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     

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。     

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。     

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。     

甄氏外瓶霉致暗色丝孢霉病

报告1例甄氏外瓶霉感染所致的暗色丝孢霉病.患者男,59岁.右小腿外踝上方肿块伴瘙痒5个月,皮损为一直径约3 cm的暗红色斑块.皮损组织病理PAS染色见分隔菌丝,组织块马铃薯葡萄糖琼脂(PDA)培养基上25℃培养长出黑色酵母样菌落,分离菌株最高生长温度37℃,小培养下见分枝分隔菌丝,环痕产孢,分生孢子聚集在环痕梗顶端,分离菌株ITS1区测序结果与甄氏外瓶霉98.54%同源.根据分离菌株生物学特点,菌株被确定为甄氏外瓶霉.     

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，外瓶的霉素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。     

系统性红斑狼疮伴棘状外瓶霉所致暗色丝孢霉病一例

患者女,27岁,右下肢结节、溃疡6个月,有系统性红斑狼疮病史2年。皮损脓液直接镜检可见分支、分隔链状菌丝和孢子,组织病理检查显示棕黄色菌丝、孢子。沙氏葡萄糖琼脂培养基（SDA）培养出深绿色绒状菌落,微量培养可见分支、分隔菌丝和棘状环痕孢梗,DNA序列分析属于棘状外瓶霉。菌株不能液化明胶,可在25 ~ 39 ℃环境下生长,对伊曲康唑、两性霉素B、特比萘芬敏感。动物实验发现免疫抑制小鼠感染比正常对照组严重。依据临床特征、组织病理学检查、真菌培养及基因鉴定结果,该例患者确诊为系统性红斑狼疮伴棘状外瓶霉所致的暗色丝孢霉病。     

临床常见毛孢子菌的鉴定

为了探讨临床常见毛孢子菌（Trichosporon spp.)的鉴定方法，在形态学、营养生理学试验的基础上对临床常见的毛孢子菌－皮瘤毛孢子菌（Trichosporon inkin)、皮毛孢子菌（Trichosporon cutaneum)及阿萨希毛孢子菌（Trichosporon asahii)进行核糖体核糖核酸（rRNA)基因（rDNA)的内转录间区（ITS）扩增，用限制性内切酶Cfr3I、NcoI对聚合酶链反应（PCR）产物进行限制性片段长度多态性（RFLP）分析及序列测定，发现用Cfr13I做酶切，皮毛孢子菌的RFLP带型和阿萨希毛孢子菌、皮瘤毛孢子菌的不同；用NcoI做酶切，皮瘤毛孢子菌的带型与另两者不一致，序列分析的结果与形态学、营养生理学试验一致。结果提示，温度试验、API 20C、RFLP及序列测定可用于鉴定临床常见的毛孢子菌。     

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.     

Development of an oligonucleotide probe specific for Trichophytonrubrum

A species-specific DNA probe was developed to detect the dermatophyte species Trichophyton rubrum. The selected oligonucleotide sequence is derived from the highly variable internal transcribed spacer 2 region of the ribosomal DNA operon. The specificity of the non-radioactive labelled oligonucleotide probe was tested against related dermatophytes, other eukaryotic microorganisms and against human DNA. No cross-hybridization was found, and hybridization signals were invariably detected in all T. rubrum strains investigated. In addition, no homologous sequences were found searching the EMBL database. Experiments to establish a method for isolating DNA directly from clinical specimens gave successful amplification and hybridization products in about 30% of the samples.     

Molecular typing of Trichophyton mentagrophytes var. interdigitale isolated in a university hospital in Japan based on the non‐transcribed spacer region of the ribosomal RNA gene

Detecting intraspecies polymorphisms in fungi causing dermatophytoses is important in elucidating routes of infection and determining whether Tinea recurrence is caused by exacerbation or re‐infection. In fungi, the non‐transcribed spacer region (NTS) of the ribosomal RNA gene shows the greatest accumulation of base sequence mutations. We therefore assessed NTS sequences in 64 clinical isolates of Trichophyton mentagrophytes var. interdigitale, the second most common species of dermatophytes in Japan. These isolates were among the clinical isolates of dermatophytes in the Department of Dermatology, Kanazawa Medical University Hospital in 2006 and were obtained by morphological and molecular biological identification methods. DNA was extracted from each isolate, as well as from one isolate maintained in our department, to detect length polymorphisms at each of three variable loci, TmiS0, TmiS1 and TmiS2, of the NTS for subtyping. We observed seven patterns for TmiS0, six patterns for TmiS1 and three patterns for TmiS2. The combinations of these patterns enabled us to classify the 65 isolates into 15 types. The most prevalent, constituted 46% (30/65) of all isolates. Eleven types were new combinations, whereas the other four were previously described. These results suggest that this method may be used to determine the molecular epidemiology of T. mentagrophytes var. interdigitale in Japan, because it generated results rapidly and in a sensitive manner.     

