Rhinosporidium seeberi: Is It a Fungi or Parasite?

Rhinosporidium seeberi (R. seeberi) causes rhinosporidiosis, which is manifested as tumor-like polyps developing primarily in the nostrils and conjunctiva in human and animals. This disease is characterized by the presence of large, round-shaped mature stage and small endospores with resistance to culturing. R. seeberi was first reported in 1900 as a sporozoan parasite, but later classified as a lower fungi, although its morphological similarity with aquatic parasites were also noticed. According to 18S small-subunit ribosomal DNA sequencing, R. seeberi belongs to a group of fish parasite DRIP clade located between the animal and fungal divergence. Histological examination is thus necessary for the definitive diagnosis of rhinosporidiosis, and the first line of treatment is usually total surgical excision and electro-cauterization of the polyp base. Among the drug therapies attempted, remission has been reported in some patients who received only Dapson treatment. This disease is endemic across India, Pakistan and Sri Lanka and occurs sporadically in other parts of The World with a common history of patients bathing in stagnant water. An outbreak in Serbia during 1992-1995 and 5 rhinosporidiosis cases from Turkey have been reported until date. Considering that rhinosporidiosis is associated with exposure to water and the agent belongs to a branch of aquatic parasites, it has been proposed that aquatic animals are the natural hosts and that the mammalian hosts acquire infection by contacting contaminated water. Therefore, there is a need for the investigation of the infection in fish besides mammalian animals as reservoirs as well as to conduct screening of antiparasitic drugs with infected fish or infected cell lines with the nearest phylogenetic relatives of R. seeberi.

An unexpected host in a soft-tissue lesion of thigh.

Rhinosporidiosis is an enigmatic entity and poses a major health problem in the developing countries of South-East Asia. A soft friable polypoid nasal mass is the most common presentation, while sparse literature is available on extranasal involvement. We describe the case of a 35-year-old female patient who presented with a slow-growing soft-tissue swelling with ulceration over the thigh. On clinical and radiological examination, a provisional diagnosis of soft-tissue neoplasm was made. After resection, histopathological sections showed a closely packed cyst with innumerable endospores. The present case report documents the rare occurrence of an incidentally detected cutaneous rhinosporidiosis causing diagnostic difficulty.

Comparative genomic analysis of the 'pseudofungus' Hyphochytrium catenoides.

Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. The Fungi successfully coupled osmotrophy with filamentous growth, and similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete 'pseudofungus'; Hyphochytrium catenoides Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration across the Pseudofungi. H. catenoides has a complex life cycle involving diverse filamentous structures and a flagellated zoospore with a single anterior tinselate flagellum. We use genome comparisons, drug sensitivity analysis and high-throughput culture arrays to investigate the ancestry of oomycete/pseudofungal characteristics, demonstrating that many of the genetic features associated with parasitic traits evolved specifically within the oomycete radiation. Comparative genomics also identified differences in the repertoire of genes associated with filamentous growth between the Fungi and the Pseudofungi, including differences in vesicle trafficking systems, cell-wall synthesis pathways and motor protein repertoire, demonstrating that unique cellular systems underpinned the convergent evolution of filamentous osmotrophic growth in these two eukaryotic groups.

Elusive treatment for human rhinosporidiosis.

OBJECTIVES: The aim of this study was to clarify the contentious taxonomic classification of Rhinosporidium seeberi, the cause of human rhinosporidiosis, which may have treatment implications. METHODS: PCR was used to amplify the internal transcribed spacer (ITS)-2 region from the genomic DNA of the aetiological agent obtained from a sample of human rhinosporidiosis lesions. The amplicon was sequenced and the organism identified using the Basic Local Alignment Search Tools (BLAST). RESULTS: Phylogenetic analysis revealed that the aetiological agent clustered along with the R. seeberi isolated from humans and also with Amphibiocystidium ranae from frogs. This organism is a member of the order Dermocystida in the class Mesomycetozoea. A patient with disseminated rhinosporidiosis did not respond to conventional therapy with dapsone and surgical excision, and treatment with amphotericin B also proved futile. CONCLUSION: An effective treatment for R. seeberi-a eukaryote belonging to the class Mesomycetozoea-is still elusive.

