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Journal of Clinical Microbiology, January 2001, p. 413-415, Vol. 39, No. 1
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.413-415.2001
LETTERS TO THE EDITOR
Causative Agent of Rhinosporidiosis
 |
LETTER |
I have read the paper by Herr et al. (7) describing
18S ribosomal DNA (rDNA) in Rhinosporidium seeberi and its
phylogenetic similarities with protoctistan fish parasites known as the
DRIP clade. For extraction of DNA, the authors either dissected
sporangia and purified them by centrifugation to remove human cells or
used 100 mg of human tissue containing R. seeberi
(7). This procedure without enzymatic digestion is
insufficient for removing human cells that are always associated with
sporangia (1, 3). Thus, contaminating human cells are the
source of 18S rDNA amplified in this study (7).
We have isolated a prokaryotic cyanobacterium, a
Microcystis sp. (a blue-green alga), from pond water samples
where patients had been bathing. The same cyanobacterium,
Microcystis, and its daughter cells termed nanocytes have
been demonstrated in clinical samples (4). After gaining
entry into the light-deprived environment in human epithelium, the
photosynthetic cells of Microcystis differentiate into round
bodies containing many Microcystis cells described by Herr
et al. (7) as sporangia and spores.
We have successfully cultured the organism isolated from round bodies
for the first time (2). Pure axenic cells were used for
extraction of DNA. Our team has compared the DNA from
Microcystis from pond water with the DNA from microbes found
in clinical samples by using PCR, cloning, sequencing, and Southern
hybridization. Our study demonstrates the presence of a prokaryotic
cyanobacterium inside round bodies (unpublished results) but not 18S
rDNA. Vanbreuseghm (12) had also suggested that R. seeberi produces precursors of chlorophyll and should be regarded
as a pathogenic alga.
In the findings of Herr et al. (see Fig. 2A in reference
7), structures labeled as nuclei do not demonstrate
a double membrane, while ribosome-like configurations are visible both
outside and inside these "nuclei." Actually, these are not nuclei
but nanocytes of Microcystis encompassing naked prokaryotic
DNA. The mitochondria illustrated in the work of Herr et al. (see Fig.
2A of reference 7) do not demonstrate two membranes.
The mitochondria with flat cristae shown in Fig. 2B of this study
(7) have two distinct membranes and are strikingly similar
to those present in human cells. Since mitochondria magnified in Fig.
2B in reference 7 do not belong to Fig. 2A, their spatial relationship
to sporangia or human cells and their source are not clear. It is
noteworthy that mitochondria were observed only in intermediate
sporangia (7), in which a large amount of host epithelial
cells is present (see Fig. 5 in reference 9). Why
were mitochondria, which are specialized for vital functions, not
observed in young and mature sporangia? Why did previous investigators
not find well-defined mitochondria? I am convinced that these
mitochondria (7) are from human cells. On the basis of
flat cristae in mitochondria in R. seeberi (see Fig. 2B in
reference 7) and in Dermocystidium (10), Herr et al. (7) have suggested a
relationship between R. seeberi and the DRIP clade. Flat
cristae are not a distinctive feature of the DRIP clade but are common
to most eukaryotes. The authors who studied the DRIP clade have
themselves stated that mitochondria in Dermocystidium are
like those in almost all animals and eumycota (10).
Herr et al. mention that I consider the causative organism an artifact
of carbohydrate waste (7). I have never stated that R. seeberi is an artifact (1). The spheres of
cellular waste (1) are now found to be polysaccharide
reserves characteristic of cyanobacteria (5). Unexplained
vacuoles, vesicles, and refractile bodies, as well as the concentric
lamellated bodies in R. seeberi (8, 9, 11),
correspond to cell inclusions (5) and photosynthetic membranes in cyanobacteria (6).
Finally, the conclusion of Herr et al. (7) that
R. seeberi is related to the DRIP clade, based on 18S rDNA
and mitochondria from human cells, is unwarranted. Supporting evidence
drawn from superficial criteria such as spherical parasites,
endospores, the inability to culture, and the aquatic habitat
(7) has very little meaning. Inadequate knowledge about
the various manifestations of the microbe, after acquisition of the
pathogenic state, may also have led to the erroneous conclusions.
| |
REFERENCES |
| 1.
|
Ahluwalia, K. B.
