Table of Content

28 February 2013, Volume 8 Issue 1

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Analysis of the growth characteristics of Aspergillus fumigatus in different temperature and nutrient media

ZHANG Jin-qing, LIU Wei, TAN Jing-wen, WAN Zhe, LI Ruo-yu

2013, 8(1):  6-9. 

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Objective To determine the growth characteristics of Aspergillus fumigatus in different nutrient media and temperature.Methods Growth curves were used to determine the optimum medium, optimum and maximum growth temperatures for two strains of Aspergillus fumigatus:colony counts were used to determine the fatal temperatures for them.Results Three different nutrient media, yeast extract medium (YG), minimal medium (MM) and yeast nitrogen base (YNB), were tested. YG medium was the optimum medium for measuring the growth curves of Aspergillus fumigatus; In YG medium, the optimum growth temperature for Aspergillus fumigatus was 37℃, and the maximum growth temperature was 52℃. The slower growth rates of strains were observed with gradually elevated temperature. Unexpectedly, the responses of these two strains to high temperature were different:one strain's growth became slower markedly at 48℃ than at 37℃ during log phase, while the other one grew at 48℃ as well as at 37℃ during log phase. The strains of Aspergillus fumigatus were dead when exposed to 65℃ lasting more than 180 min or 70℃ lasting more than 120 min.Conclusions The optimum and the maximum growth temperature for Aspergillus fumigatus were 37℃ and 52℃, respectively.The fatal condition for Aspergillus fumigatus was 65℃ lasting more than 180 min or 70℃ lasting more than 120 min.

The study of inhibiting the expression of ALP in Fusarium solani by RNA interference and establishing the gene-silenced strains

LIANG Tao, LIU Ke-feng, HE Hong, ZHAO Gui-qiu, WANG Ting, YU Wen-qian

2013, 8(1):  10-13,19. 

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Objective To inhibit the expression of ALP gene in Fusarium solani by RNA interference using constructed plasmid vectors and to identifiy the gene-silenced strains by detecting the expression and enzymic activities of ALP.Methods Two kinds of vectors pBC-hygro were transformed into Fusarium solani by dsRNAi.The strains we got were named after △ALP1,△ALP2,respectively.The reduction of mRNA in Fusarium solani was detected by RT-PCR. Enzymic activities were analysized using special culture medium.The gene-silenced strains ΔALP were screened out,and the strain virulence was assessed.Results The expression of mRNA of ALP in the gene-silenced Fusarium solani strains were significantly inhibited (F=184.67,P<0.01). Among the gene-silenced Fusarium solani strains, ΔALP2 achieved better inhibitory effect on enzymatic activities than other strains (q=5.276,5.463,P<0.01) did.Conclusions RNA interference in this study can significantly downregulate the expression of ALP gene in Fusarium solani.The results provide us the gene-silenced Fusarium solani strains for promoting animal experiments in vivo and exploring the mechanism of the ALP in keratitis caused by Fusarium solani.Thus we might find a new therapeutic method for curing the disease.

Expression of secretory aspartyl proteinase2 gene of Candida albicans in Escherichia coli and purification of its product

LI Lei, QIU Meng, ZOU Xian-biao

2013, 8(1):  14-19. 

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Objective To construct the SAP2 prokaryotic expression vector, to express and purify solvable protein, so as to lay a foundation for antibody preparation and Sap2 antigen detection.Methods After the target gene fragment SAP2 was obtained by standard PCR amplification, the SAP2 and plasmids pMAL-c2x (+) were cleaved with two restriction endonucleases, and the digestion products were connected. Ligation products were transformed into competent cell,E.coli TOP10. Then positive clones of recombined plasmid were screened and identified by DNA sequencing. After recombinant plasmid pMAL-c2x/SAP2 was transformed into E.coli strain BL21 (DE3) which was induced by IPTG subsequently. The fusion protein was purified by Amylose Resin affinity chromatography,and then was cleaved by Xa factor.Results The target gene SAP2 amplified by PCR had the same molecular size as predicted. It was inserted directionally into vector pMAL-c2x (+). Procaryotic expression plasmid pMAL-c2x/SAP2 can express soluble fusion protein MBP-Sap2 by IPTG induction 14 h later. Total 32 mg target protein Sap2 was obtained after purifying and cleaving label.Conclusion The recombined plasmid pMAL-c2x/SAP2 is successfully and highly expressed in BL21 (DE3) with soluble form. Target protein Sap2 was successfully obtained through affinity chromatography and proteinase cleavage.

