Diagnosis of congenital toxoplasmosis by using a whole-blood gamma interferon release assay

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-gamma) 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-gamma. 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-gamma 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.

FIG. 1.

IFN-γ responses after stimulation of whole-blood samples from uninfected and infected adults by T. gondii crude antigen (P < 0.0001; Mann-Whitney test). The hinges at the top and bottom of the box represent the upper (75%) and lower (25%) quartiles. The thick black line inside the box represents the median. The horizontal lines above and below the box represent the adjacent values, i.e., the most extreme values in the sample that lie between the hinges and the “inner fences,” which lie at positions 1.5 times the H spread (i.e., the distance between the upper and lower hinges) above and below the hinges. Dots represent outliers (positioned between 1.5 and 3 times the H spread); asterisks represent extreme cases (positioned more than 3 times the H spread).

FIG. 2.

IFN-γ responses after stimulation of whole-blood samples from uninfected and congenitally infected infants under 1 year of age by T. gondii crude antigen (P < 0.0001; Mann-Whitney test).

FIG. 3.

ROC curve for determining the sensitivity and specificity of the IFN-γ test. Using a threshold of one yielded a sensitivity of 94% and a specificity of 98%.

FIG. 4.

Evolution of IFN-γ levels (solid line) and specific antibody titers (dashed line), expressed in international units (IU), in a congenitally infected infant during and after the withdrawal of treatment. The arrow indicates the time of withdrawal of treatment. Months indicate the age of the infant at sample collection. IFAT, indirect fluorescent antibody technique.