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.     

非霍奇金淋巴瘤伴播散性隐球菌感染1例

患者男,16岁,非霍奇金淋巴瘤化疗5个月,发热1周,血培养新生隐球菌生长。脑脊液真菌镜检可见少量孢子,真菌培养阴性。血清及脑脊液隐球菌荚膜多糖抗原乳胶凝集试验及胶体金试验阳性。头颅核磁平扫未见明显异常,胸部增强CT可见左肺下叶后基底段结节,感染可能性大。分离菌株ITS及NL区测序比对菌种鉴定为新生隐球菌新生变种。确诊播散性隐球菌病。予两性霉素B及氟胞嘧啶强化治疗4周,后由于副作用严重,予氟康唑巩固及维持治疗1年获得痊愈,随访4年多无复发。     

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。     

以皮下结节为首发表现的播散性隐球菌感染1例

报道1例系统性红斑狼疮合并干燥综合征患者出现以皮下结节为首发表现播散性隐球菌感染。患者,女,50岁,以双下肢皮下结节、头痛为主要症状,血培养、脑脊液培养出新生隐球菌,皮肤活检病理可见大量均一、透亮的小圆形菌体真菌孢子浸润,PAS(+)、六胺银(+)。诊断为播散性隐球菌感染。先后使用两性霉素B、氟康唑、两性霉素B脂质体、氟胞嘧啶治疗1个月后症状好转。     

以皮肤受累为首发表现的播散性念珠菌病1例并文献复习

目的：播散性念珠菌病是一种致命性真菌感染性疾病,在免疫缺陷患者中发病率逐年增多,报道1例以双下肢多发皮下结节为首发表现,伴有肺及脑受累的播散性念珠菌病,并文献复习播散性念珠菌病的皮肤受累临床表现。方法患者女,37岁。因双下肢多发皮下结节6个月余就诊。有局灶节段性肾小球硬化病史,口服强的松及他克莫司2a余。取患者皮损组织行病理学检查,皮损组织、脓液、血、痰、尿、粪、脑脊液进行真菌镜检及真菌培养,并文献检索统计播散性念珠菌病皮肤受累患者临床特点。结果皮损组织病理见假菌丝,皮损组织、脓液、痰、尿、粪标本直接涂片均见假菌丝并培养出白念珠菌,CT显示肺受累,诊断为播散性念珠菌病,予抗真菌治疗,患者皮损愈合及肺部病灶部分吸收,但因自行停药,最终出现颅内播散。结论以皮损为首发表现的播散性念珠菌病临床罕见,临床诊疗中应重视应用免疫抑制剂患者皮损的组织病理及微生物检查,及早进行诊断和治疗,防止出现系统性播散,从而降低死亡率。     

儿童播散性隐球菌病1例报道及文献复习

1例主诉为“发现皮肤巩膜黄染1个月余,发热8 d”的患儿就诊于我院消化内科,经肝穿刺病理活检及脑脊液培养、脑脊液新生隐球菌抗原检测诊断为播散性隐球菌病(肝脾,胆管,脑)。以黄疸为首发症状的隐球菌病临床较少见,故报道本例患儿的诊治经过并进行相关文献复习。     

采用分子生物学技术诊断隐球菌脑膜脑炎的研究

目的采用分子检测技术对疑似隐球菌感染的脑膜炎病例进行诊断。方法收集患者的脑脊液样本,提取DNA,设计引物进行PCR扩增,采用DNA芯片技术对扩增产物进行分子检测。结果显示样本新生隐球菌阳性。结论通过ITS保守序列设计引物进行PCR扩增和DNA芯片技术对常规真菌学检查不能确定的疑似隐球菌脑膜炎患者脑脊液样本进行非培养检测,具有实验室诊断参考价值。     

