“ITS/CAL"靶位分子鉴定结合表型分析确诊狭义申克孢子丝菌致淋巴管型孢子丝菌病一例

患者女,54岁,农民.左上肢近手腕部外伤后出现豌豆大小的单一红色结节15d,后肿大、破溃,并在30 d内发展为多个成串状分布的结节.皮肤科检查:患者左上肢可见多个呈线性排列的紫红色结节,质硬;部分结节破溃,伴少许脓性分泌物.组织病理提示,感染灶呈混合炎性细胞浸润为主的化脓性肉芽肿炎症;过碘酸雪夫染色阴性,未见真菌孢子、菌丝及星状体.活检组织真菌培养阳性.根据培养物形态学分析和内部转录间隔区(ITS)及钙调蛋白(CAL)编码区靶位的分子生物学鉴定结果,确诊该病例为狭义申克孢子丝菌致淋巴管型孢子丝菌病.给予患者10％碘化钾溶液10 ml/次每日3次口服;治疗2个月后,患者自觉症状明显改善,后失访.本例报道提示联合应用表型鉴定和“ITS/CAL”靶位的基因分析能够准确将孢子丝菌复合体鉴定到种水平.     

手背深部真菌感染2例

报告2例手背深部真菌感染。例1女,46岁。右手背红斑、肿胀、脓疱半年余,脓液真菌培养鉴定为申克孢子丝菌。诊断:孢子丝菌病。例2女,53岁。左手背红斑、丘疹、脓疱4月,脓液真菌培养鉴定为裴氏着色真菌。诊断:着色芽生菌病。     

国内首见真皮毛孢子菌引起皮肤感染一例

目的 报道1例真皮毛孢子菌引起的皮肤感染。方法 取皮损作直接镜检、真菌培养和组织病理学检查,分离菌株行DNA序列分析、API 20C AUX试剂盒检测、明胶液化试验、温度试验和体外药敏试验。结果 70岁男性患者,右内踝植物刺伤后出现肿块、溃疡9个月。皮损直接镜检阴性,组织病理显示真皮内菌丝和孢子。沙氏葡萄糖琼脂培养基培养出奶黄色酵母样菌落,微量培养可见假菌丝、真菌丝、关节孢子和芽生孢子,API 20C AUX试剂盒检测结果提示为土生念珠菌,DNA序列分析属于真皮毛孢子菌。菌株不能液化明胶,可在25 ～ 40 ℃环境下生长,对两性霉素B、伊曲康唑、伏立康唑、制霉菌素敏感。伊曲康唑治疗4个月后皮损完全愈合。结论 根据其形态学特点和DNA序列分析,菌株被鉴定为真皮毛孢子菌。伊曲康唑治疗有效。     

种特异性引物鉴定申克孢子丝菌

目的 研究申克孢子丝菌的分子鉴定方法,为孢子丝菌感染的分子诊断奠定基础.方法 对22株申克孢子丝菌和12种12株暗色真菌临床分离株的核糖体DNA(rDNA)内转录间隔区(ITS)进行聚合酶链反应扩增.对10株来源于中国不同地区及1株来源于美国的申克孢子丝菌的扩增产物测序并进行分析,以获得的ITS2区序列为靶序列设计出申克孢子丝菌的种特异性引物(SSP),并用该引物扩增全部受试菌株.结果 序列分析显示,申克孢子丝菌rDNA的ITS2区相当保守,特异性引物对22株申克孢子丝菌可扩增出一条300bp的片段,其他受试菌株均为阴性.结论 应用设计出的种特异性引物,结合PCR方法,鉴定申克孢子丝菌特异、敏感、可靠,可用于临床诊断.     

