致病性外瓶霉的PCR-RFLP分类

应用PCR方法对致病性外瓶霉进行分类鉴定。以ITS3和ITS4为引物 ,对常见的7种致病性外瓶霉的模式株核糖体DNA转录间隔区(ITSⅡ)进行扩增 ,4种内切酶(HinFI、MspI、BsuRI和RsaI)酶切。各种间多态性显著 ,常规方法难以鉴别的皮炎外瓶霉和甄氏外瓶霉较易区分。PCR RFLP准确可靠 ,可用于形态及其他方法难以确定的致病菌种的鉴别。     

致病性外瓶霉的体外抗真菌药敏试验研究

目的 测定致病性外瓶霉的药物敏感性,对不同药物间最小抑菌浓度（ＭＩＣ）的差异以及各菌种间药物敏感性差异进行比较,为指导临床正确用药提供理论依据。方法 参照Ｍ２７－ａ方案中的微量稀释法。受试菌株包括１３株皮炎外瓶霉。１７株甄氏外瓶霉,１３株丛梗孢外瓶霉,１０株棘状外瓶霉及４株威尼克外瓶霉;所研究的药物为伊曲康唑,氟康唑,酮康唑,二性霉素Ｂ及５－氟胞嘧啶。结果 二性霉素Ｂ、伊曲康唑、氟康唑、酮康唑和５     

致病性瓶霉超微结构的研究

我们应用扫描电镜对疣状瓶霉、烂木瓶霉、寄生瓶霉和匐根瓶霉作了超微结构的研究.疣状瓶霉的瓶梗顶端破裂,出现典型的花瓶状的领状结构,并可见向基性连续发生的分生孢子.烂木瓶霉在光镜下见到的吸盘状、领状结构,在电镜下则见到领状结构内壁增厚,可能是连续产生分生抱子致使其内壁增生,促进领状结构轻度外翻.寄生瓶霉和匐根瓶霉均有较长瓶梗,但寄生瓶霉的较长;而匐根瓶霉瓶梗常见成簇并常有分枝.但有时需要结合培养形态来进行鉴别.     

甄氏外瓶霉和皮炎外瓶霉线粒体的DNA酶切分析

暗色孢科菌的分类目前仍以形态学为依据,致某些属间的鉴别较困难.如高氏瓶霉和甄氏瓶霉,现改为外瓶霉,曾在形态学和生物学方面有过很多争论;又如皮炎外瓶霉常与某些甄氏外瓶霉混淆.本文作者分析了15株高氏瓶霉,30株甄氏瓶霉和31株皮炎外瓶霉的线粒体(mt)DNA酶切电泳图.企图从基因分析进行分类研究.     

微量法检测致病性外瓶霉对特比萘芬的敏感性

目的 :　探讨应用美国国家实验室标准委员会 (NCCLS)推荐的微量法检测致病性外瓶霉对特比萘芬的敏感性 ,为临床治疗由外瓶霉引起的暗色丝孢霉病提供依据。方法 :　参照M 2 7A方案(1997)检测 7种 6 6株外瓶霉最小抑菌浓度 (MIC) ,其中皮炎外瓶霉 (E .d) 19株、甄氏外瓶霉 (E .j) 18株、棘状外瓶霉 (E .sp) 12株、丛梗孢外瓶霉 (E .m) 13株、威尼克外瓶霉 (E .w) 2株、鲑鱼外瓶霉 (E .sa) 1株、嗜鱼外瓶霉 (E .p) 1株 ,菌悬液终浓度 (0 .5～ 2 .5 )× 10 3 CFU/ml,孵育温度 2 7℃ ,培养时间 5～ 7天。分别应用RPMI 16 40和SDB培养基比较其MIC值的差异。结果 :　特比萘芬对 6 6株外瓶霉的MIC范围为0 .0 0 4～ 0 .5 μg/ml,小于 0 .12 5 μg/ml的菌株有 5 8株 ,占 87.9% ,MIC50 分别为E .d 0 .0 3μg/ml,E .sp 0 .0 93μg/ml,E .j0 .0 3μg/ml。E .m 0 .0 3μg/ml,E .w 0 .0 12 μg/ml。应用RPMI 16 40和SDB培养基时MIC值具有一致性。结论 :　经过改良的M 2 7A方案可以用于特比萘芬对外瓶霉的药敏实验。致病性外瓶霉对特比萘芬的敏感性较高 ,由该类菌引起的暗色丝孢霉病可用特比萘芬治疗。     

疣状瓶霉与美洲瓶霉的关系

众所周知疣状瓶霉及美洲瓶霉这两种暗色丝孢霉可引起着色真菌病,即使大多数医学真菌学家认为美洲瓶霉是疣状瓶霉的同义名,然而有些学者根据美洲瓶霉的颈圈较疣状瓶霉更深而认为这二种菌是不同的种。作者等对32株证实为美洲瓶霉或疣状瓶霉的菌种从形态学,生理学及抗原特征来研究其分类情况,其中12株可产生带或浅或深颈圈的分生孢子。这32     

外瓶霉和暗色丝孢霉病

外瓶霉是暗色真菌的一个属,可以引起皮肤、皮下组织及系统性暗色丝孢霉病,对人类的健康危害较大。随着器官移植、免疫抑制治疗的广泛使用及实验室诊断技术的提高,外瓶霉感染的病例报告日趋增加。治疗较为困难,皮肤及皮下组织感染,经手术及抗真菌药物治疗效果较好,但系统性感染者预后较差。     

