不同氨基酸对少孢节丛孢菌（Arthrobotrys oligospora）生长的影响

为了解氨基酸在玉米粉琼脂培养基上对捕食线虫性真菌--少孢节丛孢菌Arthrobotrys oligospora产生捕食器及对寄生性线虫第3期幼虫捕食情况的影响,首先需要确定加入的氨基酸对真菌的生长是否有影响,我们研究了L-组氨酸、L-广异亮氨酸、L-亮氨酸、DL-广亮氨酸、L-苯丙氨酸、DL-苯丙氨酸、L-蛋氨酸、DL-缬氨酸、L-色氨酸、L-赖氨酸、L-谷氨酸、L-缬氨酸、L-苏氨酸共13种氨基酸(以生理盐水、pH6.O的磷酸盐缓冲液和空白作对照)在玉米粉琼脂固体培养基上对少孢节丛孢菌生长的影响.结果表明,不同氨基酸对少孢节丛孢菌生长的影响表现各不相同,其中L-组氨酸、L-异亮氨酸、L-亮氨酸、DL-亮氨酸对菌丝生长促进作用明显,但同对照组比差异不显著;L-苯丙氨酸、DL-苯丙氨酸、L-蛋氨酸、DL-缬氨酸、L-色氨酸、L-赖氨酸、L-缬氨酸、L-谷氨酸(0.500 g/L浓度的溶液除外)对菌丝有一定促进作用,统计学分析差异也不显著;L-苏氨酸和L-广谷氨酸(0.500 g/L浓度的溶液)对真菌的生长有明显的抑制作用.另外,除L-谷氨酸外,不同浓度的同种氨基酸溶液对真菌生长的影响差异不显著.     

科玛嘉念珠菌显色培养基在诊断侵袭性真菌感染中的应用

目的探讨CHROMagar显色培养基检测侵袭性真菌感染的应用价值。方法将2007年7月至2008年6月湖南省肿瘤医院肿瘤病人的呼吸道标本采用CHROMagar显色培养基及沙保罗培养基进行真菌检测,CHROMagar显色培养基上生长的真菌根据菌落的颜色及形态进行鉴别,在沙保罗培养基上生长的真菌经VITEK2-Compact全自动细菌鉴定仪进行鉴定。结果660份标本在沙保罗培养基上分离出383株真菌,经鉴定其中白色假丝酵母菌为262株、热带假丝酵母菌为51株、克柔假丝酵母菌15株、光滑假丝酵母菌34株,其他真菌21株。CHROMagar显色培养基上检出360株真菌,检出率与前者的符合率为93．99％。结论科玛嘉显色培养基能作为临床快速鉴定酵母样真菌感染的方法,对侵袭性真菌感染的早期诊断有意义;但鉴定菌种有限,有一定的局限性。     

互隔交链孢霉菌在不同培养基中致敏蛋白组份的变化

目的 研究互隔交链孢霉菌在不同培养基中致敏蛋白组份的变化,为我国该菌过敏原的标准化提供理论依据.方法 用4种不同的培养基(营养肉汤培养基、察氏肉汤培养基、察氏酵母膏培养基、细菌合成培养基)培养互隔交链孢霉菌,生长14 d 后,观察霉菌生长情况,收集菌体制备粗浸液,应用SDS-PAGE和免疫印迹分析互隔交链孢霉菌粗浸液的蛋白质成份及过敏原成份,再用Bio1D软件分析各蛋白质和过敏原组份的相对分子质量.结果 在不同培养基中,互隔交链孢霉菌菌体的形态学特征各不相同.4 种培养基培养的霉菌菌体的蛋白质组份分别为4、10、11和13条蛋白带;用真菌过敏患者阳性血清免疫印迹显示4种不同培养基培养的互隔交链孢霉菌特异性过敏原分别为1、6、2和1条阳性带.其中,察氏肉汤培养基培养的菌体中阳性血清反应带最多.相对分子质量分别为83000、27 000、26000、20000、12 000和10 000.察氏肉汤培养基、察氏酵母膏培养基、细菌合成培养基培养14 d的菌体中都存在一种Mr12 000的致敏蛋白组份;并且不同培养基培养的互隔交链孢霉菌各自有其特异性致敏蛋白组份.结论 不同培养基成份能够影响互隔交链孢霉菌的形态学特征、菌体蛋白质组份和致敏蛋白组份的数量.     

