Toxoplasmosis Acquired during Pregnancy: Improved Serodiagnosis Based on Avidity of IgG

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1 691 Toxoplasmosis Acquired during Pregnancy: Improved Serodiagnosis Based on Avidity of IgG Maija Lappalainen, Pentti Koskela, Marjaleena Koskiniemi, Pirkko Ammala, Vilho Hiilesmaa, Kari Teramo, Kari O. Raivio, Jack S. Remington, and Klaus Hedman Department of Virology. Departments I and II ofobstetrics and Gynecology. and Children's Hospital. University ofhelsinki, and National Public Health Institute. Oulu, Finland; Stanford University School ofmedicine and Research Institute. Palo Alto Medical Foundation, Palo Alto. California Serodiagnostic methods were evaluated in prenatal screening for primary Toxoplasma infections acquired during pregnancy in the Helsinki area. Altogether 44,181 sera were obtained consecutively during each trimester from 16,733 mothers. All IgG-containing samples were first examined by a sensitive JL-capture (IgM) ELISA, and positive results were reassessed by IgM immunoblotting and indirect IgM ELISA. An assay measuring the avidity of toxoplasma IgG was used for the first time under screening conditions. Patients suspected to have recent toxoplasmosis were reexamined by IgA ELISA and selectively by the differential agglutination assay (HS/AC test) and IgE ELISA; 16 women with diagnostic increases in IgG titer, 36 with IgM fulfilling strict specificity criteria, and 25 with IgG of low avidity were identified. The measurement of IgG avidity was a highly specific and sensitive method suitable for verification of acute primary Toxoplasma infections during pregnancy. Toxoplasma gondii has a worldwide distribution and causes chronic infection in > 10 9 people. Congenital toxoplasmosis results from primary infection of the mother during pregnancy but, as a rule, not from reactivation of her latent infection [1]. The rate oftransmission from mother to fetus increases from 20%to ~70%with gestational age [1, 2]. Most children with intrauterine infection are initially asymptomatic; by early adulthood, however, the vast majority develop late manifestations such as chorioretinitis or neurologic defects [3-5]. Postnatal serodiagnosis is complicated by interference from transplacentally acquired maternal IgG and by variability in the expression of Toxoplasma IgM during infancy [6, 7]. Advances in the polymerase chain reaction methodology may in the future facilitate the diagnosis of congenital toxoplasmosis [8-10]. Received 22 June 1992; revised 21 September Grant support: Finnish Academy, Finnish Cultural Foundation. Finnish Insurance Institution. Finnish Medical Society Duodecirn, Orion Corporation Research Foundation. Paivikki and Sakari Sohlberg Foundation. Rhone-Poulenc Sante. Rinnekoti Research Foundation. and Sigrid Juselius Foundation. Finland; National Institutes of Health (AI ). Presented in part: II th European Meeting of Immunology. Espoo, Finland. June 1991 (abstract 15-19); 31st Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago. September-October 1991 (abstract 659); International Symposium of Recent DevelopmentsofObstetric, Perinatal. and Childhood Infections, Jerusalem, August The study was done with the approvals of the ethical committees ofboth the Helsinki University Central Hospital and the communities. Reprints or correspondence: Dr. K. Hedman. Department of Virology. University of Helsinki, Haartmaninkatu 3. SF Helsinki. Finland. The Journal of Infectious Diseases 1993;167: by The University of Chicago. All rights reserved /93/ $01.00 On account of the diversity or absence of symptoms, the detection of Toxoplasma infection during pregnancy has to be based not on clinical findings but on maternal serology [11]. However, the conventional single-serum assays do not make a clear distinction between a primary and chronic infection. The tendency of specific IgM to persist for a long time, and even at high levels, has been reported before and is not restricted to pregnancy [12-16]. According to several reports, the demonstration of specific IgA is useful in diagnosis of Toxoplasma infection. Encouraging IgA results have been obtained by using fractionated antigen preparations (P30) or whole parasites, especially in the diagnosis of congenital toxoplasmosis [17-22]. A new approach is to seek antibodies specific for the