威尔尼克外瓶霉致右手掌黑癣

报告1例威尔尼克外瓶霉致掌黑癣。患者女,24岁。右手大鱼际淡褐色椭圆形斑块4年余,无不适。皮损鳞屑真菌镜检:10%KOH涂片可见浅棕色短菌丝及出芽孢子,美蓝涂片染色可见短菌丝及孢子。皮屑培养生长缓慢,初期为灰白色至橄榄色酵母样菌落,很快变为墨绿色至黑色菌落。菌落表面可见絮状、暗灰色至绿灰色气生菌丝。对培养菌株行土豆培养基小培养观察形态学符合威尔尼克外瓶霉,用扫描电镜观察其产孢方式为环痕产孢、合轴式排列方式,菌丝两侧及顶端有环痕梗,环孢子聚集在环痕梗周围。基因测序与标准菌株GQ334387.1(GI:254802560)威尔尼克外瓶霉具有100%同源性。诊断为掌黑癣。给予5%硝酸舍他康唑乳膏每日2次外用,连用2周后皮损完全消退。随访3个月未见复发。     

PCR-RFLP技术快速鉴定八种致病丝状病原真菌的实验研究

目的 建立能快速鉴定深部丝状真菌感染病原菌的PCR-RFLP方法。方法 用真菌通用引物扩增烟曲霉、黄曲霉、土曲霉、黑曲霉、杂色曲霉、构巢曲霉、尖端赛多孢和串珠镰刀菌的ITS区,分别用HhaⅠ、HaeⅢ、HinfⅠ、TaqⅠ和MspⅠ 5种限制性核酸内切酶对PCR产物进行酶切,建立以PCR为基础的RFLP方法,然后对22株临床株和2株环境分离株进行PCR-RFLP图谱分析。结果 对PCR产物进行RFLP分析可以准确鉴定8种深部致病丝状真菌,从DNA提取到酶切分析可以在1个工作日完成。22株临床株和2株环境分离株PCR-RFLP鉴定结果与传统的形态学鉴定结果一致。结论 PCR-RFLP技术是一种能够快速鉴定丝状真菌的有效方法。     

Molecular Phylogenetics of Exophiala Species Isolated from Korea

Background

Recently, identification of fungi have been supplemented by molecular tools, such as ribosomal internal transcribed spacer (ITS) sequence analysis. According to these tools, morphological Exophiala species was newly introduced or redefined.

Objective

This study was designed to investigate the phylogenetics based on ribosomal ITS sequence analysis from clinical Exophiala species isolated in Korea.

Methods

The strains of Exophiala species were 4 clinical isolates of phaeohyphomycosis agents kept in the department of dermatology, Dongguk University Medical Center(DUMC), Gyeongju, Korea. The DNAs of total 5 strains of Exophiala species were extracted by bead-beating method. Polymerase chain reaction of ITS region using the primer pairs ITS1-ITS4, was done and phylogenetic tree contributed from sequences of ITS region from 5 Korean isolates including E. dermatitidis CBS 109154 and comparative related strains deposited in GenBank.

Results

The strains of Exophiala species were 3 strains of E. dermatitidis, 1 strain of E. jeanselmei and 1 strain of Exophiala new species. Among the 3 subtypes (type A, B, C) of E. jeanselmei, E. jeanselmei DUMC 9901 belonged to type B. Of the 2 main types of E. dermatitidis (type A, B) and 3 subtypes of E. dermatitidis type A (A0, A1 and A2), two strains (E. dermatitidis CBS 709.95, E. dermatitidis CBS 109154) belonged to A0 subtypes, 1 strain (E. dermatitidis DUMC 9902) A1 subtype, respectively.

Conclusion

Phylogenetic analysis of ITS region sequence provided useful information not only for new species identification but for the subtyping and origin of Exophiala species.     

A Case of Cutaneous Infection by Exophiala jeanselmei

A case of cutaneous infection by Exophiala jeanselmei is reported. The patient was a 45-year-old Japanese female. Her skin eruption consisted of a group of erythematous papules on her left cheek to which corticosteroid had been topically applied for nearly 10 years. Histopathologically, a granuloma formation was noted in the dermis within which were many brown spores. The isolated fungus was identified as Exophiala jeanselmei.     

皮炎外瓶霉分子鉴定的初步研究

目的：设计皮炎外瓶霉种特异性引物，并对其特异性及敏感性进行检测。方法：通过对暗色真菌核糖体DNA进行分析，设计皮炎外瓶霉种特异性引物；实验菌株包括标准株、参照株及临床分离株等皮炎外瓶霉，以及甄氏外瓶霉、裴氏着色霉、卡氏枝孢霉、疣状瓶霉等，应用常规聚合酶链反应(PCR)及快速PCR方法检测其特异性及敏感性。结果：序列分析显示皮炎外瓶霉rRNA基因转录内间隔区较为保守，特异引物对15株皮炎外瓶霉均可扩增出单一的特异性片断，将模板稀释成1万倍后仍可得到相同的结果，其他致病菌种均为阴性。结论：皮炎外瓶霉种特异性引物具有较高的特异性及敏感性，可试用于该菌种的鉴定。     

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.