E X P E R I M E N T A L S T U D I E S R E L A T I N G T O O C U L A R I N F E C T I O N I N T O X O P L A S M O S 1 S

by

LEON JACOBS

( Bethesda)

With 1 Figure and IV Tables

The occurrence of chorioretinitis in association with, or following soon after an attack of acquired systemic toxoplasmosis has been re- corded only twice. The first such case was reported by WISlN6 (1952) and the second instance was described by KAYHOE et al. (1958). The diagnosis in the first case rested on serological tests; in the second case it was confirmed by isolation of Toxoplasma from lymph node and muscle. Furthermore, in the second case the ocular lesion was controlled by pyrimethamine and sulfadiazine.

In addition to these cases, there have been at least 5 isolations of Toxoplasma from diseased eyes of juveniles and adults. JACOBS, FAIR,

& BICKERTON (1954) isolated and identified the parasite f rom the eye of a 30-year-old man which was enucleated because of pain and blindness

due to chorioretinitis of over 8 years' duration. HABEGGER (1954) isolated Toxoplasma f rom subretinal fluid of a 45-year-old woman. PJrr_Ar & TnALHAMMER (1957) succeeded in demonstrating the parasite in mice

inoculated with tissues f rom an eye enucleated due to iridocyclitis of 10 months ' duration. HOGAN et al. (1958) isolated the organism from the eye of a 20-year-old man who had suffered chorioretinitis since birth or early childhood. More recently, Dr. J. K. FmZNKEL has isolated Toxoplas- ma from an adult eye.

Except for the cases of PILl_aT & TI-IALHAMMER (1957) and HABE~ER, (1954) these demonstrations of the parasite were made from chronic cases of chorioretinitis. This is not surprising, since the enucleation of an eye is indicated only when the inflammation proceeds to painful sequelae, a process that usually requires considerable time after the appearance of the initial lesion. The case reported by HOaAN et al. (1958) is very likely one of congenital origin. In view of the fact that chorioretinitis has been associated with the acute acquired disease in the two cases cited above, there is no reason to presume that the other chronic cases from which ToxopIasma was isolated were also congenital. The serological and skin

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test data indicate, also, that the case of PILLAT & THALHAMMER (1957) was one of recently acquired toxoplasmosis without systemic: signs or

symptoms. Statistical studies, which have been summarized by FRENKEL & JACOBS

(1958), indicate a definite correlation between Toxoplasma antibodies, or skin-sensitivity, and chorioretinitis. In all, the bulk of the evidence acquired within the last 5 or 6 years presents a substantial case incrimi- nating Toxoplasma as the cause of about 30-35 9o of cases of chorioreti- nitis, or posterior uveitis, in the United States.

One difference between acquired ocular toxoplasmosis and the in- volvement of the eyes in the congenital disease is that in the former a single eye is involved more frequently than both, while the congenital disease most often shows bilateral involvement (FELDMAN, 1956). Another difference is the frequent occurrence of the acquired ocular disease with- out systemic manifestations, which happens only rarely in congenital cases. Ocular toxoplasmosis appears to be, frequently, a manifestation of the chronic stage of the infection, with low and stable serological test titers. The discrepancies can possibly be explained on the basis of newer knowl- edge of the behavior of some strains of Toxoplasma. It is the purpose of this paper to present some data from my own Laboratory and to review reports from others, that bear on these points, and that aid in understanding the chronic ocular disease. Furthermore, I wish to present some data on the chemotherapy of experimental ocular toxoplasmosis.

Parasitcmia in chronic toxoplasmosis

JACOBS & JONES (1950), RUCHMAN & FOWLER (1951) and others have described the parasitemia occurring in mice and other animals infected with virulent strains of Toxoplasma. JACOBS et al. (1953) also described the parasitemia occurring in animals infected with an avirulent strain. In either case, the usual pattern has been the development and increase in parasitemia during the acute stage of the infection; and diminution of the numbers of parasites in the blood, and their eventual disappearance, following the development of antibodies. Only rarely have parasites been reported in the blood of infected animals long after the acute infection (e.g. HELLBRi.iGGE, (1953)).

