新生隐球菌的生态学,流行病学,分子生物学及临床研究

在我国，对新生隐球菌已进行了较系统的研究，生态学方面，由鸽粪分离的环境株具有表型的多态性，包括新生变种的Ａ、Ｄ血清型及尿素酶阴性株，而这些多态性菌株均已在临床发现。分子生物学方面，Ｇ＋Ｃｍｏｌ％和核型已被进行分析。由ＰＦＧＥ分析所得到的有意义的信息是两个变种和５种血清型新生隐球菌株具有明显不同的核型谱。临床方面，研制的一种新的可同时检测酚氧化酶和尿素酶的培养基可用于该菌的临床鉴定。使用一种高渗培养     

新生隐球菌的酚氧化酶及用于菌种鉴定的研究

采用4％玉米浸汁咖啡酸琼脂(CACA)培养基。观察了具不同生物学特性的新生隐球菌的酚氧化酶活性,并对临床常见的多种酵母和酵母样真菌作了该酶的检测。结果,受试的3个变种、5种血清型和尿素酶阴性新生隐球菌均呈明确的阳性反应;150株常见酵母和酵母样真菌中43株新生隐球菌全部呈酚氧化酶阳性。107株其它酵母和酵母样真菌全部阴性。具各种不同生物学特性的新生隐球菌均特异性地产生酚氧化酶,用检测该酶的方法作该菌鉴定的特异性和敏感性均为100％,且可于72小时内得到结果。此外,结合尿素酶试验可以准确的鉴定出尿素酶阴性的新生隐球菌。     

新疆首次临床分离新生隐球菌菌株分子鉴定、基因分型及体外药物敏感性研究

目的对新疆首次临床分离的5株新生隐球菌进行分子鉴定、RAPD-PCR基因分型及体外药物敏感性研究。方法5株新生隐球菌分子鉴定采用核糖体DNA大亚基(LSU rDNA)D1/D2基因区域分子鉴定。分子分型采用引物SEQ-6结合RAPD-PCR法扩增5株新疆临床分离新生隐球菌菌株及5株由上海长征医院提供分离自上海新生隐球菌菌株,根据扩增产物带型进行基因型判定。采用临床实验室标准化协会(CLSI)的酵母微量液基稀释法(M27-A3)测定5株新疆临床分离新生隐球菌菌株对6种抗真菌药(伏立康唑、伊曲康唑、两性霉素B、特比萘芬、氟康唑、5-氟胞嘧啶)的体外敏感性。结果 5株新疆临床分离隐球菌菌株经过D1/D2区域序列分析在基因Bank中比对后鉴定为新生隐球菌。5株新疆临床分离新生隐球菌和5株上海新生隐球菌菌株经RAPD-PCR法扩增,带型显示分为A、B、C、D 4个基因型,其中新疆5株菌株A型1株、其余4株均为B型,上海菌株B型为1株,C型3株、D型1株,5株新疆临床分离新生隐球菌株对伏立康唑、伊曲康唑、两性霉素B、特比萘芬的MIC值(μg/mL)范围依次为:0.062 5~0.25、0.25~1、0.125~0.5、1~2,对氟康唑、5-氟胞嘧啶MIC值较高,MIC值范围依次为:4~16、8~32。结论新疆临床分离5株隐球菌株采用D1/D2基因序列鉴定为新生隐球菌。RAPD-PCR分型显示新疆临床分离5株新生隐球菌株以B型基因型为主。A型和B型对伏立康唑、两性霉素B敏感,对伊曲康唑、特比萘芬剂量依赖型敏感,对氟康唑、5-氟胞嘧啶耐药。     

获得性免疫缺陷综合征患者的新生隐球菌基因多态性研究

本文旨在调查2003 年1 月― 2007 年12 月分离自上海地区获得性免疫缺陷综合征( AIDS) 患者的新生隐球菌临床株的配型及基因型分布特征, 为隐球菌病的诊疗提供科学依据。首先以M13 为单引物对模板DNA 进行聚合酶链反应( PCR) 扩增, 参照标准株指纹图将临床株鉴定至基因型; 同时对12 株来自AIDS 患者的新生隐球菌临床株的内转录间隔区( ITS) 基因进行PCR 扩增、序列分析, 以CLUSTAL W1. 83 软件多重比对分析ITS序列的差别,MEGA3. 1 软件处理数据, NJ 法绘制系统进化树, Bootstrapping 法对系统进化树结果进行统计学检验, 区分新生隐球菌格鲁比变种、新生变种及格特变种菌株; 最后选用特异性引物PCR 特异性扩增相关基因, 鉴定α和a 配型。结果显示, 分离自上海地区的12 株隐球菌临床株中, 9 株( 75% ) 为VNⅠ基因型/ α配型菌株,3 株( 25%) 为VNⅡ基因型/ α配型菌株, 且ITS基因序列分析可将各临床株鉴定至变种水平。本研究提示, 分离自上海地区AIDS患者的新生隐球菌临床株存在一定的遗传多态性, 以VNⅠ基因型/ α配型菌株为主, 有少量VNⅡ基因型/ α配型菌株。     

