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.     

Taxonomic identity of caricicolousPuccinia host-alternating onPetasites in Japan

Uredinial-telialPuccinia fungi onCarex shimizuensis in Nagano Pref. and onC. dimorpholepis in Ibaraki Pref. were proven to host-alternate onPetasites japonicus by field observations and inoculation experiments. These fungi from the two localities were morphologically similar and were compared with three describedPuccinia species that host-alternate betwenCarex species andP. japonicus in Japan, i.e.,P. caricis-petasitidis, P. caricis-flabellatae andP. caricis-podogynae. The three previously described species and the newly foundPuccinia fungi were morphologically indistinguishable at all stages of the life cycle; therefore, it was concluded that three species and the two newly found fungi are taxonomically identical, in whichP. caricis-petasitidis has nomenclatural priority.     

Novel DNA markers reveal for taxonomic characterization and identification of the Fusarium fungi species

RAPD analysis for ten F. sporotrichioides strains of different geographical origin was done for DNA loci, potentially suitable as a new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three selected monomorphic fragments--products of amplification with one of standard RAPD primers were sequenced that allowed creating SCAR markers for identification of Fusarium fungi on the species group level with similar profiles of produced mycotoxins.     

Species level identification of conifer associated Ceratocystis sapstain fungi by PCR-RFLP on a beta-tubulin gene fragment

The genus Ceratocystis includes several morphologically similar species commonly found as agents of sapstain in coniferous trees. In this paper we describe a simple and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique that aids in the identification and differentiation of these fungi. PCR was used to amplify a 1.3-kb fragment of the beta-tubulin gene from C. coerulescens, C. pinicola, C. douglassi, C. resinifera, C. rufipenni, C. polonica and C. adiposa. The PCR amplicon from representative isolates was sequenced. This information was utilized to select restriction enzymes that generated species-specific RFLP patterns. This approach was tested on our collection of over 200 Ceratocystis isolates and identified the fungi with a high level of confidence, reducing the time needed to identify these species by classical methods.     

The application of immunoperoxidase staining for the detection of causative fungi in tissue specimens of mycosis I

This study was performed in order to identify the fungi of four species (Aspergillus fumigatus, Fusarium anthophilum, Candida albicans, Cryptococcus neoformans) in formalin-fixed, paraffin-embedded tissue sections by the indirect method of immunoperoxidase staining. Mature albino rabbits were immunized by formalin-killed organisms. The antibodies were prepared by precipitation at a 50% saturation of ammonium sulfate and were checked for cross-reactivities by Ouchterlony's double immunodiffusion and precipitin test. The immunoperoxidase staining was applied to the paraffin-embedded tissue sections of infected mice, human autopsy and biopsy specimens. Although each fungus was stained clearly the cell wall, cross-reactivities appeared among them, however it was possible to identify four fungi by absorption and dilution of the antisera.     

The system approach to the identification of aflatoxigenic fungi in foodstuffs and feedstuffs

Aspergillus flavus, A parasiticus, A nomius, A tamarii andA pseudotamarii are important microorganisms capable of producing aflatoxins and further mycotoxins. Aflatoxigenic Aspergillus species are morphologically similar species belonging to the Aspergillus section Flavi. The aflatoxigenic fungal strains were isolated from foods (cereals, pulses, oilseeds, dried fruit, spices), soil, air and water. Mycological analyses are based on valid standards and recommendations of the International Commission for Food Mycology (ICFM). The identification of isolated aflatoxigenic fungi in foodstuffs and feedstuffs can be proved by using classical mycological cultivation methods, diagnostic nutrient media, chemotaxonomy and molecular biological methods (PCR). The system approach to the identification of aflatoxigenic fungi combines these four methods. Thirty strains of the aflatoxigenic fungi were tested.     

A species-specific real-time PCR assay for identification of three lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica

We present a multiplex real-time PCR assay for the simultaneous identification of three morphologically similar species of lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica. Based on TaqMan MGB (minor groove binding) probes targeting the fungal internal transcribed spacer (ITS nrDNA) region, our assay unambiguously identifies known samples from all the three species, thus providing a powerful alternative to the more expensive DNA-sequencing techniques.     

Fluorescent antibody techniques for identification of Coccidioides immitis and Histoplasma capsulatum

This is a review of practical uses of immunofluorescence in detection of the two fungi in host and environment and in identification of their cultures, as well as in serologic case finding. Reagents directed at the yeast phase ofHistoplasma capsulatum have been fairly successful in differentiating this species from others, the main difficulty being the tendency to cross-react withBlastomyces dermatitidis andH. duboisii. Conjugates for the mycelial phase ofH. capsulatum tend to cross-react withSepedonium andChrysosporium, but careful absorption may yield specific reagents. Anti-yeast-phase conjugates are a valuable adjunct to cultural and clinical methods when used to detect and identifyH. capsulatum in sputum and other clinical specimens. Conjugates specific for the spherules or tissue phase ofCoccidioides immitis have yielded false negative results when applied to clinical specimens. The fluorescent-inhibition procedure is useful for serologic case finding in histoplasmosis and the same technique has shown fairly good agreement in coccidioidomycosis with complement-fixation and tube-precipitin methods. Immunofluorescence reagents for the two species have been useful in screening surgical and autopsy specimens, animal tissues, and soils.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Identification of Actinomyces israelii and Actinomyces naeslundii by Fluorescent-Antibody and Agar-Gel Diffusion Techniques

