DRUG RESISTANCE AND ACANTHAMOEBA KERATITIS: THE QUEST FOR ALTERNATIVE ANTIPROTOZOAL CHEMOTHERAPY JOHN HAyl, COLIN M. KIRKNESSI, DAVID V. SEAL! and PETER WRIGHT" Glasgow and London SUMMARY trate, dendritiform patterns and localised oedema, especially in a young person, as well as an association with Trophozoites and cysts of 20 isolates of Acanthamoeba from the cornea and fivefrom related samples were tested previous contact lens wear, should suggest the possibility in vitro for sensitivity to ten drugs (three aromatic dia­ of Acanthamoeha keratitis. This can be confirmed by iso­ midines, two aminoglycosides, two macrolides, a polyene lation and cultivation of the protozoan from corneal macrolide antibiotic, an organoarsenical and an anti­ scrapes or biopsy material.7 Once a definitive diagnosis metabolite) and two cationic antiseptics (chlorhexidine has been achieved, appropriate anti-acanthamoebal drug and polyhexamethylene biguanide, PHMB). Only chlor­ therapy will invariably be required. hexidine and PHMB showed uniform amoebacidal activ­ The literature attests to a wide variety of drugs provid­ ity. Aromatic diamidines (pentamidine isethionate, ing variable efficacy against different Acanthamoeha propamidine isethionate and diminazene aceturate) species or strains, both in riva and in vitro. In clinical prac­ generally proved effective against both forms of the tice, however, it is important to note that some of the amoeba; only pentamidine gave synergy with the bigua­ agents, for example some of the aromatic diamidines, may nide while propamidine gave an additive effect. Other merely inhibit replication or induce encystment of the drugs tested proved erratic or ineffective against differ­ tropozoite form,x rendering it quiescent as a cyst, and thus ent isolates. Chlorhexidine alone, or together with pro­ often resistant to conventional drug therapy. In such cir­ pamidine, was subsequently used in two patients with cumstances the cyst retains the potential to exacerbate the proven keratitis; the causative isolates Acanthamoeba disease on discontinuation of the drugs or, if infected tis­ were sensitive to the individual compounds and to the sue is retained, following corneal transplantation.9 combination The treatment provided resolution in vitro. In the United Kingdom, empirical combination therapy of the clinical disease; amoebae were shown to be non­ of propamidine, dibromopropamidine and neomycin has viable by histology and culture. The combination of proved efficacious in some patients. \0 Drug resistance, II as chlorhexidine and propamidine is recommended for well as allergic or toxic reactions after prolonged therapy treatment of proven Acanthamoeba keratitis. with propamidine,12 has limited the use of this combi­ Keratitis associated with Acanthamoeha infection is a nation, and has prevented its widespread acceptance in relatively rare, sight-threatening condition occurring most clinical practice. often in contact lens wearers,I where there has been Novel approaches to chemotherapy of Acanthamoeha inappropriate or inadequate disinfection of contact lens keratitis continue to be forthcoming. For example, a for­ systems." The clinical presentation of the disease is often mulation containing the cationic antiseptic polyhexameth­ mistakenly diagnosed as herpes simplex or fungal infec­ ylene biguanide at fairly low concentration, alone or in tion.3 This results in inappropriate anti-microbial agents combination with propamidine and/or neomycin, has being administered. Early features4-6 such as pain, photo­ proved very effective against both trophozoites and cysts phobia and recurrent epithelial breakdown with little infil- of Acanthamoeha derived from proven clinical cases of 1 1 From: 'Department of Bacteriology. The Royal Infirmary. Glasgow: the infection. 3. 4 Drug therapy, however, may be com­ 'Department of Ophthalmology. Tennent Institute. University of plicated by a number of factors, mostly associated with Glasgow. Glasgow. UK. failure to attend to the natural history and metabolism of Correspondence to: Professor C. M. Kirkness. FRCS (Edinburgh and Glasgow). FRCOphth. Department of Ophthalmology. Tennent the protozoan within the diseased cornea and also to the Institute. University of Glasgow GIl 6NT. UK. pharmacology of the selected drug(s) within this location. Eye (1994) 8, 555-563 © 1994 Royal College of Ophthalmologists 556 J. HAY ET AL. The purpose of this study was to suggest a more rational After several transfers, the amoebae were incubated in this approach to chemotherapy of Acanthamoeha keratitis medium for approximately 72 hours at either 25°C or based on in vitro drug sensitivity studies on cultures of 32 0c. Acanthamoeha isolated from patients and contact lens In order to determine