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Biomedical subjects

S N Arseculeratne

Publications and source records attributed to S N Arseculeratne.

At least 19 recordsLinked to original sources
PubMed2006

The effects of biocides (antiseptics and disinfectants) on the endospores of Rhinosporidium seeberi.

No data exists on the activity of biocides (antiseptics and disinfectants) on Rhinosporidium seeberi that causes rhinosporidiosis in humans and animals. On account of the inability to culture R. seeberi, in vitro, dyes were used to assess the morphological integrity and viability of biocide-treated endospores that are considered to be the infective stage of this pathogen. Evan's Blue (EvB) identifies the morphological integrity of the endospores while MTT (3-[4, 5-dimethylthiazol-2yl]-2, 5-diphenyl tetrazolium bromide) identifies metabolic activity through its reduction by cellular dehydrogenases to microscopically visible deposits of insoluble formazan. MTT-negativity has earlier been shown to correlate with absence of growth of yeast and mycelial fungi in culture and could thus indicate the loss of viability of MTT-negative rhinosporidial endospores. Hydrogen peroxide, glutaraldehyde, chloroxylenol, chlorhexidine, cetrimide, thimerosal, 70% ethanol, iodine in 70% ethanol, 10% formalin, povidone-iodine, sodium azide and silver nitrate were tested on freshly-harvested endospores and all biocides caused metabolic inactivation with or without altered structural integrity as shown by absence of MTT-staining after 3, 24 or 36 hour after exposure, while EvB stained only the endospores treated with sodium azide, ethanol, thimerosal, chloroxylenol, glutaraldehyde and hydrogen peroxide. With clinically useful biocides - chlorhexidine, cetrimide-chlorhexidine, 70% ethanol, povidone-iodine and silver nitrate, a total period of exposure of endospores to the biocide, for seven minutes, produced metabolic inactivation of the endospores. Anti-rhinosporidial antiseptics that could be used in surgery on rhinosporidial patients include povidone-iodine in nasal packs for nasal and naso-pharyngeal surgery, chlorhexidine and cetrimide-chlorhexidine on the skin, while povidone-iodine and silver nitrate could have application in ocular rhinosporidiosis.

Animals↗
PubMed2005

Nature and significance of the electron-dense bodies of the endospores of rhinosporidium seeberi: their reactions with MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and TMRE (tetramethyl-rhodamine ethyl ester).

The most conspicuous internal structures of the endospore of Rhinosporidium seeberi are the 10-16 spherical, 1.0-1.5-microm bodies that have been termed electron-dense bodies (EDB) or lipid bodies (LB); some authors have regarded them as nutritive stores of lipid or protein while others have regarded them as DNA-containing, ultimate generative units of R. seeberi. The literature is reviewed as supporting either view. We report, for the first time, (i) reactions of the endospores with the salt MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and (ii) ultrastructural appearances; and suggest that both views on the nature of spherical bodies are valid (i.e. the endospore contains both EDB, and lipid or protein bodies). Well-marked reduction of the MTT with the production of deep-purple staining was seen in a proportion of the spherical bodies, probably the EDB, suggesting that they are actively metabolizing, viable elements with dehydrogenase activity, and that these bodies are the thick-walled electron-dense bodies described as EDB and visualized in the transmission electron microphotograph illustrated in this paper. The spherical bodies showed fluorescent labeling with acridine orange and with ethidium bromide supporting the idea that they contained nucleic acids. TMRE (tetramethyl rhodamine ethyl ester), a mitochondrion-specific dye, also labeled the intra-endosporial spherical bodies. Other bodies (LB) of a similar size that were MTT-non-reducing, electron lucent, and have no organized structure, are probably the lipid or protein containing, inert, nutritive storage bodies suggested by previous authors.

Acridine Orange↗
PubMed2005

Human anti-rhinosporidial antibody does not cause metabolic inactivation or morphological damage in endospores of Rhinosporidium seeberi, in vitro.

This report describes the use of the MTT-reduction and Evan's blue-staining tests for the assessment of the viability and morphological integrity, respectively, of rhinosporidial endospores after exposure to sera from rhinosporidial patients with high titres of anti-rhinosporidial antibody. Sera from three patients, with nasal, ocular and disseminated rhinosporidiosis respectively were used, with human serum without anti-rhinosporidial antibody for comparison, with or without added fresh guinea pig serum as a source of complement. All four sera tested, with or without guinea-pig serum, had no effect on the morphological integrity or the viability of the endospores and it is suggested that anti-rhinosporidial antibody has no direct protective role against the endospores, the infective stage, in rhinosporidiosis. This finding is compatible with the occurrence of chronicity, recurrence and dissemination that are characteristic of rhinosporidiosis despite the presence of high titres of anti-rhinosporidial antibody in patients with these clinical characteristics. The possible occurrence of humoral mechanisms of immunity that involve anti-rhinosporidial antibody with cells such as leucocytes and NK cells, in vivo, cannot yet be discounted, although the presence of high titres of anti-rhinosporidial antibody in patients with chronic, recurrent and disseminated lesions might indicate that such antibody is non-protective in vivo.