EICHENWALD (1952) induced the occurrence of parasitemia in chroni- cally infected mice by injecting them with anti-Toxoplasma sera of high titer obtained from mice, guinea pigs, or rabbits. In our Laboratory, within the last two years, we have attempted to duplicate his results

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with another strain of Toxoplasma, our 113-CE, that produces chronic infections in mice. The results of this study will be reported in detail by REMINGTON et al. (to be published). Suffice it to say that we did succeed in inducing parasitemia in a small number of mice chronically infected with strain 113-CE. However, we also used another strain of Toxoplasma which was originally isolated from a rabbit by Dr. J. K. BEVERLEY of Sheffield, England, and this gave results of an entirely different character.

Mice with chronic infections with BEVERLEY strain Toxoplasma, when injected with hyperimmune serum, manifested parasitemia much more frequently than did those with 113-CE infections. However, when control mice, which received normal serum injections or no injections, were tested, they were found to have parasitemias just as frequently (Table I).

T A B L E I Occurrence of parasitemia in chronically infected mice

Strain of Toxoplasma

Serum injected

Immune Normal

Immune Normal

113-CE

Beverley

No. mice positive No. tested

4/22 0/6 0/20

% positive

18

38/49 64 7/11 64

12/24 50

In extended tests with the BEVERLEY strain, parasitemias were found as late as 9 months after original infection of mice.

Mice infected for 8 months with a strain of Toxoplasma of porcine origin also showed occasional spontaneous parasitemias.

Rabbits sometimes showed parasitemias 4 and 6 months after inocu- lation with BEVERLEY parasites. Guinea pigs, too, had occasional late parasitemias when infected with this strain of Toxoplasma. Rats, on the other hand, never showed delayed parasitemias with the BEVERLEY strain nor with 6 other Toxoplasma strains of various origin.

The parasitemias were tested by inoculating fresh mice with blood withdrawn from the infected animals. In all positive cases, only a few mice of those that received the blood became infected. The parasitemias in chronically infected animals must, therefore, be of a low order, with only a few organisms in the circulating blood. Because of the presence of high antibody levels, the circulating organisms must be protected within an infected leucocyte or wandering cell.

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Without attempting a discussion of the mechanisms involved in the production of induced or spontaneous parasitemias, it is believed that the demonstration of their occurrence offers an explanation for the ap- pearance of uveitis without any evidence of systemic infection due to Toxoplasma. Wandering ceils containing parasites could arrive in the eye and the parasites could be liberated into tissues with low antibody levels, thereby allowing invasion of susceptible cells. Thus, infection of the eye need not necessarily occur only during acute toxoplasmosis. The low number of parasites in the circulating blood would explain the occurrence of unilateral ocular involvement in the adult disease. In contrast, the infant congenitally infected in utero has both eyes involved probably be- cause of a much higher parasitemia, furnishing a greater opportunity for Toxoplasma to reach the ocular tissues. It is likely that strains of Toxo- plasma vary considerably in their ability to continue proliferation during the chronic stage, as is evidenced by the differences between the 113-CE and the BEVERLEY strains in mice.

The above discussion is, of course, subject to the qualification that, in human beings as well as animals, parasitemias may occur late in the course of the infection. At least one such instance has been recorded, by PRIOR et al. (1953). These authors isolated Toxoplasma twice from the blood of a woman with a low and stable dye test titer and no signs or symptoms of disease.