the purity of Acanthameoha cul­ accoutrements (details of which have been published pre­ tures, Giemsa staining was performed. Viability of each viously). Representatives of selected drug classes and Acanthamoeha culture was assessed using a 0.2% trypan cationic antiseptics were used singly or, where considered blue. Cyst populations were obtained by incubating tro­ appropriate, in combination with each other, in order to phozoite cultures for about 7 days at 25°C or 32 0c. Puri­ determine whether a potent acanthamoebacidal action ty and viability were determined retrospectively. as could be identified ill vitro, where the mechanism of described above, on excysted cohorts of amoebae. Only action could be established, and the effect subsequently cultures with >98% purity and viability were used for exploited in vivo. Illustrative case reports are used to high­ drug sensitivity studies. light some important aspects. Drugs MATERIALS AND METHODS Aqueous solutions (100 Ilg/mi) of drug or disinfectant Acanthamoeba and Cultivation were prepared immediately prior to use and filter-steril­ Eighteen Acanthamoeha corneal isolates, including three ised using Gelman filters with 0.22 11m pore size. Com­ from the same patient (TB) taken at different time inter­ pounds used for assessment of amoebacidal action are vals, were initially investigated. From one of these (AT), shown in Table L These agents were selected mainly as a isolates from the soft contact lens and its storage case were consequence of literature reports of their efficacy, or that also included. From another (MT), an isolate from the of related drugs or antiseptics, against Acanthamoeha, contact lens storage case and one from the water supply at either in vitro or in vim. work (the home was negative) were included. On completion of testing these isolates, another patient Drug and Antiseptic Screening (AB) presented with Acanthan10eha keratitis. Isolates Drug and antiseptic screening was performed using a from a corneal biopsy and scrape as well as a soft contact series of sterile 96-well microtitre plates containing a stan­ lens were examined. This afforded the opportunity of dardised concentration of 2 x 10" organisms per 100 IIIof assessing the in vitro findings from the 18 samples in a medium per well. One hundred microlitres of doubling clinical setting. dilutions of each compound (100-0.8 Ilg/ml) were pro­ All amoebae were maintained by routine passage on to duced vertically for each of the 12 compounds tested. Lids 1.5% high clarity bacteriological agar No. I (LAB M) were secured, then the contents of plates mixed gently for made up in amoebal saline.IS It was spread with heat-killed 10 minutes on a plate rotator prior to incubation at either Klehsiella aerogenes and moistened intermittently with 25°C or 32 0c. amoebal saline prior to incubation in air at either 25°C, Sensitivity of isolates was assessed after 48 hours of 32°C or 35°C. incubation, by recording either the lowest concentration of In order to obtain sufficient numbers of each of the 25 drug or antiseptic which resulted in complete lysis or isolates for in vitro drug screening, the surface of each degeneration of trophozoites and non-viability of result­ plate was flooded with amoebal saline and agitated in ing cysts (minimum trophozoite amoebacidal concentra­ order to permit transfer of amoebae to sterile plastic tion, MTAC) or, for cysts, the lowest concentration of test 75 cm" tissue-culture flasks (Sterlin, CelCult) containing compound that resulted in no excystment and trophozoite approximately 50-100 cm3 of a definedgrowth medium.16 replication (minimum cysticidal concentration, MCC),13 Table I. Drugs and antiseptics used in this study, their classification and proposed antimicrobial mechanism of action Agent Class Inhihitor of Chlorhexidine digluconate' (chlor)* Cationic antiseptic Membrane function Polyhexamethylene biguanide'" (phmb) Cationic antiseptic Membrane function Propamidine isethionate 1 (propam) Aromatic diamidine DNA synthesis Pentamidine isethionate" (penta) Aromatic diamidine DNA synthesis Diminazine aceturateb.5 (dim) Aromatic diamidine DNA synthesis Neomycin sulphate" (nco) Aminoglycoside antibiotic Protein synthesis Paromomycin sulphate7 (paro) Aminoglycoside antibiotic Protein synthesis Amphotericin B' Polyene macrolide antibiotic Membrane (ergosterol) biosynthesis Dirithromycin'" (dir) Macrolide antibiotic Protein synthesis Spiramycinu.1o (spir) Macrolide antibiotic Protein synthesis Cymelarsanb.1I (cymel) Organoarsenical Energy metabolism a-Diftuoromethylomithinebl2 (dfmo) Antimetabolite Substrate-enzyme reaction (inhibitor of ornithine decarboxylase) GijiFam: 'Moorfields Eye Hospital; "Prof. F. W. Jennings; 'Lilly Research Centre Ltd; "Rhone-Poulenc Ltd. Supplied