Diagnosis of Congenital Toxoplasmosis by Immunoblotting and ...

JOURNAL OF CLINICAL MICROBIOLOGY, June 1995, p. 1479–1485 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 6

Diagnosis of Congenital Toxoplasmosis by Immunoblotting and Relationship with Other Methods BERNABE F. F. CHUMPITAZI,1* ALI BOUSSAID,1 HERVE PELLOUX,1 CLAUDE RACINET,2 MICHEL BOST,3 AND ANDREE GOULLIER-FLEURET1 De´partement de Parasitologie Mycologie Me´dicale et Molećulaire, Universite´ Joseph Fourier-Grenoble I, EP J0078 Centre National de la Recherche Scientifique, 38706 La Tronche cedex, and Services de Parasitologie Mycologie,1 Gynećologie-Obste´trique,2 et Pe´diatrie,3 Ho ˆpital Albert Michallon Grenoble, France Received 4 October 1994/Returned for modification 11 November 1994/Accepted 2 March 1995

Immunoblot has been evaluated as a diagnostic method for congenital toxoplasmosis. Like enzyme-linked immunofiltration assay (ELIFA), immunoblot can be used to compare antibody patterns and to determine if the antibodies are transmitted by the mother or synthesized by the fetus or infant. Among the 48 infants tested, 27 had congenital toxoplasmosis and 21 were suspected but had none. Reproducibility, sensitivity, specificity, and positive predictive values in immunoblot for immunoglobulins (Igs) G 1 M 1 A and/or G 1 M were 90, 92.6, 89.1, and 92.4%, respectively. G 1 M immunoblot and G 1 M ELIFA have better sensitivities than the conventional IgM immunosorbent agglutination assay, IgM enzyme-linked immunosorbent assay (ELISA), IgM immunofluorescence antibody test, in vitro culture, and mouse inoculation. The novel antibodies, i.e., those synthesized by infants against Toxoplasma gondii, were of the IgG class in most cases, although a confident diagnosis could be related to the number of observed Ig classes (G 1 M, G 1 A, and G 1 M 1 A). Immunoblot has a better resolution than ELIFA. In prenatal diagnosis, immunoblot could be complementary to in vitro culture and mouse inoculation. In the other cases, early detection by immunoblot appears to give the best results when compared with the other serological methods. detection can be performed by in vitro cell culture, mouse inoculation, and PCR (20, 21) to confirm the diagnosis. For serodiagnosis, different methods based mainly on the detection of specific anti-Toxoplasma immunoglobulin M (IgM) antibodies are used: immunofluorescence antibody test (IFAT), Immunosorbent agglutination assay (ISAGA), enzyme-linked immunosorbent assay (ELISA), and enzymelinked immunofiltration assay (ELIFA). By using ELIFA, it was possible to compare immunological patterns (precipitin arcs) and to deduce the origin of antibodies, i.e., to determine whether they were transmitted by the mother or synthesized by the fetus or infant (34). The diagnosis was confirmed by the detection of anti-Toxoplasma antibodies synthesized by fetuses or infants. Immunoblot (IB) is a technique that combines electrophoresis of toxoplasmic antigens under denaturing conditions, an electrotransfer, and a specific antibody test (28, 38). The recent advances in the technology of appropriate equipments should permit a better reproducibility and handling of IB. Thus, IB can be used to compare the immunological patterns of mothers, fetuses, and infants. The aim of this study was to evaluate the IB contribution to prenatal, at-birth, and afterbirth diagnosis of congenital toxoplasmosis, in comparison with the other conventional tests used.

Congenital toxoplasmosis is the most frequent infectious condition of human fetuses in France. Its occurrence has been evaluated as being between 1 and 1.5 per 1,000 newborns (15). In 1985, the prevalence of anti-Toxoplasma antibodies in women at the time of early pregnancy was 72% (7). During gestation, routine screening of initially seronegative women permits the detection of a seroconversion that indicates infection by Toxoplasma gondii, a ubiquitous protozoan responsible for congenital toxoplasmosis. A recent study of 1,270 cases of maternal infection in treated pregnant women has shown a fetal infection rate of 7%, which varies from 1.2 to 28.9% depending on the date of initial infection (27). Other authors have evaluated the contamination risk in untreated pregnant women as ranging from 20 to 70% (24, 26). The most dangerous period is between the weeks 10 and 24 of gestation, with the risk of severe fetal infection being at its greatest during that time (brain calcification, retinochoroiditis, and risk of ventricle dilatations). Thus, therapeutic abortion is recommended only in severe cases of congenital toxoplasmosis; it is not necessary if repeated fetal ultrasound is normal and antiparasitic treatment is given (3). If congenital toxoplasmosis is diagnosed early, a pyrimethamine-sulfadoxine treatment will be given to the pregnant women. These women, and subsequently their infants, are then included in a multidisciplinary survey. Bioclinical screening of suspected children is continued for 1 year to identify additional cases that have not been diagnosed either in utero or at birth. Screening is continued for more than 1 year in case of clinical or subclinical congenital toxoplasmosis (23, 41). The diagnosis is based on ultrasound scanning, parasite detection, and serological testing (8). Ultrasound data can be a major element in detection of prenatal infection. Parasite

MATERIALS AND METHODS All the tests carried out in this study were done prospectively, except for IB, for which 18 patients were analyzed prospectively and the other 30 were studied retrospectively. IgG antibodies to T. gondii. IgG antibodies to T. gondii were detected by IFAT (1) and ELISA (VIDAS, VITEK system; BioMe´rieux, Lyon, France). The IFAT and ELISA cutoffs used were 8 and 10 IU, respectively (29). IgM antibodies to T. gondii. IgM antibodies to T. gondii antibodies were detected by ISAGA (BioMe´rieux), immunocapture ELISA (VIDAS, VITEK system), and IFAT (1). The ISAGA, ELISA, and IFAT cutoffs used were as follows: index 9 for adults (12) and 6 for fetuses and newborns, relative fluorescence at 450 nm of 0.65 (manufacturer’s indication), and a titer of 1/40, respectively (29). Criteria for seroconversion. On the basis of the monthly serological screening

* Corresponding author. Mailing address: DP3M, Faculte´ de Me´decine, Universite´ Joseph Fourier-Grenoble I, 38706 La Tronche cedex, France. Fax: (33) 76 76 56 60. 1479

1480

CHUMPITAZI ET AL.

