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.
- [show abstract] [hide abstract]
ABSTRACT: Infection of humans by Toxoplasma gondii leads to an acute systemic phase, in which tachyzoites disseminate throughout the body, followed by a chronic phase characterized by the presence of tissue cysts, containing bradyzoites, in brain, heart and skeletal muscles. This work focused on studying the antigenic regions of bradyzoite-specific proteins involved in human B- and T-cell responses. To this aim, we constructed a phage-display library of DNA fragments derived from the bradyzoite-specific genes BAG1, MAG1, SAG2D, SAG4, BSR4, LDH2, ENO1 and p-ATPase. Challenge of the bradyzoite library with sera of infected individuals led to the identification of antigenic regions within BAG1 and MAG1 gene products. Analysis of the humoral and lymphoproliferative responses to recombinant antigens demonstrated that the BAG1 fragment induced T-cell proliferation in 34% of T. gondii-exposed individuals, while 50% of them had specific IgG. In the same subjects, the MAG1 fragment was recognized by T cells from 17% of the exposed donors and by antibodies from 73% of them. A detailed analysis of the antibody response against BAG1 and MAG1 antigen fragments demonstrated that the immune response against bradyzoites occurs early after infection in humans. Finally, we provide evidence that the T-cell response against BAG1 is associated with the production of interferon-gamma, suggesting that bradyzoite antigens should be considered in the design of potential vaccines in humans.Microbes and Infection 03/2004; 6(2):164-71. · 2.92 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The diagnosis of perinatal tuberculosis (TB) is problematic because of its nonspecific presentation, the difficulty of obtaining microbiological confirmation, and the unreliability of the tuberculin skin test. Immunodiagnosis of TB has received new attention with the discovery of Mycobacterium tuberculosis-specific immunodominant antigens (early secreted antigenic target 6 [ESAT-6] and culture filtrate protein 10 [CFP-10]) that are encoded by the RD1 region of the pathogen. A whole blood assay has recently been developed to quantitatively measure interferon- gamma production by lymphocytes specific to these antigens, but its evaluation in the diagnosis of TB in infants and children has been limited to date. In addition to routine diagnostic evaluation (tuberculin skin tests, culture of early-morning gastric aspirate samples, and chest radiographs), 2 infants with suspected perinatal TB were investigated with a whole blood interferon-gamma release assay. The results of the tuberculin skin tests were negative for both patients. The findings of the chest radiographs were abnormal with features suggestive of miliary TB. A whole blood interferon- gamma release assay was performed and yielded positive results within 48 h after admission to the hospital for both patients, prompting early antituberculous treatment. M. tuberculosis was cultured after 6 weeks from gastric aspirate samples collected on admission to the hospital from both infants. At 6 months of age, both infants were thriving and had acheived normal developmental milestones. The advent of interferon- gamma release assays may prove to be useful in the evaluation of infants with suspected perinatal TB.Clinical Infectious Diseases 07/2006; 42(11):e82-5. · 9.37 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: CD4(+)CD25(+) regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4(+) or CD8(+) T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4(+)CD25(+)FoxP3(+) regulatory T cells may modulate immunity against human tuberculosis (TB). Our results indicate that the number of CD4(+)CD25(+)FoxP3(+) Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4(+)CD25(+)FoxP3(+) Treg in pleural fluid inversely correlates with local MTB-specific immunity (p<0.002). These CD4(+)CD25(+)FoxP3(+) T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-gamma and IL-10 production in TB patients. Therefore, CD4(+)CD25(+)FoxP3(+) Treg expanded in TB patients suppress M. tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.Clinical Immunology 04/2007; 123(1):50-9. · 3.77 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2010, p. 41–45
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 1
Diagnosis of Congenital Toxoplasmosis by Using a Whole-Blood
Gamma Interferon Release Assay?