Antibodies, Fungal↗
PubMed2004

Anti-rhinosporidial antibody levels in patients with rhinosporidiosis and in asymptomatic persons, in Sri Lanka.

The only report hitherto, from India in 1982, on anti-rhinosporidial antibody levels in patients with rhinosporidiosis recorded that antibody was not detected in Indian patients. The present report describes the use of the dot-ELISA assay of serum anti-rhinosporidial IgG, IgM and IgA and salivary sIgA in patients with diverse clinical presentations, in rural asymptomatic persons who had bathed in ground waters that probably harboured the causative pathogen, Rhinosporidium seeberi, and in laboratory persons who were exposed to R. seeberi. Ultrasonic extracts of purified endospores and sporangia of R. seeberi were used as antigen. The geometric mean (reciprocal) titres of serum antibody detected in patients were IgM 142.1, IgG 178.5, IgA 84.6, with ranges of 0-640, 30-960 and 0-160 respectively, salivary sIgA titres ranged from 0 to 18 with a mean of 4.6. The levels of antibody had no correlation with the site, the number of sporangia, duration and recurrence of the disease. Asymptomatic persons from the same endemic area as patients showed mean titres of IgM 89.6, IgG 69.1, IgA 95.5, with salivary sIgA titres of 3.1. Asymptomatic personnel who had been working in a laboratory where rhiniosporidial work was being done, showed mean titres of 169.6 IgM, 62.8 IgG, and 6.5 salivary sIgA. These results indicate that an anti-rhinosporidial antibody response occurs in rhinosporidial patients, as well as in asymptomatic persons who were exposed to R. seeberi in the environment. Anti-R. seeberi antibody does not appear to be protective in rhinosporidiosis since appreciable titres were present in patients with recurrent, single, multiple or disseminated lesions of long duration.

Animals↗
PubMed2002

Lymphadenitis, trans-epidermal elimination and unusual histopathology in human rhinosporidiosis.

From a study of rhinosporidial tissues of 64 human cases of ocular, urethral and nasopharyngeal disease, unusual histopathological features of 27 cases are described. Histopathological evidence of lymphadenitis in rhinosporidiosis is presented for the first time. The phenomenon of 'trans-epidermal elimination' of sporangia of the causative pathogen Rhinosporidium seeberi is illustrated and it is argued that this phenomenon is rather the pathogen's mechanism for endospore-dispersal than a non-specific defence reaction of the host as has previously been suggested. Other unusual appearances described include variations in the intensity and composition of the host-cell infiltrate in tissues from different patients and in different portions of the same tissue, pitfalls in histopathological diagnosis, and unusual appearances of the pathogen. Histopathological clues to the pathogenesis of rhinosporidiosis and mechanisms of anti-rhinosporidial immunity in the host are discussed, illustrating the probable occurrence of immunesuppressive reactions to account for the variations in the density and composition of the host-cell infiltrate and the state of the rhinosporidial sporangia--intact or degenerate--, relating these variations to the chronicity, recurrences and systemic dissemination of rhinosporidiosis.

Diagnostic Errors↗
PubMed2001

Cell-mediated immune responses (CMIR) in human rhinosporidiosis.

Cell mediated immune responses (CMIR) to Rhinosporidium seeberi in human patients with rhinosporidiosis have been studied. With immuno-histochemistry, the cell infiltration patterns in rhinosporidial tissues from 7 patients were similar. The mixed cell infiltrate consisted of many plasma cells, fewer CD68+ macrophages, a population of CD3+ T lymphocytes, and CD56/57+ NK lymphocytes which were positive for CD3 as well. CD4+ T helper cells were scarce. CD8+ suppressor/cytotoxic-cytolytic cells were numerous. Most of the CD8+ cells were TIA1+ and therefore of the cytotoxic subtype. CD8+ T cells were not sub-typed according to their cytokine profile; 1L2, IFN-gamma (Tcl); IL4, ILS (Tc2). In lympho-proliferative response (LPR) assays in vitro, lymphocytes from rhinosporidial patients showed stimulatory responses to Con A but lymphocytes from some patients showed significantly diminished responses to rhinosporidial extracts as compared with unstimulated cells or cells stimulated by Con A, indicating suppressor immune responses in rhinosporidiosis. The overall stimulatory responses with Con A suggested that the rhinosporidial lymphocytes were not non-specifically anergic although comparisons of depressed LPR of rhinosporidial lymphocytes from individual patients, to rhinosporidial antigen with those to Con A, did not reveal a clear indication as to whether the depression was antigen specific or non-specific. The intensity of depression of the LPR in rhinosporidial patients bore no relation to the site, duration, or the number of lesions or whether the disease was localized or disseminated. Rhinosporidial extracts showed stimulatory activity on normal control lymphocytes, perhaps indicating mitogenic activity. These results indicate that CMIR develops in human rhinosporidiosis, while suppressed responses are also induced.