acute stage of Toxoplasma infection. In the form ofa differential agglutination assay (HSjAC test) or an ELISA using acute stage-specific antigens, this strategy showed promise in the separation of recent infections from those acquired in the distant past [23-25]. The search for specific IgE could be another useful approach in the serodiagnosis of recently infected pregnant women [12, 26]. An assay measuring the antigen-binding avidity (functional affinity) of IgG antibodies against T. gondii was recently developed to separate the low-affinity antibodies produced at an early stage ofinfection from those with a higher binding affinity that reflect past immunity [27, 28]. By this technique, primary infection can be identified using a single serum specimen. Similar IgG avidity tests for other pathogens have shown their value as both a frontline assay and a confirmatory test in distinguishing primary from secondary infections and in assessing the time of the initial antigenic challenge [29-34]. In this study we examine, under large-

2 692 Lappalainen et al. 1ID 1993;167 (March) scale screening conditions, the ability ofthis novel serodiagnostic approach to identify primary Toxoplasma infections acquired during pregnancy. Patients and Methods Patients The study area comprises four subdivisions of the metropolitan area ofhelsinki (Espoo, Helsinki, Kauniainen, and Vantaa), with 820,700 inhabitants. We examined women who became pregnant between 1 January 1988 and 30 June Upon antenatal booking at municipal maternity centers, all pregnant women received written information about this study, with requests to present themselves for voluntary blood tests in the 10th-12th (1st trimester), 20th-22nd (2nd trimester), and 35th-37th (3rd trimester) weeks of pregnancy. The participation rate (donors of ~ I sample) among all pregnant women in the study area was 90.2%. More demographic data are given elsewhere [35]. We obtained 44,181 prenatal sera (from 16,733 mothers); complete sets of 3 sera (samples obtained during each trimester) were obtained from 70.5% of the participants. Women with findings suggesting recent Toxoplasma infection (seroconversion or diagnostic [fourfold or more] increase in IgG, positive IgM, low avidity of IgG) were referred to the Helsinki University Central Hospital for obstetric consultation and treatment. Maternal and cord blood sera were obtained at delivery. Subsequent serum sampling was scheduled for postnatal months I, 3, 6, and 12; these results will be published separately. Methods IgG. The samples of uncitrated cubital venous blood were allowed to clot, and sera were separated by centrifugation and stored frozen. The primary screening test was indirect ELISA (Toxenz-G; Northumbria Biologicals, Cramlington, UK) for Toxoplasma IgG [36]. Sera with ambiguous results were reexamined by another IgG ELISA (Toxoplasma gondii IgG EIA kit; Labsystems, Helsinki). In this assay, specific IgG was quantified by serial dilution ofthe sera to obtain end-point titers with A 405 = as the cutoff. Increase in IgG titer was considered diagnostic when it was fourfold or greater. IgM. In sera containing specific IgG, Toxoplasma IgM was first measured by a highly sensitive [37] wcapture ELISA (Toxenz M II; Northumbria Biologicals). Positive specimens, as well as a similar number ofwcapture-negative samples as a control series, were reexamined by two other IgM tests and by the toxoplasma IgG avidity test. For quantitation of Toxoplasma IgM, we used an indirect ELISA (Toxoplasma gondii IgM EIA kit; Labsystems) that has been shown to be both sensitive and specific [38, 39]. Before the assay, all sera were depleted of IgG according to the directions ofthe manufacturer. Single dilutions of sera were placed in microtiter wells in duplicate, and results were expressed as EIA units relative to standard sera: ~40 = IgM-positive; = borderline; <20 = IgM-negative. Unless otherwise stated, IgM results given here are those obtained by the indirect IgM ELISA. IgM immunoblotting was carried out with antigen (T. gondii RH strain) obtained as described [40]. Briefly, intraperitoneal exudate from infected NMRI mice was filtered; the parasites were pelleted by centrifugation, washed in PBS, and sonicated in H 2 0. Supernatant from centrifugation at 10,000 g (60 min) was run in SDS-PAGE using 12% gels, and the separated