Background: The IFA test is one of the most usual methods for detecting anti-Toxoplasma antibodies, although it has not any unique standardization. It seems that the microscopic judgment of results is an important confounder in IFA test. Therefore, we conducted the present study to clarify the role of microscopic observer, and other confounders on the test.Methods: Eighty sera were collected from patients suspicious to toxoplasmosis for detection IgG anti-T. gondii by this test. Samples were examined against different series of antigens, IgG anti-human conjugates, and observers.Results: There were no significant differences between the two series of antigens and conjugates. For the observers groups the kappa coefficient of the test results in the experts group (0.97, 0.94-1.00) were significantly higher than the less experienced observers (0.77, 0.68-0.87).Conclusion: We recommend the IFA test to be performed only in reference laboratories and by laboratory technicians that have enough experience for this test. Otherwise, we suggest the substitution of this test with other tests like ELISA for the diagnosis and epidemiological studies
