【作者】 闫洁；

【导师】 郝飞；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 足菌肿是一种热带或亚热带疾病,该病的世界分布极其不均,南纬15?和北纬30?之间的区域为足菌肿高发区,尤以南亚的印度、非洲的苏丹和南美的墨西哥发病率为最高。据苏丹喀土穆中心医院的保守统计,足菌肿的就诊率大约在300~400人/年度。而地处温带和寒带的国家发病率较低,罕见于中国。据不完全统计,1960~2009这半个世纪以来,我国共有足菌肿的报道18例,发病率不足1人/年度。足菌肿可发生于全身任何部位,其中以足部最多见,约占70%,其次为手、四肢、肩背、臀部、会阴。一些罕见部位如胸腹壁、睾丸、女阴、腭部、眼睑、眶内、鼻旁窦、脑、肺、脊柱、胆囊、手术切口处也有报道。头颈部足菌肿的发生率为3.75%,一个无法解释的现象是:发生在头面部的足菌肿,80%由放线菌引起,仅有不足20%由真菌引起。尽管头颈部足菌肿发病率低,但后果严重且非常难以治疗。根据致病菌种类的不同,足菌肿分为真菌性足菌肿和放线菌性足菌肿两种。马杜拉分支菌是真菌性足菌肿最常见的致病菌,已知的致病菌种有两种:足菌肿马杜拉分支菌和灰色马杜拉分支菌。足菌肿马杜拉分支菌在过去一直被认为是同质的,不存在多态现象。但是,随着分子生物学的发展,越来越多的证据表明足菌肿马杜拉分支菌和灰色马杜拉分支菌的异质现象非常明显,很有可能存在其它菌种。系统进化分析甚至显示足菌肿马杜拉分支菌和灰色马杜拉分支菌的进化距离很远,极大可能不属于一个种属。本课题报道了重庆地区一例罕见的发生在下颌部的真菌性足菌肿,研究该病例的意义不仅在于它发病部位的特殊性和地域的罕见性,更重要的是我们从患者下颌部黑色颗粒中分离到一株国内罕见的暗色真菌,常规真菌学无法判知其种属。经ITS区测序发现,该真菌与足菌肿马杜拉分支菌有最大的相似性,但相似性小于95%,极大可能是马杜拉分支菌属一新的菌种,菌株编号为TMMU3956。鉴于马杜拉分支菌混乱的分类学范畴,本研究的目的在于获得TMMU3956详尽的形态学、理化特性、药物敏感性、动物致病性和分子生物学等方面的资料,为证明它是马杜拉分支菌属一新的菌种提供证据,并对马杜拉分支菌的进化起源和分类学进行探讨。我们以TMMU3956为研究对象,将菌株接种在沙堡氏培养基(SDA)、马铃薯培养基(PDA)、脑心浸膏培养基(BHIA)、察氏培养基(CDA)和玉米培养基(CMA)上行平皿培养和玻片培养,8周后观察菌落宏观形态和光镜、扫描电镜下微观形态。行温度试验、同化试验观察其理化特性。采用改良的美国临床实验室标准化协会(CLSI)M-38A方案对TMMU3956进行体外药敏试验,测试药物包括氟康唑、氟胞嘧啶、伊曲康唑、两性霉素B、特比萘芬和伏立康唑。玻璃珠-盐析法提取TMMU3956的DNA,使用通用引物和足菌肿马杜拉分支菌特异性引物扩增真菌的ITS1、ITS2、D1/D2、18S和ITS1+2区,测序后进行BLAST,并选取有代表性的40种真菌性足菌肿致病菌行系统进化分析。最后用TMMU3956感染免疫正常的和免疫抑制的BALB/c小鼠和新西兰大白兔,观察动物致病性。结果显示:光镜与电镜下,该真菌至少观察到两种无性繁殖方式:瓶梗产孢和孢子囊孢子产孢。最佳生长温度为37℃,最高耐热温度为42℃。能同化葡萄糖、阿拉伯糖、木糖、纤维二糖、麦芽糖、海藻糖。对伊曲康唑、两性霉素B、伏立康唑高度敏感,特比萘芬中度敏感,对氟胞嘧啶和氟康唑耐药。未检测到BALB/c小鼠和新西兰大白兔致病性。18S和ITS1+2区与足菌肿马杜拉分支菌的相似性分别为100%和93%,足菌肿马杜拉分支菌特异性探针无法扩增TMMMU3956。系统进化分析显示TMMU3956与足菌肿马杜拉分支菌遗传距离最近,同属于子囊菌门核菌纲;而灰色马杜拉分支菌属于子囊菌门腔菌纲,与TMMU3956相去甚远。根据TMMU3956的形态学、理化特性、rDNA的序列分析及系统进化分析,我们认为该菌株有别于足菌肿马杜拉分支菌和灰色马杜拉分支菌,支持该菌株为马杜拉分支菌属一新的致病菌种。更多还原

【Abstract】 Mycetoma is a chronic granulomatous subcutaneous infection caused by true fungi (eumycetoma) or bacterial actinomycetes (actinomycetoma). The pathogens involved are found in the environment in certain types of soil and are directly inoculated into the subcutaneous tissues, commonly in the foot, through minor trauma or a thorn prick. Mycetoma has a prolonged, progressive, and indolent course and, if untreated, ultimately leads to destruction of deeper tissues and bone, resulting in deformity and disability which may necessitate amputation. The triad of a subcutaneous painless mass, sinuses, and grains discharged through the sinuses is the hallmark of mycetoma.Mycetoma has a worldwide but uneven distribution. The disease is endemic in tropical and subtropical regions and the African continent has the highest prevalence. Eumycetoma prevails in the mycetoma belt that stretches between the latitudes of 15? south and 30? north. The belt includes Sudan, Somalia, Senegal, India, Yemen, Mexico, Venezuela, Columbia, Argentina, and other countries. In Sudan, An admission of 1231 mycetoma cases to outpatient clinics throughout the country within a period of 2~5 years was observed. The disease has also been extensively reported in India. But it is seldom encountered in Western countries. Most of the imported cases involved immigrants who probably contracted the infection in their home countries. This complicates the clinical diagnosis since it is frequently unexpected. Reports on