proteins were transferred to nitrocellulose. The strips of sensitized nitrocellulose were subsequently treated with 5% defatted milk and 0.2% Triton X-100 in PBS, then with patients' sera at a dilution of 1:50. The bound IgM antibodies were marked with peroxidase-conjugated rabbit anti-human IgM (I: 100; Dakopatts, Glostrup, Denmark) and visualized by diaminobenzidine HCl (100 JLg/mL) plus H (0.0 I%). The criterion for positivity was simultaneous staining ofthe protein bands at and 25 kda [41-44]. Among our 79 seronegative controls, none showed an IgM reaction against this doublet ofbands (data not shown). Avidity ofigg. The avidity ofigg specific for T. gondii was measured by a protein-denaturing immunoassay (avidity ELISA) as described [27]. Briefly, serially diluted sera were placed in microtiter wells coated with the T. gondii antigen. After withdrawal ofthe sera, the antibodies attached to the antigen were eluted with a protein denaturant (6 M urea), and the proportion of residual to total antigen-bound IgG was quantified immunoenzymatically. During the first year, we used our own preparations of coated microtiter plates, and during the second, commercially coated IgG ELISA plates (Labsystems). The two preparations gave similar results. IgG avidity results ~15% were considered low; 16%-25%, borderline; and >25%, high. An increase in avidity in paired samples (within 200 days) was considered significant if it was simultaneously twofold or more and of ~ 10% in magnitude. The reproducibility ofthe avidity assay was evaluated by two pools of 5 sera. Pool A contained samples drawn during the acute phase of primary toxoplasmosis, and pool B, sera representing past immunity [27]. The interassay reproducibility was calculated from the results of45 successive "field" experiments during 5 months: the mean IgG avidity results in pools A and B were 10.5%and 49.8%,respectively, and the coefficients of variation were.20 and.15. For intraassay reproducibility, 10 parallel examinations were done. The respective coefficients ofvariation were.16 and.10. IgA andige ELISA andhs/ac test. We determined specific IgA antibodies from the mothers with suspected primary toxoplasmosis during pregnancy by a commercial double-sandwich EIA using the P30 antigen of T. gondii (Toxo A Ab EIA; Clonatee, Paris). The results in this test are expressed as IgA indices: >.70 = positive, = borderline, and <.50 = negative. IgE ELISA [12, 26] and the HS/AC test for acute stage-specific IgG antibodies [24, 25] were done at the Research Institute, Palo Alto Medical Foundation. Results The first screening test was a sensitive Toxoplasma IgG ELISA. Among 3178 IgG-positive women, the sensitive p, capture ELISA gave a positive IgM result for 226 women, who were all studied further. In the indirect IgM ELISA,

3 JID 1993;167 (March) Serodiagnosis of Toxoplasmosis during Pregnancy 693 Table 1. Persistence of Toxoplasma IgM (indirect ELISA) , Time after first IgM-positive 50 sample No. of 40 Group pregnancies 6 months 12 months IgG seroconverters 10 3/10 (30) 2/10 (20) IgG avidity ~ I5% II 6/10 (60) 5/10 (50) IgG avidity> 15% 15 10/13(77) 11/13 (85) Total 36 19/33 (58) 18/33 (55) "#. 30 ~ Cl ~ 20 NOTE. Data are no. with Toxoplasma IgM/no. followed (%). only 36 of the J,L-capture-positive women were positive. For these, immunoblotting confirmed that the IgM ELISA reactions were specific for toxoplasmic proteins (bands of35 and 25 kda). Among the 240 J,L-capture-negative (IgO-positive) controls, 8 (3%) were positive in IgM immunoblotting. We had complete sets of 3 sera during pregnancy from 88 subjects who were IgM-positive during the first trimester by J,L-capture ELISA. The third-trimester sera of these women, obtained on average 24 weeks after the first-trimester samples, were still positive by J,L-capture ELISA in 80 cases (91%). Of the 36 women positive in the indirect IgM ELISA, 58% maintained this result for 6 months and 55% for 12 months (table 1). All the 240 J,L-capture IgM-negative (IgO-positive) control subjects had high (>25%) avidity of Toxoplasma IgO antibodies. The relationship between IgO seroconversions, positive results in the two IgM ELISAs, and low IgO avidity among the 226 J,L-capture-positive women is illustrated in figure 1. D M(Jl)+ ~M+ ~AVI-! IIGtt Figure 1. In women with suspected primary Toxoplasma infection