Antigens, Bacterial↗
PubMed2001

Cell-mediated immune responses (CMIR) to Rhinosporidium seeberi in mice.

There is no published data on Cell Mediated Immune Responses in experimental animals to Rhinosporidium seeberi the causative agent of human and animal rhinosporidiosis. The quantitative mouse foot-pad model was used to assay the Delayed-Type Hypersensitivity (DTH) cell-mediated immune response to extracts of purified endospores and sporangia of R. seeberi. Histological examination was used to confirm that the foot-pad reactions were compatible with DTH reactions in the mouse. We report that sonically disintegrated rhinosporidial endospores/sporangia induced DTH responses in the foot-pads of sensitized mice which were comparable in intensity and histological profile to that induced by sheep red blood cells in SRBC sensitized mice. Anti-rhinosporidial antibody was also induced. Filtrates of the soluble antigens in sonicated suspensions failed to evoke a DTH-foot-pad (DTH-FP) response in sensitized mice although an anti-rhinosporidial antibody response to this preparation was detected. Prolonged pre-treatment with sonicated suspensions of endospores and sporangia resulted in a decrease of DTH reactivity as compared with reactions following pre-treatment of a shorter duration.

Animals↗
PubMed1999

Purification of the endospores and sporangia of Rhinosporidium seeberi on Percoll columns.

Human rhinosporidial tissue was used as the source of the various developmental stages of Rhinosporidium seeberi--endospores with electron dense bodies, juvenile, and immature sporangia. After homogenisation in phosphate buffered saline (PBS) and removal of tissue fragments by centrifugation, the rhinosporidial bodies were isolated on centrifuged Percoll columns with gradients of densities or on triple-layered columns of varying density. The separated bands, after repeated washing in PBS gave bodies free from human tissue as shown on Leishman and PAS staining and indirect immunofluorescence with rabbit and human patients' anti-rhinosporidial sera. Sonicates of these bodies were tested on agarose gel for precipitation with antisera, and on SDS-PAG electrophoresis and Coomassie Blue staining. Percoll columns were shown to be capable of isolating these stages of R. seeberi, free from human tissue and contaminating bacteria.

Animals↗
PubMed1999

In vitro studies on the mechanisms of endospore release by Rhinosporidium seeberi.

Studies of Rhinosporidium seeberi have demonstrated that this organism has a complex life cycle in infected tissues. Its in vivo life cycle is initiated with the release of endospores into a host's tissues from its spherical sporangia. However, little is known about the mechanisms of sporangium formation and endospore release since this pathogen is intractable to culture. We have studied the in vitro mechanisms of endospore release from viable R. seeberi's sporangia. It was found that watery substances visibly stimulates the mature sporangia of R. seeberi to the point of endospore discharge. The internal rearrangement of the endospores within the mature sporangia, the opening of an apical pore in R. seeberi's cell wall, and the active release of the endospores were the main features of this process. Only one pore per sporangium was observed. The finding of early stages of pore development in juvenile and intermediate sporangia suggested that its formation is genetically programmed and that it is not a random process. The stimulation of R. seeberi's sporangia by water supports the epidemiological studies that had linked this pathogen with wet environments. It also explains, in part, its affinities for mucous membranes in infected hosts. The microscopic features of endospore discharge suggest a connection with organisms classified in the Kingdom Protoctista. This study strongly supports a recent finding that placed R. seeberi with organisms in the protoctistan Mesomycetozoa clade.

Histocytochemistry↗
PubMed1999

Immunolocalization of an endogenous antigenic material of Rhinosporidium seeberi expressed only during mature sporangial development.

