申克孢子丝菌酵母相和菌丝相cDNA消减文库的构建

目的 筛选与申克孢子丝菌酵母相与菌丝相双相转换相关的差异表达基因,为探讨其双相转换的分子的机制奠定基础.方法 应用抑制性消减杂交技术,构建高特异性的申克孢子丝菌菌丝相（mycelium,M）和酵母相（yeast,Y）的正反cDNA消减文库,并对其差异表达的基因进行生物信息学分析.结果 M＋Y文库获得751条表达序列标签,平均长度为690.98 bp,经拼接后获得101条非冗余序列.Y＋M文库获得875条表达序列标签,平均长度为575.9 bp,拼接获得249条非冗余序列.经BLASTN比对,这些差异表达基因中,某些结构基因和功能不明的细胞分子类基因可能与双相转换有关.结论 成功构建了高特异性的申克孢子丝菌菌丝相和酵母相的正反cDNA 消减文库,为进一步筛选申克孢子丝菌的双相转换基因奠定了基础.     

1株多重耐药申克孢子丝菌感染的治疗

报道1例由多重耐药申克孢子丝菌引起的面部孢子丝菌病。患者61岁男性,面部皮疹2 a余,先后口服伊曲康唑、特比萘芬和氟康唑治疗16个月无效。患者皮损经真菌镜检和培养确诊为孢子丝菌病,分离的申克孢子丝菌体外药敏试验显示其菌丝相和酵母相对上述三种药物均不敏感。给予患者口服10%碘化钾溶液治疗3个月获得痊愈。     

特比萘芬治愈儿童鼻翼孢子丝菌病1例

目的报道1例发生在4岁儿童鼻翼的孢子丝菌病。方法刮取病变组织接种到沙堡弱培养基培养,并对培养出的病原菌进行形态学鉴定和ITS区序列分析。结果病变组织在沙堡弱培养基25℃培养长出黑褐色菌落,25℃小培养后光镜及扫描电镜下见菌丝细长,分枝分隔,合轴式产孢形成的成簇和沿菌丝两侧排列,分生孢子卵圆形。提取真菌总DNA用PCR方法扩增ITS序列,测序后登录GeneBank进行比对,该菌与申克孢子丝菌菌株KMU3360ITS区碱基序列一致性达99%,鉴定为申克孢子丝菌。内服联合局部应用特比萘芬治疗6个月后皮损完全消退。结论确诊1例儿童鼻翼的孢子丝菌病,内服联合局部应用特比萘芬治疗儿童孢子丝菌病疗效良好。     

申克孢子丝菌体外药敏试验研究进展

申克孢子丝菌是孢子丝菌病的致病真菌,作为一种双相型真菌,室温培养为菌丝相,37℃则为酵母相。哪一种生长状态的药敏试验结果更准确可靠、并能指导治疗尚有争议。就此对其体外药敏试验的影响因素、试验方案的改良以及试验条件的探索等研究进展作一综述。     

球形孢子丝菌致婴儿固定型孢子丝菌病1例及相关实验研究

报道1例由球形孢子丝菌所致的婴儿固定型孢子丝菌病。患儿女,3个月,因左眼下内侧皮损2个月就诊,皮损脓液标本进行真菌培养,对培养获得菌株进行形态学、生理学和分子生物学鉴定,并进行药物敏感性检测。真菌培养阳性,镜下可见典型的套袖样菌丝。钙调蛋白基因序列分析鉴定为球形孢子丝菌。药敏试验显示特比萘芬和伊曲康唑对该菌株的菌丝相最低抑菌浓度（minimal inhibitorycon centration,MIC）分别为0．5μg／mL和0．5μg／mL;对该菌株的酵母相的MIC值分别为0．25μg／mL和0．5μg／mL。给予患者口服特比萘芬32．5mg／d治疗10周后皮损消退呈瘢痕化修复。依据临床及实验室检查确诊该病例为球形孢子丝菌所致固定型孢子丝菌病,特比萘芬治疗本病例显示较好疗效。     