氟康唑的体外抑菌试验

氟康唑(Fluconazole,Flu)系双三唑类广谱抗真菌药,与两性霉素B和酮康唑(keto)相比具有优良的药动学特性,疗效高,毒副作用小,对白念珠菌和新型隐球菌等深部真菌所致各种感染具有卓著的治疗和预防作用。 我们在培养基,pH,菌量,方法不同的条件下按常规药敏法试验,分别测定了Flu和keto对26种(51株)深部和浅部致病真菌(如念珠菌,新型隐球菌,曲霉菌,球孢子菌,皮炎芽生菌以及须毛、红毛癣菌等)的MIC和MBC值。其结果表明:(1)国产Flu对所试各     

小鼠皮下感染着色芽生菌病皮损中局部细胞免疫功能研究

目的 利用小鼠皮下着色芽生菌病模型,研究在不同的病程发展阶段其T细胞免疫功能的变化.方法 足垫局部皮下注射法建立小鼠的裴氏着色霉感染的着色芽生菌病模型,通过免疫组织化学技术,检测正常小鼠足垫皮肤皮损局部细胞因子IFN-γ、IL-4、IL-10、TNF-α的表达并作为对照组,观察免疫正常组和环磷酰胺免疫抑制处理的免疫抑制组小鼠分别在感染上述真菌后7 d、30 d时,皮损局部细胞因子水平变化,并与对照组比较.结果 在着色芽生菌病小鼠模型中,第7天时,免疫正常组IL-4、TNF-α和IL-10较正常对照组均出现了显著的升高(P＜0.01),表现为Th1和Th2型细胞免疫均增强的模式,并以Th2型为主;30 d时IL-10表达显著下降(P＜0.01),与同期正常对照组比较差异无统计学意义(P=0.52),IFN-γ、TNF-α水平比7 d时显著升高(P＜0.01),表现为Th2型细胞免疫减弱Th1型占主导的模式.免疫功能受损组第7天时IL-10、IL-4的水平升高与其他两组相比差异均有统计学意义(P＜0.01),IFN-γ表达水平则明显下降(P＜0.01),TNF-α的表达与正常对照组相比差异无统计学意义(P=0.39),表现为Th2型细胞免疫增强Th1功能受抑模式;30 d时,IL-10表达水平较7 d时显著减少,但仍然高于同期的免疫正常组和正常对照组,IFN-γ、TNF-α水平显著升高(P＜0.01),但却低于免疫正常组,表现为Th2型免疫模式渐弱Th1功能逐渐增强模式.结论 在不同免疫状态下小鼠着色芽生菌病感染的过程中,随着病情好转存在由Th2型细胞免疫为主导转化为Th1型细胞免疫功能占主导的过程,免疫抑制下主要表现为Th1反应受抑制,Th1型细胞因子在控制着色芽生菌病的发展过程中具有关键性的保护作用,Th2型细胞因子则可能与感染的发展进程相关.     

外用红色诺卡氏菌细胞壁骨架提高脂肪间充质干细胞活性修复糖尿病创面北大核心

背景:根据目前临床上外用红色诺卡氏菌细胞壁骨架对宫颈糜烂的治疗应用及其免疫作用,考虑到其对慢性创面治疗的可能性。目的:探讨外用红色诺卡氏菌细胞壁骨架对脂肪间充质干细胞体外增殖、凋亡的影响,以及对脂肪间充质干细胞体内移植存活率和皮肤愈合的影响。方法:以质量浓度为10 mg/L外用红色诺卡氏菌细胞壁骨架作用于脂肪间充质干细胞,采用CCK-8法检测细胞活力,EDU试剂盒检测细胞增殖能力;在脂肪间充质干细胞中加入50%蔗糖诱导细胞凋亡,给予外用红色诺卡氏菌细胞壁骨架干预后采用FITC-PI流式细胞凋亡试剂盒、TUNEL凋亡试剂盒检测细胞凋亡情况。第3代脂肪间充质干细胞中加入10 mg/L外用红色诺卡氏菌细胞壁骨架5 mL,共培养48 h,用荧光染料CM-Dil标记后以皮内注射方式注入糖尿病小鼠创缘皮肤,LB983活体成像系统检测细胞存活率,苏木精-伊红染色、Masson染色和免疫荧光染色验证慢性创面第14天愈合情况。结果与结论:外用红色诺卡氏菌细胞壁骨架可提高脂肪间充质干细胞的活力及增殖能力,并抑制其凋亡;在体内条件下外用红色诺卡氏菌细胞壁骨架可以增加脂肪间充质干细胞的存活率,糖尿病小鼠创面愈合速度更快。     