Chemotherapeutic studies

JACOBS, MELTON, & COOK (1956) reported, in abstract form, the pro- duction of anterior uveitis in rabbits by injection of an avirulent strain of Toxoplasma into the anterior chamber, and the effects of drugs on such lesions. The regimen most effective in partially controlling the uveitis was pyrimethamine at 20 mg/kg combined with sulfadiazine at 40 mg/kg. Pyrimethamine or sulfadiazine alone at dosages of 40 mg/kg were not as effective (Table II). Parasites could be isolated from the eyes of control rabbits, or those treated with the drugs singly, but not from those that received the combination therapy (Table III). In later studies, it was found that pyrimethamine alone at 100 mg/kg was as effective as the combination of pyrimethamine and sulfadiazine at 20 mg and 40 mg/ kg in controlling experimental anterior uveitis in the rabbit (Table IV). We have also confirmed the report of BEVERLEY, BEATTIE 8Z FRY (1954) who used a similar system, on the effectiveness of diaminodiphenyl sulfone at 100 mg/kg. In general, these data have contributed some ex-

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T A B L E I I

Summary of effect of treatment of experimental anterior uveitis in rabbits

Treatment No. of Total possible Total of grades rabbits grade of of uveitis

uveitis* Day 4 Day 7

O.D. O.S. O.D. O.S.

Pyrimethamine 40 mg/kg Sulfadiazine 40 mg/kg Pyrimethamine

20 mg/kg 3- Sulfadiazine 40 mg/kg

32 32

32

11 2 17 3

5 1

14 4 24 8

7 0

Inocula were: O.D. 5000 parasites of strain l l3 -CE O.S. 500

* Uveitis was graded from 1 3- to 43- depending on the degree ofhyperemia and edema of the iris, the amount of exudate, and the extent of perilimbal injection.

TABLE III Demonstration of parasites in rabbits under treatment for experimental

anterior uveitis

Treatment Rabbit n o .

Day tested

Grade of uveitis

Results of inoculation of eye tissues into mice

Pyrimethamine 10 mg/kg 517 14 1 3- 3- 521 15 43- 3-

Sulfadiazine 20 mg/kg 524 15 4 3- 3-

Pyrimethamine 20 mg/kg3- ~ 520 14 0 - - Sulfadiazine 40 mg/kg ~ 530 15 1 3- - -

None I 518 14 43- 3-

I 523 15 43- 3-

T A B L E I V

Effects of treatment with pyrimethamine or Diamino-diphenyl sulfone at 100 mg/kg on experimental anterior uveitis of rabbits

Treatment No. of Total grades No. of rabbits with rabbits of uveitis uveitis graded

0 13- 23- 33- 4 +

Pyrimethamine DDS None

5 2

16

1 2 1 2 2

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perimental basis to various reports of successful clinical use of drugs in human toxoplasmic uveitis.

More recently, however, HOGAN et al. (1958) have published studies on the treatment of posterior uveitis in guinea pigs, which may cast some doubt on the value of pyrimethamine in ocular toxoplasmosis. HOGAN used dosages comparable to those used in the treatment of human beings (0.8 mg pyrimethamine/kg body weight daily), and found them ineffec- tive. We believe that the reason for this is the very rapid elimination of pyrimethamine by rodents, as compared with human beings. Bio-assays of the serum of rabbits at various periods following the oral administra- tion of a single dose of 100 mg pyrimethamine per kg body weight have yielded curves such as that in Fig. 1. It can be seen that even 5 hours after

3 . 0

~r sa

sart~'a

2.5

2.0

1.5

loO

o.~

bouts 3 5

Fig. 1

Pyrimethamine concentrations in rabbits after a single oral doses of 100 mg/kg

the dose, the blood level of pyrimethamine is relatively low. At 24 hours, the drug is no longer detectable. These data explain not only the need for high drug dosages in rabbits to control ocular toxoplasmosis, but also the ability of these animals to survive amounts of the drug that would be ahrlost certainly lethal to monkeys and man (ScHMrDT et al. (1953). A loading dose of 100 nag pyrimethamine twice on the first day of treatment, followed by 50 mg b.i.d, thereafter produced a serum drug level of 2.5 gamma per ml in human beings (KAUFMAN & CAr.DWELL, 1959). Since Cook & JACOBS (1958) found pyrimethamine effective in tissue culture

