The Chemotherapy of Rhinosporidiosis: A Review:- Arseculeratne SN. Review Article 21 Chemotherapy of Rhinosporidiosis: a Review S. N. Arseculeratne, MBBS, Dip.Bact., D.Phil. ABSTRACT
Even though rhinosporidiosis was first identified in 1892, the published literature contain limited
information on options for the chemotherapy of this disease. The absence of methods for in vitro culture of
Rhinosporidium seeberi has restricted the development of in vitro drug susceptibility assays. A new
method of 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H tetrazolium bromide (MTT) reduction was recently
introduced to assess the viability of rhinosporidial endospores, the putative infective stage of R. seeberi.
Using this modification for the microscopy of target endospores, eight antimicrobial agents have been found
to be effective anti-rhinosporidial therapeutic agents (in order of decreasing potency: imidocarb diproprionate,
diminazine aceturate, cycloserine, dapsone, trimethoprim-suphadiazine, ketoconazole, sodium stibogluconate,
and amphotericin B). Fifty-percent inhibitory concentration (IC50) values of less than 100 μg/mL were
regarded as indicating therapeutic efficiency. In vitro determinations of the time-course of action of dapsone
revealed more rapid inactivation than clinical responses suggested, probably because of the in vivo
pharmacokinetics of dapsone that delay an access of the drug into the pathogen. While dapsone remains the
best clinically documented anti-rhinosporidial agent, it is suggested that combination drug therapy may be
advantageous, as in the treatment of tuberculosis, to forestall development of drug resistance by R. seeberi.
The early use of anti-rhinosporidial medical therapy, especially in the absence of surgery and to preempt the
dangerous complications especially in ocular rhinosporidiosis, is emphasized. (J Infect Dis AntimicrobINTRODUCTION
wide range of animal species, and has been reported
Rhinosporidiosis was first observed in a human
from 70 countries. It is highly endemic in South Asia
nasal polyp by Malbran in Argentina in 1892. Seeberi,
also in Argentina, identified the pathogen and
The major sites of rhinosporidiosis are the upper
Wernicke named it Rhinosporidium seeberi in
respiratory tract (the nasal cavity and the nasopharynx),
1900.1,2 Rhinosporidiosis occurs in humans and in a
the eye and its adnexae, the urethra especially in males,
Department of Microbiology, Faculty of Medicine, University of Peradeniya, Sri Lanka.
Received for publication: October 30, 2009.
Reprint request: S. N. Arseculeratne, MBBS, Dip.Bact., D.Phil., Department of Microbiology, Faculty of Medicine, University of
Keywords: Rhinosporidiosis, Rhinosporidium seeberi, MTT-reduction assay, chemotherapy
the skin, and rarely in disseminated sites including
found4 that the salt, 3-[4, 5-dimethyl-2- thiazolyl]-2, 5-
various viscera. The bone and cartilage are eroded,
diphenyl–2H tetrazolium bromide (MTT), could be used
and the granulomatous polyps especially in the
to assess the viability of the endospores of R.seeberi.
nasopharynx become very vascularized making
This salt is used in lymphoproliferative assays5, and in
studies of fungal viability after exposure to antimicrobial
agents.6 Viable endospores of R. seeberi are reactive
growths has been noted in animals and in humans but
with MTT4, resulting in the formation, by MTT-
is rare, hence medical and/or surgical intervention is
reduction, of a deep purple-coloured formazan that
necessary. Radiotherapy has been shown to have no
stains the cytoplasm and some spherical bodies of the
effect.3 The mainstay of treatment is surgical excision
endospores. These bodies have been identified as the
of the polyps, even though the recurrence is common.
electron dense bodies (EDBs),4 that are extensively
Since the early decades of the twentieth century,
described in the literature on R. seeberi. Endospores
several drugs and proprietary preparations have been
have been found not to be capable of MTT-reduction
used, mainly in individual cases, the clinical outcomes
after treatment with the standard microbial-
have been either variable or inconclusive. A major
inactivating agents, heat (100°C), 10 percent formol-
problem with most of these ‘trials’ has been that the
saline, and sodium azide. Slow freezing to minus
period of observation after surgery has been far too
20°C also rendered the endospores non-reactive with
short. Other shortcomings have been unsuitable routes
of administration such as topical application and a
adopted as a method for the determination of the
lack of histological studies of rhinosporidial tissues
viability of rhinosporidial endospores.
from treated patients except after dapsone therapy.