recommend in France, seroconversion is defined when a toxoplasmic serology changes from negative to positive. Seroconversion can also be suspected when an IgM-positive serology is significantly higher than the previous one, with negative or slightly positive levels of IgG antibodies to T. gondii. The date of the infection is determined by comparing these data with the standard kinetics of IgG and IgM antibodies during the infection (29). Patients. The 46 pregnant women under study and infected by T. gondii during pregnancy as determined by IFAT, ELISA, and ISAGA had a mean age of 29.2 6 5.2 years (range, 19 to 41 years). Two of them had twins. Therefore, among the 48 offspring, 4 infants were from mothers infected by the time of conception (periconceptional), 11 were from mothers infected during the first trimester of pregnancy, 14 were from mothers infected during the second, 9 were from mothers infected during the third, and 10 were from mothers infected at dates that were not possible to determine precisely. When indicated by the physician and according to the date of infection, fetal blood (FB) and amniotic fluid (AF) samples were collected between weeks 19 and 25 of gestation. In addition, nine placentas (PL) were tested for detection of T. gondii. Infants were usually monitored for 1 year in if they were suspected of having congenital toxoplasmosis and for more than 1 year if they were proven to have congenital toxoplasmosis. Biological samples. For IB, 60 serum samples from mothers (MS), 14 amniotic fluid samples, 15 fetal blood samples, 21 cord blood samples (CB), and 24 serum samples from infants (IS) were obtained from 46 mother-fetus or mother-infant pairs before, during, and after birth (from days 2160 to 800). Between 3 and 11 samples were taken in each case. Complementary tests. The absence of maternal contamination of fetal blood was tested by agglutination of anti-I and anti-i antibodies, which ensures its specific nature. In addition, the lactate dehydrogenase (LDH) and g-glutamyl transpeptidase (g-GT) cutoffs were 370 and 50 UI/liter, respectively. The cutoffs were calculated from data by Desmonts et al. (11), by adding two standard deviations to the mean value. In vitro culture. Parasite multiplication was carried out in human embryonic lung fibroblast MRC5 cells (BioMe´rieux) on glass coverslips (Flobio, Courbevoie, France) deposited in 24-well culture plates (Nunc) at 378C and in a wet atmosphere composed of 95% air and 5% CO2. To Eagle’s basal medium (BioMe´rieux) and modified Eagle’s medium (BioMe ´rieux) were added 20 ml of Ultroser-G per liter of penicillin (100 mg/liter), streptomycin (100 mg/liter), kanamicin (50 mg/liter), colistin (40 mg/liter), and amphotericin B (5 mg/liter); one medium was used to grow the MRC5 cells to confluence ($3 days) and one was used for parasite multiplication, respectively. The AF, FB, and PL were sown within a week of MRC5 confluence. An IFAT was carried out with rabbit polyclonal anti-T. gondii serum (30 min at 378C; dilution, 1:400 in phosphatebuffered saline [PBS]–10% fetal calf serum [pH 7.2]) and then with a fluorescein anti-rabbit IgG conjugate (Institut Pasteur, Paris, France) (30 min at 378C; dilution, 1:100 in PBS–0.002% Evans blue [pH 7.2]). The reading was performed with a fluorescence microscope (10). The test was considered positive when a T. gondii division was observed. Mouse inoculation. OF1 SWISS female mice (30 g) were given injections of AF, FB and PL when possible. After 6 weeks, toxoplasmic serological tests were carried out in mice, and T. gondii cysts were sought in mouse brains by light microscopy. A positive serological test was considered a possible criterion and detection of a parasite was considered a confident criterion of infection, except when only the PL result was positive (since it may block T. gondii and the parasite may not be able to infect the fetus). Antigens. ELIFA antigens were prepared from 1010 T. gondii tachyzoites of the RH strain. The peritoneal exudates from the infected mice were suspended in 154 mM NaCl at 378C. Following mechanical lysis, cell trypsinization (0.05% trypsin for 15 min at 378C) and centrifugation (15 min at 300 3 g and 48C), the first antigen fraction (S1) was obtained after three cycles of freezing (2808C) and thawing (378C). The S1 supernatant was separated after centrifugation (20 min at 600 3 g) and stored at 48C. The insoluble material was resuspended with 7 ml of 145 mM PBS (pH 7.2)–0.002% Tween 80, subjected to two ultrasound cycles with amplitudes of 4 and 30 (five times for 30 s each, separated by a 1-min pause), and cooled in ice. After a 3-h freezing-thawing cycle, the second antigen fraction (S2) was obtained after centrifugation (20 min at 600 3 g). S1 and S2 were mixed to obtain ELIFA antigens. The protein content was 5 mg/ml. Antigens for IB were prepared from 1010 T. gondii tachyzoites of the RH strain. The peritoneal exudates from the infected mice were suspended in 154 mM NaCl at 378C. After mechanical lysis of cells by passage through needles with different diameters (0.8, 0.5, and 0.45 mm), the cellular suspension was filtered on a 3-mm polycarbonate filter and then washed three times by centrifugation (10 min at 300 3 g and 48C) in 145 mM PBS (pH 7.2). Parasites were lysed in a final solution concentration of 5% sodium dodecyl sulfate and heated for 3 min at 1008C. The solution was filtered through a 0.22-mm-pore-size Millipore filter. The protein content was 25 mg/ml. IB and ELIFA antigens were distributed in 100-ml aliquots and stored at 2808C until used. ELIFA. The method of Pinon et al. (34) was modified as follows. Briefly, ELIFA antigen was deposited linearly (15 ml) on the absorbent side of a cellulose-acetate membrane (Sartorious; 25-160) previously saturated with 0.05 M Tris-glycine buffer (pH 8.8). The excess antigen was then removed from the membrane and placed on an appropriate carrier in a electrophoresis tank (SEBIA). AF was concentrated 10-fold on Minicon ultrafiltration cells (Amicon,

