Table of Content

28 June 2016, Volume 11 Issue 3

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论文

Cryptococcus traversal across the Blood-Brain Barrier though S100A10-dependent activation of urokinase-plasmin system

QIU Tian wen, ZHU Hong-mei, WEN Hai

2016, 11(3):  129-134. 

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Objective To find if Cryptococcus traversal across the blood-brain barrier though S100A10-dependent activation of urokinase-plasmin system.Methods ①The mouse brain microvascular endothelial cells bEnd.3 and Cryptococcus neoformans B3501 were co-incubated.Then the expression of S100A10 gene and UPA genewere tested.②Expressionof S100A10 and UPA in S100A10 normal and silent groups of endothelial cellsInfected with lentivirus were also compared.③The abilities of Cryptococcus S100A10 normal and silent groups traversal across the transwell blood-brain barrier model were compared.Urokinase-related activation of plasminogen was tested by the chromogenic substrate assay.Results ①In bEnd.3 incubated with Cryptococcus neoformans B3501,Real-time PCR display high expression of mRNA of S100A10 and UPA;Western-blot show S100A10 protein and UPA protein overexpression.②While S100A10 gene been silenced,S100A10 and UPA expressions were suppressed.③Cryptococcal traversal across the transwell in vitro BBB model in S100A10 silenced group was less than that of controlgroup.The diffences on 8 h,16 h,24 h were especially statistically significant,P<0.05.The activation of urokinase and plasmin was also lower than that of control group.The diffences of plasminogen activation on 8 h,16 h and 24 h in two groups,the diffences of urokinase activation on 16 h and 24 h in two groups,were statistically significant,respectively.Conclusion Cryptococcus traversal across the blood-brain barrier might be though S100A10-dependent activation of urokinase-plasmin system.In the meantime,the S100A10 gene and UPA gene of brain microvascular endothelial cells were highly expressed.

Evaluation of antifungal drugs using a Caenorhabditis elegans-Candida albicans infection model

BI Shuang, HU Dan-dan, JIANG Yuan-ying, WANG Yan

2016, 11(3):  135-139. 

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Objective To investigate the reliability and stability of Caenorhabditis elegans-Candida albicans infection model in the application of antifungal drug evaluation,and to evaluate the value of this model in antifungal drug development.Method C.albicans was ingested by C.elegans and caused a persistent lethal infection to establish this infection model.Common antifungal drugs includingfuconazole,amphotericin B,caspofungin and 5-fluorocytosine,and farnesol,a quorum sensing molecule secreted by C.albicans,were used to treat the infected nematodes.The survival rate of the nematodes was observed to evaluate the activities of antifugal agents.Results C.albicans infected C.elegans,formed hyphae piercing the cuticle of the nematodes and resulted in the death of the nematodes.Fluconazole,caspofungin and 5-fluorocytosine inhibited the hyphal formation of C.albicans in different degrees in this in vivo model,improving the survival of C.elegans in a dose-dependent manner.Amphotericin B also showed antifungal effect in the C.elegans-C.albicans model,while at high concentrations as 16 or 32 μg/mL,the toxicity of amphotericin B was exhibited.Farnesol inhibited the hyphal formation of C.albicans,but showed weak protection effect to C.elegans.Conclusion The C.elegans-C.albicans infection model is reliable for antifungal drug evaluation.Moreover,this model has high-throughput,low-cost,time-saving properties,and is worthy to be widely used in antifungal drug development.

Optimization of the protoplast-mediated transformation system of Penicillium marneffei

CHEN Chun-mei, FENG Jiao, CHEN Zhi-wen, XIAO Xing, JIANG Min-min, SHI Ming-lan, HE Li-ya, CAI Wen-ying, XI Li-yan, LI Xi-qing

2016, 11(3):  140-144. 

