1、第四节 真菌感染检查,检验特点,形态学检查为检测真菌的重要手段 抗原检测适合血清和脑脊液中隐球菌、念珠菌、荚膜组织胞浆菌。 血清学诊断适用于深部真菌感染。,检测程序,标本,直接镜检,抗原检出,分离培养,不染色,乳酸酚棉蓝染色,墨汁染色,氢氧化钾消化后涂片镜检,二相性真菌,脑心浸液,脑心浸液,沙氏培养基,病原性真菌,血琼脂平板,观察菌落性状和菌丝孢子形态,显微镜检查-湿片法或直接涂片高倍视野镜检，见有菌丝孢子或单细胞真菌（有诊断价值，但不能确定真菌种类）,真菌的菌落-真菌分类重要的依据,真菌的菌落一般是指在一定基质上，接种某种真菌的一个孢子或一段菌丝，经过培养，向四周蔓延生长出丝状的群体称为真菌
2、菌落。菌落呈放射状生长，因而菌落外周的生命力最旺盛。在不同成分的培养基上和不同条件下培养，同一种真菌，形成的菌落也有差别。因此，菌落的形态观察是指在固定的条件下，菌落所表现的形状、大小、色泽和结构等。不同的真菌菌落所表现特征差别显著，它为真菌分类工作提供了重要的依据。,菌落颜色,除酵母菌的菌落比较简单，丝状真菌的菌落形态各种各样。常以下述特征加以描述。 其颜色的多样很难用色泽描述，常借助于色谱加以鉴别。很多真菌产生多种颜色的色素，使菌落的背面染有颜色，有的甚至分泌可溶性色素，扩散到全部基质中去。,菌落质地,气生菌丝构成表面菌丝体质地。底部菌丝体埋伏于培养基中或紧贴培养基表面向四周蔓延， 由底部
3、体直接生出分生孢子梗，菌落外观呈短茸毛状， 由底部菌丝体生出气生菌丝，再由它生出分生孢子梗，因而往往有缠绕的气生菌丝团，一般菌落较厚，呈絮状。 也有部分气生菌丝扭结成绳索、还有分生孢子梗自底部菌丝成束地生长，在菌落的外观上成粒状或粉状。 有些真菌产生子实体或菌核，则菌落表面呈颗粒结构。 有的菌落出现同心环或辐射状沟纹。 有的真菌只在菌落中间区域生出分生孢子头，边缘菌丝则不生育。 菌落的大小也大有不同，有些种的菌落可蔓延扩展到整个培养基，而另一些种的菌落则局限生长。,真菌菌落外观结构描述,菌落表面平滑或具皱纹、致密或疏松、同心环或辐 射状沟纹等；菌落质地绒毛状、毡状、棉絮状、羊毛状、束状、绳索状
4、、粉粒状、明胶状或皮革状等；菌落的边缘可呈全缘、锯齿状、树枝状或纤毛状等；菌落高度扁平、丘状隆起、中心部分突起或凹陷等。,193 patients (392 samples); 43 with ICSensitivity SpecificityMannan (ag) 40 % 98 %Antimannan (ab) 53 % 94 %ag and/or ab 80 % 93 %,（一）抗原：甘露聚糖 Detection of Mannan/Antimannan in Serum of Patients with Invasive Candidiasis,Sendid B et al, J Cl
5、in Microbiol 1999;37:1510-7,（二）抗原：半乳甘露聚糖 Serial Aspergillus Galactomannan Screening,n = 362 high-risk neutropenic patients Sandwich ELISA 2 times weekly, 11.7% positive n = 30 proven IA n = 9 probable IA n = 264 with no IA Sensitivity 89.7% Specificity 98.1% PPV 87.5% NPV 98.4%,Maertens J et al, Blo
6、od 2001;97:1604-10,GM抗原检测用于侵袭性曲霉病诊断,与临床诊断和疗效反应有良好的一致性。 检测方法有ELISA、放射免疫分析（RIA）和乳胶凝集试验等，但敏感性低。 ELISA检测24只兔(王莉，等。2003 )敏感性和特异性：血清 95% 78%尿液 90% 51.4%,检测曲霉GM抗原的ELISA试验,GM抗原ELISA检测方法 （Platelia试剂盒）,48例患者：确诊IA 4例，3例阳性；高度怀疑IA 13例，8例阳性；可疑IA 31例，2例阳性。,王莉，等。临床皮肤科杂志，待发表。,灵敏性为64.7% 特异性为93.5% 假阳性率为6.5% 假阴性率为35.3%
7、,（三）新生隐球菌乳胶凝集试验,（四）-1,3葡聚糖,使用仪器及配套试剂盒能快速地检测出血液和体液中真菌-1,3葡聚糖的实际含量，对真菌的早期诊断具有重要意义。,-1,3-D-葡聚糖检测阴性是否需要作进一步的 复查？,Furuya的报道即使是确诊的深部真菌感染患者,-1,3-D-葡聚糖检测也有可能是阴性,因此单次-1,3-D-葡聚糖检测阴性并不能排除深部真菌感染的可能，必須在疾病的过程中重复进行-1,3-D-葡聚糖检测。,Furuya T et al. Usefulness of -D-glucan measurement for diagnosis of deep mycosis, Jnp
8、J Antibiot 1993; 46(6):437-443,确诊深部真菌感染病人病史調查,临床表现及影像学检查:,1)发热、咳嗽、痰血10余天,2) 抗生素治疗效果不佳,3) 胸部CT检查,发现左肺阴影,支氣管鏡活組織檢查:,找見真菌,真菌分子生物学的鉴定方法,核酸碱基GC比分析 限制性片段长度多态性 Southern印迹分析 脉冲场凝胶电泳（PFGE） PCR 随机扩增多态性 (RAPD) DNA片段测序等,分子流行病学研究,Yu J，et al. Mycopathologia, 2004.,引物OPI07 RAPD扩增结果,引物OPK20 RAPD扩增结果,IGS区扩增结果,真菌感染检查