J. CLIN. MICROBIOL. Paris, France) before deposition. AF, FB, CB, MS, and IS, were applied in 15-ml drops. The first electrophoresis was run at 150 V for 3 h 15 min. Sera were reapplied, and electrophoresis was repeated. After this and a 10-min membrane stabilization, they were cut from the membrane, washed for 5 min in 154 mM NaCl–0.5% TFD4 (Franklab, Saint Quentin-en-Yvelines, France) with agitation, and placed in filtration cells (Plastimarne, Epernay, France). Goat anti-human IgG and anti-human IgM labeled with peroxidase (Institut Pasteur) were diluted 1:15,000 and 1:7,500, respectively, in 145 mM PBS (previously filtered through a 0.22-mm-pore-size filter)–0.1% bovine albumin–0.1% Tween 20 (pH 7.2). The tank was filled with the last buffer (2 min at 5.8 ml/min). Then, the filtered conjugate was added to the cells (30 min at 0.4 ml/min) before performing two more steps with PBS (10 min at 5.8 ml/min) and 10 mM Tris-HCl (pH 7.6), respectively. A Biomed minimonitor drove the various steps. Reading was performed after membrane submersion in a solution containing 10 mg of 3,39diaminobenzidine and 40 ml of 30% H2O2 in 40 ml of 10 mM Tris-HCl (pH 7.6). The reaction was stopped by three 5-min washes in distilled water. The ELIFA was positive if at least one novel precipitation arc was observed from the fetus or infant and if the specificity was different from that of the mother or identical but with a higher concentration. Immunoblot. A modified version of the procedure described by Remington et al. (38) was used. Briefly, the T. gondii antigen was denatured for 3 min at 1008C with b-mercaptoethanol plus sodium dodecyl sulfate and separated by electrophoresis in a 12.5% polyacrylamide minigel at 250 V for 45 min at 158C on the PHAST-SYSTEM (Pharmacia, Saint Quentin-en-Yvelines, France). After being blotted on 0.45-mm-pore-size Hybond nitrocellulose sheets (Amersham, Les Ulis, France) for 45 min at 25 mA and 68C on the PHAST-SYSTEM, the membranes were fixed for 15 min at 378C and stored at 2208C until used. The nitrocellulose sheets were saturated in 145 mM PBS–5% nonfat milk (pH 7.2) for 20 min at 378C. Samples (MS, FB, AF, CB, and IS) from mothers and fetuses or from mothers and infants were diluted 1:20 in 145 mM PBS (pH 7.2)–5% nonfat milk–0.01% Triton X-100 and added to the nitrocellulose sheets for 1 h at 378C with mechanical agitation. After four 2-min washes in PBS, goat anti-human IgG (1:1,500), IgM (1:1,500), and IgA (1:750) phosphatase alkaline conjugates (Calbiochem, France-Biochem, Meudon, France) diluted in 145 mM PBS–5% nonfat milk–0.01% Triton X-100 (pH 7.2) were added, and the mixtures were incubated for 1 h at 378C. After a new cycle of washing, bands were visualized with 0.15 mM nitroblue tetrazolium plus 0.15 mM 5-bromo-4-chloro-3-indolyl phosphate in phosphate buffer (pH 9.5) (100 mM Tris, 100 mM NaCl, 100 mM MgCl2, 0.025% Triton X-100) for 5 min. The reaction was stopped by two washes in distilled water for 5 min each. IBs were considered positive when at least one clear specific IgG band was present in fetal or infant samples either not recognized by MS or with the same specificity but higher concentration (16). For IgMs and IgAs, the same criterion was used for FB, CB, and IS collected before day 10. From day 10 on, any IgM or IgA band present in sera was considered positive. MS were used as control and as comparative patterns for IgGs, IgMs and IgAs. The IB was considered positive in mothers when at least one clear specific IgG and/or IgM and/or IgA band was present. The reproducibility of this test was calculated from the data obtained after performing the IB test twice at two different dates, in all the samples obtained from 20 of the 48 cases. Diagnosis criteria used. Detection of T. gondii in AF and/or FB (in vitro culture, mouse inoculation) was considered a confident criterion, but the presence of T. gondii in PL was only a criterion of suspicion. In serological testing, the detection of novel (synthesized by fetus) IgM anti-T. gondii antibodies by IFAT, ELISA, and ISAGA in FB, CB, and IS, free of maternal blood, until day 5 was considered a confident criterion. The same criterion applied to ELIFA novel IgG and/or IgM anti-T. gondii antibodies in any sample. High levels of LDH and g-GT in FB were considered nonconfident criteria. From day 10, contamination of IS by maternal blood was excluded as a reason for detection of novel IgM anti-T. gondii antibodies. A persistent positive level of IgG anti-T. gondii antibodies between 6 months and 1 year confirmed a diagnosis of congenital toxoplasmosis (Tables 1 and 2). Statistical testing. To compare the data obtained, x2 and percent comparison tests were used.

RESULTS Dates of infection. The data obtained are shown only by case and not by sample. Among the 48 cases, 27 newborns had proven congenital toxoplasmosis (CT1) and 21 did not (CT2). Of the 27 CT1 newborns, 3 babies were born of mothers infected during the first trimester of pregnancy, 9 were born of mothers infected during the second, and 7 were born of mothers infected during the third; for 8, the dates of infection were not possible to determine precisely. The 27 CT1 newborns were diagnosed as indicated in Table 1. Of the 21 CT2 newborns, 4 were born of mothers infected by the time of conception (periconceptional), 8 were born of mothers infected during the first trimester of pregnancy, 5 were born of mothers

VOL. 33, 1995

SERODIAGNOSIS OF CONGENITAL TOXOPLASMOSIS

1481

TABLE 1. Condensed results for 27 cases of proven congenital toxoplasmosis Case no.

IgMa

Human fibroblast culturea

Inoculation in micea

g-GT

LDH

1 2 4 5 6 7 8e 9 10 11 12 14 23 24 26 27e 28 29 30 31e 32 33 34e 35 36 37e 44

2 1 1 2 2 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 2 2 1 2

NDd 1 ND 1 ND ND 1 ND 2 ND ND 1 ND ND 2 2 2 ND ND ND 1 ND 1 1 1 ND 2

ND 1 ND 1 2 2 1 1 2 ND ND 1 ND ND 1 2 1 ND ND ND 1 ND 1 1 1 ND 1

ND ND ND ND ND ND 2 ND ND ND 1 ND ND ND 1 2 2 ND ND ND ND ND 1 ND ND ND 2

ND ND ND ND ND ND 2 ND ND ND 1 ND ND ND 2 2 2 ND ND ND ND ND 1 ND ND ND 2

ELIFA Igb

G 2 M M G, G, G 2 G G, 2 2 G G, 2 G 2 G G G, 2 G 2 G G G, 2

M M

M

M

M

M

IB Igb

G M G, G, G, G, G, M G, G, G, G G, G, G, 2 M G, G, G, G, G, M G 2 G, G

Clinical signsc

rc (left eye) M, M, M M, M,

A A

ic and rc (left eye)

A A

M A A M, A M, A M M M M A M

rc ic

ic (suspicion)