隐球菌感染体外血脑屏障模型的构建与应用

目的构建体外血脑屏障模型,并检测隐球菌不同菌株穿越血脑屏障的能力。方法本研究应用商品化的小鼠脑微血管内皮细胞系b END.3构建体外血脑屏障模型,并验证该模型应用于隐球菌穿越血脑屏障机制研究的可行性。通过构建模型,以非致病性的酿酒酵母作为阴性对照,比较新生隐球菌不同血清型标准株及基因缺陷株穿越体外血脑屏障能力的差异。结果跨膜电阻值(TEER)检测提示体外血脑屏障模型构建成功。检测结果显示酿酒酵母作为阴性对照穿越血脑屏障效率最低,新生隐球菌血清A型标准株H99穿越细胞屏障效率最强,血清D型标准株JEC21穿越细胞屏障效率显著低于H99。较之H99,黑色素酶缺陷株lac1裣穿越体外血脑屏障模型的效率没有显著差异;尿素酶缺陷株ure1裣效率显著下降(P0.05),约为标准株H99通过率的59.9%;荚膜缺陷株cap59裣突破体外血脑屏障模型效率最低,约为标准株H99的18%(P0.001)。结论隐球菌中枢系统感染体外模型成功构建。新生隐球菌突破血脑屏障的能力与其血清型以及荚膜、尿素酶等毒力因子的表达密切相关。     

播散性隐球菌病1例及其实验研究

目的 播散性隐球菌病1例临床及实验研究.方法 患者男,72岁,红皮病1年2个月,口服醋酸泼尼松治疗,双下肢出现结节、溃烂6个月.皮损组织病理、皮损组织真菌培养、尿素酶试验、PCR扩增测序比对明确诊断,同时做胸部及脑部CT.结果 皮损组织病理显示为感染肉芽肿改变,可见大量圆形和椭圆形酵母细胞.皮损组织真菌培养可见酵母样菌落生长,菌株尿素酶试验阳性,ITS区测序比对鉴定为新生隐球菌grubii变种.血清隐球菌荚膜多糖抗原乳胶凝集试验阳性(++++).胸部CT显示左下肺后基底段空洞性病灶.依据临床及实验室检查确诊为由新生隐球菌grubii变种引起的 播散性隐球菌病.给予患者静滴氟康唑400 mg/d治疗2周,之后改口服300 mg/d治疗,3个月后结节性皮损全部消退,胸片显示左肺陈旧性病变,血清隐球菌荚膜多糖抗原乳胶凝集试验阳性(++).治疗15个月后,血清隐球菌荚膜多糖抗原乳胶凝集试验仍阳性(++).结论 对该病例的临床和实验室研究为临床明确诊断和治疗提供了依据,确定菌种需要进行分子生物学研究.     

隐球菌感染体外血脑屏障模型的构建与应用CSCD

目的构建体外血脑屏障模型,并检测隐球菌不同菌株穿越血脑屏障的能力。方法本研究应用商品化的小鼠脑微血管内皮细胞系b END.3构建体外血脑屏障模型,并验证该模型应用于隐球菌穿越血脑屏障机制研究的可行性。通过构建模型,以非致病性的酿酒酵母作为阴性对照,比较新生隐球菌不同血清型标准株及基因缺陷株穿越体外血脑屏障能力的差异。结果跨膜电阻值(TEER)检测提示体外血脑屏障模型构建成功。检测结果显示酿酒酵母作为阴性对照穿越血脑屏障效率最低,新生隐球菌血清A型标准株H99穿越细胞屏障效率最强,血清D型标准株JEC21穿越细胞屏障效率显著低于H99。较之H99,黑色素酶缺陷株lac1裣穿越体外血脑屏障模型的效率没有显著差异;尿素酶缺陷株ure1裣效率显著下降(P<0.05),约为标准株H99通过率的59.9%;荚膜缺陷株cap59裣突破体外血脑屏障模型效率最低,约为标准株H99的18%(P<0.001)。结论隐球菌中枢系统感染体外模型成功构建。新生隐球菌突破血脑屏障的能力与其血清型以及荚膜、尿素酶等毒力因子的表达密切相关。     