Rhinosporidium seeberi Nuclear Cycle Activities Using Confocal Microscopy.

Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 µm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 µm cell-walled endospores, each containing a 2-4 µm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development.

Rhinosporidiosis in African reed frogs Hyperolius spp. caused by a new species of Rhinosporidium.

We report the identification of a new Rhinosporidium species (Dermocystida, Mesomycetozoea) infecting amphibian hosts, while showing a species specificity for African reed frogs of the genus Hyperolius. Large dermal cysts (sporangia) of R. rwandae sp. nov. were observed in 18% of H. lateralis and similar cysts in 0.7% of H. viridiflavus surveyed. Fully developed R. rwandae cysts are about 500 to 600 µm in diameter and sealed from the frog tissue by a thick chitinous wall. Some cysts were filled with numerous round-oval basophilic microspores of 8 to 12 µm diameter. With the exception of legs, nodules were visible over the complete torso surface including the vocal sac of males, but the most affected skin region was the area around the cloaca. Behavior, condition, and lifespan of infected frogs do not seem to be distinct from that of healthy individuals. The mode of infection remains unknown, but we hypothesize that the infectious life stage reaches the dermis via the intraepidermal ducts of the skin glands. Molecular evidence places the new frog pathogen as a sister species of the human pathogen R. seeberi.

Taxonomía y filogenética de Rhinosporidium seeberi, un patógeno para el ser humano y los animales: revisión crítica / The taxonomy and phylogenetics of the human and animal pathogen Rhinosporidium seeberi: a critical review

Rhinosporidum seeberi es el agente etiológico de la rinosporidiosis, una enfermedad de las membranas mucosas y, con menos frecuencia, de la piel y otros tejidos. Debido a que se resiste a crecer en los medios de cultivo desde hace más de 100 años, la identidad taxonómica de R. seeberi ha sido motivo de controversia. Tres nuevas hipótesis en una larga lista de puntos de vista similares han sido introducidas: 1) la cianobacteria Microcystis es el agente etiológico de la rinosporidiosis, 2) R. seeberi es un patógeno eucariota en los Mesomycetozoa, y 3) R. seeberi es un hongo. La literatura revisada sobre los estudios realizados con microscopia electrónica, los datos histopatológico y, más recientemente, los datos de varios estudios moleculares, apoyan fuertemente la idea de que R. seeberi es un patógeno eucariota, pero no un hongo. La semejanza morfológica propuesta por algunos de que R. seeberi es similar a los miembros de los géneros Microcystis (bacteria), Synchytrium y Colletotrichum (hongos) es meramente hipotética y no tiene el rigor científico necesario para validar el sistema propuesto. Un aspecto fundamental en contra de la teoría procariota es la presencia de núcleos descrita por numerosos autores y que actualizamos en esta revisión. Además, las características ultra-estructurales de los géneros Microcystis y Synchytrium y de sus ciclos celulares no han sido encontradas en la fase parasitaria de R. seeberi. La amplificación por PCR de una secuencia del rADN 16S típica de las cianobacterias en muestras de casos de rinosporidiosis, aunque interesante, será considerada en esta revisión como una anomalía debido a la contaminación con el medio ambiente (Microcystis) o tal vez como una adquisición endosimbiótica de plastidios a partir de cianobacterias ancestrales. Así pues, aunque R. seeberi podría poseer ADN procariota, esto no demuestra necesariamente que R. seeberi sea una cianobacteria. La clasificación de R. seeberi dentro de los hongos es insostenible. El aislamiento de un hongo, los análisis de ADN realizados, y la ausencia de controles apropiados son los problemas más importantes de esta teoría. Más estudios serán necesarios para validar la adquisición de plastidios procariotas en R. seeberi, y otros temas que requieren un cuidadoso escrutinio(AU)