M, A

a

IgM antibodies to T. gondii. For further information, see Table 3. Ig (G 1 M 1 A) antibodies to Toxoplasma. rc, retinochoroiditis; ic, intracranial calcifications. d ND, not done. e Patients 8 and 37 had recurrent toxoplasmosis as indicated by serological testing. Patient 27 had a spontaneous abortion. Patient 31 had a temporary muscular hypotonia, and patient 34 had a therapeutic abortion. b c

infected during the second, and 2 were born of mothers infected during the third; for 2 the dates of infection were not possible to determine precisely (Table 2). Nonspecific markers. LDH and g-GT data are shown in Tables 1 and 2. Contamination of FB by mother’s blood was not observed in this study. Of the six blood samples obtained from infected fetuses (means 6 two standard deviations, 408 6 290 UI/liter for LDH and 128 6 298 UI/liter for g-GT), two had elevated levels of LDH and four had elevated levels of g-GT. However, of the six negative samples (311 6 88 UI/liter for LDH and 58 6 90 UI/liter for g-GT), one had elevated levels of LDH and another had elevated levels of g-GT. Prenatal diagnosis. The best antenatal diagnoses of congenital toxoplasmosis with FB was obtained with Ig(G 1 M 1 A and/or G 1 M) IB and mouse inoculation, with a common sensitivity of 71.4% (five of seven). Ig(G 1 M) ELIFA, IgM ELISA, and IgM ISAGA allowed the diagnosis of three (42.9%), three, and four (57.1%) cases, respectively. The best antenatal diagnoses from AF was obtained with mouse inoculation, with a sensitivity of 75% (six of eight). With cell culture, the sensitivity was 42.9% (three of seven). Early diagnosis of congenital toxoplasmosis before birth and at birth was evaluated according to the different types of samples examined (Tables 3 and 4). Diagnosis at birth. Mouse inoculation gave a sensitivity of 100% (four of four) with placentas. The highest sensitivity in neonatal diagnosis with CB was obtained with Ig(G 1 M 1 A and/or G 1 M) IB, with 66.7% (8 of 12). IgM ISAGA, Ig(G 1 M) ELIFA, and IgM ELISA permitted the diagnosis of 10 of

18, (55.6%), 6 of 15 (40%), and 5 of 18 (27.8%) cases, respectively. Early diagnosis after birth. A total of 12 cases were diagnosed by IB in the first 4 months after birth, compared with 11 by IgM ISAGA in the first 3 months, 10 by ELIFA in the first 10 months, and 7 by IgM ELISA in the first 5 months. Overall IB and ELIFA data in serodiagnosis. Among the 48 cases, 32 (66.7%) were consistent by both IB and ELIFA. Figures 1 and 2 show IgG, IgM, and IgA anti-T. gondii prevalences obtained by IB and ELIFA. The number of bands obtained by IB was larger than the number of ELIFA arcs for the same samples. Antibodies were directed against T. gondii antigens with high and low molecular weights: from 18,000 to 185,000, from 18,000 to 170,000, and from 22,000 to 185,000 for IgG, IgM, and IgA, respectively. Nevertheless, most of them are in the 18,000 to 135,000 range. Within this range, the most prevalent for were 30,000 and 35,000 IgG, IgM, and IgA; 22,000 and 90,000 for IgG and IgM; 18,000 and 75,000 for IgG; 135,000 for IgM; and 115,000 for IgA. Reproducibility, sensitivity, specificity, and positive predictive value. The reproducibility of IB has been evaluated at 90% (95% confidence interval 5 68 to 99%). From the statistical point of view, the present study revealed no significant differences between IB and ELIFA sensitivities. In addition, the number of IB bands observed was larger than the number of ELIFA arcs. Results are listed in Table 5. False-positive results by IB. To characterize IB in the serodiagnosis of congenital toxoplasmosis, five false-positive cases are described concisely (Table 2). In these cases, the sizes of

1482

CHUMPITAZI ET AL.

J. CLIN. MICROBIOL.

TABLE 2. Condensed laboratory results for 21 cases of noncongenital toxoplasmosisa Case no.

IgMb

Human fibroblast cultureb

Inoculation in miceb

g-GT

LDH

ELIFA Ig

IB Ig

3 13 15 16 17 18 19 20 21 22 25 38 39 40 41 42 43 45 46 47 48

2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2

2 ND ND ND ND 2 2 2 2 2 ND 2 ND ND 2 ND 2 2 2 2 ND

2 ND ND ND ND 2 1 2 ND 2 ND 2 ND ND 2 ND 2 2 2 2 ND

NDc ND ND ND ND 1 ND 2 ND 2 ND 1 ND ND ND ND ND 2 ND 2 ND

ND ND ND ND ND 2 ND 2 ND 1 ND 2 ND ND ND ND ND 2 ND 2 ND

2 G 2 2 2 2 2 2 2 2 2 2 G 2 G 2 2 2 2 2 2

G 2 2 A 2 M 2 2 2 2 2 M, A 2 A 2 2 2 2 2 2 2

a

In these cases, IgG toxoplasmic serologies have been monitored for up to one year until they were under the respective cutoffs. IgM anti-Toxoplasma antibody. For further information, see Table 4. c ND, not done. b

bands were 58,000 for IgG, 80,000 and 27,000 for IgM, and 54,000 for IgA. In addition, in two false-positive cases the molecular weights were 230,000 for IgM and 210,000 for IgA. False-negative results by IB. Two cases of false-negative results were revealed at the end of the present study. Cases 27 and 36 were diagnosed by ELIFA but not by IB (Table 1). Termination of pregnancy. Among the study cases, one spontaneous abortion was observed at week 23 of gestation. The fetus was confirmed to be infected by T. gondii. The serological test was positive by ELIFA, and the LDH level was 352 UI/liter. In another patient with a severe case of congenital toxoplasmosis, a therapeutic abortion was performed. In vitro culture and mouse inoculation results were positive with FB and AF. LDH and g-GT levels in FB were high: 647 and 352 UI/liter, respectively. Two novel IgM anti-Toxoplasma bands were detected by IB in FB. After the therapeutic abortion,

