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

Disclosures

Emerging Infectious Diseases. 2000;6(3) 

In This Article

Results

R. seeberi
Inferred from the 18S rRNA Gene

Consensus PCR of the 18S rRNA gene with DNA from a digest of an R. seeberi-infected canine nasal polyp produced amplification products of the expected size visible on gel electrophoresis (primer pairs F1-fw/F2-rev = ~500 bp and F1-fw/F3-rev = ~1000 bp) (data not shown). No amplification product was detected by using control tissue and reagents. On the basis of the initial phylogenetic assessment of these sequences, primers Dermo-fw and Dermo-rev were designed for amplification of a more complete portion of the R. seeberi 18S rRNA gene. Our phylogenetic analysis of this gene suggests that R. seeberi is a member of the DRIPs clade of aquatic protistan parasites (Figure 2). The nearest evolutionary neighbors of R. seeberi for which a sequence is available are members of the Dermocystidium genus, which infect salmon and trout.

Figure 2A, 2B. Phylogeny of Rhinosporidium seeberi and the DRIPs clade of protists (Ichthyosporea). A. Phylogenetic tree inferred from the 18S rDNA sequences of R. seeberi and other selected eukaryotes by using a maximum likelihood algorithm; 1,350 masked positions were used for analysis. Bootstrap values were generated from 100 resamplings. The bar, which represents 0.1 base changes per nucleotide position, is a measure of evolutionary distance. B. Phylogenetic tree using the data from A, but with pruning and grouping to show the broader evolutionary position of the DRIPs clade.

Figure 2A, 2B. Phylogeny of Rhinosporidium seeberi and the DRIPs clade of protists (Ichthyosporea). A. Phylogenetic tree inferred from the 18S rDNA sequences of R. seeberi and other selected eukaryotes by using a maximum likelihood algorithm; 1,350 masked positions were used for analysis. Bootstrap values were generated from 100 resamplings. The bar, which represents 0.1 base changes per nucleotide position, is a measure of evolutionary distance. B. Phylogenetic tree using the data from A, but with pruning and grouping to show the broader evolutionary position of the DRIPs clade.

A PCR assay specific for R. seeberi was developed. Primers were created by aligning 18S rDNA sequences from R. seeberi, members of the DRIPs clade, Saccharomyces cerevisiae, and humans. The Rhinosporidium primers (Rhino-fw, Rhino-rev) each have three nucleotide mismatches with the sequences from the nearest phylogenetic relatives in the Dermocystidium genus and multiple other mismatches with fungal and human 18S rDNA sequences. An assay sensitivity of 1-10 gene copies was demonstrated by using a dilution series of cloned R. seeberi 18S rDNA. The specificity of the assay was assessed with DNA from human lymphocytes, S. cerevisiae, D. salmonis, and the Rosette agent. No amplification was detected when these DNA samples were used in the specific PCR assay, although product was amplified from these samples with either ß-globin primers (lymphocytes) or broad-range 18S rDNA primers (F1-fw/F2-rev) (Table).

The original DNA sample from the infected canine nasal polyp yielded a product of the expected size with the Rhinosporidium-specific PCR assay (Figure 3). Purified DNA from the tissue blocks of human nasal polyps resected from three patients with rhinosporidiosis also yielded positive results in this PCR assay, with visible bands seen on gel electrophoresis (RS 1-3) (Figure 3). Direct sequencing of these PCR products demonstrated complete identity over the 377 bp with the cloned sequence from the canine polyp. DNA samples from 23 nasal polyp specimens resected from 12 patients without rhinosporidiosis were subjected to both Rhinosporidium-specific and ß-globin PCR assays. These uninfected polyps failed to yield visible amplicons after gel electrophoresis of the Rhinosporidium-specific PCR reactions (data not shown). However, all these samples yielded visible amplification products after ß-globin PCR, demonstrating that amplifiable DNA was present, without substantial PCR inhibition. These results confirmed that the presence of the putative R. seeberi 18S rDNA sequence correlated with the presence of disease (rhinosporidiosis).

Figure 3. Agarose gel electrophoresis of Rhinosporidium-specific PCR products. The specific amplification product is 377 bp. No amplification product is seen in the negative control samples consisting of water (reagent-only control), digestion buffer (DB), or lymph node tissue control (Tis Cnt). The human rhinosporidiosis samples (RS1-3) and the original canine nasal polyp show visible amplification products.