mycetoma have come from the USA, Ceylon, Germany, Egypt, Turkey, Philippines, Japan, Lebanon, Thailand, Iran, Netherlands, and Saudi Arabia. What is more, the disease didn’t spare China. Between the decades of 1960~2009, 18 cases have been reported in China, of which 9 are eumycetoma and 9 are actinomycetoma.Mycetoma can affect any part of the body, and lesions are not restricted to the limbs only. Most cases are usually seen in the feet (70%), followed by hands (12%), then legs and knee joints. In highly endemic areas, other parts of the body might become affected as well. These include the arm, head and neck, thighs, and the perineum. Rarely, the chest and abdominal walls, facial bones, mandible, paranasal sinuses, eyelid, vulva, orbit, scrotum, and old surgical incisions might also be affected. The incidence of mycetoma infections of the head and neck is 3.75%. For unexplained reasons 80% craniofacial mycetoma was due to actinomycetes. Although the craniofacial mycetoma is rare, when affected it is the most difficult to treat. They can lead to serious complications arising from extension of swelling through the cranium that puts pressure on the brain. If the patient does not seek early medical care, the disease may run a rapidly fatal course.This study presents an extraordinary case of craniofacial eumycetoma extending from gum to lower jaw in a 27-year-old Chinese male. Our case is worth reporting not only by its rareness in China but also by its unusual affected site. Moreover, we have isolated a distinctive dematiaceous fungus from clinical specimens of this patient. By sequencing of the internal transcribed spacer (ITS) region, we found that it had maximum sequence identity with Madurella mycetomatis, one of the main microorganisms causing black-grain fungal mycetoma, but the similarity was less than 95%. It is proposed that the fungus is probably a novel species of genus Madurella and coded as TMMU3956 tentatively.Madurella species are well known agents responsible for eumycetoma. Two species are recognized, M. mycetomatis and M. grisea. The homogeneity of M. mycetomatis has been reported previously, at least in Sudan. By sequencing the ITS region with the ITS4 and ITS5 primers and using large-scale random amplification of polymorphic DNA(RAPD), those authors suggested that this fungus had a clonal origin. However, the same group that suggested a clonal origin of M. mycetomatis strains from Sudan has recently reported polymorphism within this species by using amplified fragment length polymorphism (AFLP). Those authors even suggested a possible relationship with clinical data such as lesion size. In the present study, the genus Madurella has been proven to be heterogeneous on the basis of rDNA small subunit (SSU) and ITS sequencing data. de Hoog et al. found that the ITS1+2 sequences of four M. mycetomatis clinical strains, CBS201.38, from Indonesia, CBS248.48, from New Mexico, USA, CBS216.29, from Italy, and CBS217.55, from Argentina, have significant difference (average 5.3%) and supposed that the sequence variability at ITS locus of‘M. mycetomatis’might be related to the geographical area and the climatic environment. In addition, another investigation showed that 7 out of 15 