Ocular Toxoplasmosis - A Laboratory View Chatterton JMW* Evans R Ho-Yen DO Scottish Toxoplasma Reference Laboratory Microbiology Department Raigmore Hospital Inverness IV UJ * corresponding author
Introduction Ocular toxoplasmosis is usually a clinical diagnosis. Laboratory investigations are undertaken to support the clinical diagnosis or help when the clinical presentation is atypical. Ocular toxoplasmosis is usually considered to be due to infection acquired congenitally but may be the result of postnatal infection. Less commonly ocular symptoms may be associated with acute toxoplasma infection. In acute infection antibody levels are raised and specific IgM will be detected. In reactivated infection (usually congenital) antibody levels are often not raised and IgM is not detected. Serological tests in these patients are not diagnostic and are used to confirm previous exposure to Toxoplasma gondii or to exclude the disease. The sensitivity and specificity of the tests used is important. Antibody levels may be low even during episodes of acute ocular disease. At the Scottish Toxoplasma Reference Laboratory a cutoff of iu/ml in the dye test is used. The dye test is the gold standard test and measures all types of immunoglobulin. A positive result is useful in supporting a clinical diagnosis of ocular toxoplasmosis and a negative result is useful in excluding the disease. Toxoplasmosis is a notifiable disease and the Scottish Toxoplasma Reference Laboratory is responsible for reporting cases of toxoplasmosis. Ocular patients with a positive dye test cannot be reported on serological evidence alone. For those patients with ocular symptoms and a positive dye test laboratories are sent questionnaires to forward to the clinician caring for the patient. This report correlates the questionnaire responses and the serological results to assess the laboratory diagnosis of ocular toxoplasmosis. Materials and methods Questionnaires The Scottish Toxoplasma Reference Laboratory receives specimens from throughout Scotland and Northern Ireland. For those patients with ocular symptoms and a positive dye test ( iu/ml) referring laboratories are sent questionnaires to forward to the
clinician caring for the patient. Information is requested on the clinical diagnosis and the usefulness of serological testing in confirming the diagnosis. Laboratory tests All serum samples are screened using the in-house IgG enzyme linked immunosorbent assay (EIA), in-house biotin avidin IgM toxoplasma assay and a modification of the Sabin-Feldman dye test. The results of these tests influence the choice of further tests. The tests available include the Abbott AxSym IgM, in-house IgG avidity EIA, IgG Western blotting, and the immunosorbent agglutination assay (ISAGA) for toxoplasma specific IgM, IgA and IgE. Polymerase chain reaction (PCR) techniques are available for use on other specimen types, such as aqueous or vitreous fluid. The questionnaire responses and the laboratory results for patients investigated for ocular toxoplasmosis between April 4 and June 8 were analysed. Results Between April 4 and June 8, 9 patients were investigated for ocular toxoplasmosis. Toxoplasma dye test results < iu/ml were obtained in 9 (5.5%) patients excluding a diagnosis of ocular toxoplasmosis. Toxoplasma dye test results iu/ml were obtained for 5 patients which would support a clinical diagnosis of ocular toxoplasmosis. There was a female to male ratio of.: (9 females and males);.5 % (9/5) were < years old and 59.9% (5/5) were < 5 years old (Table ). Of the 5 patients, 8 (7.%) had a raised dye test result ( 5 iu/ml) and three (.%) had serological evidence of current toxoplasma infection (raised dye test 5 iu/ml, specific IgM and low IgG avidity detected). Toxoplasma gondii DNA was detected by PCR in vitreous fluid samples from two patients.
TABLE : Age and dye test result in 5 patients investigated for ocular toxoplasmosis Patients no. (%) Patients no. (%) Age years < - - -4 4-5 5-6 6-7 7-8 8 5 4 7 48 7 4 9 (.9) (9.5) (4.6) (8.9) (4.6) (6.) (.5) (.8) (5.5) Dye test result iu/ml 4 8 5 65 5 5 5 5 9 78 44 6 8 (.9) (8.7) (9.9) (5.4) (.8) (7.4) (6.) (.9) (.) Questionnaires were returned on 95/5 patients (7.7%); 57 females and 8 males (.5: ratio). Of these 4 (45.%) were considered clinically to have ocular toxoplasmosis and 5 (54.7%) were considered clinically not to have ocular toxoplasmosis. The ratio of females to males was more pronounced in patients with ocular toxoplasmosis (/;.:) than in those without (7/5;.8:) but this was not significant (P.9, Fisher s exact test, two tailed). Questionnaires were not returned for the three patients with serological evidence of current infection but were returned for the two positive PCR patients. The age and dye test results in the 4 patients with clinical ocular toxoplasmosis and 5 patients considered not to have ocular toxoplasmosis are given in Table. Significantly more patients in the ocular toxoplasmosis group were <5 years old 5/4(8.4%) compared to 5/5 (48%) (P., Fisher s exact test, two tailed). Significantly more patients in the ocular toxoplasmosis group were < years old, /4 (.%) compared to /5 (5.7%) (P.7, Fisher s exact test, two tailed). There was no significant difference in the range of dye test results between patients with and without clinical ocular toxoplasmosis (Table ). Three patients considered not to have ocular toxoplasmosis had raised dye test results 5 iu/ml (Table ). 4
