无绿藻病的现状与发病原因分析

目的了解我国无绿藻病的发病现状及流行病学特征。方法通过对国际及国内已报道的无绿藻病相关文献的分析,归纳其病因、发病机制、临床表现、诊断治疗及预后,并对病原菌的分类、鉴定及体外药敏试验作描述。结果根据众多对无绿藻病相关文献的综合分析,导致人与动物致病的无绿藻现被确认为有3个种：大型无绿藻、中型无绿藻及小型无绿藻。而与人类疾病相关的仅是中型及小型两种。无绿藻病的临床表现主要分为3类：皮肤及皮下组织感染、滑膜炎及其纤维组织炎、系统性感染。结论随着各种原因导致的免疫缺陷患者的增多,无绿藻病的发病在全球有上升趋势,此病呈慢性、无痛性,未发现有自愈倾向。我国无绿藻病的诊断率可能远低于实际感染率,无绿藻病的治疗至今尚无明确的方案及标准。     

小型无绿藻的分离及形态特征

无绿藻（Ｐｒｏｔｏｔｈｅｃａ）是一种少见的条件致病菌^［１］,可引起人畜共患的无绿藻病。在其所致人类疾病中,主要有三种临床类型：皮肤损害、局限性感染、系统性无绿藻病^［２］。本文于１９９７年从一名女患者面部丘疹处的皮肤结节活检,分离培养出一株无绿藻菌,并进行了分离培养鉴定,利用光学显微镜、电子显微镜对其形态特征和超微结构进行观察,并通过直接组织压片和组织病理切片观察和生化试验特性,确定为一例由小型无绿藻（Ｐｒｏｔｏｔｈｅｃａｗｉｃｋｅｒｈａｍｉｉ     

无绿藻的表型及其分子生物学鉴定

目的通过表型及分子生物学方法,正确鉴定无绿藻及其变种。方法回顾、总结与分析无绿藻的形态与结构、表型特征及生理生化及分子生物学鉴定、对常见抗真菌药物的敏感性及组织病理学特征。结果通过表型及分子生物学技术成功鉴定2株来自脑脊液及淋巴结的无绿藻:中型无绿藻碳水化合物变种及中型无绿藻波多黎各变种,并展示相关照片。结论无绿藻病的症状尚无特异性,其诊断主要依靠真菌学检查。标本的直接镜检、真菌培养及组织病理检查是主要手段。对无绿藻菌种的鉴定除了菌落形态、镜下结构外(含内孢子的孢子囊是无绿藻属的重要特征),糖类、醇类的同化利用,温度试验,结合分子生物学鉴定将有助于菌种的鉴定。     

无绿藻的研究进展:从基础到临床

无绿藻是一种直径约3 ～ 30 μm的单细胞生物,广泛存在于自然界和动物体表及体内,属于条件致病性真菌.目前主要通过直接镜检、真菌培养、组织病理学检查及分子生物学等手段对无绿藻进行鉴定.现已发现无绿藻属包括五个种,其中对人有致病性的仅为中型无绿藻基因型2、小型无绿藻和P.blaschkeae,其致病机制可能与外伤和免疫力低下有关.随着研究的深入,越来越多的无绿藻病被临床确诊.根据不同的类型及其临床表现,对无绿藻病的治疗也有所区别.为了提高对无绿藻这一条件真菌及其致病性的认识,该文对其生物学特性、鉴定方法、致病性、临床表现等研究进展做一简要综述.     

小型无绿藻致血流感染1例及文献复习

患者,男,44岁,农民。全身皮肤散在红色斑疹、左小腿皮肤溃烂伴发热。在皮肤伤口分泌物及血液中均培养出酵母样菌落,菌落涂片显示不同大小的球形孢子囊,具有桑葚样外观。最终确诊为无绿藻菌感染,予以氟康唑、伊曲康唑治疗后死亡。本病例包含的数据及其文献复习将增加医务工作者对该病原体的认识。     

无绿藻分子生物学鉴定与分型研究新进展

以基因序列为鉴定基础的分子生物学方法以其客观、可重复性好等优点,被认为是分子鉴定无绿藻的"金标准"。该文对近年来应用在无绿藻鉴定和分型方面的实时定量PCR(Real-Time Quantitative PCR,RT-PCR)技术、单链构象多态性(Single Stranded Conformation Polymophism,SSCP)分析、限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)分析、高分辨率熔解曲线(High Resolution Melting,HRM)分析、质谱(Mass Spectrometry,MS)分析等分子生物学新技术和新方法进行综述。     

谈谈绿藻的两种进化树

植物学家普遍地认为具双鞭毛的衣藻型或类似衣藻型的单细胞绿藻是原始型的绿藻。按照传统的观点,绿藻门基本包括三条进化路线:一条是群体的(或团藻的)路线,藻体为游动的单细胞或群体,营养时期无细胞     

全球近10年无绿藻病例报道文献的回顾

该文系统性地回顾分析了人类无绿藻病在近10a来的发病率及其趋势,并对该病的流行病学、临床表现、诊断标准、治疗方案及其预后的新特点进行了总结。全球至今已报道190例无绿藻病,约33%的无绿藻病例在近10a内发生;系统性无绿藻发病率在近10a病例数与皮肤型无绿藻病基本持平。我国大陆地区总共报道有9例无绿藻病,大约占我国(包括港澳台地区)已有报道的无绿藻病例总数的50%。无绿藻病的诊断主要依靠标本的真菌学及组织病理学检查。两性霉素B及其脂质体仍然是该病治疗最常用且最有效的药物。提示无绿藻病在近10a中发病率明显上升,且系统性无绿藻发病率显著升高。     