335

at a level of 0.25 ~,/ml for 4 days, it appears that effective drug levels can be obtained in the treatment of ocular toxoplasmosis. In additional tests of pyrimethamine in posterior uveitis in guinea pigs, dosages of 50 mg pyrimethamine and 200 mg sulfadiazine per kg body weight have success- fully eliminated Toxoplasma from the eye. The data on rabbits and guinea pigs are to be published in detail by JACOBS, KAUFMAN, & MELTON. Studies by CHOI (1958) on the penetration of pyrimethamine into the rabbit eye following the intravenous injection of small doses do not conflict with our observations. On the whole, the experimental data thus far available indicate that levels of the drug attainable in human beings by administration of small doses can penetrate the inflamed eye and can destroy proliferating Toxoplasma. BE~c~RLEY (1959) has also reported that this drug, given over a long period, results in a reduction in number of cysts of Toxoplasma in chronically infected mice. Thus, there is some indication that it may have usefulness in chronic recurrent uveitis, due to occasional rupture of encysted parasites, as well as in the progressive form of the disease characterized by continually smoldering infection and presumably due to continued activity of proliferating parasites.

Summary

Only a few cases of toxoplasmosis have been reported in which ocular lesions have appeared during the acute stage of systemic disease. However, statistical studies indicate a definite correlation between uveitis and sero- logical evidence of past exposure to Toxoplasma. Recent laboratory studies have revealed that, with some strains of the Toxoplasma, persistent activity and late parasitemias are found. Thus an explanation is possible for the appearance of uveitis in patients who show no clinical evidence of acute infection and whose dye test titers are relatively low and stable.

An account will be presented of the frequency of parasitemias in chro- nic infections in experimental animals. Also, a review will be given of chemotherapeutic studies of experimental ocular toxoplasmosis in rabbits and guinea pigs.

R6sum6

La litt~rature ne rapporte que de rares cas de toxoplasmose ofa les 16sions oculaires apparaissent au stade aiguE de la maladie g6n6rale. Cependent les travaux statistiques d6montrent clairement l'existence de relations entre l'uv6ite et des infections toxoplasmiques ant6rieures, dont la preuve fut apport6e par l 'examen s6rologique. De r6centes ex-

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p6riences de laboratoire ont d6cel6 que certaines souches de toxoplasmes se conservent longtemps apr6s la p6riode de virulence. C'est ainsi qu'on peut s'expliquer les uv6ites chez des personnes qui ne montrent aucun sympt6me clinique d'une infection aigu6 et dont les titres du dye-test sont relativement faibles et stables. L'auteur discute la fr6quence de parasit6mie chez les animaux de laboratoire affect6s d'une maladie chronique et donne une vue g~n6rale sur les r6sultats du traitement chimioth6rapeutique de la toxoplasmose oculaire du lapin et du cobaye.

Zusammenfassung

Nur wenige F~ille yon Toxoplasmose sind berichtet worden, bei denen Augensch/idigungen w~ihrend des akuten Stadiums der Allgemeinkrank- heit auftraten. Doch zeigen statistische Arbeiten eine sichere Korrelation zwischen Uveitis und dem serologischen Nachweis friiherer Toxoplas- moseinfektion. Neuere Laboratoriumsuntersuchungen haben gezeigt, dab bei manchen St~imrnen von Toxoplasma die Aktivit~it lange bestehen bleibt und sprite Parasit/imien gefunden werden. So kann das Auftreten von Uveitis bei solchen Patienten erkl~irt werden, die keine klinischen Zeichen akuter Infektion aufweisen und bei denen die Farbtest-Titer verh/iltnism~iBig niedrig und stabil sind.

Es wird fiber die H/iufigkeit der Parasit~imien bei chronischen Effekten yon Versuchstieren berichtet und auBerdem eine Ubersicht fiber die che- motherapeutischen Studien der experimentellen Augentoxoplasmose von Kaninchen und Meerschweinchen gegeben.

REFERENCES

BEWRLEY, J. K. A. (1958) A Rational Approach to the Treatment of Toxoplasmic Uveitis. Trans. Amer. ophthal. Soc. 78, 109-121.

- - , BEATTIE, C. P. & FaY, B. A. (1954) Experimental Toxoplasmosis of the Uveal Tract. Brit. J. Ophthal. 38, 489-496.

