Table of Content

28 December 2016, Volume 11 Issue 6

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论文

Purification and C-terminal domain comparison of heat shock protein 90(HSP90) in Candida albicans and human

MENG Jing-shun, WU Jian-hua, ZHAO Jing, WANG Yu-lian, XUE Wei

2016, 11(6):  321-326. 

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Objective To express and purify Candida albicans HSP90 (CaHSP90) and human HSP90 (hHSP90) and to compare the different effects of C-terminal domain on their oligomeric forms in vitro.Method The encoding sequences for wild type CaHSP90,hHSP90 and their C-terminal enzyme digested base sequences were amplified via PCR and the products were cloned into pET-22b⁺ vector using NdeI/XhoI cloning sites.The plasmids were expressed in E. coli BL21 (DE3) cells and the His-tagged proteins were purified through Ni²⁺-NTA column and Superdex-200 column chromatography on FPLC. The molecular weights and oligomeric forms of these proteins were analyzed by SDS-PAGE and size exclusion chromatography (SEC).Results All plasmids were successfully constructed by DNA sequencing and the proteins were pure enough for SEC analysis.The results showed that wild type CaHSP90 and hHSP90 have similar oligomeric forms,while their C-terminal mutants were different.Conclusion The C-terminal domain of CaHSP90 and hHSP90 have different effect on their oligomeric forms in vitro and might be a potential target for CaHSP90 inhibitor.

Molecular identification and genotyping and in vitro drug sensitivity study of the first five clinical isolates of Cryptococcus neoformans in Xinjiang

Yultuz·Xohrat, Kadirye·Kasim, WANG Xiao-dong, DENG Shu-wen, Prida·Abliz

2016, 11(6):  327-331. 

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Objective For the first time to perform the molecular identification,RAPD-PCR genotyping and in vitro drug sensitivity study of 5 clinical strains of Cryptococcus neoformans in Xinjiang.Methods Molecular classification using primer SEQ-6 from big subunit ribosomal DNA (LSU rDNA) D1/D2 gene region and RAPD-PCR method were applied in 5 strains of clinical isolated Xinjiang Cryptococcus neoformans strains and 5 Cryptococcus strains provided by Shanghai Changzheng hospital.Sensitivity in vitro of 5 clinical isolated Xinjiang Cryptococcus neoformans strains of the above 6 kinds of antifungal drugs (Voriconazole,itraconazole,amphotericin B,terbinafine,fluconazole,5-fluorine cytosine)were judged according to clinical laboratory standards institute (CLSI) trace liquid dilution method of yeast (M27-A3).Results The 5 strains of Xinjiang were identified as Cryptococcus neoformans through D1/D2 region sequence analysis.All 10 strains are divided into A,B,C,D 4 genotype by RAPD-PCR.One Xinjiang strain was type A,the rest 4 strains were type B;Among 5 Shanghai strains,1 strain was type B,3 strainswere type C,1 strain was typeD.For the 5 Xinjiang clinical Cryptococcus neoformans strains,to the MIC value ranges of voriconazole,itraconazole,amphotericin B and terbinafine were as following:0.062 5-0.25 μg/mL,0.25-1 μg/mL,0.125-0.5 μg/mL,1-2 μg/mL,respectively;MIC value ranges of fluconazole,5-fluorine cytosine MIC value were much higher as:4-16 μg/mL,8-32 μg/mL,respectively.MIC values of genotype A and B to voriconazole were 0.062 5-0.125 μg/mL.Conclusion The 5 Xinjiang clinical isolates of Cryptococcus were identified as Cryptococcus neoformans using D1/D2 genes analysis.RAPD-PCR revealed these clinical Cryptococcus neoformans strain in Xinjiang with priority given to type B genotypes.Type A and type B isolates weremore sensitive to voriconazole,amphotericin B,itraconazole and terbinafine.

Comparison of azoles MICs against Candida spp.determined by the broth microdilution method at different incubation time

GUO Li-na, XU Zhi-peng, WANG Yao, WANG He, ZHAO Ying, XIAO Meng, XU Ying-chun

2016, 11(6):  332-336. 

