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P.21-27-inf-19-51-review article.pmdThe Chemotherapy of Rhinosporidiosis: A Review:- Arseculeratne SN.
Review Article 21
Chemotherapy of Rhinosporidiosis: a Review
S. N. Arseculeratne, MBBS, Dip.Bact., D.Phil.
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 Antimicrob INTRODUCTION
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 imizol showed 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.
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 were berenil (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 References
in R. seeberi. The strains obtained from human and Arseculeratne SN, Kumarasiri R, Sumathipala S, animal rhinosporidiosis have shown genetic variations, Atapattu DN, Eriyagama NB, Balasooriya P, Fernando and the possibility exists also of variation of drug R. The determination of the sensitivity of the susceptibility based on genotype and which might endospores of Rhinosporidium seeberi to seven anti- explain the variation of clinical responses to some drugs microbial drugs using MTT-reduction as an indicator including amphotericin B and antimony compounds that of endospore-viability. J Infect Dis Antimicrob Agents have been noted in the literature. With mycobacterial infections, notably tuberculosis, a development of Arseculeratne SN, Mendoza L. Rhinosporidiosis.
resistance to a single drug, as in the early days of In: Mertz WG, Hay RJ, eds. Topley & Wilson’s prolonged anti-tuberculosis chemotherapy, prompted the Microbiology & Microbial Infections. Vol. 5: Medical use of multiple drugs to forestall development of drug- Mycology. 10th ed. London: Arnold, 2005:436-75.
resistance of M. tuberculosis. At present, there are Satyanarayana C. Rhinosporidiosis with a record of no data on the development of resistance in R. seeberi 255 cases. Acta Otolaryngol 1960;51:348-66.
to dapsone. As dapsone is the single most common Arseculeratne SN, Atapattu DN. The assessment of drug used in the chemotherapy of rhinosporidiosis, it the viability of the endospores of Rhinosporidium might be prudent to use combinations of several seeberi with MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5- medications. This could be dapsone combined with diphenyl-2H-tetrazolium bromide). Mycol Res one or more of the drugs mentioned above, that have been recently shown1 to have in vitro anti-rhinosporidial Mosmann T. Rapid colorimetric assay for cellular activity. Cycloserine which is administered for several growth and survival: application to proliferation and The Chemotherapy of Rhinosporidiosis: A Review:- Arseculeratne SN.
cytotoxicity assays. J Immunol Methods 1983;65:55-63.
Nair KK. Clinical trial of diaminodiphenylsulfone Levitz SM, Diamond RD. A rapid colorimetric assay of (DDS) in nasal and nasopharyngeal rhinosporidiosis.
fungal viability with the tetrazolium salt MTT. J Infect Job A, Venkateswaran S, Mathan M, Krishnaswami H, Jahn B, Martin E, Stueben A, Bhakdi S. Susceptibility Raman R. Medical therapy of rhinosporidiosis with testing of Candida albicans and Aspergillus species dapsone. J Laryngol Otol 1993;107:809-12.
by a simple microtiter menadione-augmented 3-(4,5- Kunel’skaia VI, Chelidze ND, Berchenko VI. Current dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium possibilities of the diagnosis and treatment of bromide assay. J Clin Microbiol 1995;33:661-7.
rhinosporidiosis of the nose. Vestn Otorinolaringol Cushion MT, Chen F, Kloepfer N. A cytotoxicity assay for evaluation of candidate anti-Pneumocystis carinii Rajam RV, Viswanathan GS, Rao AR, Rangiah PN, agents. Antimicrob Agents Chemother 1997;41:379-84.
Anguli VC. Rhinosporidiosis-A study with report of a Rex JH, Pfaller MA, Rinaldi MG, Polak A, Galgiani JN.
fatal case with systemic dissemination. Indian J Surg Antifungal susceptibility testing. Clin Microbiol Rev Sekar B, Elangeswaran N, Jayarama E, et al. Drug Arseculeratne SN, Atapattu DN, Balasooriya P, susceptibility of Mycobacterium leprae: a retro- Fernando R. The effects of biocides (antiseptics and spective analysis of mouse footpad inoculation results disinfectants) on the endospores of Rhinosporidium from 1983 to 1997. Lepr Rev 2002;73:239-44.
seeberi. Indian J Med Microbiol 2006;24:85-91.
Mahakrisnan A, Rajasekaram V, Pandian PJ. Dis- Eriyagama NB, Arseculeratne SN. Further obser- seminated cutaneous rhinosporidiosis treated with vations on the effects of biocides (antiseptics and dapsone. Trop Geogr Med 1981;33:189-92.
disinfectants) on the endospores of Rhinosporidium Venkateswaran S, Date A, Job A, Mathan M. Light seeberi, as assayed by the MTT-reduction test. J Infect and electron microscopic findings in rhinosporidiosis Dis Antimicrob Agents 2007;24:125-32.
after dapsone therapy. Trop Med Int Health 1997;2: Kutty MK, Teh EC. The morphological response of Rhinosporidium seeberi to Amphotericin B. Malaysian Bhanu TS. New drug regime for rhinosporidiosis.
Bhomaj S, Das JC, Chaudhuri Z, Bansal RL, Sharma P.
Woodard BH, Hudson J. Rhinosporidiosis: ultra- Rhinosporidiosis and peripheral keratitis. Ophthalmic structural study of an infection in South Carolina.
Ho MS, Tay BK. Disseminated rhinosporidiosis. Ann Kameswaran S. Surgery in rhinosporidiosis.
Experience with 293 cases. Int Surg 1966;46:602-5.
Allen FR, Dave ML. The treatment of rhinosporidiosis Karunaratne WAE. Rhinosporidiosis in Man. London: in man based on the study of sixty cases. Indian Med Senaratne T, Senanayake S, Edussuriya K, Wijenayake Atav N, Goksan T, Ural A. The first case of P, Arseculeratne S. Ocular rhinosporidiosis with rhinosporidiosis met with in Turkey. Ann Otol Rhinol staphyloma formation: The first report in Sri Lanka. J Infect Dis Antimicrob Agents 2007;24:133-41.
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