液体菌种培养时间对灵芝液体发酵的影响

目的 探讨灵芝液体菌种培养时间对灵芝液体发酵的影响。 方法 将灵芝菌块接种于液体摇瓶菌种培养基中，置于25℃恒温培养箱内静置培养24h，然后分别恒温振荡培养4、5、6d，分别命名为4号、5号、6号液体菌种，把各号液体菌种转接于液体培养基中继续培养，观察不同液体菌种发酵过程中发酵液pH值、菌丝量和胞外粗多糖含量的变化。 结果 在发酵过程中4号菌种的发酵液pH值先下降后上升，其他2个菌种变化不明显。在液体发酵第7天时，5号菌种发酵液中的菌丝量明显高于4号、6号，达到10．2mg／ml。在液体发酵第4天时，4号与5号液体菌种发酵液中胞外多糖的含量最多，6号液体菌种则推迟2d。发酵液中胞外多糖含量以5号最高，4号次之，6号最低。 结论 液体菌种培养时间对于灵芝发酵液中菌丝量有明显影响，而对胞外粗多糖含量无明显影响；6d是灵芝液体菌种最佳培养时间长度。     

妇科门诊患者常见阴道炎发病情况及护理

目的：研究临床上妇科阴道炎患病情况以及患病后的护理。方法回顾分析2013年3月~2014年2月我院妇科门诊接诊的6218例妇女的阴道分泌物检测(包括滴虫、假丝酵母菌菌丝、pH值和孢子)，并统计分析所得资料，针对炎症进行护理。结果阴道炎总的检出率为26.12%，其中真菌性阴道炎为18.61%，细菌性阴道炎为5.44%，滴虫性阴道炎为2.07%。全年四个季节中，春季阴道炎的发病率最高为29.77%。结论阴道炎的发病率与季节有关。阴道炎是妇科门诊最常见的疾病，我们要正视阴道炎，尽早发现，科学的治疗和针对性的护理，争取早日康复，预防及减少复发。     

沙门氏菌单克隆抗体研究 Ⅰ.杂交瘤细胞系的建立

用沙门氏菌O,H,Vi抗原免疫BALB/c小鼠,采用细胞融合技术,获得分泌抗沙门氏茵0-2,4,7,8,9,3,10,11和H-a,b,c,d,i及Vi抗原的单克隆抗体杂交瘤细胞系29株。所产生的抗体17株属IgM,7株为IgG3,3株为IgG1,1株为IgG_(2a),1株未测;轻链除3株为λ链外,其余为κ链。将杂交瘤细胞所诱生的腹水,组成一套(11种)沙门氏菌诊断用单克隆抗体试剂,经用沙门氏菌属和其它肠道菌属的代表菌株作鉴定,证明具有良好的特异性和敏感性、可供初步诊断伤寒,副伤寒和鼠伤寒用。与目前常规生产的家兔诊断血清相比,具有简化生产过程,节约家兔与培养基及省时省力等优点,值得在生产上和使用上推广。     

五株虫生真菌对白纹伊蚊成蚊及幼蚊致病力的研究

随着蚊虫对传统化学杀虫剂耐药性的出现,生物灭蚊途径得到越来越广泛的关注。本实验选取生物防治中常用的5株野生型虫生真菌菌株,包括4株野生型白僵菌Beauveriabassiana及1株绿僵菌Metarhiziumanisopliae,测定不同浓度的孢子悬液对白纹伊蚊幼虫和等量的固体培养基上的孢子粉对白纹伊蚊成蚊的逐日累积校正死亡率,通过比较累计校正死亡率的大小来判断各株菌株对蚊虫的毒力。实验结果显示。菌株GIM3．428、GIM3．45、GIM3．528、GIM3．436和GIM3．46对白纹伊蚊幼虫和成蚊均具有致病性,通过1×10‘个孢子／mL的孢子悬液处理白纹伊蚊幼虫10d,幼虫的累计校正死亡率分别为（46．7±2．3）％、（45．0±4．0）％、（41．7±2．3）％、（35．0±4．0）％和（25．0±4．0）％;而用等量孢子粉感染成蚊,成蚊10d的累计死亡率分别为（37．84-3．1）％、（51．1±4．1）％、（38．9±4．1）％、（30±2．7）％和（17．8±3．1）％。我们筛选出了对白纹伊蚊的幼虫和成蚊的杀灭效果较为理想的白僵菌GIM3．45和GIM3．428菌株,为生物杀蚊剂的开发提供理论依据     