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Objective To investigate the agreement between the 24 h and 48 h azoles MICs against Candida species by broth microdilution method.Methods Antifungal susceptibility testing was carried out by broth microdilution method against 653 Candida isolates and MICs were obtained at 24 h and 48 h incubation for assessing of essential agreement (EA),categorical agreement (CA) and categorical error (Error).Results Most of Candida isolates tested could obtain sufficient growth after 24 h incubation.The overall EAs of 24 h and 48 h MICs were excellent with the three azoles for most of the species,fluconazole (91.9%),voriconazole (92.0%) and itraconazole (95.6%);the overall CAs were wonderful with fluconazole (96.9%),voriconazole (90.4%) and itraconazole (88.2%),depending on the species type tested.Very major errors (VMEs) were absent in our study,minor errors were reported most of the time.The ME/MiE for the total 653 Candida isolates were fluconazole (1.7%/2.9%),voriconazole (2.0%/10.6%) and itraconazole (1.5%/13.0%).Conclusion These data suggested that the performance 24 h MIC readings gave results similar to those of 48 h MICs and reading MICs at 24 h incubation would shorten the MIC determination of azoles and avoid errors due to trailing misinterpretation.

Effect of Pseudomonas aeruginosa lipopolysaccharide on biofilm formation of Candida albicans

DENG Juan, SUN Wei, SU Jian-rong

2016, 11(6):  337-340. 

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Objective To explore the effect of Pseudomonas aeruginosa lipopolysaccharide on Candida albicans biofilm formation.Methods XTT reduction assay was used to elucidate the effect of Pseudomonas aeruginosa lipopolysaccharide on biofilm formation of Candida albicans.Results These date are indicative that Pseudomonas aeruginosa lipopolysaccharide modulate Candida biofilm formation in a time-dependent and concentration-dependent manner.Conclusion Pseudomonas aeruginosa lipopolysaccharide could modulate Candida albicans biofilm formation by inhibit the hyphal development.

Inhibitory effects of chloroform extracts of Longdan xiegan decoction against hyphae formation of Candida albicans isolated from VVC

WANG Xia, WU Da-qiang, SHI Gao-xiang, DUAN Qiang-jun, SHAO Jing, WANG Tian-ming, WANG Chang-zhong

2016, 11(6):  341-347. 

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Objective To investigate the effects of chloroform extracts of Longdan xiegan decoction (CELX) against hyphae formation of Candida albicans clinical strains isolated from Vulvovaginal Candidiasis (VVC).Methods Microdilution method was used to determine the minimal inhibitory concentrations (MICs) of Longdan xiegan decoction extracts against C. albicans isolated from VVC. Solid agar and Semi-solid agar were utilized to evaluate colony morphology and invasive growth of C. albicans,respectively.XTT and Fluorescence microscope assay were applied to determine the metabolic activity of hyphae of C. albicans treated by CELX for 6 h.Plate assay counted CFU variation.Quantitative Real-time PCR (qRT PCR) was adopted to analyze the expression of hyphae-specific genes including ALS3,SSA1,SUN41,HWP1,UME6 and ECE1.Results The MICs of CELX against C. albicans strains were between 32 and 64 mg/L,which were better than MICs of total extract,petroleum ether extract,ethyl acetate extract,buty alcohol extract and water extract.CELX with concentration of 256 mg/L inhibited hyphae formation and influenced colony morphology significantly,with the wrinkle in solid agar and invasion in semi-solid agar being blocked.The expression of hyphae formation related genes,ALS3,SSA1,SUN41,HWP1,UME6 and ECE1 were down-regulated by 81.6%,60.2%,69.8%,73.7%,49.5%,52.8%.Conclusion Our results suggested that CELX could inhibit hyphae formation through repress hyphae-relative genes expression to reduce the invasive activity against patient.

Re-evaluation of the traditional Chinese medicines (TCMs) with antifungal activities recorded in ‘Zhong Yao Da Ci Dian’

WU Jing, WANG Liang, CAO Yong-bing, YAN Lan, JIANG Yuan-ying

2016, 11(6):  348-353. 

