Effect of Testing for IgG Avidity in the Diagnosis of Toxoplasma gondii Infection in Pregnant Women: Experience in a US Reference Laboratory

1248 Effect of Testing for IgG Avidity in the Diagnosis of Toxoplasma gondii Infection in Pregnant Women: Experience in a US Reference Laboratory Oliver Liesenfeld, 1,2,3 Jose G. Montoya, 1,2 Sandra Kinney, 1 Cynthia Press, 1 and Jack S. Remington 1,2 1 Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto, and 2 Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California; 3 Department of Medical Microbiology and Immunology of Infection, Institute for Infection Medicine, Benjamin Franklin Medical Center, Free University of Berlin, Berlin, Germany The usefulness of testing for IgG avidity in association with Toxoplasma gondii was evaluated in a US reference laboratory. European investigators have reported that high-avidity IgG toxoplasma antibodies exclude acute infection in the preceding 3 months. In this US study, 125 serum samples taken from 125 pregnant women in the first trimester were chosen retrospectively, because either the IgM or differential agglutination (AC/HS) test in the Toxoplasma serologic profile suggested or was equivocal for a recently acquired infection. Of 93 (74.4%) serum samples with either positive or equivocal results in the IgM ELISA, 52 (55.9%) had high-avidity antibodies, which suggests that the infection probably was acquired before gestation. Of 87 (69.6%) serum samples with an acute or equivocal result in the AC/HS test, 35 (40.2%) had high-avidity antibodies. Forty women were given spiramycin, to prevent congenital transmission, and 7 (17.5%) had high-avidity antibodies. These findings highlight the value of testing a single serum sample obtained in the first trimester of pregnancy for IgG avidity. Infection with Toxoplasma gondii during pregnancy may lead to severe sequelae in the fetus [1]. At present, definitive serologic diagnosis of acute infection during gestation can be made only if paired serum samples reveal a significant increase in titer most often of IgG antibodies [2]. Such increasing titers are not commonly seen in US pregnant women, because no systematic serologic screening to detect seroconversion is done. Although successful screening programs have been used in France and Austria for many years [3, 4], in the United States, usually only a single serum sample is submitted for testing, frequently late in gestation. Because IgM antibodies may persist for months or even years after acute infection [5, 6], their greatest value is in determining that a pregnant woman was not infected recently. A negative result virtually rules out recent infection, unless serum samples are tested so late after infection that IgM antibody has disappeared or so early after the acute infection that an antibody response has not occurred or is not yet detectable [1, 2]. When Received 28 July 2000; revised 10 January 2001; electronically published 16 March 2001. Financial support: National Institutes of Health (grant AI-04717); Infectious Disease Fellowship from the German Ministry of Research and Technology to O.L. Reprints or correspondence: Dr. Jose G. Montoya, Research Institute, Palo Alto Medical Foundation, Ames Bldg., 795 El Camino Real, Palo Alto, CA 94301 (gilberto@leland.stanford.edu). The Journal of Infectious Diseases 2001;183:1248 53 2001 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2001/18308-0010$02.00 results are positive, a number of additional serologic tests, including those for the detection of IgG [7, 8], IgA [9], and IgE [10] antibodies, and a combination of these tests, the Toxoplasma serologic profile (TSP) [11, 12], can help discriminate between recently acquired and distant infection. The differential agglutination (AC/HS) test most closely approaches a single reference standard test for the discrimination of recently acquired and distant infection [8]. However, at present in the United States, this test is done only by the Toxoplasma Serology Laboratory, Palo Alto Medical Foundation (TSL-PAMF; Stanford, CA), because the required antigen preparations are not commercially available. In addition, as is true for IgM antibodies, an acute pattern in the AC/HS test may persist for 11 year [8]. Recently, several tests for avidity of toxoplasma IgG antibodies have been introduced to help discriminate between recently acquired and distant infection [13 20]. The commercial tests are available in Europe but are not licensed in the United States. Results are based on the measurement of functional affinity of specific IgG antibodies [21]. IgG affinity, which initially is low after primary antigenic challenge, increases during subsequent weeks and months by antigen-driven B cell selection. Protein-denaturing reagents, including urea, are used to dissociate the antibody-antigen complex. The avidity result is determined by using the ratios of antibody titration curves of urea-treated and -untreated sample. We investigated the usefulness of testing for avidity of IgG antibodies in a US toxoplasma serology reference laboratory