无绿藻对小鼠Th17细胞分化的影响

目的 研究无绿藻是否能够影响小鼠Th17细胞的分化,影响Th17细胞分泌相关的细胞因子.方法 体外培养无绿藻,用尼龙毛柱法分离培养小鼠脾脏T淋巴细胞,按照1∶5的比例将两者在transwell培养板上共培养,培养2h、4h、8h、12 h、24 h、48 h,分别收集T淋巴细胞,留取培养上清液.流式细胞技术检测Th17细胞表型,ELISA法检测培养上清液中IL-17的水平.结果 从共培养2h开始,随着共培养时间的延长Th17细胞所占比例逐渐降低,共培养8h后趋于稳定（P＜0.05）;从共培养2h开始,培养上清液中IL-17的水平逐渐升高,8h后出现下降,24h后趋于稳定（P＜0.05）.结论 在与T细胞共培养的条件下,无绿藻抑制了小鼠Th17细胞的分化,但是在最初2～8h可以促进Th17相关细胞因子IL-17的释放.     

下肢小型无绿藻感染1例

患者,男,39岁,8年前体检时发现全血细胞减少,行骨髓、流氏细胞术等检查确诊阵发性夜间血红蛋白尿(paroxysmal nocturnal hemoglobinuria, PNH)后,长期服用“环孢素、泼尼松”治疗,2个月余前左下肢部位皮肤划破后出现破溃伴红肿热痛,取创面组织及分泌物行病理检查与病原学检测,选用哌拉西林/他唑巴坦经验性抗感染治疗。在确诊为小型无绿藻所致左下肢软组织感染,药敏试验提示其对两性霉素B敏感后,更改抗菌药物为两性霉素B静滴：首日1 mg试针,第2～4日10 mg,第5～10日剂量增加至25 mg,每日一次。10天后因肌酐升高,将用量减至15 mg,隔日一次,同时进行创口护理,经对症治疗1个月后复查,患者左下肢皮肤破溃无明显炎性分泌物,创面已基本愈合。     

Simple new test for rapid differentiation ofPrototheca stagnora fromP. wickerhamii andP. zopfii

A simple new test to differentiatePrototheca wickerhamii andP. zopfii fromP. stagnora by determining susceptibility to neomycin is described. Susceptibility determined using a 30 µg neomycin disk provides a rapid and reliable means of distinguishingP. wickerhamii andP. zopfii fromP. stagnora.     

DEGRADATION OF ADENINE BY PROTOTHECA ZOPFII (CHLOROPHYTA)1

The purines 6-amino-2-hydroxypurine and 6-amino-8-hydroxypurine, not normally associated with purine degradation in algae, were isolated by high-pressure liquid chromatography from cell extracts of Prototheca zopfii Krüger grown with adenine as the sole nitrogen source .     

An ultrastructural survey of the genus Prototheca with special reference to plastids

An ultrastructural investigation of six different species of Prototheca showed that all of them contained starch grains enclosed in double-membrane-bounded structures recognized as plastids. It is concluded that these unicellular species of Prototheca must be considered as non-photosynthetic algae.     

A rapid real-time PCR/DNA resolution melting method to identify Prototheca species

Aim: The study describes the development of a simple and rapid tool to identify yeast‐like microalgae belonging to the genus Prototheca. Methods and Results: The method, based on two‐step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non‐pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype‐specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. Conclusions: The assay two‐step Real Time PCR is accurate, robust, cost‐effective and faster than auxonographical, biochemical or conventional molecular biology methods. Significance and Impact of the Study: the rapid and high throughout two‐step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.     

Activity of biogenic silver nanoparticles against isolates of Prototheca species from bovine mastitis

Prototheca spp. cause numerous infections in a wide variety of species, including treatment-unresponsive mastitis. Thus, the search for an effective therapy is essential. Silver nanoparticles are compounds with high therapeutic potential. This study aimed to evaluate the susceptibility profile and morphological changes in Prototheca spp. treated with biogenic silver nanoparticles (Bio-AgNP). The algaecide activity was evaluated in microplates by microdilution method, resulting in a MIC₅₀ of 30 μg ml⁻¹ and a MIC₉₀ of 60 μg ml⁻¹. Scanning electron microscopy demonstrated changes in the surface of Prototheca bovis cells following treatment. The algaecide activity of Bio-AgNP suggests a therapeutic potential as a novel approach for the control of Prototheca spp. in bovine mastitis.     

Effects of some factors on protoplast formation of a microalga,Prototheca zopfii

The effects of experimental factors on protoplast formation of Prototheca zopfii Kru¨ger in 0.85 m NaCl using Macerozyme R-200 were studied based on a fractional factorial experimental design. The rate of protoplast formation was mainly affected by the incubation temperature and the age of algal cells. The optimal condition for the maximum protoplast yield was determined based on a response surface model. These were: mid-logarithmic phase cells and Macerozyme concentration of 4% at a temperature of 35°C.     

Immunohistochemistry combined with periodic acid-Schiff for bovine mammary gland with protothecal mastitis

Immunohistochemistry (IHC) of bovine cytokeratin combined with periodic acid-Schiff (PAS) was applied to study the pathogenesis, localization and distribution of Prototheca zopfii in bovine mammary protothecosis. The standard immunohistochemical procedure using anti-bovine cytokeratin was employed before and after PAS staining to optimize this combined method. The best results were obtained when IHC procedures were performed first. Most of the epithelial cells reacted strongly with the pancytokeratin antibody. Protothecal cell walls stained well with PAS. Algal organisms were present within the lumen and between the epithelial lining and basement membrane of the affected alveoli, but not inside the positive mammary epithelial cells. This combined staining method resulted in clear alveolar epithelial detail and good contrast between the epithelial cells and algae, and contributed to studying the pathogenesis of P. zopfii in mammary protothecosis.