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Objective This study aims to optimize the protoplast-mediated transformation system of Penicillium marneffei,it would provide a good platform for the gene function research of P.marneffei.Methods We inserted the selectable marker pyrG gene (from A.nidulans) into P.marneffei strain ligD (pyrG-,ligD-) by protoplast method,screened positive transformant by medium without uracil,and used PCR to verify the restructuring.Through changing some important effecting factors including enzymolysis period,concentration of plasmid,protoplast fusion agents PTC and screening culture medium to find out the optimal protocol.Results As a result,The positive transformants growed well in the media without uracil,and PCR analysis showed that pyrG was inserted into the transformants,the appropriate conditions for protoplast-mediated transformation of P.marneffei were as follows:enzymatic time is 6 h;PTC:60%PEG-4000,100 mmol/L Tris-HCl pH8.0,100 mmol/L CaCl₂;0.6 mol/L sucrose screening culture medium;add 2.5 μg plasmid per 100 μL protoplast (10⁶~10⁷/mL).The transformation efficiency was about 68 transformants/μg plasmid DNA under these conditions.Conclusions Protoplasts-mediated transformation in P.marneffei was successfully optimized,this method could get high transformation efficiency and provide a good platform for P.marneffei gene function research.

A rare case of Aspergillus terreus endogenous endophthalmitis in a nonimmunocompromised patient and laboratory research

ZHAO Ying, WANG Peng, SUN Hong-li, YANG Qi-wen, WANG Yao, WANG He, XU Ying-chun, ZHAO Yu-pei

2016, 11(3):  145-148. 

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Objective To report a rare case of Aspergillus terreus endogenous endophthalmitis in a nonimmunocompromised patient and laboratory research.Methods A vitreous biopsy was taken for culture and direct smear microscopy,and further identified the isolate from culture positive plates by phenotype observation and molecular methods.In vitro antifungal susceptibility test was done by E-test method.Results Fungal spores and septate hyphae were found in vitreous direct smear.Tiny hair like colonies growed after 7 days in culture.The isolate was final identified as Aspergillus terreus according to morphology of the fungus and the results of phenotypic and molecular identification.The isolate's MICs (μg/mL) to itraconazole,voriconazole,amphotericin B were 0.25,0.125,2,respectively.Conclusion Early diagnostic and therapeutic vitrectomy was useful to obtaining sufficient samples for organism identification and also to reduce organism load and inflammatory mediators in the eye.Reporting the direct smear results timely to clinical physician is very meaningful for the initial selection of antimicrobial agents.For the clinical isolated filamentous fungi with atypical phenotype,ITS sequence analysis and other molecular methods can help to identify the fungal pathogen promptly.

In vitro activity of triterpene saponins from tea plant (Theaceae) against Candida species

XIA Xiao-ping, YE Hong, ZHENG Cai-hong, FENG Chun-hong, LIU Long-xiao

2016, 11(3):  149-152. 

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Objective To explore the antifungtal activity of TS from Camellia sinensis L.against Candida.Methods The minimal inhibitory concentration (MIC) was tested by broth microdilution method based on CLSI (Clinical and Laboratory Standards Institute).The minimal fungicidal concentration (MFC) was tested by agar plate dilution assay.The ultrastructure of Candida albicans treated with TS was observed by transmission electron microscopy.Result The range of inhibitory concentration of the antifungal activity against Candida albicans were from 0.08 mg/mL to 0.32 mg/mL.The minimal fungicidal concentration against Candida albicans was 0.64 mg/mL.Candida albicans treated with TS showed that the cell wall shrinked and ruptured,the cell nucleus had the abnormal morphology and the cell organelles were imcomplete as well as vacuoles were formed under TEM.Conclusion TS has antifungal activity against Candida species in vitro.

In vitro antifungal activity of sertaconazole against clinical isolates of Candida spp.

WANG Qian, WU Wei-wei, ZHANG Hao, WAN Zhe, LI Ruo-yu, WANG Ai-ping, LIU Wei

2016, 11(3):  153-155. 

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Objective To investigate the in vitro antifungal activity of sertaconazole against the clinical strains of Candida spp.isolated from China.Methods Minimal inhibitory concentration (MIC) of sertaconazole against 112 strains were tested by using CLSI (Clinical and Laboratory Standards Institute)broth microdilution method M27-A3.Results The overall geometric mean 24 h MIC of sertaconazole was 0.18 μg/mL with a MIC range of ≤0.03~16 μg/mL,MIC₅₀ and MIC₉₀ of sertaconazole were 0.125 μg/mL,1 μg/mL,respectively.In the same time,the geometric mean MIC was 0.080 μg/mL for C.albicans,0.158 μg/mL for C.glabrata,0.318 μg/mL for C.parapsilosis,0.425 μg/mL for C.tropicalis,1.414 μg/mL for C.krusei.Conclusion Sertaconazole is active against pathogenic Candida spp..

