A keratitis rat model for evaluation of anti-Acanthamoeba polyphaga agents.
OBJECTIVE
To develop a rat model of chronic Acanthamoeba polyphaga keratitis suitable for pharmacologic assessment of therapeutic agents.


METHODS
An A. polyphaga isolate (ATCC #50495) was grown in peptone-yeast extract-glucose medium. Five-weeks-old, Sprague-Dawley male rats were injected with 10(3) or 10(4) trophozoites in the left cornea stromal layer. A subconjunctival injection of 0.14, 0.28, or 0.57 mg long-acting betamethasone was performed weekly. At the end of experiments, rats were killed; the superficial corneal epithelium gently scraped and cultured; and globes histologically examined. Topical polyhexamethylene biguanide (PHMB), hexamidine diisethionate, and miltefosine (hexadecylphosphocholine) were administered topically as eye drops 3 times a day at concentrations of 0.02%, 0.1%, and 0.01% respectively. In vitro minimal inhibitory concentration (MIC) and fractional inhibitory concentration values were measured in A. polyphaga cultures.


RESULTS
In infected eyes, lesions consisted of the sequential appearance within 2 weeks of edema, infiltrates, and/or abscesses. On day 35 postinfection, a combination of 10(4) parasites with a regimen of 0.28 mg/week betamethasone resulted in the highest ratio of rats with abscesses. Presence of A. polyphaga was confirmed histologically and inconsistently in cultures. In rats optimally prepared as said earlier, agents were administered on day 6 postinfection. A combination of PHMB and hexamidine diisethionate exerted a synergistic effect and was more effective than PHMB, hexamidine diisethionate, or miltefosine alone. In vitro, PHMB (MIC = 14.6 microM) and hexamidine diisethionate (MIC = 555 microM) exerted a synergistic effect (fractional inhibitory concentration = 0.06), and miltefosine exhibited antiamoebal activity (MIC = 27.4 microM).


CONCLUSIONS
In this study, a rat model of chronic A. polyphaga keratitis was obtained and found suitable for assessment of pharmacologic agents. It provides an in vivo approach of drug resistance, pathogenicity, and physiopathologic mechanisms of chronic amoebic keratitis.
Vasseneix
Caroline
C
Laboratoire de Parasitologie, ADEN EA-3234, CHU Charles Nicolle, Rouen, France.
Gargala
Gilles
G
François
Arnaud
A
Hellot
Marie France
MF
Duclos
Célia
C
Muraine
Marc
M
Benichou
Jacques
J
Ballet
Jean Jacques
JJ
Brasseur
Gérard
G
Favennec
Loïc
L
D000048:Acanthamoeba / Q000187:drug effects / Q000302:isolation & purification; D015823:Acanthamoeba Keratitis / Q000188:drug therapy* / Q000469:parasitology / Q000473:pathology; D000287:Administration, Topical; D000818:Animals; D000981:Antiprotozoal Agents / Q000627:therapeutic use*; D001550:Benzamidines / Q000627:therapeutic use; D001623:Betamethasone / Q000627:therapeutic use*; D001645:Biguanides / Q000627:therapeutic use; D002908:Chronic Disease; D003319:Corneal Stroma / Q000187:drug effects / Q000469:parasitology / Q000473:pathology; D004195:Disease Models, Animal; D004341:Drug Evaluation / Q000379:methods; D004359:Drug Therapy, Combination; D005938:Glucocorticoids / Q000627:therapeutic use*; D008297:Male; D021261:Parasitic Sensitivity Tests; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D013047:Specific Pathogen-Free Organisms
D000981:Antiprotozoal Agents; D001550:Benzamidines; D001645:Biguanides; D005938:Glucocorticoids; D010767:Phosphorylcholine; C031233:polihexanide; C018961:hexamidine; C039128:miltefosine; D001623:Betamethasone