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Objective To investigate the antifungal activity of 127 types of the traditional Chinese medicines (TCMs) with the so-called antifungal activities recorded in ‘Zhong Yao Da Ci Dian’ against Candida albicans,Cryptococcus neoformans and Aspergillus fumigatus in vitro.Methods The portion from each TCM by 75% alcohol extracts were determined for antifungal activity against C. albicans, C. neoformans and A. fumigatus by using the standard CLSI M27-A3 and M38-A2 broth microdilution assays,and further to study the antifungal spectrum and inhibition of hyphae.Results The ethanol extracts from 17 TCMs exhibited the inhibitory activity against C. albicans with MICs ranged from 15.625 to 2 000 μg/mL.34 kinds of ethanol extracts from TCMs had the inhibitory effect against C. neoformans.5 ethanol extracts from TCMs had the inhibitory activity of inhibiting A. fumigatus.Conclusion The antifungal activities of TCMs which has been recorded in ‘Zhong Yao Da Ci Dian’ are measured in vitro by the consistent standard method for the first time,providing the experimental basis for investigation of new antifungal agents.

Hyalohyphomycosis caused by Sarocladium implicatum: first case report in China

CHEN Ping, ZHOU Ya-bin, SUN Ting-ting, WANG Xing-jia, LI Dong-ming

2016, 11(6):  354-356. 

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Objective To report the first case of hyalohyphomycosis caused by Sarocladium implicatum (Acremonium implicatum) in China.Methods A 69-year-old female patient came to us with an nodule on the left forearm for 9 months.Tissue was biopsied for microbiological and histopathological studies.Pathology showed inflammation,necrosis and granulation with fungal element in tissue.A positive fungus was recovered that was later identified as Sarocladium implican (Acremonium implicatum) according to morphology and DNA sequencing identification.Results This case was diagnosed as hyalohyphomycosis according to the finding of uncolored fungal elements in histopathological examination,with the positive fungal culture of the tissue.The isolate grown on culture was demonstrated belong to S. implicatum by DNA sequence analysis.She was cured after 3 months treatment with oral kalium iodidum solution and topical sertaconazole nitrate cream.Conclusion This is the first case of hyalohyphomycosis caused by S. implicatum in China confirmed both by culture and by pathological methods.Morphological characteristics and sequence of the ITS region of ribosomal DNA confirmed the identification of the isolate.Combination of kalium iodidum and sertaconazole is effective.

A case of cutaneous mycosis caused by Arthrinium phaeospermum

CHEN Xue-wen, ZHU Hong-mei, WEN Hai

2016, 11(6):  357-360,365. 

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Objective Report of a case of fungal infection caused by Arthrinium phaeospermum and the relevant mycological study.Methods A-29-woman had suffered symmertrical infiltrative erythema for several years.Direct microscopy,culture,microculture,strain identification,drug susceptibility test in vitro,animal experiment were performed.Results While direct microscopy was negative,culture was positive and Arthrinium phaeospermum was identified.The typical characteristics of this isolate were observed with Medan staining,microculture and scanning electron microscopy.This isolate was resistant to itraconazole and fluconazole,but was susceptible to caspofungin,micafungin,posaconazole,ravuconazole,voriconazole and terbinafine.Animal experiment shows the very Arthrinium phaeospermum was able to cause disease.Conclusion This was a rare dermatomycosis caused by Arthrinium phaeospermum.Mycological examinations,drug susceptibility test in vitro,and animal experiment would have active guidance meaning and applied value for further cases.Abnormal immune status might be a risk factor for such cases.

A survey on indoor and outdoor airborne fungal species distribution in a general hospital located in Shanghai, China

HONG Nan, FANG Wen-jie, LIU Jia, CHEN Min, PAN Wei-hua, LIAO Wan-qing

2016, 11(6):  361-365. 

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Objective To monitor the characteristics of airborne fungi distribution around a Chinese general hospital during Autumn in Shanghai,for scientific basis for clinical prevention and treatment of invasive fungal infections.Methods In a randomly selected general hospital in Shanghai,collecting air samples in 26 indoor and 7 outdoor environments using cyclone-based bioaerosol sampling method and precipitation method,simultaneously.The samples were cultivated,separated and purified on the Sabouraud Agar with chloramphenicol and then identified in molecular technique.All of the data obtained was analyzed statistically.Results The genus Penicillium (24.14%) was prominent in the fungal population structure in indoor environments of the enrolled hospital.In contrast,the genus Cladosporium (34.09%) has the advantage in outdoor environments.The proportion of the genus Penicillium in indoor environments (24.14%) is significantly higher than that in outdoor environments (11.09%).Conclusion Although the level of medicine in Shanghai remains leading position in China and the investigated hospital has been conducting disinfection work according to regulations,there is still a high diversity of fungal species distribution found in the surveyed hospital,which contains common opportunistic pathogenic fungi such as Aspergillus fumigatus,etc.Therefore,continue to reinforce the research on environmental surveillance and infection control of general hospital is of significance.