1992.
New interpretations in rhinosporidiosis, enigmatic disease of the last nine decades.
J. Submicrosc. Cytol. Pathol.
24:109-114[Medline].
|
| 2.
|
Ahluwalia, K. B.
1999.
Culture of the organism that causes rhinosporidiosis.
J. Laryngol. Otol.
113:523-528[Medline].
|
| 3.
|
Ahluwalia, K. B., and S. Bahadur.
1990.
Rhinosporidiosis associated with squamous cell carcinoma of the tongue.
J. Laryngol. Otol.
104:648-650[Medline].
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| 4.
|
Ahluwalia, K. B.,
N. Maheshwari, and R. C. Deka.
1997.
Rhinosporidiosis: a study that resolves etiologic controversies.
Am. J. Rhinol.
11:479-483[Medline].
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| 5.
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Allen, M. M.
1984.
Cyanobacterial cell inclusions.
Annu. Rev. Microbiol.
38:1-25[CrossRef][Medline].
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| 6.
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Golecki, J. R., and G. Drews.
1982.
Supramolecular organization and composition of membranes, p. 128-138.
In
N. G. Carr, and B. A. Whitton (ed.), The biology of cyanobacteria. Blackwell Scientific Publications, Oxford, England.
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| 7.
|
Herr, R. A.,
L. Ajello,
J. W. Taylor,
S. N. Arseculeratne, and L. Mendoza.
1999.
Phylogenetic analysis of Rhinosporidium seeberi's 18S small subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade.
J. Clin. Microbiol.
37:2750-2754[Abstract/Free Full Text].
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| 8.
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Kannan-Kutty, M., and E. C. Teh.
1974.
Rhinosporidium seeberi: an electron microscopic study of its life cycle.
Pathology
6:63-70[Medline].
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| 9.
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Kennedy, F. A.,
R. R. Buggage, and L. Ajello.
1995.
Rhinosporidiosis: a description of an unprecedented outbreak in captive swans (Cygnus spp.) and a proposal for revision of the ontogenic nomenclature of Rhinosporidium seeberi.
J. Med. Vet. Mycol.
33:157-165[Medline].
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| 10.
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Ragan, M. A.,
C. L. Goggin,
R. J. Cawthorn,
L. Cerenius,
A. V. C. Jamieson,
S. M. Plourdes,
T. G. Tand,
K. Soderhall, and R. R. Gutell.
1996.
A novel clade of protistan parasites near the animal fungal divergence.
Proc. Natl. Acad. Sci. USA
93:11907-11912[Abstract/Free Full Text].
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| 11.
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Thianprasit, M., and K. Thagerngpol.
1989.
Rhinosporidiosis, p. 64-85.
In
M. R. McGinnis, and M. Borgers (ed.), Current topics in medical mycology, vol. 3. Springer Verlag, New York, N.Y.
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| 12.
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Vanbreuseghm, R.
1973.
Ultrastructure of Rhinosporidium seeberi.
Int. J. Dermatol.
12:20-28[Medline].
|
| | | | |
Karvita B. Ahluwalia
Cell Biology and EM Section
Department of Biophysics
All India Institute of Medical Sciences
New Delhi-110029, India
Phone: 0091-11-6593640
Fax: 0091-11-6862663
E-mail: aluwalia{at}medinst.ernet.in
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| |
AUTHORS' REPLY |
We thank Dr. Ahluwalia for the opportunity to address her position on
the prokaryotic nature of Rhinosporidium seeberi (4, 5). We differ with the arguments presented in her letter
regarding our phylogenetic analysis of R. seeberi
(7) and her assertion that this hydrophilic pathogen is a
cyanobacterium and not a Mesomycetozoan. Our position is based on our
studies (7) and a report by Fredericks et al.
(6) which confirmed our phylogenetic analysis.