during pregnancy (n = 42), relationship between IgO seroconversions (ott; n = 13), positive results in both IgM ELiSAs [M+, indirect; M(JL)+, wcapture; n = 36], and low IgO avidity (AVI.; n = 25). Disc areas reflect numbers of women with findings and overlapping indicates concordance between assays TIME OF OBSERVED SEROPOSITIVITY (DAYS) Figure 2. Maturation of toxoplasma IgO avidity after IgO seroconversion (n = 13). IgG Seroconverters IgM. Of3178 seropositive women, 13 had IgO seroconversions (no IgO in the first sample). They all became IgMpositive in J,L-capture ELISA. By indirect ELISA, 10 seroconverters (76%) turned IgM-positive, 2 had borderline results, and 1 remained negative. The IgM-negative sample had been taken 13 weeks after the preceding IgO-negative sample. Among the IgM-positive IgO seroconverters, 20% had IgM by indirect ELISA after 12 months (table 1). Avidity ofigg. Of the seroconverters, 11 (85%) had low and 2 had borderline avidity in their first IgO-containing samples (figure 2). The 2 sera with borderline avidity had been obtained exceptionally late, 15 and 27 weeks after the IgO-negative sera. Of the low-avidity sera, 10 had been collected shortly (~ 13 weeks) after the preceding IgO-negative sera and 1 after a 32-week interval. Among the women with low avidity after IgO seroconversion, 10 (91%)reached borderline or high levels by 6 months after the first seropositive sample (figure 2). The avidity of I seroconverter continued to be low until her last sample, taken 11 months after seropositivity was observed. A significant increase in IgO avidity was encountered in 77%of the serocon verters. IgA. Altogether 100 serum samples from the 13 patients with IgO seroconversion during pregnancy were tested by IgA ELISA. All 13 became IgA positive (mean IgA index [±2 SD], 2.1% [±3.6%]). After 6 months of follow-up, 10 (77%), and after 12 months, 9 (69%) still had IgA. Women with Initially Low Avidity of IgG Avidity ofigg. Among the 226 wcapture IgM-positive women, 25 had low (~15%) avidity of IgO: 11 of the seroconverters plus 14 initially IgO-positive women, ofwhom 3 (patients 1, 6, and loin table 2) had significant (fourfold or

4 694 Lappalainen et al. lid 1993; 167 (March) , ~30 ~ C1 :> < DAYS Figure 3. Maturation of initially low (~15%) Toxoplasma IgO avidity (n = 14). *, Toxoplasma IgO disappeared within 6 months. more) increases in IgG titer. Among these 14 initially seropositive subjects with low avidity, II could be followed for at least 6 months: 9 gained borderline avidity within 3 months and I by 10 months (figure 3). The other patient lost reactivity in Toxoplasma IgG ELISA before delivery, at an avidity of II.7% (figure 3), but continued to express a high level of Toxoplasma IgM. A significant increase in IgG avidity occurred in 79% of the initially seropositive patients with low avidity. IgM. After a follow-up of 12 months, 50% ofthese subjects still exhibited IgM by the indirect ELISA (table I). IgA. Altogether, 91 sera from the 14 initially seropositive mothers with low avidity ofigg were tested for IgA: 13 women were initially IgA-positive, and I was in the borderline range. The mean IgA index of this group was 2.2% (±4.3%). In this group, the positive IgA finding persisted for 6 months in 10 (83%) of 12 and for 12 months in 7 (58%). The IgA results among IgG seroconvertersand those initially seropositive with low avidity were almost identical. HS/AC test and IgE ELISA. Serum specimens from 13 of the 14 initially seropositive mothers with low avidity were tested with the HS/AC test and IgE ELISA (table 2). The first-trimester sera were used in 12, while only a second-trimester sample was available for I. Each first-trimester sample showed an acute pattern in the HS/AC test, as did the second-trimester sample. The IgE ELISA was positive in 9 (69%) ofthe 13. IgM-Positive Mothers with High or Borderline Avidity of IgG Avidity ofigg. All the initially IgM-positive women with high (or borderline) IgG avidity retained high avidity beyond 6 months offollow-up: No relapses to the diagnostic (low) avidity range occurred. The avidity of2 started at the borderline range and appeared to mature, raising the possibility of recent infection (figure 4). However, our criteria for significance ofavidity maturation were not met by any subject in this group. IgM. Among the IgG- and