1例皮肤播散型孢子丝菌病的分离菌株的基因鉴定

孢子丝菌病是由申克孢子丝菌及其卢里变种感染引起的皮肤、皮下组织和附近淋巴系统的亚急性和慢性感染,偶可播散至全身引起多系统损害。该病遍布全世界,我国孢子丝菌病主要是由申克孢子丝菌引起。孢子丝菌一般生存在土壤和植物上,人的皮肤接触带菌植物或土壤后可引起感染。临床上主要表现为固定型和淋巴管型孢子丝菌病,皮肤播散型孢子丝菌病少见。现认为机体感染申克孢子丝菌引起的不同的临床型别与机体免疫状态、申克孢子丝菌的致病力及其基因差异相关[1-2]。我     

申克孢子丝菌感染诊治1例

通过1例16岁女孩由申克孢子丝菌导致的皮下孢子丝菌病,口服伊曲康唑治疗有效,回顾申克孢子丝菌的实验室检验方法 ,以此来提高孢子丝菌感染的诊断水平。     

申克孢子丝菌体外药敏试验研究近况

申克孢子丝菌是孢子丝菌病的致病菌,如何选择高效敏感的抗真菌药物对于有效治疗孢子丝菌病具有重要意义,抗真菌药物体外药敏试验是评价抗真菌药物抗真菌活性的重要手段。综述了申克孢子丝菌体外药敏试验的研究近况,展望了研究及应用前景。     

鼻部固定型孢子丝菌病1例

报道1例猫抓后引起的固定型孢子丝菌病.患者男,16岁,皮损表现为鼻翼部位的增生物,其上覆有脓痂.临床上易与细菌感染混淆,但根据患者的病史、临床表现、病理、真菌镜检及培养诊断为申克孢子丝菌引起的孢子丝菌病.患者在应用7个月的碘化钾结合特比萘芬软膏外用治疗后,皮损完全消失.     

申克孢子丝菌菌丝相至酵母相转化的实验研究

目的观察申克孢子丝菌菌丝相向酵母相转化的形态学变化并初步研究连续传代后菌株转化为酵母相的百分率。方法将95株申克孢子丝菌临床株于脑心浸液琼脂培养基上连续传代至酵母相,利用显微镜及血细胞计数板计数菌丝和孢子比例并记录显微镜下形态。结果 95株申克孢子丝菌中,经1次传代即成功转化有23株,经2次传代有14株,经3次传代有10株,经4次传代有6株,4次传代后总计55.8%实验菌株转化为酵母相。结论部分申克孢子丝菌由菌丝相向酵母相转化需经过连续多次传代,连续传代增加了菌丝相至酵母相的转化率。     

1株多重耐药申克孢子丝菌感染的治疗

报道1例由多重耐药申克孢子丝菌引起的面部孢子丝菌病。患者61岁男性,面部皮疹2 a余,先后口服伊曲康唑、特比萘芬和氟康唑治疗16个月无效。患者皮损经真菌镜检和培养确诊为孢子丝菌病,分离的申克孢子丝菌体外药敏试验显示其菌丝相和酵母相对上述三种药物均不敏感。给予患者口服10%碘化钾溶液治疗3个月获得痊愈。     

皮内与腹腔接种不同来源菌株对实验性小鼠孢子丝菌病的影响

目的将不同来源的申克孢子丝菌菌株接种于免疫抑制处理后的小鼠皮内及腹腔,造成其实验性感染,观察其感染情况,研究不同菌株及不同接种途径对小鼠孢子丝菌病的影响。方法分别在小鼠皮内和腹腔注射孢子丝菌菌悬液0.1ml,约含1×107个孢子,观察3周。观察结束时处死小鼠取其血、脑、肺、肝、脾、肾、肠系膜、睾丸、腹膜进行真菌培养和组织病理检查。结果皮内和腹腔接种不同来源菌株的小鼠各器官感染率各不相同。播散株皮内接种组、固定株腹腔接种组、播散株腹腔接种组的各器官平均感染率分别为16.05%、18.89%、58.73%。播散株腹腔接种组与其他二组之间差异均有显著性,脾和睾丸是最易受累的器官。结论不同来源菌株可能毒力不同,播散型菌株具有更强的侵袭性。脾脏可能在宿主抗孢子丝菌感染中具有重要作用。     

