从野生蟾蜍分离致病性着色真菌的研究

目的 从野生蟾蜍分离致病性着色真菌。方法 在着色芽生菌病高发区和无病区分别捕捉野生蟾蜍,取内脏制成悬液分离着色真菌。结果 在着色芽生菌病高发区山东章丘,354只野生蟾蜍中有151只分离出着色真菌,占42.7%,共计分离出着色真菌11种,162株,其中疣状瓶霉49株,外瓶霉33株,瓶霉属19株,甄氏外瓶霉19株,卡氏枝孢12株,可疑卡氏枝孢3株,喙枝孢7株,裴氏着色霉5株,毛样枝孢4株,棘状外瓶霉3株,枝孢属3株,小尾枝孢1株,未鉴定4株。而在无病区山东莱阳,45只蟾蜍中仅有4只分离出非致病性着色真菌,占8.9%。结论 着色芽生菌病流行区野生蟾蜍真菌的分离阳性率显著高于非流行区,且分离出多种致病性着色真菌,提示野生蟾蜍带菌与着色芽生菌病的流行相关。     

几种常见暗色致病真菌的耐热实验

观察并记录经37℃、45℃、55℃处理10、30、60min后裴氏着色真菌、紧密着色真菌、疣状瓶霉、卡氏枝孢霉、皮炎外瓶霉、甄氏外瓶霉、假阿利什菌、链格孢和孢子丝菌在沙氏培养基上的生长情况。所有受试菌种的菌悬液经37℃、45℃不同时间处理后再培养的生长情况未受影响；在55℃处理后除皮炎外瓶霉、链格孢外其余菌种在55℃不同时间处理后生长均受不同程度影响。可通过耐热试验来鉴别皮炎外瓶霉与甄氏外瓶霉；局部温热引起的局部血循环加速，血管扩张，局部细胞因子释放、各种效应细胞等可能也参与了对着色真菌及其它几种常见深部致病真菌的杀灭过程。     

裴氏着色真菌种特异性DNA探针的筛选

通过斑点杂交和Ｓｏｕｔｈｅｒｎ印迹技术，首次从已构建的裴氏着色真菌基因组ＤＮＡ库中筛选出种特异性ＤＮＡ探针。首先借助斑点杂交技术，筛选出与紧密着色真菌、皮炎万氏霉、疣状瓶霉、卡氏枝孢霉和甄氏外瓶霉及细菌、人白细胞等无同源性的二个ＤＮＡ片段；再经Ｓｏｕｔｈｅｒｎ印迹分析，确认其中的Ｂｆ片段（３．１ｋｂ）具有良好的裴氏着色协菌种特异性，并初步显示具有多态性特征。     

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。     

以蛋白SDS-PAGE图谱为依据的致病性暗色丝孢科真菌系谱学研究

目的　研究对人类致病的主要暗色丝孢科真菌裴氏着色霉、紧密着色霉、疣状瓶霉、皮炎瓶霉、卡氏枝孢霉、甄氏外瓶霉间的系谱格局。方法　采用薄层扫描方法对上述6种30株重要致病性暗丝孢科真菌可溶性全细胞蛋白单向SDS PAGE图谱结合基型趋异方法进行分析。结果　得出其系谱格局为{ [P. verrucosa(F. pedrosoiF. compacta) ]W. dermatitidis[E. jeanselmeiC. carrionii] }。结论　显示(F. pedrosoi和F. compacta,E. jeanselmei和C. carrionii,以及Fonsecaea和Phialophora之间有密切关系。     

287株致病性着色霉菌实验室观察与分析

目的　研究山东省章丘县及其周围地区着色霉菌病的菌种组成。方法　采用葡萄糖蛋白胨琼脂培养基进行试管法培养 ,将培养阳性菌落分别进行葡萄糖蛋白胨琼脂、葡萄糖玉米粉琼脂、马铃薯葡萄糖琼脂钢圈小培养 ,观察其分生孢子梗形态 ;应用扫描电镜观察了卡氏枝孢霉的产孢方式。结果　共鉴定出 2 83株卡氏枝孢霉、2株裴氏着色霉、1株疣状瓶霉及 1株未定瓶霉。结论　山东省章丘县及其周围地区着色霉菌病的致病菌种以卡氏枝孢霉为主。这一结果进一步证实我国北方该病致病菌种主要为卡氏枝孢霉。     