IgM-seropositive women with initially high or borderline (» 15%) avidity, 85%retained IgM by the indirect ELISA for I year (table I). IgA. We examined 97 sera from 15 IgM-positive mothers with borderline or high IgG avidity. The mean IgA index ofthis group was 1.8% (±4.0%). Altogether, 9 mothers (69%) retained a positive IgA result for 6 months and 8 (62%) for 12 months of follow-up. HS/AC test. Serum specimens from 15 IgM-positive mothers with initially borderline or high IgG avidity were examined with the HS/ AC test. The first-trimester sera were used in 13 mothers, while in 2 only a second-trimester sample was available. Among 12 with initially high avidity, II exhibited nonacute patterns, and 3 mothers with borderline IgG avidity showed acute patterns in the HS/AC test. One mother with initially high avidity had an acute HS/AC pattern both during pregnancy and also in a follow-up sample obtained 12 months postnatally. Predictive Values of Avidity Results We wanted to assess precisely the predictive values oflowand high-avidity results during early pregnancy. However, since absolute diagnostic criteria for single sera do not exist, we sought the avidity result during the third trimester and determined the serologic status from the first-trimester sample. If the third-trimester sample was missing, the result at delivery was used. Low avidity. There were II women with low IgG avidity during the last trimester, of which 10 were IgG seroconverters. The IIth woman had given no first-trimester serum. Her second-trimester sample had IgM in both ELISAs, very low avidity ofigg, and a diagnostic increase in IgG titer. By all available criteria, this woman had had a primary Toxoplasma infection during early pregnancy. The positive predictive value ofa low-avidity result for Toxoplasma infection during --8 months was 100% (11/II ). High avidity. Each ofthe 75 women with high avidity in the last trimester was already seropositive in the first trimester, 71 ofthem with IgG ofhigh (>25%) avidity, I with low, and 3 with borderline. In these subjects, almost halfof whom had been positive by wcapture ELISA, the predictive value of a high-avidity result against toxoplasmosis within 5 months (the average interval between the first- and third-trimester samples) was 100% and within 8 months (from conception to the third-trimester sample), --95% (71/75). Discussion Specific IgG of low avidity proved to be an excellent single-serum indicator of recent primary Toxoplasma infection,

5 JIO 1993;167 (March) Serodiagnosis of Toxoplasmosis during Pregnancy 695 Table 2. Serologic findings in 14 mothers with initially low avidity of IgG. Patient, IgG Avidity day titer WIgM IgM* IgA HS/AC (%)t IgE 1, Acute NO + + Acute 10.4 NO 2, Acute NO + + NO 28.4 NO NO + + NO 49.8 NO 3, /- Acute Not acute 11.7 NO 202 <200 NO + NO NO 4, /- + Acute NO +/- + NO 11.9 NO /- NO 14.0 NO 5, Acute NO + + NO 28.5 NO NO + + NO 43.3 NO 6, NO 6.6 NO /- + Acute Not acute 21.1 NO NO + NO 41.4 NO 7, Acute /- + Acute 18.8 NO NO + NO 31.1 NO 8, Acute 11.0 NO Acute NO 21.2 NO NO + + NO 25.0 NO 9, NO Acute NO + Not acute 26.4 NO NO + NO 31.8 NO 10, Acute 1.8 NO Acute /- + NO 6.6 NO NO + + NO 30.6 NO 11, Acute 14.3 NO Acute NO NO 21.0 NO NO NO 36.4 NO 12, NO 15.0 NO NO +/- + NO 43.7 NO NO +/- NO NO 29.3 NO 13, Acute NO 32.0 NO NO + NO 36.9 NO 14, Acute /- NO NO 24.0 NO NO + NO 43.6 NO NOTE. NO = not done; +/- = borderline. * Indirect IgM ELISA. t Ratio (%) of end-point titers (with/without urea elution). as already suggested by previous studies [27, 45]. Under our demanding screening conditions, its positive predictive value was 100%, as assessed by the third-trimester sera coupled to retrieval of the first-trimester antibody status. This serodiagnostic marker also had a high sensitivity, intermediate between the two IgM ELISAs. With the avidity technique we were able to show that among women presenting with Toxo- plasma IgM at the time ofpregnancy, a high proportion had not actually been infected recently. Concerning the role of IgM, most investigators would agree that the most telling detection of IgM antibody by a sensitive test is its absence: A woman with IgG but without IgM is extremely unlikely to have acquired toxoplasmosis recently [I]. Since specific IgM antibodies may be detectable

