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Original Investigation | Laboratory Sciences

Development of a Practical Complete-Kill Assay to Evaluate Anti-Acanthamoeba Drugs

Regis P. Kowalski, MS, M(ASCP)1; Salwa Abdel Aziz, MD1; Eric G. Romanowski, MS1; Robert M. Q. Shanks, PhD1; Amy C. Nau, DO1; Leela V. Raju, MD1
[+] Author Affiliations
1The Charles T. Campbell Ophthalmic Microbiology Laboratory, UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, the Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
JAMA Ophthalmol. 2013;131(11):1459-1462. doi:10.1001/jamaophthalmol.2013.5062.
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Importance  Acanthamoeba keratitis is a debilitating eye disease that requires effective topical drug therapy. Currently, there is no standard in vitro test to evaluate anti-Acanthamoeba drugs.

Objective  To develop a practical in vitro complete-kill assay to assess anti-Acanthamoeba drugs.

Design and Setting  Isolates of Acanthamoeba strains (n = 15) evaluated in a clinical laboratory. An in vitro laboratory assay was created to determine whether polyhexamethylene biguanide, 0.02%, chlorhexidine digluconate, 0.02%, hexamidine diisethioonate, 0.1%, and voriconazole, 1.0%, were effective in completely killing 15 different isolates of Acanthamoeba at time points of 24, 48, and 72 hours in comparison with a saline control. Each 0.5-mL volume of drug was inoculated with 0.1 mL of Acanthamoeba cysts (range, 1-3 × 106/mL) (determined with a hemacytometer) and allowed to incubate at 30°C. At the time points listed, 0.05 mL from each treatment group was inoculated onto nonnutrient agar overlaid with Enterobacter aerogenes. The plates were microscopically examined for growth 1 and 2 weeks after inoculation. At 2 weeks, all plates were subcultured onto a fresh medium. At another 7 days, the growth in subculture at each time point was graded “1” for growth and “0” for no growth.

Main Outcomes and Measures  The cumulative grades of 3 time points (range, 0-3) for each drug and isolate were nonparametrically compared to determine differences in growth between the drugs. The “kill” incidence rates over the 3 time points were also compared.

Results  In vitro testing determined that antiacanthamoebal efficacy (determined by the median growth grade and the kill incidence rate) was more prominent for hexamidine diisethioonate (median growth grade, 0.0; kill incidence rate, 93% [14 of 15 isolates]) and polyhexamethylene biguanide (median growth grade, 0.0; kill incidence rate, 80% [12 of 15 isolates]) than for chlorhexidine digluconate (median growth grade, 1.0; kill incidence rate, 40% [6 of 15 isolates]), voriconazole (median growth grade, 2.0; kill incidence rate, 13% [2 of 15 isolates]), and saline (median growth grade, 3.0; kill incidence rate, 0% [0 of 15 isolates]).

Conclusions and Relevance  The complete-kill assay appears to provide separation in the effectiveness of different antiamoebic drug solutions. This assay may be helpful for guiding topical Acanthamoeba therapy and providing a practical method to evaluate and screen new anti-infectives in the treatment of Acanthamoeba keratitis.

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