In vitro interactions of itraconazole with caspofungin or terbinafine against Sporothrix globosa

TAN Jing-wen, LIU Wei, LIU Wei-xia, ZHANG Jin-qing, SUN Yi, WAN Zhe, LI Ruo-yu

2013, 8(1):  20-25. 

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Sporotrichosis is a common fungal infection with limited therapeutic strategies.Combination of synergistic drugs could provide new angle for treating this disease. We used the standard broth microdilution and checkerboard method to evaluate the in vitro efficacy of combining itraconazole with caspofungin or terbinafine against the yeast and mycelial phases of Sporothrix globosa. Fractional inhibitory concentration index (FICI) was used to interprete the result as following: synergistic if the FICI is ≤0.5; indifferent if the FICI is >0.5 but ≤4 and antagonistic if the FICI is >4. For yeast phase, the synergic interaction of itraconazole combined with caspofungin or terbinafine was 93.94% (31/33) and 60.61% (20/33), respectively; for mycelial phase, the synergic interaction of itraconazole combined with caspofungin or terbinafine was 96.97% (32/33) and 84.5% (20/33), respectively. Antagonism was not observed. The in vitro efficacy of combining itraconazole with caspofungin or terbinafine showed perfect synergy against S.globosa, suggesting a potential therapeutic choice for treatment of Sporotrichosis.

Comparison of in vitro activities of itraconazole and hydroxyl-itraconazole

GUO Li-na, XU Ying-chun, CHU Yun-zhuo, LV Huo-xiang, CHEN Dong-mei, TONG Ming-qing, LIU Yong, XI Li-yan, SHI Yi, YU Yun-song, ZHOU Xin

2013, 8(1):  26-29,34. 

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Objective The in vitro activities of itraconazole and hydroxyl-itraconazole against clinically important fungi isolates were tested and compared.Methods The yeast isolates were obtained from blood and normal sterile body fluids, and the Aspergillus isolates were from deep respiratory specimens. The yeast isolates were identified by CHROMagar medium, API 20C and microscopy for Aspergillus strains. Broth microdilution testing was carried out according to CLSI document exactly.Results From 10 hospitals, we collected 338 pathgentic fungi, including 281 strains of Candida spp, 37 strains of Aspergillus spp, 18 strains of Cryptococcus neoformans and 2 other yeasts isolates. Itraconazole exhibited good activities to most yeast isolates. The susceptible rate of Candida albicans,C.tropicallis,C.parapsilosis,C.krusei were 83.8%,70.8%,78.6%,38.5%, respectively;more than 50% C.krusei isolates were susceptible dose dependent; respectively. Itraconazole has less susceptibility to C.glabrata.To Cryptococcus neoformans,Aspergillus fumigatus,A.flavus, the MIC₅₀/MIC₉₀ were 0.5/0.5、1/1、1/1 μg/mL, respectively. The antifungal activity of H-ITZ is similar to ITZ with against isolates tested.Conclusion Itraconazole has good activity against most pathgentic fungi tested, resistant isolates increased among C.glabrata;the major metabolite of itraconazole has in vitro antifungal activity.

Primary cutaneous zygomycosis caused by Rhizomucor variabilis in China：a review of 13 cases

SHANG Yi, FANG Wei, LIAO Wan-qing

2013, 8(1):  30-34. 

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Objective To investigate the epidemic and clinical characteristics of primary cutaneous zygomycosis due to Rhizomucor variabilis in China since 1991.Methods Thirteen cases of cutaneous mucormycosis caused by Rhizomucor variabilis were searched from the electrical database, and all the clinical information was collected and evaluated.Results Of the 13 case records, 7 were female while 6 were male.Median age was 34?17 (5-65) year and the course ranged from 5 months to 16 years. Most cases were mainly reported in Yangtze River valley. The major clinical manifestations were local erythema, papula, and/or nodule, which might spread into infiltrating plaque associated with ulceration and necrosis. Six patients had defined mucocutaneous damage histories before the onset. All the lesions primarily occurred in exposure parts of the body (10 in the face and 3 in the upper limb) and all the diagnoses were made by mycological and histopathological examinations.The major treatments were amphotericin B and azole drugs.Eight patients were cured, and two had poor results while three lost follow-up.Conclusions Primary cutaneous zygomycosis due to Rhizomucor variabilis is severe disfiguring disease. Enhancing the understanding of its clinical characterisctics will contribute to the early diagnosis and timely treatment, and amphotericin B should be considered as a preferred choice.