Dr. Ahluwalia's statement that the 18S small-subunit (SSU) rDNA that
we isolated from R. seeberi's endospores and sporangia came
from DNA of human origin is groundless and reflects little understanding of molecular microbiological procedures. An experienced molecular biologist would have readily noted that the human 18S SSU
rDNA nucleotide sequences and our sequence (GenBank accession no.
AF118851) are unrelated. In fact, as per our BLAST (Basic Local
Alignment Search Tool) analysis, our sequence was not found in the
human genome. Moreover, the sequence of R. seeberi published by Fredericks et al. (GenBank accession no. AF158369) (6), from a dog with rhinosporidiosis, is identical to our sequence. Both of
these groups proved beyond doubt that their 18S SSU rDNAs came from the
sporangia and endospores of R. seeberi. Fredericks et al.
(6) did not contaminate their samples with canine DNA; neither could our sequence (7) possibly be of human
origin. These two independent reports on the phylogenetic connection of R. seeberi with the Mesomycetozoans clearly indicate that
Dr. Ahluwalia's prokaryotic theory is incorrect. There can be no
reasonable doubt that R. seeberi is a eukaryotic organism
not only on the basis of the two cited DNA studies (6, 7)
but on the demonstration by numerous investigators of the presence of
prominent nuclei and mitochondria in the sporangia of this pathogen
(8, 9).
Dr. Ahluwalia's evolving concepts regarding the nature of R. seeberi have ranged widely during the past 8 years. In 1992, she
concluded, on the basis of light microscopy, electron microscopy, and
cytochemical studies of tissue from subjects with rhinosporidiosis, that "The round body is a unique structure composed of both plant and
human material that is self-assembled in response to specific function
pertaining to its elimination from the tissue" (1). In a
follow-up paper (2), she stated that "This study
provides unequivocal evidence against involvement of fungus in
rhinosporidiosis." "Two carbohydrates, namely defective
proteoglycans synthesized intracellularly and an exogenous
polysaccharide ingested through diet of tapioca constitute indigestible
material in NB (nodular bodies) and scw (spheres of cellular waste)."
In a 1994 publication, Ahluwalia et al. (3) concluded that
"The so-called fungal sporangium was shown to be a unique body
organized in host tissue for elimination of two indigestible
carbohydrates, starch (possibly from tapioca) and defective
proteoglycans (precursor of mucus)." The authors went on to say that
"Dietary dry tapioca and chronic inflammation in undernourished
individuals could lead to rhinosporidiosis."
From this bizarre concept, Ahluwalia et al. in 1997 (4)
went on to publish a new hypothesis on the etiologic agent of
rhinosporidiosis. In this study, she announced that "We have been
able to isolate the cyanobacterium Microcystis aeruginosa
from water samples of ponds and rivers where patients of
rhinosporidiosis were bathing. It is likely that this cyanobacterium is
the causative agent of this disease." Finally, in 1999 (5), she announced that "Observations based on
laser-scanning confocal microscopy, light and electron microscopy
confirm that a cyanobacterium Microcystis sp. is the causative agent of the disease. Rhinosporidiosis is the first human
disease shown to be caused by a cyanobacterium." Surprisingly, controls such as samples from normal people inhabiting the same areas
where the water samples were collected and cultures of endospores and
sporangia free of bacteria obtained after multiple washes and
filtration steps were not included. It would also be interesting to
probe M. aeruginosa with sera from patients with
rhinosporidiosis, a key experiment that was also absent in Ahluwalia's
studies. The mere isolation of the ubiquitous blue-green bacterium
M. aeruginosa from pond and river waters in India does not
have any significance with respect to the infections caused by the
eukaryotic protist R. seeberi. Thus, the lack of controls in
Dr. Ahluwalia's experiments is the source of her errors. This lapse
undoubtedly led Dr. Ahluwalia to conclude that the mere isolation of
M. aeruginosa from infected tissues was enough to prove her conjecture.
We disagree with Dr. Ahluwalia's statement that there are no other
studies depicting mitochondria in R. seeberi. At least two
independent studies showed these organelles within R. seeberi's sporangia (8, 9). In an effort to further
clarify our finding, we have included Fig.