新生隐球菌的AFLP分析

目的评估AFLP-DNA指纹技术在新生隐球菌分类中应用情况。方法新生隐球菌基因组DNA用双酶酶切,双链接头连于其酶切末端,用与接头和酶切位点互补的引物扩增DNA片段,其产物在高分辨的变性聚丙酰胺凝胶上电泳分离,然后进行银染。结果分析来自5种血清型和临床分离株的18株新生隐球菌,可见有30多条大小在30～500bp的DNA-AFLP指纹,相同的血清型有不同的指纹图谱,来自同一患者不同病期的两株分离株和来自同一患者患者的不同部位的两株分离株都显示出相同的带型。结论显示了AFLP的高分辨率,是适用于新生隐球菌流行病学调查的有力工具。     

利用微卫星标记研究新生隐球菌分子流行病学

目的 研究国内隐球菌临床分离株的遗传多态性和分子流行病学.方法 选择与新生隐球菌遗传相关的9个微卫星标记,分析这9个位点从1993 ～2009年国内分离到的新生隐球菌临床株遗传背景、来源及变异程度.结果 116株被研究的隐球菌临床分离株,主要归属于3个微卫星复合物(MC2,MC3和MC12),其中大部分为MC2(103株).8株菌株属于目前为止未被国内外认识的新复合物(MC12).结论 利用微卫星DNA多态性研究新生隐球菌分子流行病学有较大的应用价值.     

一株尿素酶阴性的新生隐球菌

本文报告从鸽粪标本中分离出的一株尿素酶试验阴性、咖啡酸玉米吐温琼脂培养呈棕色菌落的酵母样真菌。根据该菌袜的生理、生化特性及致病力,认为是尿素酶阴性的新生隐球菌。此外,应注意这种菌在自然界中的存在和对人类造成感染的可能。这是关于从自然环境中分离出尿素酶阴性新生隐球菌的首次发现和报告。     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Chromosome length polymorphism in Cryptococcus neoformans clinical and environmental isolates

A protocol for intact DNA preparation from the basidiomycetous yeast Cryptococcus neoformans has been developed and applied to karyotyping C. neoformans isolates displaying different degrees of capsule formation. A total of 46 strains have been analyzed: 23 (50%) isolated from environmental samples (pigeon droppings), all of them belonging to C. neoformans var. neoformans; and 23 (50%) from clinical samples (human and veterinarian) including 10 isolates of C. neoformans var. neoformans and 13 isolates of C. neoformans var. gattii. Our results showed a global genome size ranging from 14.2 to 20.9 Mb for variety neoformans and from 7.9 to 16.8 Mb for variety gattii. The karyotype diversity was very high for variety neoformans (29 different patterns for the 33 analyzed strains) and lower for variety gattii (six different patterns for 13 strains). No grouping among variety neoformans strains from the same origin was found indicating very high genome diversity for this variety, irrespectively of the origin of the strains.     

Molecular typing of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul

In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by Cryptococcus neoformans. This pathogen is a ubiquitous environmental basidiomycetous encapsulated yeast, commonly found in soil and avian excreta. The present study investigates further the population structure of clinical and environmental C. neoformans isolates from south Brazil. One hundred five clinical and 19 environmental (pigeon excreta and Eucalyptus spp.) isolates from the Brazilian state Rio Grande do Sul were characterized based on morphological, biochemical, molecular and serological data. The majority of the clinical and environmental isolates analyzed belonged to C. neoformans var. grubii serotype A (89.5 and 52.6%, respectively), were mating type alpha (98.1 and 94.7%, respectively) and were phospholipase-positive (94.3 and 73.7%, respectively). PCR-fingerprinting with the microsatellite-specific primer M13 and the minisatellite-specific primer (GACA)(4) grouped the majority of the isolates into the molecular type VNI (89.5 of the clinical and 52.6% of the environmental isolates). Our results add considerable new information to the few available data on ecology, molecular biology and epidemiology of C. neoformans in the southern region of Brazil.     