Rhinosporidum seeberi is the etiologic agent of rhinosporidiosis, a disease of mucous membranes and infrequent of the skin and other tissues of humans and animals. Because it resists culture, for more than 100 years true taxonomic identity of R. seeberi has been controversial. Three hypotheses in a long list of related views have been recently introduced: 1) a prokaryote cyanobacterium in the genus Microcystis is the etiologic agent of rhinosporidiosis, 2) R. seeberi is a eukaryote pathogen in the Mesomycetozoa and 3) R. seeberi is a fungus. The reviewed literature on the electron microscopic, the histopathological and more recently the data from several molecular studies strongly support the view that R. seeberi is a eukaryote pathogen, but not a fungus. The suggested morphological resemblance of R. seeberi with the genera Microcystis (bacteria), Synchytrium and Colletotrichum (fungi) by different teams is merely hypothetical and lacked the scientific rigor needed to validate the proposed systems. A fundamental aspect against the prokaryote theory is the presence of nuclei reported by numerous authors and updated in this review. Moreover, Microcystis's and Synchytrium's ultra-structural and key cell cycle traits cannot be found in R. seeberi parasitic phase. The PCR amplification of a cyanobacteria 16S rDNA sequence from cases of rhinosporidiosis, while intriguing, will be viewed here as an anomaly due to contamination with environmental Microcystis or perhaps as an endosymbiotic acquisition of plastids from cyanobacteria ancestors. Thus, even if R. seeberi possesses prokaryote DNA, this does not prove that R. seeberi is a cyanobacterium. The placement of R. seeberi within the fungi is scientifically untenable. The isolation and the DNA analysis performed in a fungal strain, and the lack of appropriate controls are the main problems of this claim. Further studies are needed to validate R. seeberi's acquisition of prokaryote plastids and other issues that still need careful scrutiny(AU)

The taxonomy and phylogenetics of the human and animal pathogen Rhinosporidium seeberi: a critical review.

Rhinosporidum seeberi is the etiologic agent of rhinosporidiosis, a disease of mucous membranes and infrequent of the skin and other tissues of humans and animals. Because it resists culture, for more than 100 years true taxonomic identity of R. seeberi has been controversial. Three hypotheses in a long list of related views have been recently introduced: 1) a prokaryote cyanobacterium in the genus Microcystis is the etiologic agent of rhinosporidiosis, 2) R. seeberi is a eukaryote pathogen in the Mesomycetozoa and 3) R. seeberi is a fungus. The reviewed literature on the electron microscopic, the histopathological and more recently the data from several molecular studies strongly support the view that R. seeberi is a eukaryote pathogen, but not a fungus. The suggested morphological resemblance of R. seeberi with the genera Microcystis (bacteria), Synchytrium and Colletotrichum (fungi) by different teams is merely hypothetical and lacked the scientific rigor needed to validate the proposed systems. A fundamental aspect against the prokaryote theory is the presence of nuclei reported by numerous authors and updated in this review. Moreover, Microcystis's and Synchytrium's ultra-structural and key cell cycle traits cannot be found in R. seeberi parasitic phase. The PCR amplification of a cyanobacteria 16S rDNA sequence from cases of rhinosporidiosis, while intriguing, will be viewed here as an anomaly due to contamination with environmental Microcystis or perhaps as an endosymbiotic acquisition of plastids from cyanobacteria ancestors. Thus, even if R. seeberi possesses prokaryote DNA, this does not prove that R. seeberi is a cyanobacterium. The placement of R. seeberi within the fungi is scientifically untenable. The isolation and the DNA analysis performed in a fungal strain, and the lack of appropriate controls are the main problems of this claim. Further studies are needed to validate R. seeberi's acquisition of prokaryote plastids and other issues that still need careful scrutiny.

Molecular evidence for multiple host-specific strains in the genus Rhinosporidium.