The method is straightforward. The endospores
Clinical reports on rhinosporidiosis still indicate
are incubated with various antimicrobials or other
there is a lack of information on the drug susceptibility
agents in aqueous suspension for selected periods
of R. seeberi, and on effective anti-rhinosporidial
at room temperature (28°C). The endospore
chemotherapy. Data on drug susceptibility of other
specimens used are taken from freshly harvested
members of the Class Mesomycetozoea to which R.
surgical specimens of rhinosporidial nasal or
seeberi belongs, is also lacking.
nasopharyngeal polyps. If the inactivating agent
under investigation contains serum, the treated-
Assessment of the susceptibility of R. seeberi to
endospores are washed repeatedly to remove any
antimicrobial drugs
serum that could inhibit MTT reduction7 before the
There is a contrast with the history of successful
MTT is added. The mixtures are incubated at 37°C
treatment of bacterial and fungal infections with
for 3 hours, and the endospores (after brief, low-
medications after laboratory culture and other
speed centrifugation) are examined under oil
techniques. Similar methods for R. seeberi have not
immersion at 1,000 magnification. This modification
been possible. This is because R. seeberi is still not
of the original MTT method eliminates the need for
cultivable in vitro, and hence in vitro methods based on
extraction of the formazan by acid-ethanol, as in
the multiplication of the organism have not been
lymphoproliferative assays, and its quantitation by
available. This problem was overcome when it was
spectrophotometry. The original method is liable to
The Chemotherapy of Rhinosporidiosis: A Review:- Arseculeratne SN.
cause spuriously elevated optical densities of
percent inhibitory concentrations (IC50).1 These studies
t h e extracted formazan due to contamination
included several effective generically named anti-
microorganisms or even host’s cells from the
rhinosporidial biocides10 including hydrogen peroxide,
glutaraldehyde, chlorohexenol, chlorhexidine, cetrimide,
MTT-reduction method depends on the action of
thiomerosal, 70 percent ethanol, iodine in ethanol, 10
dehydrogenases in the mitochondria in the cytoplasm
percent formalin, povidone-iodine, sodium azide, and
of the endospores to produce the microscopically
silver nitrate. The effective seven brand-named
observable deposits of the deep-purple formazan; these
biocides included11 Bacillo-floor, Bactolin, Bodedex,
bodies have been described in the cytoplasm but have
Cutasept, Korsolex, Sokrena, and Sterilium. The
apparently not been looked for in the EDBs which are
extreme susceptibility of the endospores to biocides was
capable of reduction of MTT. The advantages of the
striking, and the targets of their action were the intra-
direct microscopic assessment of the intensity of
endosporial contents mainly the EDBs, and the
MTT reduction by the endospores, rather than by
spectrophotometric quantitation of extracted formazan,
are (1) the intra-endosporial sites of MTT - reduction
Drugs used in humans
could be readily visualized microscopically as the
Antimicrobial drugs that have been found to
cytoplasm and the EDBs, (2) inevitable contamination
have significant in vitro anti-rhinosporidial activity
of the endospore suspension by nasal microorganisms
(IC50 of ≤ 100 μg/mL) include those used in human
and their resultant contribution to the formation of
as well as animal treatment: amphotericin B, dapsone,
formazan is by passed, (3) the method is simple and
ketoconazole, trimethoprim-sulphadiazine, and sodium
cheap, (4) it results in accurate estimation of the intensity
stibogluconate.1 The IC50s (with number of trials in
of MTT reduction by counts of the endospores that
parenthesis) of these drugs were (in decreasing order
have been morphologically damaged and that have been
of potency) dapsone 29.7 (10), trimethoprim-
inhibited from reducing the MTT, moreover, the results
sulphadiazine 38.4 (9), ketoconazole 51 (8), sodium
from this in vitro MTT-reduction method, as distinct
stibogluconate 55.7 (7), and amphotericin B 57.1 (8)
from conventional in vitro methods that depend on
μg/mL. The intra-endosporial targets were the EDBs,
microbial culture, can be extrapolated to the in vivo
while the endospore walls remained unaffected except
situation by demonstrating a correlation between in vitro
at very high concentrations. All five drugs were
endospore-static rather than endosporicidal, and were
non-lytic on the endospores. The two drugs for use in
Susceptibility of R. seeberi to some antimicrobial
animals, berenil and imizolshowed greater activity with
IC50 values of 13 (5) and 9 (1) μg/mL, respectively. It
Biocides
is noteworthy that berenil has also been used in human
The modified MTT-reduction method has been
trypanosomiasis and babesiosis, and imizol in Lyme
used to assess the anti-rhinosporidial activity of 14
disease. Cycloserine, a drug used as anti-tuberculous
biocides (antiseptics and disinfectants) at concentrations
medication, was recently found (Arseculeratne 2009,
used in hospital and laboratory practice10-11, and also
unpublished data) from 12 trials to have a mean (± SD)
eight antimicrobial drugs with quantification of their 50-
These in vitro results were correlated well with
oral dose used was 100 mg/day for durations from 6
the data from clinical studies from the only drugs on
which clinical information was available. These drugs
include amphotericin B, antimony compounds,
Ketoconazole
ketoconazole, and dapsone. Dapsone has had most
Only one report exists on the use of ketoconazole.