TABLE : Age and dye test result in 4 patients with clinical ocular toxoplasmosis and 5 patients considered clinically not to have ocular toxoplasmosis Ocular toxoplasmosis Yes No. patients No No. patients Ocular toxoplasmosis Yes No. patients No No. patients Age years < - - -4 4-5 5-6 6-7 7-8 8 9 8 9 8 4 7 5 5 Dye test result iu/ml 4 8 5 65 5 5 5 6 4 6 9 Overall, for /4 (76.7%) patients with ocular toxoplasmosis, serological results were considered helpful in making the diagnosis; four of these patients having dye test results of between -4 iu/ml and only two patients having dye test results of 5 iu/ml (Table ). Surprisingly, for /5 (%) of patients without ocular toxoplasmosis the serological results were considered helpful in making the diagnosis (Table ). Of the two patients with ocular toxoplasmosis confirmed by PCR the serological results were considered helpful for one patient but not the other. Both had dye test results of 65 iu/ml with no detectable IgM. 5
TABLE : Comparison of the dye test result and whether or not the serological results helped confirm the diagnosis in 4 patients with a clinical diagnosis of ocular toxoplasmosis and 5 patients considered clinically not to have ocular toxoplasmosis Patients Dye test result iu/ml No. with ocular toxoplasmosis 4 8 5 65 5 5 5 Total Serology helpful for Yes No 4 8 diagnosis No. without ocular toxoplasmosis Serology helpful for diagnosis Yes No 4 5 5 4* *Information on the usefulness of serology was not given for two patients Discussion Over the four-year period, 5 patients with ocular symptoms were found to have positive toxoplasma serology results which would support a clinical diagnosis of ocular toxoplasmosis. Only three patients had evidence of current infection which confirms that most cases are reactivated infection. Of the 95 questionnaires returned, 4 (45.%) confirmed that the patient had ocular toxoplasmosis. If this were reflected in the 5 patients, then 4 cases of ocular toxoplasmosis should have been reported. There were more females than males in the patients with ocular toxoplasmosis (ratio.:). Ocular toxoplasmosis was confirmed in patients aged from < years to > 8 years but there were significantly more in the younger age groups < years and < 5 years. These findings are consistent with our previous study in 999 which found a female to male ratio of.6: in patients with ocular toxoplasmosis and showed that ocular toxoplasmosis was most frequently diagnosed in the age band -. Ocular toxoplasmosis was diagnosed in patients with dye test results from 6
5 iu/ml. This confirms the need to use a very sensitive and specific test to avoid false negative results. With screening tests with a threshold of >8 iu/ml, six ocular toxoplasmosis patients would have been missed. Higher dye test results are not necessarily diagnostic and three of the seven patients with dye test results of 5 iu/ml were clinically considered not to have ocular toxoplasmosis. The direct detection of Toxoplasma gondii DNA in ocular fluid by PCR is confirmation of active ocular disease. Toxoplasma DNA was detected in vitreous samples from two patients but experience has shown that these invasive samples are rarely available for testing. It must also be remembered that a negative PCR result does not exclude ocular toxoplasmosis and sensitivities as low as 8% have been reported. 4 This study has shown that for /4 (76.7%) patients with clinical ocular toxoplasmosis the clinicians found the demonstration of specific antibody useful in confirming the diagnosis. This probably reflects how typical the clinical picture is with serology most useful when the presentation is atypical. It is difficult to understand how the demonstration of specific toxoplasma antibodies could help confirm another diagnosis in the /5 patients who did not clinically have ocular toxoplasmosis. It is well recognised that negative toxoplasma serology is useful in excluding ocular toxoplasmosis., Indeed it has been claimed that the only useful serology result is a negative result. The value of demonstrating previous exposure to toxoplasma in the investigation of ocular disease should not be dismissed too readily. This study shows that clinicians can also find positive serology results useful in confirming a diagnosis of ocular toxoplasmosis. Acknowledgements We are grateful to our users for their tremendous cooperation and support. We would like to thank the National Services Division of NHS National Service Scotland and Health Protection Scotland for their continued support. We would like to thank Ms Elena Duff for her assistance with the questionnaires and clinicians and laboratories for kindly completing and returning them. 7
References. Joynson DHM and Guy EC. () Laboratory diagnosis of toxoplasma infection. In: Toxoplasmosis: A comprehensive clinical guide. (Ed. DHM Joynson and TG Wregitt) pp96-8. Cambridge University Press.. Ho-Yen DO, Chatterton JMW, Evans R, Ashburn D, Joss AWL. Scottish Toxoplasma Reference Laboratory: users guide. SCIEH Weekly Report ; 4(): 7-7. Available from: http://www.hps.scot.nhs.uk/ewr/redirect.aspx?id=889.. Chapman DJ, Ashburn D, Ogston SA and Ho-Yen DO. The relationship between ocular toxoplasmosis and levels of specific toxoplasma antibodies. Epidemiology and Infection 999; : 99-. 4. Villard O, Filisetti D, Roch-Deries, Garweg J, Flament,Candolfi E. Comparison of enzyme-linked immunosorbent assay, immunoblotting and PCR for diagnosis of toxoplasmic chorioretinitis. Journal of Clinical Microbiology ; 4:57-54. Available from: http://jcm.asm.org/cgi/reprint/4/8/57. 8