Synergistic effect of rifapentine with fluconazole against FLC-resistance Candida albicans

WANG Yu-lian, ZHANG Lu-lu, QIN Yu-lin, CAO Yong-bing, WU Jian-hua

2016, 11(3):  156-159. 

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Objective To explore the in vitro synergistic effect of rifapentine with fluconazole on FLC-resistance Candida albicans.Methods The minimal inhibitory concentrations (MIC) of rifapentine and fluconazole against Candida albicans were detected by method of Clinical and Laboratory Standards Institute (CLSI) M27-A2 microdilution.The effects of rifapentine and fluconazole against Candida albicans in vitro were valued by broth microdilution checkerboard technique system.Results In the antifungal susceptibility assay,there was no antimicrobial activity of rifapentine single used against the 12 strains of Candida albicans (MIC>1 024 μg/mL).The combined of rifapentine and fluconazole didn't display synergistic effect on the FLC-sensitive Candida albicans (FICI>0.5),however,it had a significant synergistic effect on the FLC-resistance Candida albicans (FICI<0.1).Conclusion Rifapentine alone demonstrated no antifungal activity,but rifapentine can significantly enhance the effect of fluconazole against FLC-resistance Candida albicans.

Strain distribution and drug susceptibility results analysis of 1 135 Candida strains

DENG Lin, KONG Qing-tao, CHEN Jun, SANG Hong

2016, 11(3):  160-165. 

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Objective To explore the characteristics and drug resisitance of Candida infection in our hospital from Jan.2012 to Oct.2014,to provide aetiological basis for clinical rational use of antifungal agents.Methods Retrospective investigation and analysis method were used in this study.Results A total of 1 135 Candida strains were detected,most of which were C.albicans,followed by C.tropicalis,C.glabrataa,C.parapsilosis and C.krusei.Mainly from the older population,Candida distribution had obvious difference in differentage groups (P<0.05);Men more than women (P<0.05).With sputum specimen was the most,urine second,blood in the third it showed obvious difference in specimen source distribution (P<0.05).Candida infections were mainly from patients with digestive diseases and lung disease.The sensitivity to fluconazol,voriconazole,itraconazole and amphotericin B had statistically difference in different Candida (P<0.05) while the sensitivity to fluorouracil was not statistically significant (P>0.05).Conclusion Candida albicans is the main pathogen of fungal infection followed by tropical Candida.Elderly patients and lung disease patients should guard Candida infections.Fungal isolation,identification and efficacy monitoring are very important to guide clinical rational drug using.Clinical antifungal therapy should be based on the results of strain identification and antifungal susceptibility test.

Distribution and drug resistance analysis of nosocomial deep Candida infection pathogens

QIN Xiao-feng, WANG Ying, WU Jian-hua, GU Jun

2016, 11(3):  166-168. 

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Objective To understand the nosocomial deep Candida infection types,pathogenic strain distribution and the susceptibility to common antifungal agents,to provide reliable basis for clinical diagnose and treatment.Methods The fungi were isolated and cultured by using the conventional methods and identified by CHRO mager medium and API-20C AUX test.The drug susceptibility test was determined by ROSCO disc diffusion method.Results Totally 2 725 strains of Candida spp.were isolated,including Candida albicans (64.73%),Candida tropicalis (13.64%),Candida glabrata (10.57%) and Candida rusei (2.35%).The Candida spp.were isolated from the sputum (45.39%),the urine (22.06%) and the throat swab (16.66%) respectively.The four Candida species were sensitive to amphotericin B and 5-fluorocytosine.Conclusion Candida albicans was still the most common Candida spp.in the nosocomial deep Candida infections.The drug resistance rates of amphotericin B and 5-fluorocytosine were the lowest,while there was a genus difference in drug resistance of the azole drugs.

