Effects of Drug Therapy Ontoxoplasma Cysts in an Animal
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Effects of Drug Therapy on Toxoplasma Cysts in an Animal Model of Acute and Chronic Disease Paula D. Gormley, Carlos E. Pavesio, Darwin Minnasian, and Susan Lightman PURPOSE. TO evaluate the effects of drug therapy on the clinical course of acute acquired Toxo- plasma retinochoroiditis and on the number of Toxoplasma cysts present in the brain and ocular tissues in the hamster animal model. METHODS. The Syrian golden hamster animal model of Toxoplasma retinochoroiditis was used. In acute disease, systemically administered atovaquone was compared with conventional therapies (pyrimethamine combined with sulfadiazine; clindamycin; and spiramycin). The clinical course of the ocular disease was determined with retinal examination and photography of the fundus. The number of Toxoplasma cysts remaining after treatment was evaluated in aliquots of brain homog- enate and in retinal tissue. The effect of atovaquone on cerebral Toxoplasma cyst count was also studied in chronic disease. RESULTS. None of the drugs administered altered the course of the acute disease, judged by clinical examination. Atovaqvione alone significantly reduced the number of cerebral Toxoplasma cysts after acute disease. Atovaquone also significantly reduced the cerebral Toxoplasma cyst count in chronic disease. CONCLUSIONS. Tissue cysts are believed to be responsible for reactivation of Toxoplasma retinocho- roiditis. Atovaquone has the potential to reduce the risk of recurrent disease. (Invest Ophthalmol Vis Set. 1998;39:1171-1175) cular toxbplasmosis is the most common cause of Hydroxynaphthoquinones were first developed (The Well- human retinochoroiditis1"3 and accounts for as much come Foundation, Beckenham, Kent, UK) as antimalarial drugs Oas 55% of all cases worldwide.4 Treatment of the when shortage of quinine during World War II prompted disease presents a problem to ophthalmologists, and there is search for alternative preparations. The early drugs were inef- much debate about the most effective treatment regimen.5 fective in human disease because of poor absorption and rapid Clinical experience has shown that the current treatment does metabolism, but continued research resulted in the synthesis of not seem to alter the natural progression of the disease,6 and atovaquone in the 1980s. The drug proved more active against there is no evidence that the available drugs are effective strains of Plasmodium falciparum in vitro and in vivo.12 against the tissue cysts that are believed to be responsible for Unlike the earlier hydroxynaphthoquinones, atovaquone did the disease's recurrence.7 Evaluation of these drugs in the not undergo rapid hepatic metabolism. The drug was subse- treatment of cysts in patients is complicated by ethical consid- quently shown to be active against the related parasite T. erations and retinal biopsy cannot be justified in this condition. gondii. The hydroxynaphthoquinones are potent inhibitors of The Syrian golden hamster has recently been described as the mitochondrial electron transport chain, competing with a reliable animal model of Toxoplasma retinochoroiditis.8 This the electron carrier ubiquinone13 at the ubiquinone-cyto- model is used in the present study to evaluate and compare the chrome c reductase region (complex III). effect of currently available drugs on the clinical course of acute acquired Toxoplasma retinochoroiditis and on the num- ber of Toxoplasma cysts remaining in the cerebral tissues after MATERIALS AND METHODS infection. Atovaquone, a new drug that is effective against the tachyzoite and the tissue cysts of Toxoplasma gondii in vivo Maintenance of Toxoplasma Stocks 9 11 and in vitro, " was evaluated in acute and chronic infections. The avirulent ME49 cyst-forming strain of T. gondii maintained Atovaquone is a hydroxynaphthoquinone, /rarcs-2[4-(4-chloro- in mouse brain was kindly supplied by Jack Remington, Palo phenyl)cyclohexyl]-3-hydroxy-l,4-naphthalenedione, with an Alto Medical Research Foundation, (Palo Alto, CA). For main- empiric formula C22H19O3C1. tenance of the stocks, a chronically infected mouse was killed, and its brain tissue was gently homogenized with 1 ml phos- phate-buffered saline, by mortar and pestle. A 0.2-ml aliquot of From the Institute of Ophthalmology and Moorfields Eye Hospital, this suspension was inoculated intraperitoneally onto each of London, United Kingdom. the adult BALB/c mice (weight, 22-24 mg), which were used Supported by Frost TFC Charitable Trust, Research into Eye Dis- ease Fund. for maintenance of the stocks. Submitted for publication May 9, 1997; revised November 7, 1997; accepted January 28, 1998. Experimental Animals Proprietary interest category: N. Reprint requests: Susan Lightman, Institute of Ophthalmology and Adult female outbred Syrian golden hamsters (B and K Univer- Moorfields Eye Hospital, City Road, London ECIV 2PD, UK. sal; The Field Station, Grimston, Aldborough, Hull, UK), each Investigative Ophthalmology & Visual Science, June 1998, Vol. 39, No. 7 Copyright © Association for Research in Vision and