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1 Antibody tests for ocular toxoplasmosis Emily D. Maloney and Herbert E. Kaufman* The recently modified fluorescence test for antibodies to toxoplasmosis is safe and relatively simple to perform. It appears to detect antibodies early in the course of acute systemic infection but is less sensitive than either the dye test or hemagglutination test in detecting chronic infection. Although hemagglutinating antibodies develop later than dye test antibodies in the course of acute systemic toxoplasmosis, the hemagglutination test correlates very well in ivith serum from patients toith subacute or chronic infection, such as that seen in ocular disease. Since most of the proved cases of ocular toxoplasmosis have low dye s that might well be undetected in the less sensitive FI test, this test appears to be inadequate for the study of ocular toxoplasmosis. Since ocular toxoplasmosis appears to be responsible for 0 to 80 per cent of posterior chorioretinitis, its diagnosis is of extreme importance in ophthalmology. In addition to the clinical appearance of the diseased eye, serologic testing is the prime diagnostic aid. The work to be reported below is a comparison of some present serologic testing procedures. It will be apparent from this study that the diagnosis of ocular toxoplasmosis demands special consideration and imposes unique requirements for an adequate test. The most widely used test for detecting antibodies to Toxoplasma is the methylene blue dye test of Sabin and Feldman.- This test, however, requires the use of live and potentially dangerous organisms, which, after staining, must be examined micro- From the Uveitis Laboratory, Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Mass. This work was supported by Research Grant B-06 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service, Bethesda, Md. "Present address, J. Hillis Miller Health Center, University of Florida, Gainesville, Fla. 6 scopically and counted to determine the proportion of organisms in each serum dilution which is unstained. A hemagglutination test (HA) which uses a dead antigen has been developed by Jacobs and Lunde ; however, it requires erythrocytes that have recently been treated with tannic acid and sensitized. This test has been modified by the use of formaldehyde-treated erythrocytes, ' fi permitting storage of the cells after tanning or sensitization and facilitating the preparation of the reagent. The HA test is less difficult to perform than the dye test and is reported to give comparable s. > i Goldman 0 has developed a test of fluorescence (FI) for Toxoplasma antibodies which, like the hemagglutination test, uses a killed antigen that can be stored for at least one month. In the FI test Toxoplasma antibody conjugated with fluorescein is diluted until it barely causes staining of formaldehydekilled organisms. If serum containing unlabeled antibody is added to the fluorescent antibody a competition for antigenic sites inhibits the specific staining. It is possible to make serial dilutions of serum until the dilution is found which barely inhibits the Downloaded From: on 07/8/08

2 Volume Number Antibody tests for ocular toxoplasmosis 7 staining, and this is the. Although this test requires the use of ultraviolet microscopy and may sound difficult, it is, in fact, simple to perform, and s are easily determined. Because the problem of testing for toxoplasmosis of patients with uveitis is so difficult and this test is so reproducible and relatively simple, an evaluation of its utility seemed desirable. In order to study the various methods, sera were subjected to various combinations of these tests. Methods The methylene blue dye test is performed as described by Frenkel and Jacobs. 7 This method is more sensitive than that employed by some laboratories, 8 but is the one used in the testing of most proved cases of ocular toxoplasmosis in adults. The hemagglutination test was done as described previously. ' The fluorescence test was carried out with the recent modifications suggested by Goldman and was done exactly as he described. 0 Conjugated immune rabbit serum from which nonspecific fluorescence had been absorbed through a diethylaminoethyl alcohol column was employed as the testing reagent. Results One hundred and seventy-eight sera were tested by the dye and FI tests and in most cases the fluorescence s were lower than the dye test s (Table I). Sixty-seven per cent of the sera with positive dye s at 6 were negative in the FI test. Forty-eight per cent with dye s of, and 9 per cent with s of were also negative by the FI test. The FI test, therefore, not only was less sensitive than the dye test, but antibody present in low could escape detection. FI tests of 6 sera that had been tested previously by hemagglutination with formaldehyde-treated erythrocytes and the dye test were performed (Tables II and III). The FI was generally lower than either the HA or the dye test although the HA and the dye s were generally the same or within one fourfold dilution. Two sera with an HA of were negative with the FI test, with an HA of were negative to Table I. Relationship between dye and fluorescence s 6,0, Table II. Relationship between hemagglutination and fluorescence s 6,0, ' 7 9 Table III. Relationship between dye and hemagglutination s 6,0, ,0,096 FI testing, and of with an HA of was negative by the FI test, indicating that low antibody concentrations as measured by the dye test would be detected more reliably by the HA test than the FI test. The sera considered above (in which low DT s were missed by FI test) were submitted to the Uveitis Laboratory of the Massachusetts Eye and Ear Infirmary and were, in the main, from patients suspected of having ocular toxoplasmosis. The bulk of the patients with antibodies were, therefore, in the chronic phase of infection. Downloaded From: on 07/8/08