We investigated the immunolocalization of Rhinosporidium seeberi's antigens using sera from individuals infected with R. seeberi and tissue from Sri Lankan patients with rhinosporidiosis. The tissues were fixed in LR white resin, thin sectioned fixed onto nickel grids and evaluated by transmission electron microscopy for the presence of R. seeberi's sporangia. The tissue samples were reacted with the patients's sera and then labeled with protein A colloidal gold (PACG) for immunolocalization. It was found that the PACG had fixed to antibodies that specifically recognized an internal electron lucent layer situated immediately under the mature sporangium's wall. Strikingly, the endospores, the juvenile and intermediate sporangia did not undergo PACG labeling. This study found that the expression of this antigen occurs only in the final developmental stages of R. seeberi's mature sporangia. Our data may explain why circulating antibodies to R. Seeberi were not detected before in studies that used endospores as antigen in immunoassays. This is the first report in which an antigenic material with a potential role in the immunology of rhinosporidiosis has been detected.

Antigens, Fungal↗
PubMed1999

Phylogenetic analysis of Rhinosporidium seeberi's 18S small-subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade.

For the past 100 years the phylogenetic affinities of Rhinosporidium seeberi have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from R. seeberi. Using phylogenetic analysis, by parsimony and distance methods, of R. seeberi's 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium), near the animal-fungal divergence. Our phylogenetic analyses also indicate that R. seeberi is the sister taxon of the two Dermocystidium species used in this study. This molecular affinity is remarkable since members of the genus Dermocystidium form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of R. seeberi to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group's phylogenetic association within the Eucarya.

DNA, Ribosomal↗
PubMed1998

A pseudo-cryptococcal artefact derived from leucocytes in wet India ink mounts of centrifuged cerebrospinal fluid.

Wet India ink mounts of cerebrospinal fluid (CSF) are useful in the laboratory diagnosis of cryptococcal meningitis. Pseudo-cryptococcal artefacts in such mounts have been attributed to leucocytes in CSF but their mode of formation has not been explained. This report describes the reproduction of such an artefact in cryptococcus free CSF-leucocyte mixtures that had been subjected to high speed centrifugation. The viscosity of DNA that could provide a morphological pseudo-capsule, and the yellow-green fluorescence of the pseudo-capsular material on staining with acridine-orange, suggest that lymphocytic nuclear DNA, which possibly leaked out after damage to the lymphocyte membrane by centrifugation, was responsible for this artefact.

Artifacts↗
PubMed1994

Survey of mycotic and bacterial keratitis in Sri Lanka.

Over a two-year period (1976-1977 and 1980-1981), 66 cases of bacterial and mycotic cases of keratitis were diagnosed in the Eye Clinic of the General Hospital in Kandy, Sri Lanka. The clinical and microbiologic aspects of these cases are described. Noteworthy was the first known human case caused by Paecilomyces farinosus, a geophilic species, commonly encountered as an insect parasite throughout the world. The bacterial and the other fungal etiologic agents isolated and identified were: Pseudomonas aeruginosa, Streptococcus pneumoniae, Aspergillus flavus, A. niger, Fusarium oxysporum, and Lasiodiplodia theobromae. In vitro the fungi showed sensitivity in decreasing order to flucytosine, nystatin, amphotericin B and econazole. Due to the out-patient status of the patients, their in-vivo response to treatment was not assessable.

Corneal Ulcer↗
PubMed1993

Ileal perforation in typhoid: bacteriological and immunological findings.

In typhoid perforation patients, Salmonella typhi was isolated from blood in 4%, ileal contents in 23%, peritoneal pus in 13% and from mesenteric lymph nodes in 71%. While isolation of S. typhi was made from patients with less than 4 days of chloramphenicol therapy, cultures were negative from these sites after 5 days of therapy; however, S. typhi appeared to remain viable in the lymph nodes even after such therapy. All isolates of S. typhi were sensitive to chloramphenicol. Significant SAT titers (0 > or = 1/240) were obtained in only 7/21 (33%) of patients. The perforated group had lower geometric mean titers (0-1/138; H-1/46), when compared to matched patients with uncomplicated typhoid fever (0-1/476; H-1/148). This difference was significant (0- p < 0.005; H- p < 0.0025). The two groups (uncomplicated and perforated) showed no significant difference in total serum IgG, IgM and IgA or isohemagglutinin levels, indicating that the apparent hyporeactivity was not due to a generalized humoral immunodeficiency. Mesenteric lymph node histology showed hyporeactivity in both the T cell and B cell zones. These findings are discussed with the suggestion that S. typhi-specific host immunological hyporeactivity could be an explanation for these observations and a basis for the pathogenesis of perforation. Aerobic cultures of the peritoneal pus gave 39 isolates from 25 patients; the predominant isolates were Escherichia coli (24) and Klebsiella pneumoniae (12). On no occasion was S. typhi the predominant isolate. Gentamicin and kanamycin were the only two antibiotics which were consistently effective in vitro against the aerobic isolates from peritoneal pus.

Humans↗