34例HIV患者新生隐球菌感染分子流行病学及临床特点分析

目的了解HIV患者新生隐球菌感染的分子流行病学及其临床特点,为HIV患者新生隐球菌感染的预防和治疗提供依据。方法收集首次分离自HIV患者的新生隐球菌34株,回顾性分析患者一般资料;VITEK MS质谱仪进行菌种鉴定,ATB Fungus3测定新生隐球菌对5种抗真菌药物的MIC值;利用PCR对特异性引物扩增,确定变种和交配型;多位点序列分型(MLST)对菌株进行分子遗传学分析。结果 34株新生隐球菌绝大部分分离自中年男性,且主要来自脑脊液(73.5%)标本;初次脑脊液压力平均为(27.26±11.52)mmH2O,CD4细胞计数中位数28cells/μL(3~163cells/μL),脑脊液白细胞中位计数32(2~110)×106/L,蛋白质定量中位数为362 mg/L(160~2 730 mg/L),葡萄糖含量中位数为2.28mmol/L(1.50~5.98mmol/L);所有菌株对5种抗真菌药均敏感,且所有菌株均为Aα、VNⅠ型;MLST分析共检出3种ST型,ST5(n=32)、ST32(n=1)和ST186(n=1)。结论近两年本地区HIV合并新生隐球菌感染主要以中年男性多见,常规实验室检查缺乏特异性,对临床常用抗真菌药物耐药性不强,ST5是其感染的主要克隆系。     

着色真菌monophora引起皮肤着色芽生菌病1例

目的报道1例由着色真菌monophora引起的皮肤着色芽生菌病。方法取皮损皮屑标本进行真菌直接镜检和培养,同时取活检进行真菌培养和组织病理学检查。对真菌培养阳性菌株进行形态学鉴定、温度试验和放线菌酮耐受试验,PCR扩增测序。结果KOH涂片检查可见较多圆形厚壁棕色硬壳细胞。组织病理学显示为慢性肉芽肿样改变;PAS和银染色可见到圆形厚壁的硬壳细胞。真菌菌落生长缓慢,呈橄榄色到黑色。小培养可见大量棕色菌丝、分支分隔,分生孢子梗主要为喙枝孢型,分生孢子棕色,椭圆形或卵圆形,单细胞。温度试验37℃生长,38℃不生长。0．01％、0．05％和0．1％放线菌酮均能耐受。扩增真菌rDNA的ITS区得到645bp的片段,经序列分析与裴氏着色真菌monophora变种ITS区比对,100％一致。结论据真菌学形态结构特征以及DNA序列分析菌种被鉴定为着色霉monophora。     

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。     

Three galactose inducible promoters for use in C. neoformans var. grubii

Cryptococcus neoformans is the causative agent of cryptococcal meningoencephalitis, most frequently occurring in immunocompromised individuals. There are three varieties of C. neoformans, var. grubii, var. neoformans, and var. gatti. Worldwide var. grubii is the most prevalent clinical isolate. However, few tools for the study of essential genes in var. grubii exist. Here we describe three endogenous inducible promoters for use in the study of this important opportunistic pathogen. We identified eight potential homologs of S. cerevisiae galactose genes in var. grubii. We found that GAL1, GAL7, and UGE2 were regulated by glucose and galactose and can be used successfully during mating. Our analysis indicated these promoters should prove to be excellent tools for analysis of genes in var. grubii.     

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.     

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Isolated from Passerine and Psittacine Bird Excreta in the State of Paraná, Brazil

Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts, generally associated with AIDS. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. The occurrence of C. neoformans and Cryptococcus gattii in bird excreta in the state of Paraná in Brazil was determined in this study. A total of 141 samples of Passerine and Psittacine excreta from captive birds were collected. Additionally, 25 clinical samples from Hospital de Clínicas, in the state of Paraná were also analyzed. The determination of molecular and mating type of the isolates was performed by PCR fingerprinting, multiplex PCR, and mating type PCR. Cryptococcus neoformans var. grubii (VNI) was isolated from 36 (25.53%) of Passerine and Psittacine excreta samples. Almost all clinical samples, except one (C. gattii VGI), were classified as C. neoformans var. grubii (VNI). All environmental and clinical isolates were mating type alpha. These findings reinforce that, besides pigeon excreta, the excreta of these birds can also be a reservoir of C. neoformans in domestic and public environments and is of zoonotic importance to immunocompromised patients.     