Emmanuelle Chapey,2Martine Wallon,1,2Gise `le Debize,3Muriel Rabilloud,4and Franc ¸ois Peyron1,2*
Hospices Civils de Lyon, Service de Parasitologie, Ho ˆpital de la Croix Rousse, F-69317 Lyon, France1;
Universite ´ Claude Bernard Lyon 1, Service de Parasitologie, Faculte ´ de Me ´decine Lyon Sud, F-69372 Lyon,
France2; Hospices Civils de Lyon, Laboratoire d’He ´matologie, Ho ˆpital de la Croix Rousse,
F-69317 Lyon, France3; and Hospices Civils de Lyon, Service de Biostatistique, F-69424 Lyon,
Universite ´ Claude Bernard Lyon 1, F-69622 Villeurbanne, and CNRS, UMR 5558,
Laboratoire Biostatistique Sante ´, F-69495 Pierre-Be ´nite, France4
Received 25 September 2009/Returned for modification 29 October 2009/Accepted 9 November 2009
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-?) 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-?. 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-? 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 par-
asite, is an important cause of morbidity and mortality in con-
genitally infected individuals. Maternal infection may have se-
rious 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 adoles-
cence (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 af-
fects test performance (25), and at birth, the tests cannot de-
tect more than 75% of infected babies (20). Because Toxo-
plasma-specific IgG crosses the placenta, its presence in the
blood of newborns cannot be considered a marker of congen-
ital infection. Maternally transferred IgG usually disappears
within 6 to 12 months (20). Therefore, uninfected infants born
to mothers who seroconverted during pregnancy have to un-
dergo 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 clin-
ical 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 inter-
feron (IFN-?) (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 stimu-
lation of lymphocytes by T. gondii antigen in congenitally in-
fected children has been reported previously (18, 27). Re-
cently, Guglietta et al. (13), using synthetic peptides, detected
age-related impairment of the specific T-cell response to par-
asitic antigen in congenital infections. Conversely, a recent
publication reported that evaluation of T-cell immunity is im-
portant for an early and accurate diagnosis of congenital toxo-
plasmosis (3). By detecting CD25 expression by flow cytom-
etry, 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-? release assay for the diagnosis of congenital toxo-
plasmosis. In this clinical setting, the quantity of blood re-
quired is limited, and we therefore looked at the possibility of
separating plasma from blood cells in order to conduct sero-
logical tests (the “gold standard”) and the IFN-? assay with the
MATERIALS AND METHODS
Patients. (i) Group 1. Patient group 1 was used in a pilot study to check the
validity of the technique before the testing of infants. It included 172 patients
consulting in the outpatient department at Ho ˆpital de la Croix Rousse, Lyon,
France, and pregnant women tested for toxoplasmosis serology as part of the
French national program of prevention. Fifty-eight subjects were free of infec-
tion (with negative serological tests), and 114 either had chronic infection or had
recently seroconverted (with positive tests).
* Corresponding author. Mailing address: Service de Parasitologie,
Ho ˆpital de la Croix Rousse, 103 Grande Rue de la Croix Rousse,
69117 Lyon, France. Phone: (33)4 72 07 18 72. Fax: (33)4 72 07 18 73.
?Published ahead of print on 18 November 2009.
The viability of assaying specific IFN-? secretion with blood cell pellets resus-
pended in medium was first investigated using T. gondii antigens at final con-
centrations of 1.5, 3, and 6 ?g/ml. Among these concentrations, 3 ?g/ml yielded
the best results with respect to sensitivity and specificity (data not shown).
Therefore, this concentration was used for the study. Samples processed 24 h
after withdrawal were likely to exhibit a decreased IFN-? response compared to
samples processed within 24 h after withdrawal. Results were validated when
phytohemagglutinin (PHA)-stimulated wells yielded a positive value and when
the optical density (OD; calculated as the mean plus 2 standard deviations after
Box-Cox transformation) in wells containing phosphate-buffered saline (PBS)
was below 0.75. Under these conditions, for 479 tests performed in the study,
results from 25 (5%) were invalidated due to either a lack of positive response
to PHA stimulation or spontaneous secretion of IFN-?.
(ii) Group 2. Group 2 consisted of 62 infants under 1 year of age who were
born to mothers who seroconverted during pregnancy. For all these subjects,
maternal seroconversion and ante- and postnatal treatment were fully docu-
mented. Congenital toxoplasmosis was diagnosed in 17 cases on the basis of
positive PCR results for amniotic fluid, the presence of IgM or IgA in peripheral
blood, a positive mother/newborn comparative Western blot, or the presence of
specific IgG after 1 year of life. Infected patients were given a 1-year course of
pyrimethamine-sulfonamide (Fansidar). According to our protocol, all patients
underwent both a clinical and ophthalmological examination and serological
testing every 3 months during the first year. Infection was ruled out for 45
children who had negative tests in utero and at birth and had negative serology
results at 1 year of age.
