Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

Acanthameba meningitis with successful outcome.

We report a rare cause of sub acute meningitis in a 15-yr-old immunocompetent female child with successful outcome. The etiological agent was Acanthameba. The child was sucessfully treat with combination of Ketoconazole. Rifampicin, cotrimoxa zole and for a period of 9 month.

In vitro effect of artesunate against Acanthamoeba spp.

The in vitro effects of artesunate, the antimalarial agent, and metronidazole against Acanthamoeba spp were studied. Acanthamoeba Group II and Acanthamoeba polyphaga-like were isolated from natural water courses in Buri Ram Province, northeastern Thailand. The trophozoites were axenically cultured in PPYG medium and treated with artesunate in a concentration of 5-700 microg/ml. Artesunate showed its ability to inhibit the growth of acanthamoeba trophozoites: 54% at 50 mg/ml (after six days of exposure) and 93.2% at 100 microg/ml (after two days). The 500-700 microg/ml concentration caused inhibition on the first day of more than 93.2%; excystation did not occur in drug-treated medium. The present study shows that artesunate is amebastatic rather than amebicidal in an axenic culture of trophozoites at the highest concentration of 100 microg/ml. Metronidazole, in concentrations of 5-1,000 microg/ml, had no effects on either trophozoites or cysts.

Action of DL-alpha-difluoromethyl ornithine on Acanthamoeba culbertsoni.

The multiplication of A. culbertsoni in the peptone medium was not inhibited by 10-20 mM concentration of alpha-difluoromethyl ornithine (DMFO) while a partial and transient inhibition of cell multiplication was observed by 10-20 mM DFMO in proteose peptone, yeast extract, glucose (PYG) medium. Ornithine decarboxylase (ODC) activity in the cells and cell free extracts was strongly inhibited by DFMO, excluding enzyme refractoriness and impermeability of cells for DFMO as the possible causes of DFMO resistance. The presence of polyamines in the peptone and PYG media as well as uptake of polyamines by the amoebae has been demonstrated. The growth and multiplication of A. culbertsoni in chemically defined medium was not affected by 1-5 mM DFMO while 10-20 mM DMFO yielded partial inhibition. A lowering of diaminopropane levels and enhancement of spermidine levels was observed in DFMO inhibited cells and level of ODC was drastically reduced in the inhibited cultures. Uptake of polyamines from the growth media may partly account for DFMO resistance of A. culbertsoni. Alternative mechanisms for DFMO resistance are indicated.

Action of methylglyoxal bis (guanyl hydrazone) and related antiprotozoals on Acanthamoeba culbertsoni.

Methylglyoxal bis(guanyl hydrazone) (MGBG) and the related diamidine compounds berenil and pentamidine inhibited multiplication of A. culbertsoni. The growth inhibition by MGBG (2.5 mM) in the peptone medium was accompanied by the disappearance of spermidine and a marked reduction in the level of diaminopropane. MGBG and berenil completely inhibited growth in a chemically defined medium at 1 mM and 1-2 microM concentration, respectively. However, there was no decrease in the polyamine levels in the early stages of growth inhibition by these agents. Uptake of putrescine, spermidine and spermine by A. culbertsoni has been demonstrated but addition of exogenous polyamines did not reverse the growth inhibitory action of MGBG and berenil. Inhibition of S-adenosylmethionine decarboxylase and decrease in polyamine synthesis do not seem to be the primary targets for the antiamoebic action of MGBG and berenil.

Development of improved media for axenic cultivation of Acanthamoeba culbertsoni, Singh and Das 1970.

Several varieties of peptone supported growth of A. culbertsoni to different extents reaching a maximum cell density of 1-2 X 10(6)/ml. Proteose peptone and tryptone also yielded good growth when combined with thiamine and vitamin B12. A combination of proteose peptone with glucose, yeast extract and salts promoted excellent growth of A. culbertsoni with cell density reaching 1-2 X 10(7) cells/ml; tryptone and one of the indigenous peptones also yielded comparable growth when substituted for proteose peptone in this medium. Casamino acids also supported good growth of amoebae and requirement of yeast extract could be met by a combination of thiamine, vitamin B12 and biotin. Bacto peptone did not support good growth of this amoeba but supplementation of peptone with casamino acids or amino acid mixture improved the growth supporting capacity of the medium. Development of several media with or without glucose will aid in cultivation of A. culbertsoni, studies on its metabolism as well as screening of potential drugs.