Antifungal susceptibilities of 36 isolates from tinea pedis evaluated with CLSI M38-A2 and M27-A3 schemes

BU Xiao-lin, FAN Juan, HU Xiao-li, JIANG Jian-hua, QU Zi-wei, GU Jun

2016, 11(3):  169-173. 

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Objective To investigate the antifungal characteristics of fluconazole (FCZ),itraconazole (ITR),voriconazole (VCZ),ketoconazole (KETO),terbinafine (TER),amphotericin B (AmB),miconazole (MIC),5-fluorocytosine (5-FC) to thirty six isolates from tinea pedis.Methods A total of fifty five fungal samples from suspected patients with tinea pedis were examined by microscopy,culture and identification.Minimum inhibitory concentrations (MICs)were determined by Clinical and Laboratory Standards Institute (CLSI)M38-A2 and M27-A3 scheme.Results Thirty six strains of pathogenic fungi were isolated,which were positive both in microscopic examination and culture.They were identified as eight species:Trichophyton rubrum,Trichophyton mentagrophytes,Microsporum flavescens,Trichophyton floccosum,Microsporon lanosum,Candida albicans,Candida glabrata,Candida krusei,and two categories:Dermatophyte and Candida.Dermatophyte and Candida were evaluated with CLSI M38-A2 scheme and M27-A3,respectively.The MICs of eight kinds of antifungal agents to eight kinds of fungi were compared.There were significant differences in MICs of eight kinds of fungi using FCZ,ITR,VCZ,KETO,TER,AMB,MIC and 5-FC (P<0.05).Comparison of the MICs of Dermatophyte and Candida,there were significant differences using FCZ,VCZ,KETO,TER,AMB,MIC,5-FC (P<0.05),except for ITR (P>0.05).The mean rank of TER against Dermatophyte was minimum.Conclusion Eight antifungal agents had significant different antifungal characteristics against eight kinds of fungi.Except ITR,others had significant differences of antifungal characteristics between Dermatophyte and Candida.Comparatively,TER showed the best effect against Dermatophyte.

The analysis of risk factors and the immune status in Penicillium marneffei in patients without human immunodeficiency virus infection

XIE Ya-li, LI Yuan-yuan, HU Cheng-ping, DENG Peng-bo, LIU He-ping, WANG Qiong

2016, 11(3):  174-177. 

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Objective To analyze the risk factors and the immune status in Penicillium marneffei,for improving the clinical diagnosis.Methods A case admitted to Xiangya Hospital Central South University,diagnosed as Penicillium marneffei,was retrospectively analyzed and the related literature was reviewed.Databases including PubMed,Wanfang and Chinese National infrastructure (CNKI) were searched by using the keyword "Penicillium marneffei".Then the risk factors and possible immune mechanism were summarized.Results The patient,middle-age male,was a forest worker,presented with intermittent fever,cough,dyspnea,emaciation,and frequent enlargement of left supraclavicular and mediastinal lymph nodes.Although without any underlying disease,the patient was found to have a history of contact with wild animal.Imageology showed diffuse pulmonary lesions.G,GM test were dramatically accelerated,and HIV test was negative.Anti-tuberculosis treatment was invalid.Penicillium marneffei was proved by fungal culture from percutaneous lung biopsy tissue and pathological diagnosis.The syptoms and imageology of the patient,after 4months of sequential treatment of Amphotericin B and itraconazole,were relieved and improved.From databases above,91 cases of HIV-negative Penicillium marneffei were retrieved.Among the cases above,45 cases were reported with underlying diseases.Conclusions Penicillium marneffei,associated with the history of contacting with reservoir host,the life or journey in epidemic region,exposure to certain environment,climate and humidity,could happen in patients without HIV,while with underlying diseases,such as connective tissue diseases,hematological system disease,organ transplantation,and so on,or those happened with gene mutation,related to antimycotic immunity.Patiens,described as before,should be alerted for their possibility of diagnose as Penicillium marneffei. The sequential treatment of Amphotericin B to itraconazole turned out to be effective.

Etiologic analysis of 9 566 cases of superficial mycosis in Shanghai region

ZHU Jun-hao, HAN De-min, ZHAO Ying, LI Li, ZHANG Qiang-qiang

2016, 11(3):  178-180. 