(iii) Group 3. Group 3 comprised 124 congenitally infected patients enrolled
at ages 1 to 30 years. All underwent regular annual clinical and ophthalmological
examinations. Thirty-one of them presented ocular lesions, and 8 had cerebral
calcification at the time of inclusion in the study.
Serological investigations. T. gondii-specific IgG and IgM antibodies were
detected using the Enzygnost toxoplasmosis enzyme-linked immunosorbent as-
say (ELISA; Siemens Healthcare Diagnostics, Marburg, Germany). A dye test
(P. Thulliez, Institut de Pue ´riculture, Paris, France) was performed on samples
with discordant results.
T-cell stimulation. (i) Preparation of soluble T. gondii antigen. T. gondii
parasites of the RH strain were harvested from the peritoneal cavities of OF1
mice (Charles River, L’Arbresle, France). Ascites fluid was collected 2 days after
infection; parasites were washed in PBS and disrupted by four freeze-thaw cycles
and underwent three sonications for 20 s each. The suspension was filtered
through 0.2-?m-pore-size membranes and stored at ?20°C until use.
(ii) Blood samples and stimulation. Samples of 1 ml of peripheral blood were
drawn into Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ) that con-
tained lithium heparin anticoagulant. Tubes were centrifuged at 1,600 ? g for 15
min at room temperature. Plasma was collected for serological investigations and
replaced by the same volume of RPMI medium (Sigma-Aldrich, St. Louis, MO).
Aliquots of 300 ?l of diluted blood were cultured in sterile propylene tubes
(Eppendorf AG, Hamburg, Germany) in the presence of different concentra-
tions of T. gondii antigens. Positive and negative controls consisted of PHA at a
final concentration of 20 ?g/ml and PBS, respectively. All cultures were incu-
bated for 24 h at 37°C in 5% CO2in a humidified atmosphere. Culture super-
natants were collected from each tube after centrifugation at 1,600 ? g for 15 min
at room temperature and stored at ?40°C until the IFN-? assay was carried out.
IFN-? assay. IFN-? was assayed in duplicate using a commercial ELISA kit
(AbCys, Paris, France). The mean OD of the PBS controls was subtracted from
the mean OD of antigen-stimulated samples. The amount of IFN-? released,
expressed in picograms per milliliter, was obtained by converting the OD using
the standard curve from the kit.
Ethical aspects. The study was approved by the local ethical committee.
Informed consent was obtained from patients or legal guardians.
Statistical analysis. The IFN-? responses in infected and uninfected patients
were compared using the Mann-Whitney test for the different groups. A P value
under 0.05 was considered statistically significant.
For the group of infants under 1 year, the empirical receiver operating char-
acteristic (ROC) curve was built and the area under the curve (AUC) was
estimated using the nonparametric method. The 95% confidence interval was
calculated using a logistic transformation of the AUC. For the retained positivity
threshold, the sensitivity and specificity were estimated with their exact 95%
FIG. 1. IFN-? 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).
42CHAPEY ET AL.J. CLIN. MICROBIOL.
Group 1. Fig. 1 shows that IFN-? secretion in the 114 in-
fected patients in group 1 was significantly higher than that in
the 58 uninfected patients (P ? 0.0001). The test scored a
sensitivity of 96% and a specificity of 91%. Among patients
who displayed negative serology, five scored weak-positive
IFN-? values ranging from 1.5 to 23.2 pg/ml. All five had
nonspontaneous IFN-? secretion (i.e., no detectable secretion
of IFN-? in negative controls) and did not present an inflam-
matory syndrome. Two of these subjects were pregnant women
who presented an atypical profile of seroconversion with the
presence of IgM without IgG despite repeated testing. Both
were considered to be recently infected on the basis of the
presence of IgM and were given spiramycin treatment. Among
patients who tested positive for toxoplasmosis, five had a neg-
ative IFN-? response despite a positive PHA test. No evidence
of anergy or immunosuppression was found in the patients’
Group 2. From the first test at inclusion in the study, 17
infected infants displayed a significantly higher level of IFN-?
