The use of MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide]-reduction as an indicator of the effects of strain-specific, polyclonal rabbit antisera on Candida albicans and C. krusei.

There is only scanty data on the effects of specific antibody, with or without complement, on Candida albicans or Candida krusei in cell-free systems in vitro, although previously published work has shown that specific antibody mediates anti- Candida immunity in vivo by inhibition of adherence to host cells or surfaces and by the promotion of phagocytosis and intra-phagocytic killing. The MTT (3-[4, 5-dimethyl-2-thiazolyl] -2, 5-diphenyl -2H- tetrazolium bromide)-reduction method as a test of the viability of fungi was used to investigate the effect of complement, normal serum and immune serum on these two species of Candida that are of increasing importance as opportunistic pathogens. We report that normal rabbit serum or strain-specific, polyclonal anti- Candida rabbit antibody, with or without guinea pig complement, did not cause the reduction of total cell-mass or of the viability of either C. albicans or C. krusei, in vitro as determined by the MTT-reduction test. Complement alone without specific antibody, also, had no such effect on these two Candida species.

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.

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.

Recent advances in rhinosporidiosis and rhinosporidium seeberi.

Rhinosporidiosis and its causative pathogen Rhinosporidium seeberi have been known for over a hundred years. Yet unresolved enigmas in rhinosporidiosis include the mode of infection, mechanisms of spread, mechanisms of immunity, some aspects of histopathology e.g. the significance of transepidermal elimination of sporangia, the cause of the variation in cell infiltration patterns in rhinosporidial tissues and their correlations with immune status, and the absence of the Splendore-Hoeppli reaction which is well-marked in invasive, classical mycoses. This review describes the main features of rhinosporidiosis and discusses recent work which clarifies some of these enigmas. Recent work included in this review are molecular biological classification of R.seeberi among the hydrophilic organisms of the former DRIP clade, establishment of a method for the purification of the developmental stages, and some aspects of the immunology of R.seeberi with reference to mechanisms of immune evasion - antigenic variation, host immunoglobulin binding, immune deviation in relation to the chronicity, recurrence and dissemination seen in rhinosporidiosis. The mechanism of endospore release from the sporangium has been described. Some problems involved in the resolution of enigmas that persist are briefly discussed.

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.