Sequence length required for homologous recombination in Cryptococcus neoformans

Cryptococcosis is a major threat to immunocompromised individuals. Isolates of Cryptococcus neoformans var. grubii and var. neoformans are responsible for most of the infections in the United States and Europe. In depth analysis of the virulence phenotype of this organism requires the generation of specific gene disruptions. The minimum sequence requirements for efficient homologous recombination has not been determined in Cryptococcus. To investigate the flanking DNA length requirements for efficient homologous recombination in variety grubii, the rates of homologous recombination of constructs with different lengths of flanking sequence at two loci, CAP59 and CNLAC1, were examined. Five gene disruption constructs were prepared for each locus with symmetric lengths of sequence homologous to the target gene with approximately 50, 100, 200, 300 or 400bp flanking the selectable marker for hygromycin resistance. In addition, two asymmetric constructs with 50bp on one side and 400bp on the other side were generated for each locus. Overall, symmetric constructs with 300bp or more of flanking sequence on each side and the asymmetric constructs were efficiently targeted for gene disruption by homologous recombination in C. neoformans var. grubii. With one exception, the rate of recovery of homologous recombinants using the longer or asymmetric constructs as targeting vectors was greater than five percent of total transformants. Symmetrical constructs with 100bp or less of homologous flanking sequence did not efficiently generate targeted gene disruptions because the rate of homologous recombinants was less than or equal to 1%.     

Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.     

Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans

Cryptococcus neoformans (Cn), causal agent of fungal meningoencephalitis, has three varieties with variable host predilection. To explore mechanisms for these pathogenic differences, we have characterized Cu,Zn SOD gene (CnSOD1). A Saccharomyces cerevisiae sod1Delta mutant was complemented with Cn var. grubii yeast expression library. The complementing clone had an ORF of 462 bp and the deduced 154 aa sequence showed 61% identity with S. cerevisiae SOD1 and 53-65% with other eukaryotic SOD1s. Cn var. grubii CnSOD1 cDNA was used to clone corresponding cDNAs from var. neoformans and var. gattii. ORFs from three varieties revealed 20-29% differences in deduced aa (s) with a significant 6% non-synonymous aa substitution between Cn var. grubii and Cn var. gattii. Cosmid library screening and PCR cloning were used to obtain genomic SOD1, which was split by five introns with identical placements and a typical 5' splice junction sequence, GTNNGY. These introns also showed a large nt variation among the three Cn varieties. Phylogenetic analyses revealed CnSOD1 to be in a group distinct from other eukaryotic SOD1s and with a significant divergence of the var. grubii from var. gattii. The CnSOD1 -deduced protein was modeled based on the crystal structure of S. cerevisiae SOD1, which showed an excellent fit. Most of the non-synonymous aa substitutions occurred on the outside of the molecule and these may contribute to differences in antigenicity among the three varieties. Notably, Cn var. neoformans and var. gattii Cu,Zn SOD had three substitutions of glycine (Gly26, Gly92 and Gly123 for Asn26, Ser92 and Ser123) that may contribute to the observed lower thermostability of this enzyme vis-a-vis Cn var. grubii. This is the first nucleotide and structural comparison of a protein-encoding gene from the three Cn varieties, which may provide a framework for future studies on the role of Cu,Zn SOD in Cn pathogenesis.     

Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa

Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis--an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm.     

Atypical Micromorphology and Uncommon Location of Cryptococcosis: A Histopathologic Study Using Special Histochemical Techniques (One Case Report)

Here we report an unusual case of disseminated cryptococcosis in a patient with AIDS. Although typical Cryptococcus neoformans micromorphology was observed in tongue biopsy, cervical lymph node examination revealed atypical histopathologic findings. These included pseudohyphae, chains of budding yeasts and structures resembling germ tubes. Cryptococcus neoformans infection in supraclavicular lymph nodes was also confirmed by culture. The importance of using special histochemical techniques—Mayer’s mucicarmine stain for mucicarminophilic capsule and Grocott’s silver stain—in the diagnosis of cryptococcosis is reinforced.