6 696 Lappalainen et al. lid 1993;167 (March) ~ 30 ~ Cl ~ DAYS Figure 4. Follow-up of Toxoplasma IgG avidity in Toxoplasma IgM-positive mothers with high or borderline initial avidity (n = 15). in both the acute and chronic phases of toxoplasmosis, the need for confirmatory evidence ofan acute infection is crucial. So far the retrospective diagnosis of recently acquired Toxoplasma infection in non symptomatic individuals has been difficult because of the lack of reliable single-serum techniques. An accurate result is particularly important for pregnant women: The diagnosis must be made early so that appropriate therapeutic decisions can be made. Because the level ofspecific IgG antibodies as such is an unreliable indicator of recent infection [I], mothers presenting with antibodies in their first sample are the greatest challenge in diagnosis and therapy. In our study, each individual who had low-avidity IgG was a seroconverter or HSjAC-positive. The 14 initially seropositive women with low avidity in the first sample resembled the IgG seroconverters closely in three important features: IgM and IgA profiles and maturation of IgG avidity. The avidity ELISA, in contrast to the IgM and IgA ELISAs, was able to distinguish acute from chronic Toxoplasma infections; in our sera, IgA (p30 antigen) de facto persisted longer than did IgM by the least sensitive assay. There were 16 patients with infection during pregnancy verified by seroconversion or a diagnostic increase in IgG titer compared with II seropositive patients without such markers but with initially low avidity. Besides the antibody profiles, these numbers of patients support the conclusion that women in both groups had acquired Toxoplasma infections recently: Because our sequential sera spanned only the period between the first- and lasttrimester samples, diagnostic increases in antibody titer occurring during early pregnancy would have been missed by conventional IgG serology. On the other hand, the IgG avidity test should cover not only the intersample period but also the preceding weeks during early pregnancy. If the disease is rare, even a highly specific screening test will yield false-positive results, perhaps more often than correct diagnoses [46]. Therefore, it would be hazardous to screen large populations of asymptomatic individuals with any single test for a diagnosis such as toxoplasmosis that could necessitate long-term medication during pregnancy or abortion. To minimize such errors, we used a combination of assays. In general, the purpose ofthe first, sensitive assay is to select a study group of reasonable size that still includes all suspects. The second, confirmatory test has to be specific. For the latter purpose, measurement ofigg avidity was suitable because it had a very high sensitivity and excellent specificity, superior to any IgM assay tested. To identify each subject with conventional markers of infection, we positioned the avidity assay behind the IgG and IgM tests. Because the avidity test is dependent on the presence ofantibody of the IgG class, the paucity ofspecific IgG during very early illness would favor the simultaneous examination for specific IgM. Moreover, even as a straightforward modification ofindirect ELISA [27], the avidity ELISA consumes more antigen (eight wells per sample) and was more cost-effective after a simple, highly sensitive screening test. In the light of our results, we recommend as primary tools in the first trimester an IgG assay in combination with a sensitive IgM test. The role of the IgG avidity result is to confirm (low avidity) or dispute (high avidity) the diagnosis. Follow-up of seronegative women could be based primarily on IgG serology. For the initially seropositive mothers with low avidity, additional evidence for infection during early pregnancy can be obtained by increases in the titer or avidity of IgG. Acknowledgments We thank Susanna Aro, Lea Hedman, Helena Matikainen, and Teija Tekkala for expert technical assistance, Heikki Isotalo and Heikki Lehvaslaiho for help in computerizing the data, and Jean Margaret Perttunen and Ross Stephens for revising the manuscript. References I. Remington JS, Desmonts G. Toxoplasmosis. In: Remington JS. Klein JO. eds. Infectious diseases of the fetus and newborn infant. 3rd ed. Philadelphia: WB Saunders. 1990: Frenkel JK. Toxoplasmosis. 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