对流免疫电泳检测着色真菌病患者血中抗体

着色真菌病血清学诊断重要环节是抗原选择。菌丝作抗原常引起暗色真菌间交叉反应。有报道用4～6周裴氏着色真菌培养上清液作抗原,其实验结果与卡氏枝孢霉无交叉反应,并有助于疣状瓶霉与卡氏枝孢霉的鉴别。本文作者用卡氏枝孢霉培养     

着色芽生菌单孢子悬液制备方法的改进

在对着色芽生菌病致病菌种的生物学特征、菌种分类和药敏观察等方面的实验研究中,制备单孢子悬液是其重要的一环。既往所采用的吹打、研磨等方法,常不能获得满意结果。我们参考了有关文献介绍的方法［１,２］并加以改进,结果满意,兹介绍如下。１材料和方法１．１材料：采用Ｈ－１混合器,血球计数板,定性滤纸（中速、为１２．５ｃｍ）;马铃薯葡萄糖琼脂（ＰＤＡ）培养基（自配）,葡萄糖和琼脂均购自上海化学试剂公司;菌株：卡氏枝孢霉６株,疣状瓶霉３株,裴氏着色霉２株,紧密着色霉２株,均为本科真菌室保存菌株。其中６株卡氏枝…     

着色芽生菌病致病菌DNA含量的流式细胞术测定

应用流式细胞术（FCM）比较4种7株着色芽生菌病致病菌间DNA含量的差异。研究用卡氏枝孢霉4株，疣状瓶霉、裴氏着色霉、紧密着色霉1株，制备稳定生长阶段各菌株单孢子悬液，碘化丙啶（PI）一步插入法荧光染色，用人淋巴细胞作为内参标准进行DNA含量测定，80％以上的细胞处于DNA合成周期的G0/G1期，统计学处理显示4种致病菌DNA含量两两比较存在显著性差异，而卡氏枝孢霉种内两两比较DNA含量无显著性差异。FCM有望为医学真菌学提供一种先进、有效的研究方法和途径。     

着色芽生菌病:巴西亚马逊地区325例的回顾性研究

着色芽生菌病是一种侵犯皮肤和皮下组织的慢性、化脓性、肉芽肿性疾病。该病在世界上分布广泛。主要流行于热带和亚热带。病原菌由一组密切相关的黑霉所致 ,各菌在培养中的显微镜下表现各异 ,但其组织相只有一种 :硬壳小体。最常见的病原菌为裴氏外瓶霉、疣状瓶霉、卡氏枝孢霉 ,而水喙枝孢霉罕见。该研究分析了亚马逊地区在过去 5 5年 (1 942～ 1 997)中发生的 3 2 5例着色芽生菌病病例。根据患者年龄、性别、皮损部位、职业等分类 ,更新了Ｓｉｌｖａ于 1 968年在Ｐａｒ偄州进行的有关该病的系列研究 ,并探讨与当地的生态 流行病学的联系。…     

Chromoblastomycosis

Chromoblastomycosis, together with phaeohyphomycosis and mycetoma, makes up the disease entities caused by the dematiaceous fungi. Most cases of chromoblastomycosis are caused by five genera of fungi: Fonsecaea compactum, Fonsecaea pedrosoi, Phialophora verrucosa, Cladosporium carrionii, and Rhino-cladiella aquaspersa. The disease has a cosmopolitan distribution but predominates in rural, agricultural settings. Clinically, chromoblastomycosis is hallmarked by verrucous nodules at the site of fungal implantation. Treatment involves surgical excision of the affected area, antimycotic agents, physical modalities such as temperature manipulation, or a combination of these.     

A case of chromoblastomycosis with an unusual clinical manifestation caused by Phialophora verrucosa on an unexposed area: treatment with a combination of amphotericin B and 5-flucytosine

Chromoblastomycosis is a cutaneous and subcutaneous mycotic disease caused by the dematiaceous (black) fungi. Five species of fungi are known generally to be the cause: Fonsecaea pedrosoi, Phialophora verrucosa, Cladosporium carrionii, F. compacta and Rhinocladiella cerphilum. In infected tissue they can appear as pigmented sclerotic bodies, commonly called 'copper pennies', which are pathognomonic of chromoblastomycosis. The infection usually occurs through traumatic skin inoculation, with the majority of lesions occurring on the feet and legs of outdoor workers. We report a patient in whom the lesions had begun on the right breast, which is an unexposed area, without a history of trauma. A uniform, reliable treatment does not exist but our patient was mycologically cured with the use of amphotericin B and the subsequent combination of 5-flucytosine and itraconazole.     