attention with detailed descriptions of the inflammatory
Kunelskaia and colleagues19 found systemic keto-
and healing responses of the host and the effects on
conazole, topical clotrimazole, and surgery effective in
the treatment of nasal rhinosporidiosis.
Neither timethoprim-sulphadiazine nor sodium
Amphotericin B
stibogluconate (or its commercial preparation
Kutty and Teh12 found amphotericin B to have
pentostam) have apparently been used clinically in
caused arrest of the development of the pathogen,
preventing the recurrence of disease during a three-
Drugs that were not effective in the MTT-
year follow-up period, and ultrastructural damage to R.
reduction test in vitro included penicillin G, streptomycin,
seeberi was marked. Topical amphotericin B on
gentamicin, ciprofloxacin, metronidazole, pentamidine,
corneal and nasal rhinosporidiosis have been found to
pyrazinamide, isoniazid, and rifampin. Rajam and
be successful13, however, Ho and Tay14 found the
colleagues20 and Satyanarayana3 found pentamidine
intravenous drug to be ineffective in the treatment of
ineffective in the therapy of rhinosporidiosis. Other
drugs found to be ineffective in vitro1 have not been
used in the therapy of rhinosporidiosis. Antimony compounds
Allen and Dave15 used the antimony compound
Drugs for veterinary use
“Neostibosan” in 18 patients with nasal rhinosporidiosis,
Two drugs tested by the MTT-reduction method
with a satisfactory outcome in only three patients. In
wereberenil(diminazine aceturate) and imizol
another case report, there was no recurrence noted after
(imidocarb dipropionate). Both drugs were found to
one year after Neostibosan therapy and surgery on nasal
be more effective with IC50 values (with number of
rhinosporidiosis.16 In some in vitro studies1, the
trails in parenthesis) of 13 (5) and 9 (1) μg/mL,
pentavalent antimony compound, sodium stibogluconate,
respectively than the drugs used mainly in human
had the highest mean IC50, (55.7 μg/mL) from seven
treatment.1 There is a limited experimentation with
studies, compared with 29.7 μg/mL from 10 studies with
Imizol because the import was stopped. In vivo tests for drug susceptibility Mycobacterium leprae, like R. seeberi, is
Nair17 noted a significant reduction of recurrence
uncultivable in vitro, but limited multiplication occurs in
rates from 93 percent to 39 percent in dapsone-
the foot-pad of the mice. This model has been used to
untreated and -treated patients during three years,
investigate the susceptibility of M. leprae to dapsone.21
respectively. Job and colleagues18 concluded that
R. seeberi while also being uncultivable in vitro is,
medical therapy alone could replace surgery. The usual
however, unable to induce rhinosporidiosis in the foot-
The Chemotherapy of Rhinosporidiosis: A Review:- Arseculeratne SN.
pad of the mice, even though this site has been used
sporangia. Impermeability of sporangia to drugs,
to investigate the immune responses to R. seeberi,
postulated by Woodard and Hudson25 as being the cause
especially cell-mediated immune responses of mice
of failure of drug therapy, has been discounted.1
to intra-foot-pad injections of viable rhinosporidial
Clinical applications
The applicability of anti-rhinosporidial therapy
The time-course of inhibitory effects of dapsone
using medication can be considered in two scenarios
on R. seeberi in vivo and in vitro
(a) presurgical or postsurgical and (b) solely medications.