than uninfected infants (P ? 0.0001) (Fig. 2). According to the
ROC curve, values above 1 pg/ml were considered to be pos-
itive (Fig. 3). Under these conditions, 16 of 17 infected babies
produced positive IFN-? tests. Among these, five infants were
diagnosed with subclinical congenital infection between 3
weeks and 3 months of life, and in two cases, this was the only
positive criterion for congenital infection. The mothers had
seroconverted at different periods during pregnancy, and all
were given antenatal treatment for toxoplasmosis. The false-
negative test corresponded to an 8-month-old baby who was
FIG. 2. IFN-? 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-? test. Using a threshold of one yielded a sensitivity of 94%
and a specificity of 98%.
VOL. 48, 2010CONGENITAL TOXOPLASMOSIS AND IFN-? ASSAY 43
under treatment when included in the study and displayed
positive test results after the withdrawal of treatment. Among
45 uninfected infants, 44 yielded a negative response. In this
clinical setting, the sensitivity and specificity of the test were
94% (confidence interval, 69.2 to 99.7%) and 98% (confidence
interval, 86.8 to 99.9%), respectively.
The follow-up findings (96 test results) for all 62 infants were
consistent with the diagnoses except that two of the babies
displayed transitory negation of the test result when under
treatment. Interestingly, at the same time, they displayed neg-
ative serology. After withdrawal of treatment, they presented a
dramatic increase in both IFN-? and IgG titers (Fig. 4).
Group 3. All of the 172 congenitally infected subjects had a
positive test result, with IFN-? levels ranging from 9 to 2,285
pg/ml (median, 168.5 pg/ml); there were nonsignificant corre-
lations between IFN-? levels and lymphocyte counts (r ? 0.06;
P ? 0.5) and between IFN-? levels and IgG titers (r ? 0.08;
P ? 0.6). No difference between subclinical infection and
patent infection was observed (data not shown).
We investigated an in vitro assay to measure IFN-? re-
sponses of T cells that had been stimulated by crude T. gondii
antigens. The test requires only 1 ml of whole blood and is
therefore well suited for application to newborns. Moreover, in
addition to the IFN-? assay with positive and negative controls
(PHA and PBS samples, respectively), serological tests can be
carried out with the same blood sample. The overall rate of
invalidated test results was 5% due to either spontaneous se-
cretion of IFN-? in control tubes or a lack of positive response
in PHA tubes, and the invalid results were observed mainly for
adults. The assay was first validated for adults. Seropositive
patients with either acute or chronic infection exhibited signif-
icantly higher levels of IFN-? than uninfected patients (Fig. 1).
Five samples without spontaneous secretion of IFN-? were
determined to be false positives when the results were com-
pared with IgG serology. Two were from pregnant women who
presented an atypical profile but were nevertheless considered
to have had recent seroconversions and were treated. Evalua-
tion of IFN-? may help to interpret an ambiguous serological
status which may be encountered in pregnant women. A su-
perantigen effect of T. gondii antigen in uninfected mice, but
not in humans, has been reported previously (7). The low
number of false positives in our study does not favor this
phenomenon. It has been demonstrated previously that T. gon-
dii antigen can elicit marked T-cell proliferation in seronega-
tive subjects. This proliferation appeared to be polyclonal and
may result from cross-reactivity with another pathogen such as
a coccidian (21). Purification of the antigen may improve the
specificity of the test.
The five patients with false-negative results were not im-
munosuppressed, and all responded positively to PHA.
Their lymphocyte counts fell within normal values, but no
CD4 phenotyping was performed. All of them presented a
chronic toxoplasmic infection, and their lack of response was
not due to immunosuppression associated with active disease,
as reported previously for tuberculosis patients (9). Other fac-
tors such as CD4?CD25?FoxP3?regulatory cells suppress
specific immunity in patients with active tuberculosis (2). In-
terestingly, T. gondii infection has been reported to reduce
FoxP3 mRNA expression in pregnant mice (12). Whether this
phenomenon is relevant in humans has not yet been demon-
For infants under 1 year of age born to mothers who were
infected during pregnancy, the results of the first test (Fig. 2)
yielded a sensitivity and specificity of 94 and 98%, respectively.
