to C.albicans and C.glabrata.The MICs would increase with the exposure time,which show specificity to drug and Candida species.

Study on molecular epidemiology of Cryptococcus neoformans species using microsatellite marker

PAN Wei-hua, LIAO Wan-qing, WEN Hai, ZHAO Jin, Ferry Hagen, Teun Boekhout

2011, 6(5): 281-284.

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Objective To study the molecular epidemiology of clinical isolates of Cryptococcus neoformans in China.Methods Totally 116 clinical strains of Cryptococcus neoformans from 1993 to 2009 were isolated and the epidemiology was analyzed by microsatellite genotyping(STR).Results All the 116 strains of C.neoformans belonged to three microsatelite complexes(MCs),with the majority of MC2(n=103),and eight isolates belonged to an unknown microsatellite complex which was labeled as MC12.Conclusions STR is valuble in mocular epidemiological study on Cryptococcus neoformans.

Fungal rhinosinusitis casused by Scedosporium apiospermum:a case report and literature review

WANG Yan-ling, SUN Ji-mei, ZHOU Xiu-zhen, ZHANG Zhi-jie, ZHENG Wei, CUI Bing, LIU yong

2011, 6(5): 285-289.

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Objective To report a case of rhinosinusitis caused by Scedosporium apiospermum and to study the laboratory methods and the in vitro sensitivity to five kinds of antifungal drugs.Methods The mass cut off from the rhinosinusitis patient was prepared for microscopy(10% KOH and Gram dying),fungal culture(SDA) and molecular biological identification.In vitro drug sensitivity was determined by E-test.Results The isolate was identified as Scedosporium apiospermum.The MICs of five kinds of drugs were 0.064 μg/mL(voriconazole),1.500 μg/mL(caspofungin),16.000 μg/mL(fluconazole),>32 μg/mL(amphotericin B) and >32.000 μg/mL(5-flucytosine) respectively.Conclusions Fungal rhinosinusitis caused by Scedosporium apiospermum is rare in China and easily confused with tumor.Laboratory examination is important for correct diagnosis.Nasal endoscopic operation with antifungal treatment based on drug resistance test has therapeutic effect.

A case of primary cutaneous cryptococcosis in the face

GE Hong-fen, WANG Gui-zhi, ZANG Yun-shu, TANG Zhan-li, HAN Sha-sha, WANG Jun

2011, 6(5): 298-300.

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A case of primary cutaneous cryptococcosis in an immunocompetent-woman was reported.Erythema(3 cm×5 cm)with clear border and ulcers in the middle has been existed in the left face for about half a year.Granulomatous infiltration with mauve bodies were found by PAS staining.Cryptococcus spores were identified by alcian staining,mucicarmine staining and immunohistochemistry staining.The patient was diagnosed as primary cutaneous cryptococcosis and was cured with itraconazole(400 mg/d daily).

A case of hemorrhagic onychomycosis due to Aspergillus Sydowii

SHANG Pan-pan, FENG Xiao-ju, LI Dong-ming

2011, 6(5): 301-302.

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A 48-year-old storekeeper was sufferred from erythema in both toe nails for more than one year.White and green fluffy mould colony grew on the Sabouraud dextrose agar(SDA)with chloramphenicol at 25℃,which was identified as Aspergillus sydowii by morphology and rDNA sequence analysis.The patient was cured with terbinafine(250 mg daily for 1 month) after three months.

Child kerion due to Trichophyton rubrum:a case report

WANG Xue-lian, KONG Qing-tao, WANG Gao-feng, LIU Fang, SANG Hong

2011, 6(5): 303-304.

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To report one case of child kerion due to Trichophyton rubrum.A 5-year-old child had a history of abscess and scab with pruritus and pain on the hind head for 15 days.Trichophyton rubrum was identified by microscopy and culture.The child was cured by combined antifungal therapy.

Preservation of pathogenic filamentous fungi

WU Jing, LI Dong-ming, G.S.de Hoog, YAO Yi-jian

2011, 6(5): 305-307.

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Objective To explore a practical and effective method for fingi preservation.Methods Seventy-three strains of pathogenic filamentous fungi were preserved on Potato dextrose agar slants at 4℃ and in freezing tubes at-80℃,overlaid with 10%(v/v) glycerol after purification.Results The survival ratioes were 98.6% at -80℃ and 100% at 4℃ after one-year preservation.Conclusions These two methods are simple,convenient,and valueble in preservation of pathogenic filamentous fungi.

DNA sequence analysis in identification and classification of pathogenic fungi

ZHAO Zheng-juan, TIAN wei, ZHAO Jing-jun

2011, 6(5): 316-320.

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With the increasing incidence of fungal infections,molecular biological methods with quickness and accuracy are superior to phenotypic methods in diagnosis,in which DNA sequence analysis has the ability to identify the pathogenic fungi to species.Applications of DNA sequence analysis in fungal identification and classification were reviewed in this article.

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