A misdiagnosed case of invasive aspergillosis

GUO Kai, XIANG Zhen-zhen, SANG Jun-jun, CHEN Li

2016, 11(6):  372-375. 

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We reported a case of invasive aspergillosis.A male patient,32 years old,had undergone an endovascular repair of abdominal aortic aneurysm in department of vascular surgery.The result of intraoperative frozen pathology of abdominal mass revealed Castleman's disease.For further treatment,the patient transferred to our department.PET-CT revealed malignant lymphoma.Examination of bone marrow aspirate showed no abnormal sign.Histopathological findings revealed features of fungal infection in lymph nodes and abdominal mass.Despite Micafungin therapy,the patients died of multiorgan failure.We extracted fungal DNA from fungal pathology.ITS sequencing confirmed the fungus as Aspergillus fumigatus.

A case of lymphocutaneous sporotrichosis

JIANG Yuan, KONG Qing-tao, ZHANG Zheng, ZENG Qiu-qiong, SANG Hong

2016, 11(6):  376-377. 

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To report one case of lymphocutaneous sporotrichosi.A 79-year-old woman presented to our clinic with a 2-month history of painful erythematous nodules linearly arranged on her left upper limbA biopsy specimen and PAS staining of her left upper limb demonstrated changes of pyogenous granulomatous inflammation and fungal spores.Sporothrix was identified by fungal culture and ITS sequencesThe patient was treated with oral itraconazole 0.2 g twice daily.The lesions subsided after 1-month treatment.

Comparison of fluorescent staining method and KOH wet method for diagnosis of superficial fungal infections

CHEN Rui-jun, TONG Tao, LI Chao, WU Xian, CHEN Qiong, CHEN Xiao-nan, XU Ming, CHEN Bo-rui

2016, 11(6):  378-380. 

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Objective To explore the application value of fluorescent staining method for superficial fungal microscopic examination.Methods One hundred cases with suspected fungal infection were collected in the department of dermatology.Fluorescent staining method and KOH wet method were used respectively to detect these samples,and the results were compared.Results A total of 56 cases of fungal positive were detected with fluorescent staining method,while there were only 39 cases of fungal positive with KOH wet method.The positive detection rate of fluorescent staining method was higher than that of KOH wet method (χ²=5.79,P<0.05).Conclusion The fluorescent staining method is a rapid and accurate method to detect fungi.It is worth to be used in the department of dermatology for the early diagnosis of fungal infection.

A multicenter study on curative effect of 1% luliconazole cream in treatment of candidal balanoposthitis

GAO Fang-ming, YI Jiang-hua, YE Xing-dong, XIAO Chang-qing, GAO Ai-li, LIU Yu-mei, DAI Xiang-nong

2016, 11(6):  381-384. 

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Objective To investigate the curative efficiency and safety of luliconazole 1% cream in treating candidal balanoposthitis.Methods A total of 70 outpatients from department of Dermatology & STD were randomly divided into two groups.Among them,38 were given luliconazole 1% cream once a day (study group) while the others were given ketoconazole cream once a day (control group).The effect was evaluated based on scores of relief for symptoms,skin signs,and mycology inspection,while the safety was evaluated according to the side effects.Results The mycological clearance rate of 1% luliconazole cream was significantly higher than that of 2% ketoconazole cream one week after treatment (P<0.05),but there was no significant difference of clinical efficacy and mycological clearance rate between the two groups two weeks after treatment (P>0.05).No significant difference was observed between the two groups in evaluation scores of clinical effect,symptom and skin sign aspects.Conclusion The curative effect and safety for 1% luliconazole cream were comparable to those of 2% ketoconazole cream in treating candidal balanoposthitis.It is worth popularizing and applying clinically.