Ophthalmology 1171 Downloaded from iovs.arvojournals.org on 09/28/2021 1172 Gormley et al. IOVS, June 1998, Vol. 39, No. 7 weighing 100 g to 120 g, were used in the experiment. Animals activity. The solubility of atovaquone was improved with 50 were kept in single cages at an average room temperature of ml/1 chemophore and 50 ml/1 acetone (provided by V. An- 20°C in natural light. They were fed pellets (HMB; Special Diet drews, Pharmacy Department, Moorfields Eye Hospital, Lon- Services, Witham, UK) and drank tap water ad libitum from a don, UK) and the final concentration was 160 /i-g/ml, measured drinking bottle. All hamsters had normal findings on physical by immunoassay (performed by Martin French, Wellcome Re- and ophthalmic examination and were free of serum antibod- search Laboratory, Beckenham, UK) The average daily number ies to T. gondii at the beginning of the experiment. of milliliters of water drunk by each animal was measured and All procedures used were in accordance with the Animal the drug supply changed weekly. After a 4-week treatment (Scientific Procedures) Act of 1986 and conformed to the course, all hamsters received fresh drinking water (no drug) for ARVO Statement for the Use of Animals in Ophthalmic and an additional 2 weeks. The hamsters were then killed in a Vision Research. carbon dioxide chamber, and their brains and eyes were re- moved. The fresh brain was used immediately for cerebral cyst Antimicrobial Agents counts, and the eyes were fixed in 10% formaldehyde for The drugs used were pyrimethamine (Sigma, St. Louis, MO), histologic examination. sulfadiazine sodium (May and Baker, Dagenham, UK), clinda- mycin phosphate (Upjohn, Crawley, UK), vancomycin (Eli Treatment Protocol for Chronic Infection Lilly, Basingstoke, UK), spiramycin (kindly supplied by Celso Reis, Gerencia de Pesquisas Medicas, Rhodiafarma, Sao Paulo, The study group consisted of 16 hamsters that had chronic, Brazil), and atovaquone (The Wellcome Foundation). inactive Toxoplasma retinochoroiditis (diagnosed by retinal examination) 12 weeks after inoculation. Eight hamsters were Induction of Toxoplasmosis in the Hamster treated with atovaquone at the same concentration as in the Animal Model previous experiment. The treatment period was 4 weeks, in A suspension of the ME49 T. gondii strain was prepared from keeping with the usual treatment period in human disease. the infected stock mouse 6 weeks after the inoculation of the Eight untreated animals served as control subjects. After 4 mouse with the parasite. According to the protocols described weeks, all the animals were killed, and their brains and eyes previously,8 each hamster was inoculated intraperitoneally were removed. The fresh brain was used immediately for with 0.2 ml mouse brain homogenate on day 0 of the experi- cerebral cyst counts, and the eyes were fixed in 10% formal- ment. dehyde for histologic examination. Clinical Course Evaluation of the Effect of Treatment Retinal examination with an indirect ophthalmoscope and a Cerebral Cyst Counts. The brain of each hamster was 30-diopter lens was performed on all hamsters on days 0, 7, 10, cut along the sagittal plane, and one half was homogenized and 14 and weekly thereafter. Fundus photographs were taken in 1 ml phosphate-buffered saline, ground by mortar and with a fundus camera. Full pupillary dilation was achieved with pestle. A 0.025-ml aliquot of homogenate was placed on a one drop each of tropicamide 1% (Alcon Laboratories, Herts, microscope slide and mounted with a coverslip (24 mm X UK) and phenylephrine 2.5% (Smith and Nephew Medical, 24 mm). The number of Toxoplasma cysts were counted in Hull, UK) instilled 30 minutes before the examination. four O.O25-ml aliquots from each hamster by light micros- copy (magnification, X100). To calculate the total number Treatment Protocol for Acute Infection of cysts in the whole brain, the sum of the number of cysts At the first clinical signs of retinal disease, the hamsters were counted in the four aliquots was multiplied by 20. Cyst randomly assigned to one of five groups of 10 animals, and counts in the brain after the different treatment protocols treatment was administered in the drinking water according to were compared using Student's f-test and one-way analysis the following dosage regimens: group 1, untreated control; of variance (ANOVA). group 2, treated with 15 mg/1 pyrimethamine and 1000 mg/1 Retinal Cyst Count. Retinal cysts were counted in sulfadiazine; group 3, treated with 400 mg/1 clindamycin; chronic infection only. Both eyes from each hamster were group 4, treated with 2000 mg/1 spiramycin; and group 5, fixed in 10% formal saline and embedded in paraffin. Thick treated with 476 mg/1 atovaquone. sections (0.3 ju,m) were cut from three levels. One section from The drug dose was calculated from the human treatment each level was stained with hematoxylin and eosin and exam- regimen correlating to milligrams per kilogram of body weight ined for Toxoplasma cysts by light microscopy (magnification, in the hamsters. The daily human treatment regimens were as follows: 50 nig pyrimethamine, 4 g sulfadiazine, 1200 mg X100).