少见致病性毛壳菌形态学及分子生物学特性研究

目的：研究毛壳菌的形态学和分子生物学特性,为临床快速准确地鉴定此类真菌奠定基础。方法：对8株3种毛壳菌在不同培养基上的形态、显微镜下形态和不同温度生长情况进行研究,同时对其rDNA的ITS区进行序列分析,并且根据其序列的差异设计球毛壳菌的种特异性引物。结果：不同种毛壳菌的培养表现及显微镜下形态有其各自的特点。8株菌在燕麦培养基上28℃时生长较好,37℃时不能生长。毛壳菌种间rDNA的ITS区序列存在较大差异,根据此差异设计出的球毛壳菌种特异性引物具有较好的特异性。结论：根据培养形态、显微镜下表现以及基因序列的差异可以对毛壳菌进行准确的鉴定。     

Case of chromoblastomycosis appearing in an Okinawa patient with a medical history of Hansen's disease

Chromoblastomycosis is one of several chronic infectious skin diseases caused by various species of dematiaceous fungi. It is clinically characterized by verrucous skin eruptions and occurs most commonly in tropical and subtropical regions. In Okinawa, a subtropical area, there have been only three reported cases of chromoblastomycosis including the present one. Direct microscopic examination of crust specimens and findings of sclerotic cells in histopathology can confirm the diagnosis, and cultures of crust and/or tissue specimens can identify the causative fungi. We herein report the third case of chromoblastomycosis in Okinawa; it arose in an 87-year-old Japanese woman with a history of Hansen's disease, who lived in a leprosarium in Miyako Island. To identify the causative agent as Fonsecaea pedrosoi, we used the polymerase chain reaction and direct sequencing analysis in addition to the usual methods, which include 20% potassium hydroxide microscopy, histopathological confirmation of sclerotic cells by periodic acid-Schiff stain, culture by Sabouraud's glucose agar, slide culture method, and observation of conidia by scanning electron microscopic examination.     

Identification of several clinical isolates of dermatophytes based on the nucleotide sequence of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA.

Nucleotide sequences of internal transcribed spacer 1 (ITS 1) in nuclear ribosomal DNA from seven morphologically unidentified dermatophyte isolates were determined. The sequences were compared with those of typical isolates of Trichophyton (T.) mentagrophytes var. interdigitale, T. rubrum, and Epidermophyton floccosum. Two of the isolates were classified as T. rubrum and the other five as T. mentagrophytes var. interdigitale. The results did not conflict with identifications using other molecular techniques, including random amplification of polymorphic DNA (RAPD) analysis and restriction enzyme analysis of mitochondrial DNAs. Thus, the nuclotide sequence of ITS 1 is possibly a good molecular marker for identification of these major anthropophilic dermatophyte species.     

实时荧光定量PCR检测石蜡标本中麻风菌DNA

目的：评价实时定量荧光PCR(Real-time PCR)法检测石蜡标本中麻风菌DNA的应用价值。方法：根据基因库(GenBank)发表的M. leprae的全基因组序列,以一段重复序列(repetitive ele￣ment,RLEP)为扩增靶序列,合成引物和探针,构建质粒pGEMT-101作为标准品,用Real-time PCR对石蜡标本进行麻风菌DNA检测,并评价其敏感性。结果：对52例麻风患者的石蜡包埋组织标本麻风菌进行了检测,不同型别麻风(LL：8;BL：10;BT：28;TT：6)的石蜡标本检测阳性率分别为100％(8/8)、80％(8/10)、78.57％(22/28)、50％(3/6)。结论： Real-time PCR方法可在石蜡标本中快速灵敏的检测到麻风菌DNA。