TABLE 3. Laboratory results of proven congenital toxoplasmosis cases before and at birth

disseminated toxoplasmosis was confirmed in the fetus by in vitro culture and mouse inoculation. Clinical congenital toxoplasmosis. Four babies had clinical evidence of congenital toxoplasmosis and one baby had a clinical suspicion (Table 1). Of the three infants with clinical ocular toxoplasmosis, all were positive by IB, two were positive by ELIFA, and one was positive by IgM ELISA. The other 20 cases were subclinical. DISCUSSION In this study, the epidemiological data concerning the prevalence of congenital toxoplasmosis are not significant because only proven cases (CT1 or CT2) were included. Therefore, this section will focus mainly on IB and its relationship with the other methods to investigate its contribution towards prenatal, at-birth and after-birth diagnosis of congenital toxoplasmosis. IB was performed with the PHAST-SYSTEM, which allowed reproducible results to be obtained since electrophoresis and blot are semiautomatic with a computer program. Only the

No. of cases with: Method

IB (G 1 M 1 A) ELIFA (G 1 M) ELISA (M) ISAGA (M) IFAT (M) In vitro culture Mouse inoculation

1a

2a

FB

FB

5 3 3 4 0 0 5

2 4 4 3 7 7 2

AF1a

AF2a

CB1 or PL1a,b

CB2 or PL2a,b

1 0 0 1 0 3 6

7 8 8 7 8 4 2

8 6 5 10 0 1 4

4 9 13 8 18 1 0

a FB1, positive fetal blood by the technique used; FB2, negative fetal blood by the technique used; AF1, positive amniotic fluid, AF2, negative amniotic fluid; CB1, positive cord blood; CB2, negative cord blood; PL1, positive placenta; PL2, negative placenta. b Cord blood was used for serological techniques, and placenta was used for cell culture and mouse inoculation.

TABLE 4. Laboratory results of noncongenital toxoplasmosis cases before and at birth No. of cases with: Method

IB (G 1 M 1 A) ELIFA (G 1 M) ELISA (M) ISAGA (M) IFAT (M) In vitro culture Mouse inoculation a b

1a

2a

FB

FB

0 0 0 0 0 0 0

8 8 8 8 8 8 8

See Table 3, footnote a. See Table 3, footnote b.

AF1a

AF2a

CB1 or PL1a,b

CB2 or PL2a,b

0 1 0 1 0 0 0

6 7 6 5 6 9 8

1 2 0 1 0 0 1

8 12 14 13 14 4 6

VOL. 33, 1995


1483

TABLE 5. Sensitivity, specificity, positive predictive value of the different techniques used, and IB agreement in diagnosis of congenital toxoplasmosis Test

FIG. 1. Prevalence of IgG, IgM, and IgA anti-T. gondii antibodies obtained by IB in the diagnosis of congenital toxoplasmosis. Bars represent the 95% confidence interval. CT1, confirmed cases; CT2, negative cases. The specificity of IB can be 100% when diagnosis is based on Ig(G 1 M), Ig(G 1 A), and Ig(G 1 M 1 A)-positive results. IgE anti-T. gondii antibody was not explored in this study.

immunological part is manual, which may partly account for the reproducibility level of IB in diagnosing congenital toxoplasmosis. In the present study, the levels of LDH and g-GT, two nonspecific prenatal paremeters, varied within the range mentioned in literature, mainly for g-GT (2, 11). To compare the sensitivity of the IB with the other techniques used, only the IgG and IgM data have been taken into account. For ELISA, IFAT, and ISAGA, only the IgM data have been included, because the IgG data generally fail to give information in the early diagnosis of congenital toxoplasmosis. It is important to compare the sensitivity of IB in prenatal diagnosis with that of other techniques. With FB, Desmonts et al. (11) reported sensitivities of 66.7 and 44.4% by mouse inoculation and IgM ELISA, respectively. Using AF, Desmonts et al. (11) found a similar sensitivity to ours by mouse inoculation (77.8%). It is interesting to compare the sensivities of these methods with that of a recent molecular biologyrelated technique, PCR. Several authors have evaluated the sensitivity of this method in prenatal diagnosis from AF. It varied from 70 to 100% (5, 13, 19, 21). In most of these studies, fewer than 11 patients were studied (5, 13, 19). Therefore, our data are comparable to theirs in most of the cases. In the 36-case study, Hohlfeld et al. (21) suggested that FB sampling,

FIG. 2. Prevalence of IgG and IgM anti-T. gondii antibodies obtained by ELIFA in the diagnosis of congenital toxoplasmosis. Bars represent the 95% confidence interval. CT1, confirmed cases; CT2, negative cases. The specificity of ELIFA can be 100% when diagnosis is based on Ig(G 1 M)-positive results. IgA ELIFA and IgE anti-T. gondii antibodies were not tested in this study.

Positive Sensitivity Specificity predictive (%) (%)a value %

LDH g-GT In vitro culturec,d

33.3 66.6 40.0

54.5 72.7 100

50.9 77.6 100

Mouse inoculationd,e

62.5

100

100

IFAT (M) ELISA (M) ISAGA (M) ELIFA (G 1 M) IB (G 1 M 1 A)

7.4 29.6 44.4 74.1 92.6

97.8 97.8 95.7 93.5 89.1

89.6 97.2 93.5 94.2 92.4

% Agreement with IBb

58.3 (7/12) 66.7 (8/12) 69.2 (9/13) (FB) 69.2 (9/13) (AF) 91.7 (11/12) (FB) 53.8 (6/13) (AF) 50.0 (24/48) 62.5 (30/48) 72.9 (35/48) 66.7 (32/48) 100 (48/48)

a The specificity of IB can be 100% when diagnosis is based on Ig(G 1 M)-, Ig(G 1 A)-, and Ig(G 1 M 1 A)-positive results. b The ratios in parentheses correspond to the number of corresponding cases in both tests in relation to the number of total cases. For a better comparison of IB, in vitro culture, and mouse inoculation, IB agreement was calculated according to the number of samples used (FB and AF). c Results obtained from 15 CT1 and 13 CT2 cases. d In vitro culture and mouse inoculation were considered positive from fetal blood (FB) or amniotic fluid (AF) or placenta (PL). Results are grouped by case. e Results obtained from 16 CT1 and 10 CT2 cases.