8 Maloney and Kaufman Investigative Ophthalmology August 96 Although ocular disease appears to be a manifestation of chronic infection and may well escape detection with some frequency by the FI

3 8 Maloney and Kaufman Investigative Ophthalmology August 96 Although ocular disease appears to be a manifestation of chronic infection and may well escape detection with some frequency by the FI test, data on acute systemic infection suggests that FI testing may be more useful in this type of disease. Goldman and associates 0 reported acute cases in which the FI test became positive earlier although the dye test remained low. Studies of additional acute cases with a mononucleosis-like syndrome confirm this (Table IV). In order to determine the suitability of Table IV. Comparison of dye test, fluorescence, and hemagglutination s in acute toxoplasmosis Patient H. M.,0,0 C. C. 6 J. G Table V. Comparison of dye test and fluorescence s on cases of parasitologically proved ocular toxoplasmosis Patient M. F. W. G. V.I. 0. C. 0. C. 0. W. 0. W. Date sample drawn ,0,0,0 "Obtained from Dr. M. Hogan. tserum drawn months after enucleation. tpositive only when tested as undiluted serum. {Obtained from Drs. R. Wood and L. Jacobs. '! Positive t Positive t the fluorescence test in detecting ocular disease, sera from cases of parasitologically proved ocular toxoplasmosis were obtained. One serum with a dye test of,0 was negative to fluorescence testing. The remainder were positive only when tested as undiluted serum (Table V). Discussion In order to facilitate the study of ocular toxoplasmosis some alternative to the dangerous and laborious dye test is required. The hemagglutination test and its modifications provide a measurement of antibody s which correlates exceedingly well with dye test s. Although the test is simple to perform after the cells are prepared and is reliable with fresh cells, we have recently had difficulty with the preparation and preservation of formalinized cells. test antibody appears later in the course of acute systemic infection than does dye test antibody, but persists long after the acute phase in s similar to the dye test s. Although the number of acute cases studied by fluorescence test is small, FI antibodies appear to develop early in the course of infection and may provide a more prompt index of acute infection than the hemagglutination test. FI s are lower than dye test or hemagglutination s, however, and many sera with low antibody concentrations by dye test would appear negative by such a test. This failure to detect sera with low s would render the FI test of dubious value in the study of ocular disease. The majority of cases of proved ocular toxoplasmosis from which organisms have been isolated have had dye s of or below. Jacobs, Fair, and Bickerton, 9 and Pillat and Thalhammer 0 each reported a parasitologically proved case with active uveitis and a dye of before nucleation. Hogan" isolated Toxoplasma organisms from a patient with a of 8 prior to enucleation and 6 afterward, and Downloaded From: on 07/8/08

Volume Number A Antibody tests for ocular toxoplasmosis 9 Wood and Jacobs isolated organisms from two more eyes, one patient had a of during an episode of active uveitis several months before