山苍子油体外抗申克孢子丝菌的实验研究

目的探讨山苍子油对申克孢子丝菌的体外抗菌活性及其作用机理。方法纸片扩散法定性检测山苍子油对申克孢子丝菌的体外抗菌活性,CLSI-M27-A微量液基稀释法定量检测其对14株临床分离申克孢子丝菌(包括固定型和淋巴管型各7株)的最小抑菌浓度(MIC),以氟康唑作为质控药物。电镜观察山苍子油作用前后申克孢子丝菌超微结构的改变。结果纸片扩散法1周后可见清晰的抑菌环,山苍子油对皮肤固定型株和皮肤淋巴管型株的MIC范围均为156.25～1250μg/ml,差异无显著性(P>0.05)。电镜观察显示山苍子油作用24h后申克孢子丝菌的菌丝干枯、断裂,孢子壁疏松、碎裂;胞膜及胞壁结构模糊,胞浆皱缩,细胞器及细胞核结构模糊。结论山苍子油对临床分离的皮肤型孢子丝菌有较强的抗菌活性,且其对固定型株和淋巴管型株的抗菌活性无显著性差异。电镜结果提示山苍子油可能通过破坏菌胞膜,影响其通透性,进而扰乱细胞正常代谢而起到抗菌作用。     

特比萘芬治愈儿童鼻翼孢子丝菌病1例

目的报道1例发生在4岁儿童鼻翼的孢子丝菌病。方法刮取病变组织接种到沙堡弱培养基培养,并对培养出的病原菌进行形态学鉴定和ITS区序列分析。结果病变组织在沙堡弱培养基25℃培养长出黑褐色菌落,25℃小培养后光镜及扫描电镜下见菌丝细长,分枝分隔,合轴式产孢形成的成簇和沿菌丝两侧排列,分生孢子卵圆形。提取真菌总DNA用PCR方法扩增ITS序列,测序后登录GeneBank进行比对,该菌与申克孢子丝菌菌株KMU3360ITS区碱基序列一致性达99%,鉴定为申克孢子丝菌。内服联合局部应用特比萘芬治疗6个月后皮损完全消退。结论确诊1例儿童鼻翼的孢子丝菌病,内服联合局部应用特比萘芬治疗儿童孢子丝菌病疗效良好。     

实验室确诊的孢子丝菌感染51例

孢子丝菌病由申克孢子丝菌感染而引起,由于一些医师缺乏对该疾病临床表现的认识及实验室检查手段的欠缺,易造成误诊。为了提高对该病的诊断率,减少因误诊给患者带来的痛苦,我们选择了2005年在外院误诊的病例51例,后经我院真菌实验室和病理室检查纠正原诊断的患者情况进行报道。     

Effect of potassium iodide on the immune response in the sporotrichosis.

Potassium iodide (KI), the specific treatment for sporotrichosis, apparently does not have a direct action on Sporothrix schenckii. The spontaneous healing and the variability of the clinical presentation in the disease have strengthened the idea that the KI rather interacts with the immune response of the host. The phagocytic process is inefficient in individuals with sporotrichosis in whom the microbicidal mechanism of halogenation fails to control the disease. There is evidence that blocking of free radicals decreases in the presence of KI. Humoral and cellular immunity are present in sporotrichosis but its participation is uncertain; it is yet to be determined if in this mycosis the KI influences other processes or factors of immune response.     