The taxonomic relationship of Rhinosporidium seeberi with other organisms remained controversial for over a century. Recently, molecular studies have shown R. seeberi to be a protistal microbe in the newly described class Mesomycetozoea at the animal-fungal boundary. Phylogenetic analyses of R. seeberi using 18S small-subunit (SSU) rRNA genes from several hosts suggested Rhinosporidium as a monotypic genus. To test this hypothesis, the internal transcribed spacer 1 (ITS1), 5.8S, and ITS2 from eight humans, two swans, and a dog with rhinosporidiosis were sequenced. The ITS regions were amplified by PCR using a primer designed from a unique region of R. seeberi's 18S SSU rRNA genes in combination with the ITS4 universal primer. In addition, the universal ITS4 and ITS5 primers were also used. R. seeberi's ITS sequences showed differences in the numbers of nucleotides among strains. For instance, the eight human ITS sequences were uniformly similar with only a few mismatches and approximately 1,060 bp long. In contrast, sequences from one of the swans and the dog were 1,356 bp and approximately 1,147 bp long, respectively. Clustal analysis of all of the ITS sequences showed multiple 50- to 60-bp gaps and several mismatches among them. Parsimony analysis placed the Rhinosporidium ITS sequences in three well-supported sister groups according to the hosts' identities. This analysis strongly suggests that the genus Rhinosporidium may possess multiple host-specific strains. No correlation was found between this finding and the phenotypic features of R. seeberi in the studied samples.

Lacazia loboi and Rhinosporidium seeberi: a genomic perspective.

In the past five years, with the use of molecular strategies the phylogenetic affinities of the two more resilient pathogens studied in medical mycology, Lacazia loboi and Rhinosporidium seeberi were finally deciphered. These studies found that L. loboi was the sister taxon to Paraccidioides brasiliensis, and R. seeberi was closely related to protistan spherical aquatic fish pathogens, located at the point were animals diverged from the fungi, in the class Mesomycetozoea. These initial studies indicated that a molecular strategy was the ideal approach to further understand these anomalous pathogens. However, the limited amount of information gathered so far from few DNA sequences, although crucial to place these organisms in the tree of life and to take a glance to their ecological preferences, did not provide answers to other important traits. In the following pages we discuss a genomic perspective for both pathogens and the benefit that such information could generate to understand more about these two uncultivated pathogens.

Lacazia loboi and Rhinosporidium seeberi: a genomic perspective

In the past five years, with the use of molecular strategies the phylogenetic affinities of the two more resilient pathogens studied in medical mycology, Lacazia loboi and Rhinosporidium seeberi were finally deciphered. These studies found that L. loboi was the sister taxon to Paraccidioides brasiliensis, and R. seeberi was closely related to protistan spherical aquatic fish pathogens, located at the point were animals diverged from the fungi, in the class Mesomycetozoea. These initial studies indicated that a molecular strategy was the ideal approach to further understand these anomalous pathogens. However, the limited amount of information gathered so far from few DNA sequences, although crucial to place these organisms in the tree of life and to take a glance to their ecological preferences, did not provide answers to other important traits. In the following pages we discuss a genomic perspective for both pathogens and the benefit that such information could generate to understand more about these two uncultivated pathogens.

Rhinosporidiosis--a clinicopathological study of 34 cases.

Rhinosporidiosis was the commonest (68%) fungal lesion encountered during the period of 11 1/2 years from January 1987 to July 1998. Men in 2nd, 3rd, 4th decade were commonly affected. Nose and nasopharynx were the commonest (85%) sites involved followed by ocular tissue (9%). One patient had involvement of bone (tibia). Generally a lymphoplasmacytic response was observed in all cases. Polymorphonuclear leukocytic response mostly observed at the site of rupture of sporangia. Epithelioid cell granulomatous and giant cell response observed in 47% of cases. Transepithelial migration of sporangia observed in 76% of cases. Rhinosporidium seeberi could be easily identified in haematoxylin and eosin stained sections. The walls of young trophic forms are delineated well with the PAS stain and verhoeff van Gieson stain.

Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.

Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea).

Phylogenetic analysis of Rhinosporidium seeberi's 18S small-subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade.

For the past 100 years the phylogenetic affinities of Rhinosporidium seeberi have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from R. seeberi. Using phylogenetic analysis, by parsimony and distance methods, of R. seeberi's 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium), near the animal-fungal divergence. Our phylogenetic analyses also indicate that R. seeberi is the sister taxon of the two Dermocystidium species used in this study. This molecular affinity is remarkable since members of the genus Dermocystidium form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of R. seeberi to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group's phylogenetic association within the Eucarya.