Exoantigen test for differentiation of Exophiala jeanselmei and Wangiella dermatitidis isolates from other dematiaceous fungi.

Concentrated (25X) exoantigens of 105 isolates of pathogenic and saprophytic dematiaceous fungi and 3 isolates of Sporothrix schenckii were analyzed by the microimmunodiffusion method. The reagents used were nonadsorbed and adsorbed sera produced in New Zealand rabbits. One set of rabbits was immunized with soluble antigens of a 1-month-old culture of Exophiala jeanselmei (ATCC 34123), and the other set was immunized with soluble antigens from a culture of Wangiella dermatitidis (ATCC 28869). The reference antigens were 25X-concentrated exoantigens of the above cultures. This exoantigen test permitted the differentiation of E. jeanselmei and W. dermatitidis from one another as well as from other Exophiala species, Fonsecaea species, Phialophora species, Cladosporium species, Rhinocladiella species, and Sporothrix schenckii by presence or absence of lines of identity or of partial identity, or lines of nonidentity. Using adsorbed serum eliminated the problems with cross-reactivity seen with nonadsorbed serum. Thus, with an adsorbed serum as the reagent, it was possible to presumptively differentiate E. jeanselmei and W. dermatitidis from one another and from other dematiaceous fungi.     

Exoantigen test for Cladosporium bantianum, Fonsecaea pedrosoi, and Phialophora verrucosa.

Exoantigens from 10-day-old cultures of 100 isolates of pathogenic and saprophytic dematiaceous fungi were analyzed by the exoantigen test. Antisera to Cladosporium bantianum ATCC 10958, Fonsecaea pedrosoi CDC AMO-B06, and Phialophora verrucosa CDC AMO-C12 were prepared in New Zealand rabbits immunized with soluble antigens from 1-month-old cultures. Absorbed and nonabsorbed antisera and exoantigens from the same organisms were used as reference reagents. Serologic reactions were analyzed in terms of the presence or absence of lines of identity or nonidentity. These reactions allowed presumptive differentiation of C. bantianum, F. pedrosoi, and Phialophora verrucosa from other dematiaceous fungi, including Cladosporium spp. (28 isolates), Exophiala spp. (18 isolates), Fonsecaea spp. (17 isolates). Lecythophora hoffmannii (4 isolates), Phaeoannellomyces werneckii (3 isolates), Phialophora spp. (17 isolates), Wangiella dermatitidis (9 isolates), and Rhinocladiella spp. (4 isolates).     

Evaluation of the API 20C yeast identification system for the differentiation of some dematiaceous fungi.

Ninety-seven isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were used to evaluate the API 20C Yeast Identification System for the differentiation of dematiaceous fungi. Using the API 20C system, we were able to distinguish most species of Phialophora and Cladosporium and to separate L. hoffmannii from the species of Phialophora tested; X. bantiana from C. carrionii, C. resinae, and C. sphaerospermum; and W. dermatitidis from Exophiala jeanselmei and Exophiala spinifera. Ninety-two (60.1%) of 153 possible species-pair combinations were separated.     

Evaluation of proteolytic activity to differentiate some dematiaceous fungi.

A total of 123 isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were tested for proteolytic activity by using 26 different formulations of gelatin, milk, casein, and Loeffler media. Other physiological properties examined included hydrolysis of tyrosine and xanthine, sodium nitrate utilization in Czapek Dox agar, and thermotolerance. Isolates of Exophiala jeanselmei, Fonsecaea compacta, Fonsecaea pedrosoi, W. dermatitidis, and X. bantiana lacked proteolytic activity. Proteolytic activity was variable among the remaining species, depending on the type of medium used. Thermotolerance had value in distinguishing some taxa.     

