| Abstract: | ![]()
Congenital toxoplasmosis in newborns is generally subclinical, but infected infants are at risk of developing ocular lesions. Diagnosis at birth relies mainly on serological tests. Cell-mediated immunity plays the major role in resistance to infection but is not routinely investigated for diagnostic purposes. Here, we describe a simple test based on the gamma interferon (IFN-γ) response after stimulation of whole blood by crude parasitic antigens. One milliliter of heparinized blood was centrifuged; plasma was kept for routine serological tests, and pellets were resuspended in culture medium. After 24 h of culture in the presence of crude Toxoplasma gondii antigen, the cells were centrifuged and the supernatant was assayed for IFN-γ. For 62 infants under 1 year of age born to mothers who were infected during pregnancy, the sensitivity and specificity of the test were 94% (with positive results for 16 of 17 infected infants) and 98% (with negative results for 44 of 45 uninfected infants), respectively. The false-negative result was for a treated baby who gave positive results after the withdrawal of treatment. The false positive was obtained for a 3-month-old baby. For a cohort of 124 congenitally infected patients between 1 and 30 years of age, the sensitivity of the assay was 100%. We present a simple test based on IFN-γ secretion to assess cell-mediated immunity in toxoplasmosis. As only 1 ml of blood is required to investigate humoral and cellular immunity, our assay is well adapted for the study of congenital toxoplasmosis in infants. Using purified antigens or recombinant peptides may improve the test performance.Toxoplasma gondii, a ubiquitous intracellular protozoan parasite, is an important cause of morbidity and mortality in congenitally infected individuals. Maternal infection may have serious consequences for the fetus (10). In other cases, infected newborns appear to be totally asymptomatic at birth but are at risk of developing retinal diseases during childhood or adolescence (26). For these patients, the diagnosis of the disease relies mainly on the detection of specific antibodies. Toxoplasma-specific immunoglobulin M (IgM) and IgA, which do not cross the placenta, are considered to be good markers of congenital infection. However, gestational age at maternal infection affects test performance (25), and at birth, the tests cannot detect more than 75% of infected babies (20). Because Toxoplasma-specific IgG crosses the placenta, its presence in the blood of newborns cannot be considered a marker of congenital infection. Maternally transferred IgG usually disappears within 6 to 12 months (20). Therefore, uninfected infants born to mothers who seroconverted during pregnancy have to undergo regular sampling for serological testing for 1 year before congenital toxoplasmosis can be ruled out (16). Clinicians are seeking valid indicators of congenital infection to improve clinical decision making. T. gondii infection results in long-lasting cell-mediated immunity which is highly dependent upon the effector activity of T lymphocytes that produce gamma interferon (IFN-γ) (6). Surprisingly, few studies have investigated the potential role of cell immunity in diagnosis of the disease. Data in the literature are contradictory. An absence of stimulation of lymphocytes by T. gondii antigen in congenitally infected children has been reported previously (18, 27). Recently, Guglietta et al. (13), using synthetic peptides, detected age-related impairment of the specific T-cell response to parasitic antigen in congenital infections. Conversely, a recent publication reported that evaluation of T-cell immunity is important for an early and accurate diagnosis of congenital toxoplasmosis (3). By detecting CD25 expression by flow cytometry, we demonstrated previously that specific cell immunity is detectable in almost all infected patients, including newborns (11). In this study, we evaluate the performance of a whole-blood IFN-γ release assay for the diagnosis of congenital toxoplasmosis. In this clinical setting, the quantity of blood required is limited, and we therefore looked at the possibility of separating plasma from blood cells in order to conduct serological tests (the “gold standard”) and the IFN-γ assay with the same sample. |