申克孢子丝菌基因差异、致病力与孢子丝菌病临床型别的关系

目的 探讨申克孢子丝菌基因差异、致病力与孢子丝菌病不同临床型别的关系。方法 ①收集不同临床型别孢子丝菌病的申克孢子丝菌分离株并提取DNA,进行随机扩增多态DNA(RAPD)扩增。②BALB/c小鼠接种不同临床型别孢子丝菌病的分离株菌悬液,观察实验动物发病及病变情况。③发病小鼠皮肤及内脏组织病理学检查,观察接种不同临床型别孢子丝菌病的分离株菌悬液后小鼠病变内申克孢子丝菌孢子数量及分布。结果 ①不同临床型别孢子丝菌病的申克孢子丝菌分离株聚合酶链反应产物电泳带型差异较明显:播散型分离株可见1800bp、850bp、500bp、180bp,皮肤淋巴管型分离株见1400bp、800bp、700bp、500bp,皮肤固定型分离株见2500bp、1400bp、1000bp、700bp。②注射播散型孢子丝菌病分离株菌悬液的BALB/c小鼠比注射皮肤淋巴管型分离株小鼠发病早、病变部位广且死亡率高;注射皮肤淋巴管型分离株的小鼠较注射固定型孢子丝菌病分离株小鼠皮损出现早、病变范围广且严重。③实验BALB/c小鼠病变皮肤及内脏组织病理学检查显示:注射播散型孢子丝菌病分离株的小鼠病变内孢子数量明显多于注射皮肤淋巴管型分离株小鼠病变内孢子数量,而后者较注射固定型孢子丝菌病分离株的小鼠病变内孢子数量多。结论 不同临床型别孢子丝菌病的申克孢子丝菌的基因差异、致病力与孢子丝菌病不同临床型别的关系密切。     

应用PCR技术快速鉴定申克孢子丝菌的实验研究

采用Biospin真菌基因组DNA提取试剂盒提取基因组DNA,用申克孢子丝菌种特异性引物分别对21株申克孢子丝菌的基因组DNA进行PCR扩增.成功提取21株申克孢子丝菌基因组DNA,并用申克孢子丝菌种特异性引物分别从21株申克孢子丝菌基因组DNA中获得长度为320 bp的扩增产物.以申克孢子丝菌特异引物为基础的聚合酶链反应法鉴定申克孢子丝菌简便、快捷、特异,可用于临床诊断.     

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。     

皮肤淋巴管型孢子丝菌病1例

患者女,57岁。右拇指、腕部及前臂结节伴瘙痒和疼痛3月。右前臂皮损组织病理示:化脓性肉芽肿性改变。真菌培养见申克孢子丝菌生长。诊断:皮肤淋巴管型孢子丝菌病。     

石膏样小孢子菌致伪膜性阴囊癣一例

患者,男,21岁。阴囊白色斑块伴瘙痒2周。真菌培养镜检及ITS测序为石膏样小孢子菌。外用特比萘芬乳膏,4周后皮损消退,真菌学检查阴性。     

申克孢子丝菌的分子生物学研究进展

申克孢子丝菌是孢子丝菌病的致病菌 ,为双相致病真菌。发生孢子丝菌病是由于宿主体内酵母细胞增殖所致 ,同时还与宿主防御机制有关。对其基因分型、部分基因的鉴定、分子诊断方法及致病机理的分子基础等方面的研究进展进行了综述     

裴氏着色芽生菌致下肢皮肤感染一例

患者女,53岁.右下肢条带状排列损害,反复起疹30余年.损害由结节和暗红色浸润斑块覆污秽色痂组成,部分损害表面有黑点.直接镜检和组织病理可见褐色厚壁孢子,真菌培养及分子生物学鉴定病原菌为裴氏着色芽生菌.体外真菌药敏试验显示对特比萘芬及伊曲康唑敏感.联合应用特比萘芬和伊曲康唑治疗,辅以手术切除和局部热疗.治疗10个月后,脓性分泌物消失,皮损面积缩小,治愈部位留下色素沉着.对着色芽生菌病联合特比萘芬和伊曲康唑口服,辅以红光热疗及手术切除,综合疗效较好.     

