In vitro efficacies of clinically available drugs against growth and viability of an Acanthamoeba castellanii keratitis isolate belonging to the T4 genotype - PubMed (original) (raw)

In vitro efficacies of clinically available drugs against growth and viability of an Acanthamoeba castellanii keratitis isolate belonging to the T4 genotype

Abdul Mannan Baig et al. Antimicrob Agents Chemother. 2013 Aug.

Abstract

The effects of clinically available drugs targeting muscarinic cholinergic, adrenergic, dopaminergic, and serotonergic receptors; intracellular calcium levels and/or the function of calcium-dependent biochemical pathways; ion channels; and cellular pumps were tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype. In vitro growth inhibition (amoebistatic) assays were performed by incubating A. castellanii with various concentrations of drugs in the growth medium for 48 h at 30°C. To determine amoebicidal effects, amoebae were incubated with drugs in phosphate-buffered saline for 24 h, and viability was determined using trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. Of the eight drugs tested, amlodipine, prochlorperazine, and loperamide showed potent amoebicidal effects, as no viable trophozoites were observed (>95% kill rate), while amiodarone, procyclidine, digoxin, and apomorphine exhibited up to 50% amoebicidal effects. In contrast, haloperidol did not affect viability, but all the drugs tested inhibited A. castellanii growth. Importantly, amlodipine, prochlorperazine, and loperamide showed compelling cysticidal effects. The cysticidal effects were irreversible, as cysts treated with the aforementioned drugs did not reemerge as viable amoebae upon inoculation in the growth medium. Except for apomorphine and haloperidol, all the tested drugs blocked trophozoite differentiation into cysts in encystation assays. Given the limited availability of effective drugs to treat amoebal infections, the clinically available drugs tested in this study represent potential agents for managing keratitis and granulomatous amoebic encephalitis caused by Acanthamoeba spp. and possibly against other meningoencephalitis-causing amoebae, such as Balamuthia mandrillaris and Naegleria fowleri.

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Figures

Fig 1

Fig 1

A. castellanii (2 × 105 cells) was incubated with various concentrations of drugs in PYG medium at 30°C for 48 h. After incubation, A. castellanii numbers were monitored by hemocytometer counting. A. castellanii incubated in PBS (nonnutrient) exhibited no growth, while A. castellanii incubated in PYG medium (growth medium) exhibited >8-fold increase in amoeba numbers from the original inoculum. All drugs tested showed significant inhibition of A. castellanii growth at the indicated concentrations (P < 0.01; paired t test; one-tail distribution). Among various drugs tested, prochlorperazine, haloperidol, and digoxin inhibited A. castellanii growth completely at 25 μg per ml. The results are representative of at least three independent experiments performed in duplicate. The data are presented as means and standard errors.

Fig 2

Fig 2

The amoebicidal effects of various drugs were determined using trypan blue staining. Briefly, A. castellanii (5 × 105 amoebae) was incubated with various drugs (500 μg per ml) at 37°C for 24 h. After this incubation, viability was determined by trypan blue staining, and amoebae were enumerated by hemocytometer counting. At 500 μg per ml, amlodipine, loperamide, and prochlorperazine showed more than 99% A. castellanii killing. The results are representative of at least three independent experiments performed in duplicate. The data are presented as means and standard errors.

Fig 3

Fig 3

Encystation assays were performed in the presence of various drugs. Briefly, 2 × 106 amoebae were incubated with various drugs (500 μg per ml) in the presence of 50 mM MgCl2 for 1 h. After this incubation, 10% glucose was added, and the amoebae were incubated at 30°C for 72 h. After the incubation, 0.5% SDS was added to solubilize trophozoites (cysts are resistant to SDS), and cysts were enumerated using a hemocytometer. At 500 μg per ml, all drugs tested except apomorphine and haloperidol exhibited more than 90% inhibition of A. castellanii encystation. The results are representative of at least three independent experiments performed in duplicate. The data are presented as means and standard errors.

Fig 4

Fig 4

Representative effects of various drugs against A. castellanii cysts. A. castellanii cysts were scraped from nonnutrient agar plates and incubated with various drugs (500 μg per ml) for 24 h. After this incubation, the cysts were washed three times with PBS and reinoculated in fresh PYG medium at 30°C for up to 48 h. When treated with 500 μg per ml of amlodipine, prochlorperazine, and loperamide, A. castellanii cysts did not reemerge as viable A. castellanii trophozoites. Viable A. castellanii trophozoites emerged in control wells, as well as with all other drugs. The results are representative of three independent experiments.

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