This study was an attempt to develop a fluorescent-antibody (FA) test to differentiate Actinomyces israelii and A. naeslundii as an aid in their laboratory identification. Two strains of A. israelii (X522 and A601) and two strains of A. naeslundii (X454 and X600), which had received intensive study by several investigators, were used for the immunization of rabbits. Working titers, based on tests with antigens prepared from the homologous strains and from well-established heterologous strains, were determined for each labeled antibody preparation. These conjugates and their normal serum control conjugates were used separately to stain 85 cultures of Actinomyes species and 23 strains of other species that might be confused with them. Acetone-precipitated soluble antigens from these same strains were tested with different antisera in the agar-gel diffusion test. Results showed that A. israelii (X522 and A601) and A. naeslundii (X454 and X600) labeled antiglobulins, when used at their working titers, stained most strains of their homologous species. Agar-gel diffusion results showed general agreement with those of the FA tests. The two tests appear to be equal in sensitivity, but the FA test is more specific, since several cross-reactions were noted with the agar-gel diffusion test whereas no cross-reactions were obtained with the FA reagents. Agar-gel and FA studies suggest that at least two serotypes of A. israelii may be associated with human disease. Although the majority of strains tested in this study appear to belong to a common serotype, "serotype 1," two strains of an apparent second serotype, "serotype 2," were encountered. FA staining of tissue impression smears from experimentally infected mice was successful when a counterstain, Evans Blue dye, was used.     

PCR-based detection of Angiostrongylus cantonensis in tissue and mucus secretions from molluscan hosts

Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.     

DNA barcodes effectively identify the morphologically similar Common Opossum (Didelphis marsupialis) and Virginia Opossum (Didelphis virginiana) from areas of sympatry in Mexico

Two morphologically similar species of opossum from the genus Didelphis-Didelphis virginiana and Didelphis marsupialis-cooccur sympatrically in Mexico. High intraspecific variation complicates their morphological discrimination, under both field and museum conditions. This study aims to evaluate the utility and reliability of using DNA barcodes (short standardized genome fragments used for DNA-based identification) to distinguish these two species. Sequences of the cytochrome c oxidase subunit I (Cox1) mitochondrial gene were obtained from 12 D. marsupialis and 29 D. virginiana individuals and were compared using the neighbor-joining (NJ) algorithm with Kimura's two-parameter (K2P) model of nucleotide substitution. Average K2P distances were 1.56% within D. virginiana and 1.65% in D. marsupialis. Interspecific distances between D. virginiana and D. marsupialis varied from 7.8 to 9.3% and their barcode sequences formed distinct non-overlapping clusters on NJ trees. All sympatric specimens of both species were effectively discriminated, confirming the utility of Cox1 barcoding as a tool for taxonomic identification of these morphologically similar taxa.     

Novel DNA markers for taxonomic characterization and identification of <Emphasis Type="Italic">Fusarium</Emphasis> species

Ten Fusarium sporotrichioides strains from different geographic regions were analyzed by RAPD in order to detect DNA loci potentially suitable as new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three monomorphic fragments were selected from PCR amplificates obtained with one of the standard RAPD primers and sequenced. Analysis of the sequences enabled the development of specific SCAR markers for identification of Fusarium fungi at the level of species groups characterized by similar profiles of produced mycotoxins.     

Appearance of Colonies of Prototheca on CHROMagar Candida medium

The microorganisms capable of producing opportunist infections include the yeast-like organisms of the genus Candida, and the unicellular algae of the genus Prototheca, which share common features and can, therefore, lead to confusion. Their colonies are almost identical and they grow in the same culture media used routinely in mycology. CHROMagar Candida is a new chromogenic differential isolation medium that facilitates the presumptive differentiation of some of the most clinically important yeast-like organisms. To our knowledge, the use of CHROMagar Candida with Prototheca spp. has not been reported in the literature. This report describes the growth of 151 strains of Prototheca on CHROMagar Candida compared to the growth of a total of 326 well-characterized yeast organisms of the genera Candida, Cryptococcus, Trichosporon, Geotrichum, and Saccharomyces. It is clinically relevant to note that algae of the genus Prototheca (P. wickerhamii, P. zopfii, and P. stagnora) and of the genus Candida parapsilosis produced similar cream-colored colonies on CHROMagar Candida medium. Based on their growth on CHROMagar, a new species of Candida is described, C. zeylanoides, which has blue-green colonies. The colonies of two species of Trichosporon are also differentiated: the blue-green colonies of T. beigelii and the pink colonies of T. capitatum. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mycorrhizas: Gene to Function