应用PCR-RFLP进行申克孢子丝菌的分子生物学鉴定

目的　探索一种简单、快速的申克孢子丝菌的鉴定方法。方法　应用真菌通用引物ITS1和ITS4对来源于不同地区及不同临床型别孢子丝菌病的 2 8株申克孢子丝菌以及 9种其他临床上重要的真菌进行PCR扩增 ,利用限制性内切酶HaeⅢ对PCR产物进行酶切分析鉴定。结果　所有 2 8株申克孢子丝菌和其他 9种真菌均扩增出一条约 3 5 0bp的片段 ,其中 2 8株申克孢子丝菌RFLP带型一致 ,与 9种其他临床上重要的真菌RFLP带型差异较明显。 结论　PCR RFLP可以为建立一种简单、快速鉴定申克孢子丝菌的方法提供依据。     

Lymphangitic chromoblastomycosis

Chromoblastomycosis (CM), a chronic subcutaneous mycosis, is caused by several dematiaceous fungi, the most common being Fonsecaea pedrosoi. It usually occurs in the lower extremities following traumatic implantation of the organisms. We are reporting a case of chromoblastomycosis on the right lower limb in a sporotrichoid pattern caused by F. pedrosoi. The pattern was probably due to lymphatic spread that seems to be one of the rare presentations. The histopathology showed typical muriform or medlar bodies both intracellularly and extracellularly within the granuloma. Culture revealed sporulating organisms (Cladosporium and Rhinocladiella type) by a combination method, characteristic of F. pedrosoi. Our case responded well to itraconazole.     

Treatment of chromoblastomycosis with terbinafine: a report of four cases

This report is about four cases of chromoblastomycosis confirmed by direct examination, histopathology and cultures. The duration of disease infection varied from 5 to 12 years. By culture, Cladosporium carrionii was isolated in two cases and Fonsecaea pedrosoi in the other two cases. Terbinafine 0.25 g twice daily for 1 month and 0.25 g once daily for maintenance therapy were given to three patients. Terbinafine 0.25 g once daily was given to one patient. After 4-8 months of therapy, all cases were cured without relapse when followed up for 6 months. The total dosage of terbinafine was 37.5-60 g. No relevant side effects showed during treatment.     

Chromoblastomycosis

Chromoblastomycosis is a chronic subcutaneous mycotic infection caused by pigmented or dematiaceous saprophytic moulds ubiquitous in the environment. The most common etiologic agents are Fonsecaea pedrosoi and Cladophialophora carrionii, both of which can be isolated from plant debris. The infection usually follows traumatic inoculation through penetrating thorn or splinter wounds. The fungal agents develop as small clusters of cells known as muriform bodies. Several months after the injury, painless papules or nodules appear in the affected area progressing to scaly and verrucose plaques. Direct examinations of skin scrapings or histopathologic study demonstrates the typical muriform bodies. Microbiologic culture is necessary for the correct determination of the etiologic agent. Itraconazole is the treatment of choice, often in combination with surgery. Even so, results are often unsatisfactory as patients present late to medical services because of lack of funds and the fact that the disease usually affects the main family earner.     

Chromoblastomycosis: a clinical and molecular study of 18 cases in Rio de Janeiro, Brazil

Background Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by dematiaceous fungi. Methods We described epidemiological data, clinical presentation, and treatment of 18 cases of CBM diagnosed in Rio de Janeiro, Brazil. Diagnosis was obtained by mycological, histopathological findings demonstrating typical muriform cells with confirmation of isolated by DNA sequencing of the ribosomal internal transcribed spacer. Results The majority of patients were male (72.2%) ranging from 39 to 83 years old, farm laborers and construction workers. The duration of disease varied from four months to 32 years. The most common presentations were verrucous form in ten (55.6%) patients, followed by tumoral in three (16.7%) patients, primarily of moderate (55.6%) and severe (38.9%) intensity. Lower (44.4%) and upper limbs (33.3%) were the most affected sites. Fonsecaea pedrosoi isolated from 14 (77.8%), and Cladophialophora carrionii isolated from one case (5.6%). Fifteen patients (83.3%) were treated. Six patients (40%) received oral itraconazole 200–400 mg/day, five patients (33.3%) received oral itraconazole 200–400 mg/day combined with fluconazole 200 mg/day, and four (26.7%) patients were submitted to surgery. The duration of therapy varied from 12 to 48 months. Cure rate was 80% (12/15). No relapse was observed after two years of follow‐up. Conclusions Success was due to attending a center with specialized clinical care, laboratory support, and pharmaceutical care.