In vitro activities of new and established triazoles against opportunistic filamentous and dimorphic fungi.

The in vitro activities of three new triazoles were determined and compared to those of itraconazole and fluconazole against 306 clinical isolates of Blastomyces dermatitidis, Cladophialophora carrionii, Coccidioides immitis, Fonsecaea pedrosoi, Fusarium spp., Histoplasma capsulatum, Paecilomyces lilacinus, Pseudallescheria boydii and Sporothrix schenckii. Minimum inhibitory concentrations (MIC) were determined by a broth macrodilution method of the National Committee for Clinical Laboratory Standards M38-A procedure. Itraconazole (geometric mean MIC, 0.16-0.65 microg/ml), voriconazole (geometric mean MIC, 0.18-1.44 microg/ml), ravuconazole (geometric mean MIC, 0.18-1.09 microg/ml), and posaconazole (geometric mean MIC, 0.18-1.38 microg/ml), had relatively uniform values showing potent in vitro inhibitory activity against B. dermatitidis, C. carrionii, C. immitis, F. pedrosoi, H. capsulatum, and S. schenckii. The in vitro activity was variable with strains of P. boydii, P. lilacinus and Fusarium spp.     

Analysis of genes coding for small-subunit rRNA sequences in studying phylogenetics of dematiaceous fungal pathogens.

Because of their ability to display yeast-like growth forms in various environmental conditions, dematiaceous (melanized) hyphomycetes of the form-genera Exophiala, Rhinocladiella, and Wangiella have been informally termed "black yeasts." Cladistic analysis of 1,050 bp of the genes coding for small-subunit rRNA (SSU rDNA) supported a close relationship among species of these black yeasts with other dematiaceous hyphomycetes in the form-genera Fonsecaea, Phialophora, and Ramichloridium. The conventional categories of these fungi based on asexual states are not supported by phylogenetic analysis of SSU rDNA sequences. Isolates exhibiting annellidic modes of blastic conidiogenesis (e.g., Exophiala spp.) were not monophyletic and were placed as sister taxa to isolates that produce phialides or sympodulae. The results indicated very close relationships between isolates of Wangiella dermatitidis and Exophiala mansonii and between Rhinocladiella aquaspersa and Exophiala jeanselmei. This clade of dematiaceous hyphomycetes was a sister group to a clade comprising members of two orders of cleistothecial ascomycetes, Eurotiales and Onygenales. The etiological agents of chromoblastomycosis were found to be a closely related group (clade), while the agents of phaeohyphomycosis displayed a broader distribution on the SSU rDNA tree.     

Specific oligonucleotide primers for identification of Cladophialophora carrionii, a causative agent of chromoblastomycosis

Cladophialophora carrionii is one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon from C. carrionii strains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such as Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, such as Candida albicans and Cryptococcus neoformans var. neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification of C. carrionii.     

Molecular probes for diagnosis of fungal infections.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.     

New cause for false-positive results with the Pastorex Aspergillus antigen latex agglutination test.

The Pastorex Aspergillus antigen test for detection of Aspergillus galactomannan antigen in the sera of patients with invasive aspergillosis is used in many clinical laboratories. A serum sample contaminated with Penicillium chrysogenum gave a strongly positive reaction (1:128) which was heat stable, was not eliminated by pronase treatment, and was not detected by a normal rabbit globulin control. This observation was shown to be due to cross-reactions of the monoclonal antibody EB-A2 used by the kit with several airborne fungi likely to contaminate serum samples, including Penicillium chrysogenum, Cladosporium herbarum, Acremonium species, Alternaria alternata, Fusarium oxysporum, Wangiella dermatitidis, and Rhodotorula rubra.     

Thermotolerance of Wangiella dermatitidis.

A variety of diagnostic tests used by many laboratories to identify isolates of Wangiella dermatitidis (= Fonsecaea dermatitidis) were evaluated. Thirteeen isolates of W. dermatitidis were studied with respect to their ability to grow at 25, 37, 40, 45, and 50 degrees C, colonial and micromorphology, gelatin liquefaction, and hydrolysis of casein, xanthine, hypoxanthine, and tyrosine. All 13 isolates showed growth at 25, 37, and 40 degrees C but failed to grow at higher temperatures. The ability of W. dermatitidis to grow at 40 degrees C can be useful in its identification.