山苍子油体外抗申克孢子丝菌的实验研究

目的探讨山苍子油对申克孢子丝菌的体外抗菌活性及其作用机理。方法纸片扩散法定性检测山苍子油对申克孢子丝菌的体外抗菌活性,CLSI-M27-A微量液基稀释法定量检测其对14株临床分离申克孢子丝菌(包括固定型和淋巴管型各7株)的最小抑菌浓度(MIC),以氟康唑作为质控药物。电镜观察山苍子油作用前后申克孢子丝菌超微结构的改变。结果纸片扩散法1周后可见清晰的抑菌环,山苍子油对皮肤固定型株和皮肤淋巴管型株的MIC范围均为156.25～1250μg/ml,差异无显著性(P>0.05)。电镜观察显示山苍子油作用24h后申克孢子丝菌的菌丝干枯、断裂,孢子壁疏松、碎裂;胞膜及胞壁结构模糊,胞浆皱缩,细胞器及细胞核结构模糊。结论山苍子油对临床分离的皮肤型孢子丝菌有较强的抗菌活性,且其对固定型株和淋巴管型株的抗菌活性无显著性差异。电镜结果提示山苍子油可能通过破坏菌胞膜,影响其通透性,进而扰乱细胞正常代谢而起到抗菌作用。     

1例皮肤播散型孢子丝菌病的分离菌株的基因鉴定

孢子丝菌病是由申克孢子丝菌及其卢里变种感染引起的皮肤、皮下组织和附近淋巴系统的亚急性和慢性感染,偶可播散至全身引起多系统损害。该病遍布全世界,我国孢子丝菌病主要是由申克孢子丝菌引起。孢子丝菌一般生存在土壤和植物上,人的皮肤接触带菌植物或土壤后可引起感染。临床上主要表现为固定型和淋巴管型孢子丝菌病,皮肤播散型孢子丝菌病少见。现认为机体感染申克孢子丝菌引起的不同的临床型别与机体免疫状态、申克孢子丝菌的致病力及其基因差异相关[1-2]。我     

申克孢子丝菌感染诊治1例

通过1例16岁女孩由申克孢子丝菌导致的皮下孢子丝菌病,口服伊曲康唑治疗有效,回顾申克孢子丝菌的实验室检验方法 ,以此来提高孢子丝菌感染的诊断水平。     

伊曲康唑联合特比萘芬治疗孢子丝菌病疗效及其对病原真菌体外抗菌活性研究

目的 初步探讨伊曲康唑和特比萘芬联合治疗孢子丝菌病的疗效,评价两药体外联合对申克孢子丝菌菌丝相和酵母相的抗菌活性.方法 口服伊曲康唑200mg/d和特比萘芬250mg/d治疗孢子丝菌病;体外联合药敏试验采用棋盘微量稀释法,计算分数抑菌浓度(FIC)指数判定两药相互作用具有协同、拮抗或无关作用.结果 伊曲康唑和特比萘芬联...     

皮内与腹腔接种不同来源菌株对实验性小鼠孢子丝菌病的影响

目的将不同来源的申克孢子丝菌菌株接种于免疫抑制处理后的小鼠皮内及腹腔,造成其实验性感染,观察其感染情况,研究不同菌株及不同接种途径对小鼠孢子丝菌病的影响。方法分别在小鼠皮内和腹腔注射孢子丝菌菌悬液0.1ml,约含1×107个孢子,观察3周。观察结束时处死小鼠取其血、脑、肺、肝、脾、肾、肠系膜、睾丸、腹膜进行真菌培养和组织病理检查。结果皮内和腹腔接种不同来源菌株的小鼠各器官感染率各不相同。播散株皮内接种组、固定株腹腔接种组、播散株腹腔接种组的各器官平均感染率分别为16.05%、18.89%、58.73%。播散株腹腔接种组与其他二组之间差异均有显著性,脾和睾丸是最易受累的器官。结论不同来源菌株可能毒力不同,播散型菌株具有更强的侵袭性。脾脏可能在宿主抗孢子丝菌感染中具有重要作用。     

Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii

Protein kinase C (PKC) plays an important role in the control of proliferation and differentiation of a wide range of cell types, and fungi are no exception. Previous results reported by us on the effects of the phorbol ester, 12-myristate-13-acetate phorbol (PMA) and other PKC effector molecules, on dimorphism in Sporothrix schenckii suggested the presence of this enzyme in the fungus and its involvement in the control of morphogenetic transitions. The work summarized here confirms the presence of PKC in yeast and mycelium extracts of S. schenckii. Different isoforms of this enzyme were found to be present in the yeast and mycelium forms of the fungus and were identified by Western blot analysis using affinity purified anti-PKC isoforms specific antibodies: the γ and ζ isoforms were detected in both the yeast and mycelium forms of the fungus, while the β isoform was only detected in the yeast form. The presence of PKC was confirmed biochemically by measuring total enzyme activity in both forms of the fungus. No significant differences were observed for the PKC activity level recorded for both the mycelium and yeast forms of the fungus (p ≤ 0.05). These data confirm the presence of PKC activity in Sporothrix schenckii and constitutes the first evidence concerning the differential expression of PKC isoforms in the mycelium and yeast forms of a dimorphic fungus, supporting the possible involvement of this important signal transduction enzyme in the control of morphogenesis in this fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