In vitro studies on the mechanisms of endospore release by Rhinosporidium seeberi.

Studies of Rhinosporidium seeberi have demonstrated that this organism has a complex life cycle in infected tissues. Its in vivo life cycle is initiated with the release of endospores into a host's tissues from its spherical sporangia. However, little is known about the mechanisms of sporangium formation and endospore release since this pathogen is intractable to culture. We have studied the in vitro mechanisms of endospore release from viable R. seeberi's sporangia. It was found that watery substances visibly stimulates the mature sporangia of R. seeberi to the point of endospore discharge. The internal rearrangement of the endospores within the mature sporangia, the opening of an apical pore in R. seeberi's cell wall, and the active release of the endospores were the main features of this process. Only one pore per sporangium was observed. The finding of early stages of pore development in juvenile and intermediate sporangia suggested that its formation is genetically programmed and that it is not a random process. The stimulation of R. seeberi's sporangia by water supports the epidemiological studies that had linked this pathogen with wet environments. It also explains, in part, its affinities for mucous membranes in infected hosts. The microscopic features of endospore discharge suggest a connection with organisms classified in the Kingdom Protoctista. This study strongly supports a recent finding that placed R. seeberi with organisms in the protoctistan Mesomycetozoa clade.

Rhinosporidiosis: a description of an unprecedented outbreak in captive swans (Cygnus spp.) and a proposal for revision of the ontogenic nomenclature of Rhinosporidium seeberi.

Rhinosporidiosis is a mucocutaneous zooanthroponotic disease caused by Rhinosporidium seeberi, a fungal-like organism of uncertain classification with an unknown mode of transmission. Over a 3 year period, 41 captive swans (Cygnus olor and C. atratus) developed conjunctival and cutaneous polypoid lesions diagnosed as rhinosporidiosis by histopathological examination including light and electron microscopy. Investigation of this avian outbreak, the first of its kind, provides additional insight into the epidemiology of this enigmatic aetiologic agent, which has yet to be isolated and cultivated in vitro. The occurrence of rhinosporidiosis in swans supports an aquatic environment as the reservoir for R. seeberi, which is often associated with exposure to water. We report the first known occurrence of rhinosporidiosis in 41 captive mute (C. olor) and Australian black (C. atratus) swans dwelling on a lake in a Central Florida city. Additionally, we review the development stages of R. seeberi and propose a revision in its ontogenic nomenclature to reflect its probable taxonomic classification as a member of the kingdom Fungi.

Phylogenetic spectrum of fungi that are pathogenic to humans.

Recent phylogenetic studies based on ribosomal RNA sequences have confirmed that the organisms traditionally treated as fungi include those that have evolved from several different lines (multiphyletic organisms), as has been suspected. Even organisms causing disease in humans represent at least two evolutional lines. Pythium insidiosum and Prototheca species are both believed to have evolved from one line, while the rest of the pathogens have evolved from another line. P. insidiosum is more closely related to red algae and diatoms than to fungi. Prototheca species, as has been previously postulated, are closer to blue-green algae and plants than to fungi. Pythiosis and protothecosis, however, will still be dealt with by medical mycologists because of the morphological and in vivo staining characteristics of the causative organisms. Molecular genetic studies have revealed that Pneumocystis carinii can best be categorized as a fungus, although questions regarding its fungal status may remain unanswered until additional information becomes available on its life cycle, nuclear division, cell-wall chemistry, nutritional uptake pattern, and lysine biosynthetic pathway as well as the ultrastructural characteristics of its cellular components such as the Golgi complex. The phylogeny of the agents of lobomycosis and rhinosporidiosis, although they are treated as fungi, remains unknown. Although there is no in vitro culture system for Loboa loboi and Rhinosporidium seeberi at present, a molecular approach would allow us to reveal their phylogenetic relationship, and we can hope that such attempts are forthcoming.