申克孢子丝菌chs1基因生物信息分析

目的:对申克孢子丝菌几丁质合成酶基因(chs1)及其编码蛋白的结构和特性进行分析和预测。方法:利用NCBI网站、Ex PASy和CBS等工具预测申克孢子丝菌chs1基因及编码蛋白的结构和功能。结果:chs1 mRNA全长2 745 bp,编码914个氨基酸。BLAST分析显示,申克孢子丝菌与巴西孢子丝菌、蓝菌属真菌、足马杜拉分枝菌等真菌chs氨基酸序列一致性达到99%,且有保守域。预测chs1蛋白相对分子量为102 714.7,理论等电点为6.71。预测chs1编码蛋白ɑ螺旋、β折叠、β转角、无规则卷的比例分别是38.40%、17.94%、10.39%、33.26%。chs1蛋白是一种疏水蛋白,包含9个跨膜区,未发现信号肽。结论:成功预测了chs1基因及编码蛋白质的生化性质及结构特征,为下一步对其进行克隆、表达和敲除奠定基础。     

申克孢子丝菌双相体外药物敏感试验研究

目的：研究双相真菌申克孢子丝菌分别在菌丝相和酵母相两种形态下对常用抗真菌药物的敏感性。方法：分别采用Roseo纸片扩散法及微量液基稀释法对临床分离的4株申克孢子丝菌的两种形态（菌丝相及酵母相）进行体外药敏试验。测定的抗真菌药物为：两性霉素B、伊曲康唑、氟康唑、特比萘芬。结果：除AMB二者一致外，其余各抗真菌药的MIC，菌丝相30℃孵育比酵母相35℃孵育高。结论：申克孢子丝菌的生长形态（菌丝相或酵母相）及孵育温度（30℃或35℃）对其药敏试验MIC值有明显影响。     

发现于新疆的申克孢子丝菌新基因型

目的:利用多聚酶链反应随机扩增多态性DNA(polymerase chain reaction-random amplified polymorphic DNA,PCRRAPD)法对新疆地区固定型和皮肤淋巴管型孢子丝菌病致病菌进行基因分型,并与北京和哈尔滨的菌株基因型比较,探讨新疆地区申克孢子丝菌的基因特点.方法:采用...     

Sporothrix schenckii type 3D (mtDNA-RFLP): report of an osteoarticular case

Sporotrichosis is a frequent subcutaneous mycosis in Mexico and lymphocutaneous cases are the most common type. Extracutaneous or disseminated forms are exceptional and are usually seen in immunosuppressed hosts. We report the case of a 74-year-old immunocompetent male with osteoarticular involvement. The isolated Sporothrix schenckii was classified as type 3D according to restriction fragment length polymorphism analysis of the mitochondrial DNA (mtDNA-RFLP).     

申克孢子丝菌4Hppd基因结构功能的生物信息学分析

 目的:初步预测及分析申克孢子丝菌(Sporothrix schenckii，Ss.)对羟苯基丙酮酸双加氧酶基因（4Hppd）编码蛋白的结构和生物学功能特征。方法：应用生物信息学在线工具预测分析Ss.4Hppd编码蛋白的理化性质及功能特征。结果：Ss.4Hppd基因编码蛋白含对羟苯基丙酮酸双加氧酶保守域，由477个氨基酸构成，理论分子量为52.0 kD，等电点为5.98，半衰期较长，该蛋白为非分泌蛋白，定位于线粒体中。其二级结构以无规则卷曲及α-螺旋为主，另外还存在乙二醛酶功能域及丰富的磷酸化位点，与其他致病真菌同源蛋白有较高的相似度。结论：预测Ss.4HppD位于线粒体中，具有乙二醛酶功能域结构功能特点及丰富修饰位点，可为其表达、晶体结构分析及小分子抑制剂开发提供参考。     

Purification and isolation of a biologically active peptido-rhamnogalactan from Sporothrix schenckii.