鼻部固定型孢子丝菌病1例

报道1例猫抓后引起的固定型孢子丝菌病.患者男,16岁,皮损表现为鼻翼部位的增生物,其上覆有脓痂.临床上易与细菌感染混淆,但根据患者的病史、临床表现、病理、真菌镜检及培养诊断为申克孢子丝菌引起的孢子丝菌病.患者在应用7个月的碘化钾结合特比萘芬软膏外用治疗后,皮损完全消失.     

儿童孢子丝菌病的临床分析及致病菌的基因型检测

目的 对吉林省扶余县35例儿童孢子丝菌病进行临床分析并对部分菌株进行基因测定,了解该地区孢子丝菌病的致病菌菌株基因是否发生变异而致致病力增强及发病率增高.方法 收集2011年1月1日～4月30日来自扶余县就诊于我科并确诊为孢子丝菌病的患儿35例,进行临床分析;从中选取8株菌,对其核糖体保守区及内转录间隔区(ITS)、非转录间隔区(NTS)区进行基因测定,了解基因水平上的异同.结果 8株孢子丝菌菌株中5株NTS区可见较大范围的碱基缺失.结论 吉林省扶余县孢子丝菌菌株可能由于NTS区碱基较大范围缺失而致致病力增强,导致该地区短时间内发病率增高.     

申克孢子丝菌毒力因子及宿主抗感染免疫的研究进展

申克孢子丝菌是一种重要的双相型真菌,其引起的孢子丝菌病是一种常见的侵袭性皮肤感染.研究该菌毒力因子及宿主对其抗感染免疫对于深入了解其致病性及防治该病具有重要意义.该文介绍了申克孢子丝菌毒力因子及宿主对其抗感染免疫方面的研究进展.     

Isolation of ergosterol peroxide and its reversion to ergosterol in the pathogenic fungus Sporothrix schenckii

Ergosterol peroxide, a presumed product of the H_{_2}O_{_2}-dependent enzymatic oxidation of ergosterol, has been isolated from yeast forms of the pathogenic fungus Sporothrix schenckii. The substance, which may have a role in fungal virulence, has been characterized mainly using spectroscopic methods (¹H and ¹³C nuclear magnetic resonance and high resolution mass spectra). The purified compound showed a molecular formula of C_{_28}H_{_44}O_{_3}, displaying characteristic features of epidioxy sterols and was reverted to ergosterol when submitted to S. schenckii enzymatic extract. This revised version was published online in June 2006 with corrections to the Cover Date.     

Taxonomy and phylogeny of new wood- and soil-inhabiting Sporothrix species in the Ophiostoma stenoceras-Sporothrix schenckii complex

Sporothrix, one of the anamorph genera of Ophiostoma, includes the important human pathogen S. schenckii and various fungi associated with insects and sap stain of wood. A survey of fungi from wood utility poles in South Africa yielded two distinct groups of Sporothrix isolates from different geographical areas. DNA sequence and morphological data derived in this study showed that isolates in these groups represent two novel species in the S. schenckii-O. stenoceras species complex. A new species isolated from pine poles and rosebush wood and phylogenetically closely related to S. pallida is described here as Sporothrix stylites. Phylogenetic analyses also confirmed the synonymy of S. albicans and S. nivea with S. pallida. Sporothrix stylites and S. pallida also are related closely to the isolates from soil, previously treated as "environmental" isolates of S. schenckii. Soil isolates are clearly distinct from human isolates of S. schenckii. We describe the former here as Sporothrix humicola. The isolates from eucalypt poles group peripheral to most other species in the S. schenckii-O. stenoceras complex and are newly described as Sporothrix lignivora. Phylogenetic analyses of sequences of isolates from soil and wood together with those of clinical isolates showed that the human-pathogenic strains form an aggregate of several cryptic species.     

Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii

A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii. This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection. Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response. These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis. This revised version was published online in August 2006 with corrections to the Cover Date.