Substantial progress has been made toward development of molecular tools for identification and quantification of mycorrhizal fungi in roots and evaluation of the diversity of ectomycorrhizal (ECM) fungi and the phylogeny and genetic structure of arbuscular mycorrhizal (AM) fungi. rDNA analysis confirms high diversity of ECM fungi on their hosts, and for AM fungi has revealed considerable genetic variation within and among morphologically similar AM fungal species. The fungal and plant genes, regulation of their expression, and biochemical pathways for nutrient exchange between symbiotic partners are now coming under intense study and will eventually be used to define the ecological nutritional role of the fungi. While molecular biological approaches have increased understanding of the mycorrhizal symbiosis, such knowledge about these lower-scale processes has yet to influence our understanding of larger-scale responses to any great extent.     

An outbreak of fungal dermatitis and stomatitis in a free-ranging population of pigmy rattlesnakes (Sistrurus miliarius barbouri) in Florida

Between September 1997 and March 1998, a severe skin, eye, and mouth disease was observed in a population of dusky pigmy rattlesnakes (Sistrurus miliarius barbouri), at the Lake Woodruff National Wildlife Refuge in Volusia County, Florida (USA). Three affected pigmy rattlesnakes were submitted for necropsy. All snakes had severe necrotizing and predominantly granulomatous dermatitis, stomatitis, and ophthalmitis, with involvement of the subadjacent musculature and other soft tissues. Numerous fungal hyphae were seen throughout tissue sections stained with periodic acid Schiff and Gomori's methenamine silver. Samples of lesions were cultured for bacteria and fungi. Based on hyphae and spore characteristics, four species of fungi were identified from culture: Sporothrix schenckii, Pestalotia pezizoides, Geotrichum candidum (Galactomyces geotrichum), and Paecilomyces sp. While no additional severely affected pigmy rattlesnakes were seen at the study site, a garter snake (Thamnophis sirtalis) and a ribbon snake (Thamnophis sauritis) with similar lesions were found. In 1998 and 1999, 42 pigmy rattlesnakes with multifocal minimal to moderate subcutaneous masses were seen at the study site. Masses from six of these snakes were biopsied in the field. Hyphae morphologically similar to those seen in the severe cases were observed with fungal stains. Analysis of a database representing 10,727 captures in previous years was performed after the 1998 outbreak was recognized. From this analysis we determined that 59 snakes with clinical signs similar to those seen during the 1998 outbreak were documented between 1992 and 1997. This study represents the first documented report of a mycotic disease of free-ranging snakes.     

Imaging surface of gold-immunolabeled thin sections by atomic force microscopy

Atomic force microscopy (AFM) has been used to image the surface of thin sections of fungal infected plant tissue, with or without post-embedding immunocytochemical labeling with gold conjugates. Plant and fungal cells are easily identified from their size, shape and roughness. The cellular shape is similar to that observed by light or electron microscopy (LM or EM) and some internal organelles can even be individualized. The gold beads are easily observed and counted. Their dimensions varied according to the roughness of the surface, but fit with the expected sizes.     

Molecular classification and phylogenetic relationships of selected edible Basidiomycetes species

Morphological identification of edible mushrooms can sometimes prove troublesome, because phenotypic variation in fungi can be affected by substrate and environmental factors. One of the most important problems for mushroom breeders is the lack of a systematic consensus tool to distinguish different species, which are sometimes morphologically identical. Basidiomycetes as one of the largest groups of edible mushrooms have become more important in recent times for their medicinal and nutritional properties. Partial rDNA sequences, including the Internal Transcribed Spacer I-5.8SrDNA-Internal Transcribed Spacer II, were used in this study for molecular identification and assessment of phylogenetic relationships between selected edible species of the Basidiomycetes. Phylogenetic trees showed five distinct clades; each clade belonging to a separate family group. The first clade included all the species belonging to the Pleurotaceae (Pleurotus spp.) family; similarly, the second, third, fourth, and fifth clades consist of species from the Agaricaceae (Agaricus sp.), Lyophllaceae (Hypsigygus sp.), Marasmiaceae (Lentinula edodes sp.) and Physalacriaceae (Flammulina velutipes sp.) families, respectively. Moreover, different species of each family were clearly placed in a distinct sub-cluster and a total of 13 species were taken for analysis. Species differentiation was re-confirmed by AMOVA analysis (among the populations: 99.67%; within: 0.33%), nucleotide divergence, haplotyping and P value. Polymorphism occurred throughout the ITS regions due to insertion-deletion and point mutations, and can be clearly differentiated within the families as well as genera. Moreover, this study proves that the sequence of the ITS region is a superior molecular DNA barcode for taxonomic identification of Basidiomycetes.     

A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.