which is considered riskier than AF sampling, will no longer be necessary in prenatal diagnosis if the PCR assay is used on AF. In the present study, serodiagnosis by IB is better in FB sampling than in AF sampling. These conclusions are not contradictory, since PCR seeks nucleic acids derived from the parasite and IB seeks antibodies to T. gondii produce by the host. To know the exact relationship between IB diagnosis and PCR, further study is necessary. However, considering that the risk of cordiocentesis is higher than that of amniocentesis, IB can still be used at birth and after birth. Thus, taking into account technical considerations alone, IB performs at least as well in prenatal diagnosis from FB as do the other methods described in the literature. It is interesting to compare the sensitivity of IB in the diagnosis of congenital toxoplasmosis at birth with that of other methods. Our data obtained by mouse inoculation from PL samples agree with those of Desmonts et al. (11). In other studies, the sensitivities were 74.5% (6) and 87.9% (31). In newborns, IgM and IgA antibodies to T. gondii are two criteria of congenital infection, usually if the mother was infected at the end of the second or during the third trimester of pregnancy. Ig(G 1 M) ELIFA, IgM ISAGA, and IgM ELISA also permitted diagnosis at birth, while this was not possible with IgG ISAGA and IgG ELISA. Using ELIFA, Leroux et al. (25) diagnosed 7 of 16 cases, (43.8%) at birth. Their results are consistent with ours. In our study, the highest sensitivity in antenatal diagnosis from CB was obtained by IB (G 1 M 1 A and/or G 1 M), which confirms the efficiency of this method in diagnosis at birth. Congenital toxoplasmosis was diagnosed earlier after birth by IB than by ELIFA. In one case of infected twins, one baby’s serological test was positive by ELIFA 2 months before the other one’s (30). This was also observed with IB, but the difference was mainly quantitative. Interestingly, diagnosis was possible 2 to 6 months earlier by ELIFA than by IFAT, hemagglutination, or agglutination (32). In another study with ELIFA, congenital toxoplasmosis was diagnosed in 5 of 16 patients in the 3 months after birth (25). Therefore, IB could be superior to ELIFA even for early detection after birth.

1484

CHUMPITAZI ET AL.

IB has a sensitivity of the same order as or higher than that of the other techniques used in diagnosis of congenital toxoplasmosis. Using IB, Franck et al. (16) diagnosed 11 of 12 cases of congenital toxplasmosis, with a sensitivity similar to ours. In a different study, the sensitivities of ELIFA and IgM IFAT were estimated at 89 and 14%, respectively, at day 150 after birth (33). In a rabbit model, the sensitivities of cell culture, mouse inoculation, and PCR were evaluated at 25, 62, and 37% respectively (20). In other studies, the sensitivities of IgA ELISA were 89.1 and 100% (9, 44). Reported sensitivities for IgM ISAGA varied from 62 to 76.9% (4, 12, 44). Similarly, the sensitivities of IgM ELISA varied from 50 to 71% (4, 33, 44). Nevertheless, in the present study, the difference between ELIFA and IB sensitivities was not significant from a statistical point of view. The IB method also allowed us to determine the apparent molecular weight of toxoplasmic antigens detected by serum anti-T. gondii antibodies from fetuses or infants. Several years ago, Sharma et al. reported three major antigens recognized by human IgG and IgM antibodies; they had molecular weights of 6,000, 22,000, and 32,000 (42). Other specificities were also described from 6,000 to 150,000 (14, 28). Our study revealed higher molecular weights for both types of antibodies. In addition, the IB data obtained with IS, CB, and FB showed that antibody specificities and intensities could be different between mothers, fetuses, and infants, as described previously (16, 35, 37). This could allow the diagnosis of congenital toxoplasmosis. Comparable results to ours were obtained by Remington et al., who described IgG and IgM anti-T. gondii specificities from 21,000 to 94,000 and from 31,000 to 92,000, respectively (38). In another study, IgG anti-T. gondii specificities varied from 35,000 to 115,000 in sera from 12 infants with congenital toxoplasmosis (37). An IgM band up to 4,000 had a very low prevalence in infants but was present in all seven mothers under study (37). Such low-relative-mass bands were not observed in the present study. Franck et al. observed IgG, IgM, and IgA anti-T. gondii specificities from 21,000 to 97,000 in 12 cases of congenital toxoplasmosis (16). At the moment, diagnosis based only on the molecular weight of IgGs from fetuses or newborns has not been well consolidated. However, in this study, the major bands, of 22,000, 30,000, 35,000, and 90,000, may be interesting for diagnosis. In routine diagnosis, several techniques are used to confirm fetal infection. This section is devoted to false-positive results obtained by IB, taking into account the cutoffs and criteria of congenital toxoplasmosis defined above. In general, false-positive results associated with IgGs may be linked on the one hand to natural IgGs (22, 36) and on the other hand to the possible nonspecific toxoplasmic antigen interaction with IgGs. Nevertheless, in our study, the IgG false-positive results could be due to improper storage conditions (at 48C) of MS during the 1-week interval between the first and second IB tests. Thus, only the number of IgG bands from the mother was smaller in the second experiment when compared with the first one. False-positive IgM results obtained by IB in MS may be due to natural IgM, rarely to antinuclear antibodies, and mainly to rheumatoid factor, whose level increases during pregnancy (22, 36). Nevertheless, in newborns up to 6 months old, these risks are almost nonexistent (12, 22). Another, more delicate case is that maternal IgM anti-T. gondii antibody is present in the CB in 2 to 5% of cases, without indicating newborn infection. In these borderline cases, a band or an arc of a different specificity, observed by IB or ELIFA in CB, can confirm the diagnosis of congenital toxoplasmosis. In the present study, a falsepositive IgM results was observed in one case after 6 months and in the other case at day 46. The first case may be explained