我国首见由地霉引起的脓癣一例及实验研究

目的 报道我国首见由地霉所致脓癣一例。方法 患者为9岁男孩,头部出现脓癣样皮损,耳后淋巴结肿大,全面临床检查排除其他疾病。取头顶皮损表面痂皮及病发多次真菌培养、直接镜检证实为真菌病;通过真菌培养、扫描电镜、生化学实验及DNA序列分析进行菌种鉴定;通过扫描电镜及毛发受侵试验观察菌株对毛发的感染情况;通过动物试验观察菌株的致病力;观察临床抗真菌治疗效果及体外药敏实验。结果 真菌学培养均有同样菌落生长,菌落表面平坦,27℃培养边缘有菌丝生长,37℃培养为湿润的酵母样菌落。镜下见大小不一的矩形关节孢子及大量圆形或卵圆形孢子,出芽或不出芽,并可见有分支的菌丝。菌种经生化学实验等鉴定为地霉菌,DNA序列分析证实该菌属于昔维考拉地霉(Geotrichum silvicola)或其姐妹株。动物实验证明该菌有致病性。经特比萘芬治疗4周后好转,真菌学检查阴性。结论 此例地霉所致脓癣为我国首见;特比萘芬治疗可以治愈。     

Successful 2-week treatment with terbinafine (Lamisil®) for moccasin tinea pedis and tinea manuum

A new orally active antifungal agent, terbinafine, was used in the treatment of tinea pedis ('dry type' or moccasin type) and tinea manuum. Fifty-three adults over the age of 16 years with fungal infections of the feet and/or hands were treated with either oral terbinafine, 250 mg, or placebo, once daily for 2 weeks. The diagnosis of fungal infection was confirmed by examination of skin scrapings by microscopy and culture. Of these, 28 patients were evaluable for efficacy. At 8 weeks, 12 out of 14 (86%) patients who received terbinafine were mycologically negative (microscopy and culture) compared to one out of 14 (7%) patients on placebo (P less than 0.001, Fishers exact test, one-sided). At the end of the study 71% of patients in the terbinafine group were judged to have received effective therapy compared to 0% in the placebo group (P less than 0.001). Terbinafine was well tolerated, and more side-effects were seen in the placebo group.     

Deep dermatophytosis caused by Trichophyton rubrum in immunocompromised patients

In immunosuppressed patients, dermatophytosis can be more invasive, affecting the dermis and subcutaneous tissues. The authors describe the cases of two patients with kidney and heart transplanted, respectively, that developed a deep dermatophytosis caused by Trichophyton rubrum, confirmed by culture and DNA sequencing. Both patients had concomitant onychomycosis, and both were treated with itraconazole for about two months, which was interrupted due to pharmacological interactions with the immunosuppressive drugs and switched to terbinafine, leading to clinical resolution within four months. Deep dermatophytosis should be considered when dealing with immunocompromised patients, especially when a superficial dermatophytosis is present. Oral treatment is necessary and terbinafine is a preferable option in solid organ transplant recipients because it has less pharmacological interactions.     

In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine

Fifty-five strains, either authentic or ex-type, of seven Malassezia species were investigated for in vitro susceptibility to various concentrations (0.03-64.0 microg/mL) of three azole drugs, ketoconazole, voriconazole and itraconazole, as well as the allylamine terbinafine, using the agar dilution method. All strains of the seven Malassezia species were susceptible to the three azole drugs at low concentrations. M. furfur, M. sympodialis, M. slooffiae, M. pachydermatis, M. globosa, M. obtusa and M. restricta were most sensitive to ketoconazole and itraconazole, with minimum inhibitory concentrations (MICs) ranging from < or = 0.03 to 0.125 microg/mL. The recently introduced antifungal, voriconazole, was also very effective, with MIC80 values < or = 0.03 microg/mL for 80% of strains. MICs of terbinafine against the seven Malassezia species ranged from 相似文献   

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。     

从足部溃疡分离出林生地霉及其实验研究

目的 报道1例足部溃疡分离菌株的真菌学研究结果.方法 采用真菌直接镜检与培养、扫描电镜、AP120生化鉴定试验和DNA序列分析,对1例男性患者足背溃疡分离出的菌株进行鉴定,并作小鼠感染试验和药物敏感试验.结果 皮损分离菌株27℃培养见表面平坦、不湿润、无粘性的乳白色酵母样菌落;镜检见粗大、透明、分隔菌丝,有方形、矩形孢子.API20CAUX试刺盒检测,提示为帚状地霉;DNA序列分析,与林生地霉模式菌株UFMG-354-2的D₁/D₂区碱基同源序列具有99.1%的相似性,而与MTCC39474具有99.8%的相似性.该菌株对氟康唑、伊曲康唑、克霉唑、两性霉素B、制霉菌素高度敏感.小鼠接种试验显示该菌株有致病性.用伊曲康唑治疗2周后,皮损明显好转.结论 此例足部溃疡分离菌株为林生地霉,伊曲康唑治疗有效.     