M. mycetomatis strains diverged significantly from the type strain such as IP582.60, IP584.78, IP592.74 and IP1137.76 by comparison of the ITS2 regions. These strains were tentatively identified as Madurella sp. In fact, the CBS collection holds a number of strains from other climatic zones which showed more than 5% ITS difference and thus are likely to be a separate species. On the other hand, Madurella grisea were demonstrated to be more heterogeneous and closer to some Pyrenochaeta romeroi strains than to type M. grisea strains by comparison of the ITS sequences. Sequencing of the SSU rDNA gene of the authentic strain of M. mycetomatis (CBS247.48) and M. grisea (CBS331.55) revealed that M. mycetomatis was close to the genera Neurospora, Sordaria and Podospora, which all belong to the order Sordariales of ascomycetes, whereas M. grisea clustered with species of the genera Leptosphaeria, Cucurbidothis, Westerdykella and Sporormia, which are members of the order Pleosporales belong to ascomycetes. These data prove that‘Madurella’has been currently applied as an umbrella term covering partly unrelated species. The taxonomic positions of M. grisea need improvement, and the identification of new species responsible for eumycetoma is warranted.In this study, we try to provide evidences for demonstrating that our strain is different from M. mycetomatis and M. grisea and to facilitate the timely administration of effective therapy by elucidating the morphological and physiological characteristics, antifungal susceptibility, experimental animal pathogenicity and rRNA gene sequence of M. TMMU3956. In concrete, we inoculated M. TMMU3956 onto plate and slide culture of different medium. Both gross morphological and microscopic observations were made in addition to scanning electron microscopy. The temperature test between 25~42℃and assimilation assay with API AUX 20C was carried out. Antifungal susceptibility test with itraconazole, voriconazole, amphotericin B, terbinafine, fluconazole and flucytosine was performed according to a modified CLSI 38-A method. Finally, the DNA extraction was conducted by the glass beads-salting out procedure and rDNA sequences were amplified with ITS, 18S and 28S primers. A Phylogenetic analysis tree was constructed based on the ITS1+2 sequences. In addition, an animal experiment with BALB/C mice and New Zealand white rabbits was attempted.The results showed that at least two ways of asexual multiplication, the phialospores and sporangiospores, were observed. The fungus could tolerate 42°C. Assimilation of glucose, arabinose, xylose, cellose, maltose and trehalose was positive. Antifungal susceptibility test indicated that it was highly susceptible to itraconazole, voriconazole and amphotericin B, moderately susceptible to terbinafine, and resistant to fluconazole and flucytosine. No characteristic black grains or detectable infection were observed in BALB/C mice and New Zealand white rabbits. The analyses of the ITS region and the 18S ribosomal gene sequences support a new species designation.In conclusion, the new strain is different from M. mycetomatis and M. grisea. It is suggested that this strain is a novel species of Madurella responsible for eumycetoma.更多还原