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Development of a practical complete-kill assay to evaluate anti-Acanthamoeba drugs.

Abstract：

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 × 10(6)/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.

journal_name

JAMA Ophthalmol

journal_title

JAMA ophthalmology

authors

Kowalski RP,Abdel Aziz S,Romanowski EG,Shanks RM,Nau AC,Raju LV

doi

10.1001/jamaophthalmol.2013.5062

subject

Has Abstract

pub_date

2013-11-01 00:00:00

pages

1459-62

issue

eissn

2168-6165

issn

2168-6173

pii

1743098

journal_volume

131

pub_type

杂志文章

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Development of a practical complete-kill assay to evaluate anti-Acanthamoeba drugs.