球形孢子丝菌伊曲康唑耐药株在大蜡螟模型中的毒力和耐药性评价

 目的:使用大蜡螟模型评价球形孢子丝菌伊曲康唑耐药株的毒力和对伊曲康唑的敏感性。方法：使用球形孢子丝菌伊曲康唑耐药株和敏感株分别建立大蜡螟幼虫感染模型，观察大蜡螟幼虫的外观变化、身体反馈及存活情况。向感染球形孢子丝菌的大蜡螟幼虫体内分别以20 mg/kg和40 mg/kg的剂量注射伊曲康唑，检测大蜡螟幼虫的存活情况和载菌量。结果：观察终点时，大蜡螟幼虫耐药株组和敏感株组之间的生存曲线无统计学差异（ X²=2.37，P=0.124）；注射40 mg/kg的伊曲康唑后，感染球形孢子丝菌耐药株和敏感株的大蜡螟生存率分别提高40%和60%，两者之间无统计学差异（ X²=1.20，P=0.466），敏感株感染的大蜡螟菌落计数降低更为明显（t=-18.64，P <0.001）。结论：在大蜡螟模型中，球形孢子丝菌伊曲康唑耐药株的毒力和敏感株无差异，耐药株对伊曲康唑的敏感性低于敏感株。     

球黑孢霉引起白色浅表型甲真菌病一例

目的 报道1例球黑孢霉引起的白色浅表型甲真菌病.方法 取病变趾甲作直接镜检、真菌培养和组织病理学检查,分离菌株行DNA序列分析、明胶液化试验、体外药敏试验和甲板受侵试验.结果 21岁男性患者,右足拇趾甲板白色病变5个月.病变甲屑直接镜检发现孢子、菌丝和叶状分生孢子梗,组织病理显示甲板内有大量孢子及菌丝.沙氏葡萄糖琼脂培养基培养出黑色羊毛状菌落,DNA序列分析提示为黑孢霉属.菌株可液化明胶和侵犯甲组织,对制霉菌素、伊曲康唑、两性霉素B、克霉唑高度敏感.伊曲康唑冲击治疗5个疗程后,病甲恢复正常.结论 根据其形态学特点和DNA序列分析,菌株被确定为球黑孢霉.球黑孢霉引起的白色浅表型甲真菌病为国内首例报道,伊曲康唑治疗有效.     

Quantitative analysis of Malassezia in the scale of patients with psoriasis using a real-time polymerase chain reaction assay

BACKGROUND: It has been suggested that Malassezia is associated with the development of skin lesions in psoriasis because of the response of the scalp lesions in psoriasis to antifungal agents. Malassezia restricta and M. globosa are the two major members of the cutaneous Malassezia flora in patients with psoriasis, although they have not been analysed quantitatively. OBJECTIVES: This study quantified the two major cutaneous Malassezia species in psoriatic scale from different body sites using a real-time polymerase chain reaction (PCR) assay. METHODS: Scale samples were collected from lesional and nonlesional skin of 20 Japanese patients with psoriasis and fungal DNA was extracted from the samples directly. All the Malassezia species, including the two major species M. globosa and M. restricta, were quantified with high accuracy, using a real-time PCR assay. RESULTS: Colonization by M. restricta was approximately five times higher at all body sites than colonization by M. globosa. Malassezia colonization was significantly lower in patients with hyperlipidaemia than in patients with normolipidaemia. CONCLUSIONS: Malassezia restricta is the predominant species in psoriatic scale.