Immune Response Against Sporothrix schenckii in TLR-4-Deficient Mice

For many fungal diseases, macrophages are the major cell population implicated in host protection, primarily by their ability to eliminate the invading fungal pathogen through phagocytosis. In sporotrichosis, this remains true, because of macrophages’ ability to recognize Sporothrix schenckii through specific receptors for some of the fungus’ cellular surface constituents. Further confirmation for macrophages’ pivotal role in fungal diseases came with the identification of toll-like receptors, and the subsequent numerous associations found between TLR-4 deficiency and host susceptibility to diverse fungal pathogens. Involvement of TLR-4 in immune response against sporotrichosis has been conducted to investigate how TLR-4 signaling could affect inflammatory response development through evaluation of H2O2 production and IL-1β, IL-6 and TGF-β release during the course of S. schenckii infection on TLR-4-deficient mice. The results showed that macrophages are largely dependent on TLR-4 for inflammatory activation and that in the absence of TLR-4 signaling, increased TGF-β release may be one of the contributing factors for the abrogated inflammatory activation of peritoneal exudate cells during mice sporotrichosis.     

Isolation and functional characterization of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II

The N-linked glycosylation is a ubiquitous protein modification in eukaryotic cells. During the N-linked glycan synthesis, the precursor Glc(3)Man(9)GlcNAc(2) is processed by endoplasmic reticulum (ER) glucosidases I, II and α1,2-mannosidase, before transporting to the Golgi complex for further structure modifications. In fungi of medical relevance, as Candida albicans and Aspergillus, it is well known that ER glycosidases are important for cell fitness, cell wall organization, virulence, and interaction with the immune system. Despite this, little is known about these enzymes in Sporothrix schenckii, the causative agent of human sporotrichosis. This limited knowledge is due in part to the lack of a genome sequence of this organism. In this work we used degenerate primers and inverse PCR approaches to isolate the open reading frame of S. schenckii ROT2, the encoding gene for α subunit of ER glucosidase II. This S. schenckii gene complemented a Saccharomyces cerevisiae rot2Δ mutant; however, when expressed in a C. albicans rot2Δ mutant, S. schenckii Rot2 partially increased the levels of α-glucosidase activity, but failed to restore the N-linked glycosylation defect associated to the mutation. To our knowledge, this is the first report where a gene involved in protein N-linked glycosylation is isolated from S. schenckii.     

Host organism defense by a peptide-polysaccharide extracted from the fungus Sporothrix schenckii

A peptide-polysaccharide, a peptide-rhamnomannan, was isolated from the pathogenic yeast form of the fungus Sporothrix schenckii. This substance, which may play a role in fungal virulence, was tested in an animal model of systemic disease, and depression of the immune response was observed in the animals between the 4th and 6th week of infection. Concomitantly, this compound showed mitogenic activity when challenged with normal lymphocytes and was also found to be involved in the inflammatory response. These results provide further information for the understanding of fungal implantation in tissues and of the pathogenicity of this systemic mycosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variants of Sporothrix schenckii with attenuated virulence for mice

Strains of Sporothrix schenckii preserved under mineral oil were examined for virulence in BALB/c mice. The mice were inoculated with S. schenckii conidia and development of cutaneous lesions, signs of inactivity, weight loss, survival rates, number of viable yeast cells in lung and spleen, splenomegaly and organ lesions were evaluated. After intravenous injection of 7.5 x 10(6) conidia, two of five S. schenckii strains were unable to induce systemic disease and to kill the mice, only producing cord-like lesions on the tail that regressed with mouse maturation. Very small numbers of viable cells isolated from the spleen confirmed the lower invasive ability of these strains when compared with other strains studied here. These results suggest a relationship between the attenuation of virulence and the storage method under mineral oil after long periods of time.     

Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes

Vesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans , raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a trans-cell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans , Candida parapsilosis , Sporothrix schenckii and Saccharomyces cerevisiae documents that vesicles are similarly produced by additional ascomycetes. The vesicles from H. capsulatum react with immune serum from patients with histoplasmosis, providing an association of the vesicular products with pathogenesis. The findings support the proposal that vesicular secretion is a general mechanism in fungi for the transport of macromolecules related to virulence and that this process could be a target for novel therapeutics.