4 Volume Number A Antibody tests for ocular toxoplasmosis 9 Wood and Jacobs isolated organisms from two more eyes, one patient had a of during an episode of active uveitis several months before enucleation. Duke- Elder reported upon a case with a of 60, months after enucleation in which Toxoplasma-like organisms were found in microscopic sections. Of the cases reported by Jacobs, Cook, and Wilder ' which were histologically diagnosed as toxoplasmosis, 0 had s of or below. These sera were obtained after enucleation, but one patient tested within months of enucleation had s of 6 and, and one tested at months had a of. Since these s were done by a technique similar to that used above, these results suggest that at least half of the proved cases of adult ocular toxoplasmosis would not be detected by the FI test. Although Habegger " reports one case with a of,08 from whom Toxoplasma organisms were recovered from the subretinal fluid, such high s are rare among the proved cases in adults. Our present information indicates, therefore, that low dye s are the rule in the proved cases of ocular toxoplasmosis, and suggests that a test, such as the FI test, which does not reliably detect these antibodies is unsatisfactory in the study of ocular disease. REFERENCES. Remington, J. S., Jacobs, L., and Kaufman, H. E.: Toxoplasmosis in the adult, New England J. Med. 6: 80 and 7, Sabin, A. B., and Feldman, H. A.: Dyes as microchemical indication of a new immunity phenomenon affecting protozoon parasite (Toxoplasma), Science 08: 660, 98.. Jacobs, L., and Lunde, M.: test for toxoplasmosis, J. Parasitol. : 08, 97.. Maloney, E. D., and Kaufman, H. E.: The rapid and convenient detection of Toxoplasma antibodies using formaldehyde-treated human erythrocytes, Am. J. Ophth. 0: 9, Park, H. K.: Toxoplasma hemagglutination test, A. M. A. Arch. Ophth. 6: 8, Goldman, M., Gordon, M. A., and Carver, R. K.: Comparison of dye and fluorescence test s in the serologic diagnosis of toxoplasmosis (in press). 7. Frenkel, J. K., and Jacobs, L.: Ocular toxoplasmosis: Pathogenesis, diagnosis and treatment, A. M. A. Arch. Ophth. 8: 60, Goldman, M.: Symposium on toxoplasmosis, Alfred P. Sloan Foundation, November 0 to, 960, Bethesda, Md. 9. Jacobs, L., Fair, J. R., and Bickerton, J. H.: Adult ocular toxoplasmosis, A. M. A. Arch. Ophth. : 6, Pillar., A., and Thalhammer, O.: Herdfromige iridocyclitis als (einzige) Manifestation einer erworbenen Toxoplasmose, atiologisch gesichert durch kurve und tierversuch: zugleich ein beitrag zur diagnose des alters einer Toxoplasmoseinfektion durch das, diskrepanzaus wertungsverfahren und zur art der in Sabin-Feldman un komplementbindungsreaktion reagierenden antikoyster, von Graefe, Arch, ophth. 8: 0, 97. LI. Hogan, M. J. Zweigart, P. A., and Lewis, A.: Recovery of Toxoplasma from a human eye, A. M. A. Arch. Ophth. 60: 8, 98. L. Wood, R., and Jacobs, L.: Personal communication... Duke-Elder, W. S., Ashton, N., and Brihayevan Geertruyden, M.: Toxoplasmosis in the adult, Brit. J. Ophth. 7:, 9.. Jacobs, L., Cook, M. K., and Wilder, H. C: Serologic data on adults with histologically diagnosed toxoplasmic chorioretinitis, Tr. Am. Acad. Ophth. 8: 9, 9.. Habegger, H.: Toxoplasmose humaine: mise en evidence des parasites dans les milieux intra-oculaires. (Humeur aqueuse, exsudat retro-retinien), Arch. opht. : 70, 9. Discussion Dr. Ronald M. Wood, Baltimore, Md. This work is an important contribution to the understanding of the significance of the serologic procedures used for the diagnosis of ocular toxoplasmosis. Miss Maloney and Dr. Kaufman have reemphasized the fact that ocular toxoplasmosis an be proved only by isolation of the parasite, hort of isolation and in spite of serologic s r clinical appearance, diagnosis is always preumptive. Therefore, the evaluation of serologic Sts is of significance only when studied in reitionship to cases from which the parasite has een isolated. This study is of great significance because the Downloaded From: on 07/8/08

60 Maloney and Kaufman Investigative Ophthalmology August 96 authors have not only assembled and investigated a relatively large number of such cases (eye isolations), but have at the same time

5 60 Maloney and Kaufman Investigative Ophthalmology August 96 authors have not only assembled and investigated a relatively large number of such cases (eye isolations), but have at the same time compared the findings obtained by the use of the available serologic procedures. In this work which compares the dye test, hemagglutination test, and the fluorescent antibody test, the dependability of the HA test has been revealed. The superiority of the HA test over the dye test from the standpoint of simplicity, speed, and lack of infectious hazards to laboratory personnel is obvious. By demonstrating the dependability of this procedure, Miss Maloney and Dr. Kaufman have established confidence in its use as an aid to the diagnosis of ocular toxoplasmosis. This is of extreme value in that laboratories not equipped to handle the temperamental, laborious, and potentially hazardous dye test may use this procedure and supply the ophthalmologist with dependable serologic findings without undue delay. Downloaded From: on 07/8/08

A CASE OF OCULAR TOXOPLASMOSIS IN AN ADULT, THE INFECTION BEING CONFIRMED BY THE ISOLATION OF THE PARASITE FROM SUBRETINAL FLUID

Keio Journal of Medicine Vol. 10. No. 4. December. 1961 A CASE OF OCULAR TOXOPLASMOSIS IN AN ADULT, THE INFECTION BEING CONFIRMED BY THE ISOLATION OF THE PARASITE FROM SUBRETINAL FLUID HISAKICHI MATSUBAYASHI*