Job and colleagues18 studied the temporal
sequences of clinical and histological responses in
Presurgical use
humans on dapsone therapy for rhinosporidisis without
These comments relate only to dapsone since
surgery. A reduction in the lesion size was noted after
detailed studies on host and pathogen responses to
six weeks, a marked reduction at 36 weeks, and a
the other drugs shown to be effective in vitro, are not
total disappearance after one year. Mahakrishnan and
colleaques recorded a complete regression under
A serious complication of surgery in rhinosporidiosis
dapsone therapy in disseminated rhinosporidiosis
especially of the nasal and nasopharyngeal sites, is the
within 18 weeks.22 Histologically, the accentuated
profuse intraoperative hemorrhage that results from the
granulomatous responses and the arrest of the
high vascularity of the growths.26 The responses of
maturation and degeneration or absence of endospores
patients after medical therapy with dapsone without
were noted. Venkateswaran and colleagues23 found
surgery18 indicate that the disease process is arrested
no effect on the pathogen until 6 weeks after dapsone
with the increased resolution and fibrosis. It could
therapy, and they noted a reduction in the endospore
therefore be expected that presurgical dapsone would
number, and degeneration or absence of endospores
minimize both the hemorrhage by promoting resolution
after 36 weeks of therapy. Augmented host responses
of the infection, with promotion of fibrosis, as well as
were noted from the twelfth week of therapy.24 The
preventing the colonization and infection of new sites
time-course determinations of the inactivation of
after the release of endospores from the surgically-
rhinosporidial endospores by direct exposure to dapsone
traumatized polyps. Karunaratne27 has claimed that
in vitro, however, showed that the inactivation
satellite rhinosporidial polyps result after surgery from
commenced on the second day while it was complete
the ‘autoinoculation’ of the endospores, that contaminate
on the eighth day (Arseculeratne, 2008, unpublished
the adjacent mucosa during surgery. Presurgical use
data). The marked temporal difference between in
can be compared with the ‘neoadjuvant’ chemotherapy
vivo and in vitro responses is attributable to the time
of malignant neoplastic disease prior to surgery.
taken for absorption, tissue distribution, and accessibility
of orally administered dapsone to the pathogen in the
Postsurgical use
granulomatous rhinosporidial lesions which are
Colonization of normal mucosae by the endospores
surrounded by the barriers of edema, hemorrhage, cell
released from the site of excision, could conceivably be
infiltration, cystic spaces, fibrosis, and especially by
controlled by postoperative dapsone. Postoperative use
down-growths of squamous epithelia that surround the
of dapsone has been the commoner mode of treatment.
With dapsone therapy, recurrences have been reported
months in anti-tuberculous chemotherapy is, from recent
in vitro studies (Arseculeratne, 2008 unpublished data),
In view of the danger of dissemination of R.
an effective (IC50 of approximately 10 μg/mL)
seeberi, especially after surgery, with extensive
additional candidate for the multidrug chemotherapy of
histolysis of soft tissues including bone and cartilage, it
rhinosporidiosis. Whether cycloserine is synergistic with
can be considered advisable to commence medications,
however, small the original lesion appears to be.
The generation time of R. seeberi has not been
Medications can be started even before surgery. The
determined as it cannot be cultured in vitro, but this
prevention of the spread of overt ocular rhinosporidiosis
pathogen probably has a long generation time. This is
to the contralateral eye by dapsone has been
a major justification, as in tuberculosis, for prolonged
recorded.28 This emphasizes the urgency of utilizing
medications even in the absence of surgery; as ocular
rhinosporidiosis with staphyloma formation on the sclera
ACKNOWLEDGEMENT
could result in the dangerous complication of scleral
A grant from the National Science Foundation of
Sri Lanka for work quoted in this review, is thankfully
Resistance of R. seeberi to antimicrobial drugs
There are no data on antimicrobial drug resistance
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