Two cases of chromoblastomycosis caused by Cladophialophora carrionii

YU Hui-qian, LI Li-na, LI Zhen-lu, WANG Yu-ping

2013, 8(1):  40-43. 

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Chromoblastomycosis (CBM) is a chronic and more difficult-to-treat fungal infection of cutaneous and subcutaneous tissues. The most common aetiological agents are of the general Fonsecaea pedrosoi, Cladophialophora carrionii and Phialophora verrucosa, which are found as saprophytes in soil and plants. Most patients are inoculated through injuries with wood fragments and thorn brushes. The diagnosis of CBM can be confirmed by mycological tests including direct examination and cultures. Oral antifungals (itraconazole, terbinafine.etc.) combined with physical therapy such as thermotherapy, cryotherapy with liquid nitrogen are still the main treatment for CBM. Here, we report two cascs of chromoblastomycosis cased by Cladophialophora carrionii.

Tinea barbae and tinea corporis in face and neck caused by Trichophyton mentagrophytes：a case report

WANG Shu-fen, WU Wei-zhi, ZHAO Xiao-ting, LI Na, XU Xiang-hu, YU Chang-ping, ZHANG Chun-mei

2013, 8(1):  44-46. 

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A 57-year-old man had a history of erythema plaque, papules in face and neck, pustules with burning pain on upper-lip for more than 2 months. Treatment with different kinds of traditional Chinese medicine proved no effect. Direct microscopic examination of the NaOH (10%)preparation showed hyphae in the scales and moustache hair. The slide culture showed the clubshaped macroconidia with septate and grape-like crowded microconidia. Both urease test and hair penetration test in vitro were positive. The pathogen was identified as Trichophyton mentagrophytes.The patient was treated with oral itraconazole capsules and topical use of a butenafine hydrochloride gream. The lesion resolved with negative fungal check after continuous treatment for 2 months.

Interaction between Pneumocystis jirovecii and host immune defense

REN Yi, SONG Ying-gai, LI Ruo-yu

2013, 8(1):  51-54. 

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Pneumocystis pneumonia (PCP) is a form of pneumonia, caused by the yeast-like fungus Pneumocystis jirovecii (Pj). It is an important lethal factor in immunodeficient patients.Pj generally does not cause systemic infection, only multiply in the lungs. Interstitial pneumonia caused by Pj seriously damages the oxygen exchange function of the lung.Pj uses major surface glycoprotein (MSG) antigen switch to escape host immune elimination. The host initiates innate immune response through the interactions of macrophage mannose receptors with the MSG as well as through interaction of macrophage dectin-1 receptors interacting with the beta-1,3-D-glucan (BG) moiety of the organisms. Pattern recognition induces chemokines and inflammatory cytokine production, which promoting neutrophils and T-lymphocytes recruitment and activation. Cytotoxic 1 CD8⁺T cells which producing interferon-γ are critical in helping control of Pj infections. IgG may facilitate the uptake process by opsonization of the organisms. Neutrophil and non Tc1 CD8⁺T cell recruitment seems to closely correlate with lung injury in humans. The level of BG can help diagnosing PCP. The level of interleukin-8 in the bronchoalveolar lavage fluid could be a predictor of lung impairment and death from PCP.

The research process of the synergistic antifungal effect of plant compositions

SHEN Ling, JIANG Yuan-ying, CAO Yong-bing

2013, 8(1):  55-60. 

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The incidence of systemic fungal infections have increased dramatically in recent years, but now clinically available antifungal drugs are limited, and the research and development of new drugs are difficult.So combination therapy is expected to become the ideal choice for antifungal therapy. Plant compositions can play a synergistic antifungal effect with the antifungal agents in the form of a monomer or mixture.Such research is more extensive and thorough in vitro, but further study on the antifungal effects in vivo, mechanisms and clinical trials is still needed. This review focuses on the antifungal synergism of plant compositions combined with antifungal agents, so as to provide a reference for the study of the novel antifungal drugs.

Combination of antifungal therapy

WEI Bing, LIU Jin-yan, XIANG Ming-jie

2013, 8(1):  61-64. 

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Nowadays, morbidity and mortality of invasive fungal infection are increasing, especially among immunocompromised patients. However,the range of choices of existing antifungal drugs, most of which have drug toxicities and drug resistance, are limited.The combination use of antifungal drugs which has gradually applied to the clinic provides a new approach for the resistant strains infection and severe fungal infection. This review is about the effects and mechanisms of the combination therapy of different antifungal drugs.