A biologically active peptido-rhamnogalactan was isolated from disrupted Sporothrix schenckii yeast cells by sodium deoxycholate extraction and was further purified by DEAE-Sephadex A-50 chromatography. This fraction, 60.6% sugar and 3.0% nitrogen, was active in delayed hypersensitivity skin tests in sporotrichosis patients and also cross-reacted serologically with a peptido-rhamnomannan isolated from Sporothrix schenckii culture filtrates. These results suggest strong that L-rhamnose, α-1, 2 linked, is more important in determining the serological specificity of Sporothrix schenckii than the α-1, 3 linked L-rhamnose or the mannan backbone of the peptido-rhamnomannans so far studied by others.     

申克孢子丝菌基因型鉴定在临床诊断中的应用

目的：探讨申克孢子丝菌临床分离株的基因型鉴定在临床诊断中的应用。方法：使用PCR法扩增引起三种临床类型的申克孢子丝菌菌株核糖体保守区及内转录间隔区（5．8SrDNA，ITSII）的基因序列，并进行测序。结果：三株临床分离菌株核糖体保守区及ITSII区基因序列完全一致，经测定为申克孢子丝菌，该基因序列与基因库中申克孢子丝菌菌株（AB089138）相关序列的同源性达100％。结论：申克孢子丝菌的ITS区相对保守，通过对ITS段基因序列的测定可在基因水平上对孢子丝菌进行菌种鉴定，为临床快速、准确诊断和治疗提供确切依据。     

Rapid identification of Sporothrix schenckii in biopsy tissue by PCR

Background The dimorphic fungus Sporothrix schenckii is the etiological agent of sporotrichosis, an important cutaneous mycosis with a worldwide distribution. At present, it is challenging to rapidly discover and identify Sporothrix schenckii in biopsy tissues nowadays. Aims To explore new methods for rapid diagnosis of sporotrichosis. Materials and Methods We screened specific primers for Sporothrix schenckii using 50 clinical isolates from patients with sporotrichosis. DNA was extracted from the lesions of 30 cases of clinically suspected sporotrichosis using the Graham s method of CTAB and amplified by PCR using the screened specific primers. Results The primer S2‐R2 was applicable for the identification of S. schenckii from different geographic areas and clinical types with high specificity and sensitivity. Twenty‐five out of the thirty cases (83.3%) amplified using the primer S2‐R2 showed positive bands. Further positive bands were observed in 95.6% of cases tested positive by fungal culture. Conclusions Using the PCR technique and specific primers, we developed a new diagnostic method that can rapidly diagnose sporotrichosis with tissues obtained from clinical biopsies.     

四对孢子丝菌引物的特异性和敏感性评价

目的 筛选对孢子丝菌特异且敏感的引物。方法 以50株不同地区来源的孢子丝菌临床分离株及标准株的基因组DNA作为研究样本,以毛霉、烟曲霉、念珠菌临床分离菌株基因组DNA作为对照,通过特异引物PCR扩增的方法筛选国内外文献中报道的4对孢子丝菌引物。所有受试孢子丝菌菌株均出现同一扩增产物,而对照菌株未出现者记为特异性引物,随后将基因组DNA模板倍比稀释后依次行PCR扩增,记录各引物出现阳性扩增结果时的最小模板浓度,检测敏感性。结果 在相同PCR条件下,引物S2-R2、SSHF31-SSHR97及ITS3-SSP具有较好特异性,而引物SS3-SS4对孢子丝菌及念珠菌菌株均可扩增出同样大小的PCR产物。引物S2-R2的敏感性最好,基因组DNA模板浓度为5 pg/μL时即可被检出。结论 针对几丁质合成酶1基因的引物S2-R2为孢子丝菌特异且敏感的引物。     

Isolation of both Sporothrix schenckii and Nocardia asteroides from a mycetoma of the forefoot

Mycetoma is a localized primary subcutaneous infection due to fungi (eumycetoma) or aerobic actinomycetes (actinomycetoma). We report a patient who acquired an implantation infection on the forefoot following a motorcycle accident in Crete. Both Sporothrix schenckii and Nocardia asteroides were isolated simultaneously from the lesion. Under combined therapy with itraconazole and trimethoprim-sulphamethoxazole for 7 months the lesion healed completely. A combination of causative organisms in mycetomas is rare, and the combination of S. schenckii and N. asteroides together has not been reported from one lesion.     