1. In this figure an intermediate
sporangium (Fig. 1A, previously printed in reference
7) containing several nuclei, mitochondria, and a
laminated body is shown. An enlargement of Fig. 1A presents details of
the flat mitochondrial cristae of R. seeberi (Fig. 1B and
C). The presence of a laminated body indicates that this is a healthy
sporangium and that the mitochondria within the spherical body are not
of human origin, as charged by Ahluwalia, but are attributable to
R. seeberi.
View larger version (129K):
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FIG. 1.
(A) Transmission electron microscopy photograph of a
R. seeberi's intermediate sporangium, showing several
mitochondria with flat cristae (black and white asterisks) and a
laminated body (arrow). (B and C) An enlargement of the mitochondria in
panel A.
|
|
Contrary to Dr. Ahluwalia's studies, our results have already been
confirmed by other investigators (6). These researchers also concluded that R. seeberi is phylogenetically linked
with the Mesomycetozoa (DRIP) clade. In contrast, we could not
replicate her results when purified endospores and sporangia were
cultured in a variety of media. We strongly recommend that Dr.
Ahluwalia repeat our molecular studies with uncontaminated endospores
and sporangia from patients with rhinosporidiosis in India to contest our data. However, the results achieved so far by two independent laboratories could well be the requiem to Dr. Ahluwalia's prokaryotic theory of R. seeberi.
| |
REFERENCES |
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|
Ahluwalia, K. B.
1992.
Plant molecular in human disease a novel association, p. 289-292.
In
R. J. Wegmann, and M. A. Wegmann (ed.), Gene regulation and molecular aspects of muscle, liver, pancreas, connective tissue and plants, vol. 5. Peeters Press, Leuven, Belgium.
|
| 2.
|
Ahluwalia, K. B.
1992.
New interpretations in rhinosporidiosis, enigmatic disease of the last nine decades.
J. Submicrosc. Cytol. Pathol.
24:109-114.
|
| 3.
|
Ahluwalia, K. B.,
N. Sharma,
S. K. Kacker, and R. C. Deka.
1994.
Association of tapioca and chronic inflammation with rhinosporidiosis.
Indian J. Otolayngol. Head Neck Surg.
3:25-27.
|
| 4.
|
Ahluwalia, K. B.,
N. Maheshwari, and R. C. Deka.
1997.
Rhinosporidiosis: a study that resolves etiologic controversies.
Am. J. Rhinol.
11:479-483.
|
| 5.
|
Ahluwalia, K. B.
1999.
Culture of the organism that causes rhinosporidiosis.
J. Laryngol. Otol.
113:523-528.
|
| 6.
|
Fredericks, D. N.,
J. A. Jolley,
P. W. Lepp,
J. C. Kosek, and D. A. Relman.
2000.
Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.
Emerg. Infect. Dis.
6:273-282[Medline].
|
| 7.
|
Herr, R. A.,
L. Ajello,
J. W. Taylor,
S. N. Arseculeratne, and L. Mendoza.
1999.
Phylogenetic analysis of Rhinosporidium seeberi's 18S small subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade.
J. Clin. Microbiol.
37:2750-2754.
|
| 8.
|
Teh, E. C., and M. Kannan-Kutty.
1975.
Rhinosporidium seeberi: spherules and their significance.
Pathology
7:133-137[Medline].
|
| 9.
|
Thianprisit, M., and K. Thagerngpol.
1989.
Rhinosporidiosis.
Curr. Top. Med. Mycol.
3:61-85.
|
| | | | |
Leonel Mendoza
Roger A. Herr
Medical Technology Program
Department of Microbiology
Michigan State University
322 N. Kedzie Lab.
East Lansing, Michigan 48824
|
| | | | |
Libero Ajello
Department of Ophthalmology
Emory University School of Medicine
Atlanta, Georgia 30322
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Journal of Clinical Microbiology, January 2001, p. 413-415, Vol. 39, No. 1
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.413-415.2001