J. CLIN. MICROBIOL.

by natural IgM, antinuclear antibodies, or rheumatoid factor, but the second remains to be explained. The origin of false-positive IgA antibodies observed by IB is not known. Indeed, it could not be explained by natural IgAs, antinuclear antibodies, or rheumatoid factor (39, 40). A high molecular weight could not be systematically involved, because this was found in several CT1 cases. To avoid false-positive results with IB (mainly after birth), it was essential to confirm or to complete the results by another method or a further IB class prevalence analysis. Thus, it appeared that grouping IgG-IgM (IB, ELIFA)-positive, IgGIgA-positive, or IgG-IgM-IgA (IB)-positive responses corresponded to confident diagnoses. Concerning the false-negative cases revealed by IB, ELIFA may be a complementary technique to diagnose congenital toxoplasmosis. Nevertheless, in prenatal diagnosis, cell culture and mouse inoculation may also be complementary to IB. Several hypotheses have been put forward in the literature to explain the false-negative results: (i) stage-specific antibodies (18); (ii) maternally transmitted IgG competition with fetal IgM anti-T. gondii (39); and (iii) different strains (17, 43). The reasons for our false-negative results are unknown, although these results may be due to one or more of the three points mentioned above. Gross et al. (17) found a positive IB case in a child with hydrocephalus and retinochoroiditis. Using other methods, Wong et al. (44) described a positive IgE ELISA case among six patients with retinochoroiditis. All the other tests, i.e., IgE ISAGA, IgM ISAGA, and IgA ELISA, were negative. It is interesting that, in themselves, anti-T. gondii antibody levels in serum are not enough to confirm positive ocular toxoplasmosis (42). Before the exact relationship between IB diagnosis and clinical ocular congenital toxoplasmosis is clear, another study is necessary. In conclusion, IB allows the comparison of immunological patterns and the determination of the origin of antibodies, i.e., transmitted by the mother or synthesized by the fetus or infants. In addition, IB results could be similar to those of ELIFA in the diagnosis of congenital toxoplasmosis. IB, which also has a better resolution, can be carried out earlier and allows a finer analysis, even though the results may be less specific. The positive predictive value of (G 1 M 1 A and/or G 1 M) IB in the diagnosis is comparable to that of the other specific serological tests used (IgM ELISA, IgM ISAGA and G 1 M ELIFA). Confident positive criteria could be readily related to Ig(G 1 M), Ig(G 1 A), and Ig(G 1 M 1 A) anti-T. gondii antibodies obtained by IB or ELIFA. Both techniques showed better sensitivities than IgM IFAT, IgM ELISA, and IgM ISAGA and can be complementary to in vitro culture and mouse inoculation in the diagnosis of congenital toxoplasmosis. ACKNOWLEDGMENTS We thank J. Franck, F. Tossetti, S. Pozet (BioMe´rieux), M. M. Lombard, M. Hacard, E. Bouge, S. Durville, H. Hidalgo-Fricker, and M. Mertiny for their help in this work. REFERENCES 1. Ambroise-Thomas, P., J. P. Garin, and A. Rigaud. 1966. Ame´lioration de la technique d’immunofluorescence par l’emploi de contre-colorants. Application aux Toxoplasmes. Presse Med. 74:2215–2216. 2. Berrebi, A., Y. Cohen-Khallas, M. H. Bessie`res, M. Rolland, M. F. Sarramon, and A. Fournie. 1992. Valeur pre´dictive des signes non spe ćifiques d’infection foetale dans la toxoplasmose congeńitale. J. Gynecol. Obstet. Biol. Reprod. 21:791–794. 3. Berrebi, A., W. E. Kobuch, M. H. Bessie`res, M. C. Bloom, M. F. Sarramon, C. Roques, and A. Fournie. 1994. Valeur pre´dictive des signes non

VOL. 33, 1995

4.

5. 6.

7. 8. 9.

10. 11. 12.

13.

14. 15.

16. 17.

18.

19. 20. 21.

spećifiques d’infection foetale dans la toxoplasmose congeńitale. Lancet 344:36–39. Candolfi, E., M. H. Bessie`res, P. Marty, B. Cimon, F. Gandilhon, H. Pelloux, and P. Thulliez. 1993. Determination of a new cut-off value for the diagnosis of congenital toxoplasmosis by detection of specific IgM in an enzyme immunoassay. Eur. J. Clin. Microbiol. Infect. Dis. 12:396–398. Cazenave, J., F. Forestier, M. H. Bessie`res, B. Broussin, and J. Begueret. 1992. Contribution of a new PCR assay to the prenatal diagnosis of congenital toxoplasmosis. Prenatal Diagn. 12:119–128. ´ tude Couvreur, J., G. Desmonts, G. Tournier, and M. Szusterkac. 1985. E d’une se´rie homoge `ne de 210 cas de toxoplasmose congeńitale chez des nourrissons âge´s de 0 à 11 mois et de´piste´s de façon prospective. Sem. Hop. Paris. 61:3015–3019. Couvreur, J., P. Thulliez, F. Daffos, C. Aufrant, Y. Bompard, A. Gesquie`re, and G. Desmonts. 1991. Foetopathie toxoplasmique. Arch. Fr. Pediatr. 48: 397–403. Daffos, F., F. Forestier, M. Capella-Pavlosky, P. H. Thulliez, C. Aufrant, D. Valenti, and W. L. Cox. 1988. Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis. N. Engl. J. Med. 318:271–275. Decoster, A., F. Darcy, A. Caron, D. Vinatier, D. Houze de l’Aulnoit, G. Vittu, G. Niel, F. Heyer, B. Lecolier, M. Delcroix, J. C. Monnier, M. Duhamel, and A. Capron. 1992. Anti-P30 antibodies as prenatal markers of congenital toxoplasma infection. Clin. Exp. Immunol. 87:310–315. Derouin, F., M. C. Mazeron, and C. Garin. 1987. Comparative study of tissue culture mouse inoculation methods for demonstration of Toxoplasma gondii. J. Clin. Microbiol. 25:1597–1600. Desmonts, G., F. Forestier, P. Thulliez, F. Daffos, M. Capella-Pavlovsky, and M. Chartier. 1985. Prenatal diagnosis of congenital toxoplasmosis. Lancet i:500–504. Desmonts, G., Y. Naot, and J. S. Remington. 1981. Immunoglobulin Mimmunosorbent agglutination assay for diagnosis of infectious diseases: diagnosis of acute congenital and acquired Toxoplasma infections. J. Clin. Microbiol. 14:486–491. Dupouy-Camet, J., M. E. Bougnoux, S. Lavareda de Souza, P. Thulliez, M. Dommergues, L. Mandelbrot, T. Ancelle, C. Tourte-Schaefer, and R. Benarous. 1992. Comparative value of polymerase chain reaction and conventional biological tests for the prenatal diagnosis of congenital toxoplasmosis. Ann. Biol. Clin. 50:315–319. Erlich, H. A., G. Rodgers, P. Vaillancourt, F. G. Araujo, and J. S. Remington. 1983. Identification of an antigen specific immunoglobulin M antibody associated with acute Toxoplasma infection. Infect. Immun. 41:683–690. Excler, J. L., M. A. Piens, H. Maisonneuve, E. Pujol, and J. P. Garin. 1985. De´pistage de la toxoplasmose acquise chez la femme enceinte et de la toxoplasmose congeńitale chez le nouveau-ne´. Enque ˆte mene é dans les maternite´s des Hospices Civils de Lyon pour les anne és 80, 81 et 82. Lyon Med. 253:33–38. Franck, J., C. Mary, M. Laugier, H. Dumon, and M. Quilici. 1992. Apport du Western blot au diagnostic de la toxoplasmose congeńitale. Bull. Soc. Fr. Parasitol. 10:3–11. Gross, U., J. Mu ¨ller, T. Roos, L. Schrod, and J. Heesemann. 1992. Possible reasons for failure of conventional tests for diagnosis of fatal congenital toxoplasmosis: report of a case diagnosed by PCR and immunoblot. Infection 20:149–152. Gross, U., T. Roos, D. Appoldt, and J. Heesemann. 1992. Improved serological diagnosis of Toxoplasma gondii infection by detection of immunoglobulin A (IgA) antibodies against P30 by using the immunoblot technique. J. Clin. Microbiol. 30:1436–1441. Grover, C. M., P. Thulliez, J. S. Remington, and J. C. Boothroyd. 1990. Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid. J. Clin. Microbiol. 28:297–301. Hitt, J. A., and G. A. Filice. 1992. Detection of Toxoplasma gondii parasitemia by gene amplification, cell culture, and mouse inoculation. J. Clin. Microbiol. 30:3181–3184. Hohlfeld, P., F. Daffos, J.-M. Costa, P. Thuillez, F. Forestier, and M. Vidaud. 1994. Prenatal diagnosis of congenital toxoplasmosis with a polymerasechain reaction test on amniotic fluid. N. Engl. J. Med. 331:695–699.