In vitro determination of virulence factors activity associated with several Cryptococcus neoformans clinical isolates

Different phenotypic characteristics associated with virulence of the Cryptococcus neoformans species complex have shown an important role in their pathogenicity. In this study we have determined the role of phenotypically and genotypically factors of some virulence factors from clinical isolates in the two species of the complex; 35 C. neoformans and 19 Cryptococcus gattii. Growth at 37 degrees C, macroscopic and microscopic morphology, switching phenomenon, activity of 23 extracellular enzymes, variability of the colonies in agar with phloxin B; phospholipase B gene, and the mating type were determined by PCR; the molecular pattern was determined by URA5 RFLP. All isolates grew at 37 degrees C, the capsular size was greater in C. gattii (1.87 microm -/+1.47 microm) than in C. neoformans (0.83 microm -/+0.15 microm). Switching was observed mainly in isolates of C. gattii. All isolates expressed the enzyme urease, a lower activity of the proteases (Pz= 0.54), but a higher activity of the phospholipase (Pz=0.43) and phenoloxidase (Pz=0.003) was determined for C. gattii.     

Serotype and PCR-fingerprints of Clinical and Environmental isolates of Cryptococcus neoformans in Chiang Mai,Thailand

From May 1999 to April 2000, serotypes of clinical and environmental isolates of Cryptococcus neoformans were studied in Chiang Mai province, northern Thailand. Three hundred and eighty-five environmental samples, of which 100 were dove droppings, 55 pigeon droppings and 230 eucalyptus flower, were collected from 7 Amphoes in Chiang Mai. C. neoformans was isolated from 45 of 100 (45.0%) dove dropping samples, 9 of 55 (16.4%) pigeon dropping samples and 2 of 230 (0.9%) eucalyptus flower samples. Serotypes of 56 environmental isolates and 75 clinical isolates of C. neoformans,obtained during the same period, were determined by the slide agglutination test. Fifty-six environmental and 74 clinical isolates belonged to C. neoformans serotype A (C. neoformans var. grubii), and only one clinical isolate belonged to C. neoformans serotype AD. The isolation of C. neoformans var. grubii from eucalyptus flower samples suggests contamination of avian droppings. PCR-fingerprinting, using (GACA)4 as a primer, discriminated 131 clinical and environmental isolates into 2 groups (group I and II). Seventy-five clinical and 54 environmental isolates were of group I, which had two major specific bands of approximately 1,250 and 960 base pairs. Two environmental isolates, one from pigeon excreta and the other from a eucalyptus flower sample were of group II, which had two major specific bands of approximately 1,180 and 500 base pairs.     

Medium Containing Trypan Blue and Antibiotics for the Detection of Cryptococcus neoformans in Clinical Samples

A medium containing trypan blue, gentamicin, and chloramphenicol is introduced for the detection of Cryptococcus neoformans and Cryptococcus species from clinical samples. Ten recently isolated strains of Cryptococcus species as well as several clinical isolates of C. neoformans incorporated trypan blue and produced dark blue colonies on this mycological medium, whereas other common yeasts were light blue. The laboratory diagnosis of two cases of cryptococcosis was accomplished by the isolation of C. neoformans on the antibiotic-dye-containing medium. Compared to conventional media supporting large numbers of Pseudomonas aeruginosa and other gram-negative bacilli, the new medium was selective for yeasts. In one instance, the colonization of the respiratory tract by C. neoformans which led to fungemia was traced by the use of the antibiotic-dye medium. The antibiotic mixture, utilized herein, was more effective in suppressing bacteria contained in samples from patients than a medium containing cycloheximide and chloramphenicol.     

First report of isolation of Cryptococcus neoformans var. neoformans from avian excreta in Kathmandu, Nepal

This paper delineates the first report on the saprophytic distribution of Cryptococcus neoformans var. neoformans in the city of Kathmandu, Nepal. Twenty-eight samples of old and dry pigeon droppings collected from different sites in Kathmandu were investigated for the presence of C. neoformans by employing a dilution technique. The organism was isolated from seven (25%) of the specimens, representing four of the ten collection sites. All of the isolates were recovered on Pal's medium (sunflower seed agar) by observing light to dark brown coloured colonies of C. neoformans. However, no isolation could be achieved on Sabouraud medium as all the plates were badly contaminated with rapidly growing moulds. The microscopic morphology of the cultures in PHOL stain revealed circular to val, single or budding yeast cells with thin capsules. Detailed typing of all environmental strains indicated that they belonged to the variety neoformans and a mating type of Filobasidiella neoformans. The results of this study demonstrated that Pal's medium is an excellent differential medium for the screening of environmental specimens and C. neoformans var neoformans is prevalent in the environment of Kathmandu.     

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiania, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.