CHOr, C. S. (1958) Penetration of Pyrimethamine (Daraprim) Into Ocular Tissues of Rabbits. Arch. OphthaL 60, 603-611.

CooK, M. K. & JACOBS, L. (1958) In Vitro Investigations on the Action of Pyrimetha- mine Against Toxoplasma gondii. J. Parasit. 44, 280-288.

ETCHENWALD, H. (1952) New Phenomenon: Production of Recurrent Parasitemia in Chronic Toxoplasmosis by the Injection of Potent Specific Antisera and its Inhibi- tion by Corticotropin (ACTH) and Cortisone. Amer. Y. Dis. Child. 83, 73-75.

FRENr, ZL, J. K. & JACOBS, L. (1958) Ocular Toxoplasmosis. Pathogenesis, Diagnosis and Treatment. Arch. OphthaL 59, 260-279.

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HABEGGER, H. (1954) Toxoplasmose Humaine: Mise en 6vidence des parasites dans les milieux intra-oculaires (humeur aqueuse, exsudat r6troretinien). Arch. Ophtal. 14, 470-488.

HELLBROGGE, T. F., DAHME, E. & HELLBROGGE, F. K. (1953) Tierexperimentelle Beob- achtungen zur diaplazentaren Infektion der Toxoplasmen. Z. Tropenmed. Parasit. 4: 312-322.

HOGAN, M. J., ZWEIGART, V. A. t~ LEWIS, A. (1958a) Experimental Ocular Toxoplas- mosis II. Arch. OphthaL 60, 448-449.

- - , (1958b) Recovery of Toxoplasma from a Human Eye. Arch. Ophthal. 60, 548-554. JACOBS, L. & JONES, F. E. (1950) The Parasitemia in Experimental Toxoplasmosis.

J. infect. Dis. 87, 78-89. - - , FAIR, J. R. & BICKERTON, J. H. (1954) Adult Ocular Toxoplasmosis. Report of a

Parasitologically Proved Case. Arch. OphthaL 52, 63-71. - - , MELTON, M. L. & COOK, M. K. (1953) Experimental Toxoplasmosis in Pigeons.

Exp. Parasit. 2, 403-416. - - , (1956) The Production and Treatment of Acute Toxoplasmic Uveitis in the An-

terior Segment of the Rabbit Eye. Amer. J. trop. Med. 5, 375. KAUFMAN, H. E. & CALDWELL, L. A. (1959) Pharmacological Studies of Pyrimetha-

mine (Daraprim) in Man. Arch. Ophthal. 61,885-890. KAYHOE, D. E., JACOBS, L., ~BEYE, H. K. 8,; McCUELOUGH, N. B. (1957) Acquired Toxo-

plasmosis : Observations on 2 Parasitologically Proved cases Treated with Pyrimetha- mine and Triple Sulfonamides. New Eng. J. Med. 257, 1247-1254.

PILLAT, A. (~ THALHAMMER, O. 0957) Herdf6rmige Iridocyclitis als (einzige) Manife- station einer erworbenen Toxoplasmose, ~itiologisch gesichert durch Titerkurve und Tierversuch. v. Graefes Arch. OphthaL 158, 403-415.

PRIOR, J. A., COLE, C. R , DOCTON, F. L., SASLAW, S. t~ CHAMBERL.MN, D. M. (1953) Toxoplasmosis IV. Report of Three Cases With Particular Reference to Asympto- matic Toxoplasma Parasitemia in a Young Woman. Arch. intern. Med. 92, 314-320.

ROCnMAN, I. & FOWLER, J. C. (1951) Localization and Persistence of Toxoplasma in Tissues of Experimentally Infected White Rats. Proc. Soc. exp. BioL 76, 793-796.

WISING, P. J. (1952) Lymphadenopathy and Chorioretinitis in Acute Adult Toxoplas- mosis. Nord. Med. 47, 563-565.

U.S. Department of Health, Education and Welfare. National Institute of Allergy and Infectious Diseases. Laboratory of Parasitic Diseases.

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