1485

22. Konishi, E. 1993. Naturally occurring antibodies that react with protozoan parasites. Parasitol. Today 9:361–364. 23. Koppe, J. G., D. H. Loewer-Sieger, and H. De Roever-Bonnet. 1986. Results of a 20-year follow-up of congenital toxoplasmosis. Lancet i:254–256. 24. Koskiniemi, M., M. Lappalainen, and K. Hedman. 1989. Toxoplasmosis needs evaluation. Am. J. Dis. Child. 143:724–728. 25. Leroux, B., J. M. Pinon, D. Dupouy, C. Quereux, and R. Coffin. 1985. Toxoplasmose congeńitale: de´pistage et protocole de surveillance the ´rapeutique. Med. Hyg. 43:493–497. 26. McCabe, R., and J. S. Remington. Toxoplasmosis. The time has come. N. Engl. J. Med. 318:313–315. 27. Mirlesse, V., F. Jacquemard, and F. Daffos. 1993. Toxoplasmose au cours de la grossesse. Diagnostic et nouvelles possibilite´s the ´rapeutiques. Presse Med. 22:258–262. 28. Partanen, P., H. J. Turunen, R. T. A. Paasivuo, and P. O. Leinikki. 1984. Immunoblot analysis of Toxoplasma gondii antigens by human immunoglobulin G, M, and A antibodies at different stages of infection. J. Clin. Microbiol. 20:133–135. 29. Pelloux, H., P. Ciapa, A. Goullier-Fleuret, and P. Ambroise-Thomas. 1993. Evaluation du syste`me Vidas pour le diagnostic se ´rologique de la toxoplasmose. Ann. Biol. Clin. 50:875–878. 30. Pelloux, H., A. Goullier-Fleuret, J. Tous, and P. Ambroise-Thomas. 1992. Toxoplasmose congeńitale à re´ve´lation se ´rologique imme´diate ou diffe ´re é chez des jumeaux. Arch. Fr. Pediatr. 49:839–843. 31. Philippe, F., D. Lepetit, M. F. Grancher, A. Morel, and A. Henocq. 1988. Pourquoi surveiller les enfants dont les me`res ont eu une se´roconversion de toxoplasmose pendant la grossesse? Ann. Pediatr. 35:6–10. 32. Pinon, J. M., and N. Grison. 1982. Inte´reˆt des profils immunologiques compare´s ELIFA dans le diagnostic prećoce de la toxoplasmose congeńitale. Lyon Med. 248(hors Ser.):27–30. 33. Pinon, J. M., J. Poirriez, B. Leroux, D. Dupouy, C. Quereux, and J. P. Garin. 1987. Diagnostic prećoce et surveillance de la toxoplasmose congeńitale. Me´thode des profils immunologiques compare ´s. Presse Med. 16:471–474. 34. Pinon, J. M., H. Thoannes, and N. Gruson. 1985. An enzyme-filtration assay used to compare infant and maternal antibody profiles in toxoplasmosis. J. Immunol. Methods 77:15–23. 35. Potasman, I., F. G. Araujo, G. Desmonts, and J. S. Remington. 1986. Analysis of Toxoplasma gondii antigens recognized by human sera obtained before and after acute infection. J. Infect. Dis. 154:650–657. 36. Potasman, I., F. G. Araujo, and J. S. Remington. 1986. Toxoplasma antigens recognized by naturally occurring human antibodies. J. Clin. Microbiol. 24: 1050–1054. 37. Potasman, I., F. G. Araujo, P. Thulliez, G. Desmonts, and J. S. Remington. 1987. Toxoplasma gondii antigens recognized by sequential samples of serum from congenital infected infants. J. Clin. Microbiol. 25:1926–1931. 38. Remington, J. S., F. G. Araujo, and G. Desmonts. 1985. Recognition of different Toxoplasma antigens by IgM and IgG antibodies in mothers and their congenitally infected new-borns. J. Infect. Dis. 152:1020–1024. 39. Remington, J. S., and G. Desmonts. 1990. Toxoplasmosis, p. 89–195. In J. S. Remington and J. O. Klein (ed.), Infection diseases of the fetus and newborn infant—1990. The W. B. Saunders, Co., Philadelphia. 40. Rothova, A., G. S. Van Knapen, G. S. Baarsma, P. J. Kruit, D. H. LeowerSieger, and A. Kijlstra. 1986. Serology in ocular toxoplasmosis. Br. J. Ophthalmol. 70:615–622. 41. Sever, J. L., J. H. Ellenberg, A. C. Ley, D. L. Madden, D. A. Fuccillo, N. R. Tzan, and D. M. Edmonds. 1988. Toxoplasmosis: maternal and pediatric findings in 23,000 pregnancies. Pediatrics 82:181–192. 42. Sharma, S. D., J. Mullenax, F. J. Araujo, H. A. Erlich, and J. S. Remington. 1983. Western blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies. J. Immunol. 131:977–983. 43. Ware, P. L., and L. H. Kasper. 1987. Strain-specific antigens of Toxoplasma gondii. Infect. Immun. 55:778–783. 44. Wong, S. Y., M.-P. Hajdu, R. Ramirez, P. Thulliez, R. Mcleod, and J. S. Remington. 1993. Role of specific immunoglobulin E in diagnosis of acute toxoplasma infection and toxoplasmosis. J. Clin. Microbiol. 31:2952–2959.