We sought to determine by using FISH if the R. seeberi 18S rDNA sequence was linked to visible pathology in tissue. The Rhinosporidium 18S rRNA probe but not the control probe bound to R. seeberi organisms in tissue, providing further evidence that the putative R. seeberi 18S rRNA sequence is present in R. seeberi organisms (Figure 4). To test the specificity of the hybridization, smears of the fungus C. albicans, cells infected with the Rosette agent, and tissue sections of C. immitis were subjected to FISH. No specific hybridization to these organisms was detected by using the Rhinosporidium 18S rRNA probe compared with the control probe (data not shown).

Figure 4A, 4B, 4C, 4D. An oligonucleotide probe complementary to a unique region of the Rhinosporidium seeberi 18S rRNA sequence localizes to visible organisms in human nasal tissue by using fluorescence in situ hybridization. Confocal micrographs at 200X magnification. The Cy-5-labeled Rhinosporidium probe (blue) hybridizes to spherical R. seeberi trophocytes (A) and a sporangium with endospores (B). A control probe consisting of the complement of the Rhinosporidium probe labeled with Cy-5 does not hybridize to the trophocytes (C) or a sporangium with endospores (D). Images were collected in two wave-length channels: the first for Texas Red (red) or FITC (green) displays tissue architecture through autofluorescence, and the second channel for Cy-5 (blue pseudocolor) displays the probe signal.

Figure 4A, 4B, 4C, 4D. An oligonucleotide probe complementary to a unique region of the Rhinosporidium seeberi 18S rRNA sequence localizes to visible organisms in human nasal tissue by using fluorescence in situ hybridization. Confocal micrographs at 200X magnification. The Cy-5-labeled Rhinosporidium probe (blue) hybridizes to spherical R. seeberi trophocytes (A) and a sporangium with endospores (B). A control probe consisting of the complement of the Rhinosporidium probe labeled with Cy-5 does not hybridize to the trophocytes (C) or a sporangium with endospores (D). Images were collected in two wave-length channels: the first for Texas Red (red) or FITC (green) displays tissue architecture through autofluorescence, and the second channel for Cy-5 (blue pseudocolor) displays the probe signal.

Figure 4A, 4B, 4C, 4D. An oligonucleotide probe complementary to a unique region of the Rhinosporidium seeberi 18S rRNA sequence localizes to visible organisms in human nasal tissue by using fluorescence in situ hybridization. Confocal micrographs at 200X magnification. The Cy-5-labeled Rhinosporidium probe (blue) hybridizes to spherical R. seeberi trophocytes (A) and a sporangium with endospores (B). A control probe consisting of the complement of the Rhinosporidium probe labeled with Cy-5 does not hybridize to the trophocytes (C) or a sporangium with endospores (D). Images were collected in two wave-length channels: the first for Texas Red (red) or FITC (green) displays tissue architecture through autofluorescence, and the second channel for Cy-5 (blue pseudocolor) displays the probe signal.

Figure 4A, 4B, 4C, 4D. An oligonucleotide probe complementary to a unique region of the Rhinosporidium seeberi 18S rRNA sequence localizes to visible organisms in human nasal tissue by using fluorescence in situ hybridization. Confocal micrographs at 200X magnification. The Cy-5-labeled Rhinosporidium probe (blue) hybridizes to spherical R. seeberi trophocytes (A) and a sporangium with endospores (B). A control probe consisting of the complement of the Rhinosporidium probe labeled with Cy-5 does not hybridize to the trophocytes (C) or a sporangium with endospores (D). Images were collected in two wave-length channels: the first for Texas Red (red) or FITC (green) displays tissue architecture through autofluorescence, and the second channel for Cy-5 (blue pseudocolor) displays the probe signal.

Transmission electron micrographs were taken of R. seeberi trophocytes in a human nasal polyp. Multiple mitochondria were visualized, showing that the cristae had tubulovesicular morphology (Figure 5), unlike the flat cristae found in fungi.

Figure 5. Transmission electron micrograph of a mitochondrion from Rhinosporidium seeberi. The cristae of this mitochondrion (arrows) have tubulovesicular morphology. Magnification 195,000X.

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