Rhinosporidium seeberi: A Human Pathogen From a Novel Group of Aquatic Protistan Parasites

Disclosures

Emerging Infectious Diseases. 2000;6(3) 

In This Article

Discussion

In the 1890s, first Malbran and then Seeber[14] described an apparent sporozoan parasite in nasal polyps from patients living in Argentina. Seeber's teacher, Wernicke, named the organism Coccidium seeberia after the protozoal subdivision Coccidia and his pupil, Guillermo Seeber[2]. In 1923, Ashworth described the life cycle of the organism, argued that it is a fungus, and proposed the name R. seeberi[15]. Since then, the microbe has been considered a fungus by most microbiologists, although its taxonomy has been debated[1,2,16]. Using a consensus PCR approach, we amplified a unique 18S rDNA sequence from a canine nasal polyp infected with R. seeberi. To prove that this unique 18S rDNA sequence came from R. seeberi, we sought to fulfill sequence-based guidelines for microbial disease causation, since Koch's postulates cannot be fulfilled for uncultivated microbes[17]. Using a Rhinosporidium-specific PCR assay, we showed that the unique rDNA sequence present in the canine polyp was also present in three human polyp samples from patients with rhinosporidiosis. We showed the specificity of this association by demonstrating that this rDNA sequence was not present in control nasal polyps from 12 patients without rhinosporidiosis. We also used FISH to link the putative R. seeberi 18S rRNA sequence to organisms visible in tissue. Although the Rhinosporidium probe localized to R. seeberi organisms in tissue using FISH, the probe did not localize to two fungal organisms or another member of the DRIPs clade (Rosette agent), suggesting some specificity to the hybridization.

Sequence-based data also provide support for a causal relationship when a microbial genotype (e.g., phylogenetic placement) correctly predicts microbial phenotype and host response[17]. In other words, the nature of the pathogen inferred from phylogenetic analysis of its nucleic acid sequence should be consistent with the known biologic characteristics of closely related microbes and with the nature of the disease. Therefore, the nearest phylogenetic neighbors to R. seeberi could be predicted to have similarities in morphology, tissue histology, and pathogenesis. D. salmonis, the closest known relative to R. seeberi,  has a large spherical structure containing endospore-like daughter cells[18]. Histology of infected hosts shows gill inflammation and epithelial hyperplasia. The resemblance between R. seeberi and fish pathogens has been noted before. In 1960, Satyanarayana wrote in his review of 255 cases of rhinosporidiosis[1] that since R. seeberi has a morphology similar to those of some fish parasites, it "..may also be a parasite or saprophyte of fish and that man, equines, and cattle obtain the infection through water in which fish harbouring the parasite live."

A recent independent report based on amplification of 18S rDNA from two human rhinosporidiosis tissue samples also concludes that R. seeberi is a member of the DRIPs clade of microbes[19]. Our data support this conclusion and provide more evidence for a causal relationship. In addition, we describe a Rhinosporidium-specific PCR assay that can be used for detecting this organism in clinical and environmental samples. Our 18S rDNA sequence differs from the sequence determined by these investigators (GenBank AF118851) at a single position out of 1,699 common bases. We excluded from analysis sequence derived from our PCR primers, as amplicons will contain the primer sequences regardless of the target sequence as long as there is partial annealing during PCR, leading to potentially spurious conclusions about sequence in these regions. The 18S rDNA sequence of R. seeberi determined by this group includes the primer sequences.

Although we describe early trophocytes with mitochondrial cristae having vesicular ultrastructure, other investigators have found sporangia with mitochondrial cristae having a flat ultrastructure[19]. The different mitochondrial morphologies observed may be due to differences in developmental stage of the organism or in methods of tissue preparation for electron microscopy. Nevertheless, another member of the DRIPs clade, Ichthyophonus hoferi, has vesicular mitochondrial cristae. Classic fungi (Eumycota) have flat mitochondrial cristae.

Knowledge of the molecular phylogeny of R. seeberi is more than an exercise in taxonomy. For organisms such as R. seeberi, which are difficult to grow in the laboratory, phylogenetic analysis provides some insight into the characteristics of the organism that can be used to further our understanding of disease pathogenesis and epidemiology, as well as to improve diagnosis and treatment. Knowing that R. seeberi is a member of the DRIPs clade of microbes allows hypotheses to be generated about how it causes human disease by analogy, drawing on the knowledge and experience of the veterinary sciences. The separate but linked observations that rhinosporidiosis in humans is associated with exposure to water and that R. seeberi belongs to a clade of aquatic parasites lead to a testable hypothesis: the natural hosts of R. seeberi are fish or other aquatic animals, and humans acquire infection when they come into contact with water containing these fish and their parasites. Investigators should therefore look for evidence of infection in fish in ponds and rivers in disease-endemic areas. From a public health perspective, the R. seeberi-specific PCR assay can be used to study environmental sources of infection (e.g., specific bodies of water) and may provide a means of preventing disease through identification of infected water.

Conversely, knowing that R. seeberi is a member of the DRIPs clade may help us understand this important, distinct group of microbes that appear to form the deepest branch in the animal lineage. R. seeberi is a member of a newly recognized group of human and animal pathogens; the name Ichthyosporea has been proposed for this expanding taxon of microbes[20]. Little information is available about these organisms and how they cause disease. We hope that collaborations between researchers in human and animal medicine will correct this deficiency.

Multiple antimicrobials, including antifungal agents, have been used in the treatment of rhinosporidiosis, based on the belief that R. seeberi is a fungus. However, no antimicrobial agent is clearly effective. The medical treatment of rhinosporidiosis may be improved through screening antiparasitic drugs for an effect on disease in Dermocystidium-infected fish or infected cell lines.

In conclusion, phylogenetic analysis of the R. seeberi 18S rRNA gene suggests that this culture-resistant organism is not a member of the Eumycota, but rather is the first known human pathogen from a novel clade of aquatic protistan parasites that form a branch in the evolutionary tree near the animal-fungal divergence. R. seeberi-specific PCR and FISH confirm the association of this unique 18S rDNA sequence with the presence of rhinosporidiosis. This knowledge can be used to further our understanding of the natural reservoir of this organism and the risk factors, pathogenesis, and treatment of this disease. This discovery also expands our appreciation of the diversity among eukaryotic organisms that are pathogenic to humans and highlights the limitations of basing phylogenetic classification on morphology alone.

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