9、项目的选择和应用,浅部真菌直接显微镜检查 深部真菌标本培养、观察培养物性状并借助理化特性作鉴别。,Direct Examination,It is highly recommended that a direct microscopic examination be made on most this provide an immediate presumptive diagnosis for the physician, but it may also aid in the selection of an appropriate culture medium.A phase-contrast
10、 microscope is a valuable adjunct in the direct examination of specimens. The advantages include the following:1)mounts can be made and examined quickly;2)there is no need for direct staining;and3)the objects can be clearly visualized.,Tests for detection of fungal antibodies(1),Determination of ant
11、ibody and/or antigen titers may be useful in diagnosing fungal infections and when performed in a serial fashion also provide a means of monitoring the progression of disease and the patients response to therapy .,With the exceptionof antibody tests for histoplasmosis and occidioidomycosis,however,m
12、ost tests designed to detect an antibody response as a means of diagnosing invasive fungal infections lack sensitivity and specificity,are poorly standardized and are not widely available,Tests for detection of fungal antibodies(2),Tests for detection of fungal antibodies(3),Antibody tests for Candi
13、da and Aspergillus. Are often unable to distinguish active from past infection and Colonization from transient fungemia,Tests for detection of fungal antibodies(4),In addition,a negative test for fungus-specific antibodies does not rule out infection because imunocompromised patients, and some indiv
14、iduals with disseminated infection may not mount an antibody response to the infecting organism .,Tests for detection of fungal antigens(1),Detection of fungal antigens or metabolites in serum or other body fluids represents the most direct means of providing a serologic diagnosis of invasive fungal
15、 infection . Both the RIA and the EIA tests have been shown to be rapid,sensitive, specific, rapid ,and reproducible.,Tests for detection of fungal antigens(2),Although significant advances have been made in recent years,most methods for rapid detection of fungal antigens are available only in resea
16、rch laboratories . Notable exceptions are the tests for detection of the polysaccharid ride antigens of C.neoformans and H.capsulatumd .,Tests for detection of fungal -specific nucleic acid sequences(1),The use of the PCR to detect fungal nucleic acids directly in clinical material offers great promise for the rapid diagnosis of fungal infections .,Tests for detection of fungal -specific nucleic acid sequences(2),In addition to detection of fungal pathogens in clinical terid,immunologic and molecular approaches have been applied successfully to the identification of fungi in culture .,