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Objective To investigate the prevalence and pathogenic fungal species of superficial mycoses in Shanghai.Methods Culture and fungal identification were performed on clinical specimens collected from 9 566 patients with superficial mycoses in our hospital from Jan.2010 to Dec.2014.Results Nine thousand five hundred and sixty-six cases of superficial mycosis were included in this analysis,Onychomycosis 5 277 cases (55.16%),tinea corporis and tinea cruris 2 313 cases (24.18%),tinea manuum and tinea pedis 1 719 cases (17.97%),tinea capitis 257 cases (2.81%).T.rubrum,Candida,Rhodotorula among the 10 078 isolated pathogenic fungi strains were 50.43%,27.64% and 6.98%,respectively.Conclusions The major pathogenic fungi of superficial mycoses was Trichophyton rubrum in Shanghai area and the proportion of Candida and yeast-like fungus increased significantly,compared with the data during1994-1998.

Clinical observation of Heat clearing damp medicinal food fang for prevention and treatment of recurrent genital vaginal Candidal disease

ZHANG Zeng-ling

2016, 11(3):  181-184. 

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Objective To explore the effects of heat clearing damp medicinal food fang to vaginal micro ecological environment (lactobacilli number score),clinical symptoms and signs scores,the influence of the total clinical curative effect,etc in recurrent vulva vagina candida disease (recurrent vulvovaginal candicliosis RVVC) patients,and to verify whether it can effectively reduce the recurrence rate.Methods The 120 cases of recurrent vulva vagina disease patients,were randomly divided into experimental group and control group.The control group was treated with canesten vaginal tablet.The experimental group were treated with canesten vaginal tablet,combined with oral heat clearing damp herbal consolidating treatment of three menstrual cycles after intensive treatment.Curative rate after completion of the course and the recurrence rate 3 months after discontinuation were observed.Results During the treatment,no adverse drug reaction was occurred.The clinical total effective rates of two groups had no statistically significant difference (P>0.05),but the cure rate of experimental group was better than that of control group (P<0.05).In terms of improving clinical symptoms and signs of patients with RVVC,experimental group was better than the control group;in terms of increasing number of lactobacillus,experimental group's advantage was not obvious.The recurrence rate of the treatment group was lower than that of the control group.Conclusion Heat clearing damp medicinal food had good prevention and treatment value for recurrent genital vaginal candidal disease.

A case of infant kerion with dermatophytid reaction due to Trichophyton tonsurans

TANG Jiao-qing, ZHUANG Kai-wen, RAN Xin, RAN Yu-ping

2016, 11(3):  185-186,184. 

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A 5-month-old male infant had erythema around frontal hairline for 3 months.Physical examination showed large erythema with clear boundary,hair loss or broken hair,follicular pustules and skin abscesses.Based on the microscopic examination of the KOH preparation,culture and DNA extracted PCR-sequencing identification,infant kerion due to Trichophyton tonsurans was diagnosed.Treatment of oral itraconazole (33 mg/d),topical wash with 2% ketoconazole shampoo,then 1% naftifine-0.25% ketoconazole cream were administrated.During the therapeutic session,sporadic annular scaly papules and erythema were appeared over the head and face,which was considered as secondary dermatophytid reaction,be deal with by adding prednisone (5 mg/d) orally and changing topical drug with 1% luliconazole cream.Antifungal susceptibility test in vitro showed that 1% luliconazole cream is superior to 1% naftifine-0.25% ketoconazole cream.After 9 weeks treatment of itraconazole and 5 weeks of prednisone,the lesions resolved,and new hair grew back without scarring or alopecia.No recurrence after treatment was observed with one-year follow-up.

A case report of local infection by Trichosporon asahii

ZHOU Nan, ZHANG Li-sen, KE Hai-wen, YAO Shang-sheng, XIA Lai-qi, SHEN Yun-biao

2016, 11(3):  187-189. 

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A 44-years-old male patient is in hospital based on chief complaint of ulcerative wounds between bilateral feet third and fourth toe,with blisters and formation.Direct microscopic examination of pus showed infection of spores as round or oval.The diagnosis of infection caused by Trichosporon asahii was made based on the above findings.He accepted auto-skin grafting,the wound is quickly repaired in another 10 days.