The test ruled out the disease in 44 of 45 uninfected infants
and would have made IgG monitoring during the first year of
life unnecessary. The only false positive was observed for a
6-month-old baby who presented a transitory positive test re-
sult, without clinical inflammatory syndrome; further testing
was negative. Sixteen of 17 infected infants were identified by
the test. In two patients under 3 months of age, IFN-? secre-
tion was the only sign of infection and would have prompted a
decision to treat. These data demonstrated that, for infants,
the IFN-? release assay is a good marker of congenital toxo-
plasmosis, and it has also been reported previously to be ef-
fective for diagnosing perinatal tuberculosis (4); neither the
stage of pregnancy at maternal contamination nor antenatal
treatment hampered the test. The only false-negative result
was obtained for an 8-month-old, treated baby who displayed
an important rise in IFN-? after withdrawal of treatment. The
diagnosis of congenital toxoplasmosis was made on the basis of
serological tests before inclusion in the study. During the fol-
low-up with these patients, a total of 96 tests were performed.
All but two results matched the clinical statuses of the infants.
The two false negatives were obtained for treated patients who
also presented a rise in IFN-? titers after withdrawal of treat-
ment. As presented in Fig. 4, IgG titers paralleled IFN-? levels.
This antibody profile (i.e., transitory negation followed by re-
bound) is commonly observed for treated patients (14, 24).
The impact of treatment in reducing the IFN-?-specific re-
sponse in tuberculosis has been described previously (1, 9, 15)
and suggests that such tests could be used for monitoring
patient response to treatment (1). This pattern may be due to
a reduction in antigen load originating from parasitic cysts or
to a direct effect of pyrimethamine and sulfonamide on acti-
vated lymphocytes through activation of the mitochondrial
apoptotic pathway (22). A large discrepancy in drug levels
observed in babies (5) may explain why this phenomenon is not
observed in all infants. The sustained IFN-? response observed
FIG. 4. Evolution of IFN-? levels (solid line) and specific antibody
titers (dashed line), expressed in international units (IU), in a congen-
itally 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
44CHAPEY ET AL.J. CLIN. MICROBIOL.
in subclinical patients in group 3 30 years after infection is a
marker of continuous parasitic antigen production by cysts that
maintain specific effector T cells in the bloodstream. The acute
phase of the disease is characterized by the presence of fast-
replicating tachyzoites which differentiate into slow-replicating
bradyzoites under the pressure of the immune system, espe-
cially the Th1 response (17). Bradyzoites form cysts in different
tissues and persist throughout life. Both parasitic stages ex-
press specific antigens (23). During the chronic phase of the
disease, bradyzoite antigens are able to maintain an efficient
immune response (8). Interestingly, our data also demon-
strated that T cells from cyst-bearing patients could respond to
crude extracts of tachyzoites of a noncystogenic strain (the RH
strain), which suggests the lifelong presence of circulating an-
tigens common to the two parasitic stages in humans.
In conclusion, we demonstrated that evaluation of the IFN-?
response after stimulation of whole blood by crude toxoplas-
mic antigen is a simple, easily performed test that is suitable for
diagnosing congenital toxoplasmosis in newborns. This test
could reliably rule out congenital infection at birth and avoid
unnecessary anxiety and serological follow-up. Conversely,
when giving positive results, it could prompt an early decision
to treat, which is believed to reduce infection sequelae, al-
though the exact benefits are still being debated (19). The use
of purified antigens or synthetic peptides to improve the per-
formance of the test should be investigated. Additional cases
are needed in order to evaluate more precisely the sensitivity
of the test. It may also be a useful tool for screening potential
vaccine antigens in humans (8).
We thank V. Meroni (Department of Infectious Diseases, Pavia,
Italy) and G. Cozon (Ho ˆpital Lyon Sud, Lyon, France) for their com-
ments and P. Thulliez (Institut de Pue ´riculture, Paris, France) for his
None of the authors has a financial relationship with a commercial
entity that has an interest in the subject of this report.
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