Low-titer agglutinins to Sporothrix schenckii in nonsporotrichotic human and animal sera

Low-titer agglutinins to Sporothrix schenckii were demonstrated in the sera of dogs, cats, rats (Wistar), guinea pigs, hamsters, white rabbits, swine, horses, cattle, sheep, chickens, and goats as well as in the human sera of nonsporotrichotic individuals. It is hypothesized that the presence of agglutinins is due to a response to various bacteria which share common antigens with S. schenckii.     

孢子丝菌病药敏和治疗进展

孢子丝菌病是一种深部真菌病,其致病菌申克孢子丝菌为一种双相真菌,双相真菌的体外药敏试验至今仍无统一标准.饱和碘化钾溶液、伊曲康唑、特比萘芬为治疗孢子丝菌病的常用药物,临床中选择合适的抗真菌药物对治疗此病具有重要意义.体外药敏试验是评价抗真菌药物活性的重要方法,也是选择药物的依据之一.     

无毛小鼠接种申克孢子丝菌检测抗申克孢子丝菌细胞外蛋白酶抗体研究

通过申克孢子丝菌接种无毛小鼠（Ｈａｉｒｌｅｓｍｉｃｅ）来检测两种细胞外蛋白酶的血清抗体。在小鼠皮内注射申克孢子丝菌并形成结节，镜下示真菌消失，并在每周通过酶联免疫吸附试验（ＥＩＡ）检测蛋白酶Ⅰ和Ⅱ的血清抗体。结果显示抗体滴度与实验小鼠孢子丝菌病肉眼和镜下观察的变化趋势相一致。本试验证明了申克孢子丝菌在体内能产生蛋白酶Ⅰ和Ⅱ，而且蛋白酶的抗体可用于孢子丝菌病的血清学诊断。     

New strain typing method with Sporothrix schenckii using mitochondrial DNA and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique

The complete sequences of mitochondrial DNA (mtDNA) from two strains of different genotypes, American Type Culture Collection 10268 of mtDNA type 1 and KMU2025 of mtDNA type 4, were determined. These are circular molecules, 27 125 and 26 095 bp in length, respectively. The greatest difference between the two strains was found in the region encompassed by atp9 and cox2 genes, which was amplified with polymerase chain reaction (PCR) and used for preliminary restriction fragment length polymorphism (RFLP) analysis. Eight isolates of five mtDNA types were used and RFLP patterns obtained with the restriction enzyme AseI showed that this method seems to have greater discrimination power than the other PCR-RFLP typing method using internal transcribed spacer regions of nuclear DNA.     

以S2-R2为引物PCR检测石蜡切片中的孢子丝菌

目的 探讨应用特异性引物PCR方法检测石蜡切片中孢子丝菌的可行性。方法 选取30份大连及10份长春地区临床疑诊孢子丝菌病的石蜡切片标本,采用改良的微波脱蜡、液氮研磨-CTAB破壁法提取DNA,以特异性引物S2-R2进行PCR扩增,与真菌培养结果进行比对。结果 30份大连地区标本中22份见阳性PCR扩增产物（73.33%）。真菌培养阳性的22份标本中20份PCR出现阳性扩增产物（91%）,真菌培养阴性的8份标本中2 份PCR出现阳性扩增产物。10份长春地区标本7份见阳性PCR扩增产物（70%）。结论 以S2-R2为引物的 PCR适用于孢子丝菌病石蜡切片中病原菌的检测。     

In vitro activity of itraconazole in combination with terbinafine against clinical strains of itraconazole-insensitive Sporothrix schenckii

Four strains of Sporothrix schenckii were isolated from patients with cutaneous sporotrichosis who failed itraconazole (ITC) treatment. To investigate the susceptibility of these strains to ITC and terbinafine (TRB) alone and in combination in 2 growth phases in vitro, a checkerboard microdilution method was used in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI) was used in our study. The drug interaction was evaluated by assessing the fractional inhibitory concentration index (FICI). The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of 4 insensitive strains were obviously higher than those of the sensitive isolates used for comparison. The FICI analysis revealed that only 2 isolates (25%) were synergistic in yeast form. Our results indicate the failure of clinical treatments might be caused by the insensitivity to ITC of these strains.