JID 2001;183 (15 April) Toxoplasma IgG Avidity 1249 that processes serum samples primarily from pregnant women. In most cases, the physician requests information about the time of onset of the infection on the basis of results obtained from a single serum sample. Our goal was to compare results obtained in an IgG avidity test with those obtained in the IgM ELISA and AC/HS tests. Materials and Methods We retrospectively studied 125 serum samples taken from 125 pregnant women in the first trimester of gestation. The serum samples were submitted to the TSL-PAMF between September 1994 and September 1999. Serum samples had been routinely tested by the TSP, as described elsewhere [11, 12]. The TSP comprises the Sabin-Feldman dye test (DT) [7], double-sandwich IgM ELISA [22], IgA ELISA [9], IgE ELISA [10], IgE immunosorbent agglutination assay (ISAGA) [10], and AC/HS test [8]. The Sabin-Feldman DT is considered to be positive at any titer. The starting dilution was 1:16. The double-sandwich IgM ELISA method [22] has been used by our laboratory and other reference laboratories for 115 years. A result of 2.0 was interpreted as positive, 1.7 1.9 as equivocal, and!1.7 as negative. The following titers were considered to be positive, negative, and equivocal, respectively, in the tests: IgA ELISA, 2.1 and 1.4 (equivocal, 1.5 2.0); IgE ELISA, 11.8 and 1.4 (equivocal, 1.5 1.8); and IgE ISAGA, 4 and 2 (equivocal, 3). The AC/HS test was interpreted, as described elsewhere [8], by comparing titers obtained with formalin-fixed tachyzoites (HS antigen) with those obtained with acetone-fixed tachyzoites (AC antigen; figure 1). IgG antibodies formed early in infection recognize stage-specific antigens in the AC preparation, which are distinct from those formed later in infection [8, 23]. Current interpretation of results in the TSP at the TSL-PAMF is as follows: serum samples obtained within the first 2 trimesters that are positive in the DT and negative in the IgM, IgA, and IgE ELISAs and reveal a chronic pattern in the AC/HS test are typically found in patients infected before gestation. The combination of high titers in the DT, positive IgM, IgA, and IgE ELISAs, and an acute pattern in the AC/HS test is highly suggestive of a recent infection. In contrast, the presence of a positive DT and IgM ELISA but a negative, low-positive, or equivocal result in the IgA and IgE ELISAs and IgE ISAGA and an equivocal pattern in the AC/HS test is more difficult to interpret, since it suggests infection before pregnancy but does not rule out recent infection. We selected consecutive serum samples in which the TSP had 1 test result that suggested the possibility of a recently acquired infection. This approach enabled us to evaluate the discriminatory power of testing for IgG avidity. To select serum samples for testing for IgG avidity, we used the following criteria: first, positive or equivocal results in the IgM ELISA or acute or equivocal pattern in the AC/HS test or second, results in the TSP that were not clearly consistent with an infection acquired in the distant or recent past. In some cases, interpretation of the TSP did not allow for clearcut discrimination between recently acquired and distant infection, even after a follow-up sample was tested in parallel with the first serum sample. IgG avidity was measured with the T. gondii IgG avidity EIA (Labsystems), and results were interpreted in accordance with the manufacturer s instructions. In brief, serum samples were diluted in 4-fold dilutions, according to their predetermined IgG titers. Serum samples were diluted and incubated in duplicate for 60 min at 37 C int. gondii coated microstrips. We incubated 1 well of each duplicate serum sample and of the negative, low avidity, and high avidity controls in PBS/Tween; the other well of each duplicate was incubated with avidity buffer containing urea. After a wash, antigen-bound IgG was bound by conjugate applied for 1 h at 37 C and was revealed by substrate. Optical densities were read at 405 nm. For each control and serum sample, we obtained 2 titration curves (1 for each sample incubated with PBS/Tween and 1 for samples incubated with avidity buffer). The distance between curves corresponds to the avidity percentage (e.g., the greater the distance in millimeters, the lower the avidity percentage that indicates low-avidity antibodies and vice versa). We interpreted avidity results as follows:!15%, low avidity (may be seen in acute primary infection with T. gondii); 15% 30%, borderline avidity (primary infection within the past 6 months is possible); and 130%, high avidity (excludes primary infection within the previous 3 months). A high-avidity result in the first 12 weeks of pregnancy essentially rules out a recently acquired infection during gestation. A low or equivocal IgG avidity result cannot be interpreted to mean that a person was infected recently with T. gondii, because low-avidity antibodies may persist for 15 months. Thus, when the Labsystems kit is used in the first trimester, only high-avidity antibodies are clinically meaningful. When available, we used follow-up serologic test results obtained 3 weeks apart from the initial serum sample and from serum samples obtained before the initial sample submitted to the TSL- PAMF to resolve discrepant results between individual tests. All such serum samples were run in each test in parallel. Results Figure 1. Criteria for interpretation of differential agglutination (AC [acetone-fixed tachyzoites]/hs [formalin-fixed tachyzoites]) test results. We studied serum samples taken from 125 pregnant women during the first trimester. Results in the IgM ELISA were positive in 81 (64.8%), equivocal in 12 (1.0%), and negative in 25 (20%) cases; an IgM ELISA titer could not be determined in 7 samples because of high background activity. In 42 (51.9%) of the 81 serum samples that were positive by IgM ELISA, the IgG avidity result was high (table 1), which indicates that in-

1250 Liesenfeld et al. JID 2001;183 (15 April) Table 1. Comparison of results of IgM ELISA and IgG avidity test in 118 serum samples taken from pregnant women during the first trimester. Avidity result Positive (n p 81) IgM ELISA result Equivocal (n p 12) Negative (n p 25) Low 16 (19.8) 0 4 (16.0) Borderline 23 (28.3) 2 (16.7) 7 (28.0) High 42 (51.9) 10 (83.3) 14 (56.0) NOTE. Data are no. (%) of serum samples. Because of high background in IgM ELISA, 7 serum samples were not included in the comparison. fection in those women occurred before gestation. In 16 (19.8%) of the 81 serum samples, IgG avidity was low. In addition, 93 (74.4%) of 125 serum samples had positive or equivocal results in the IgM ELISA (often interpreted as being suggestive of a recently acquired infection); IgG avidity results were high in 52 (55.9%), equivocal in 25 (26.9%), and low in 16 (17.2%). In 25 serum samples that were negative in the IgM ELISA, 4 (16.0%) had low avidity, 14 (56.0%) had high avidity, and 7 (28.0%) had equivocal avidity test results (table 1). Figure 2 compares results in the IgM ELISA with those in the IgG avidity test. Mean titers of T. gondii specific IgM antibodies were 4.1 in serum samples with low-avidity antibodies, 3.2 in serum samples with borderline avidity, and 2.6 in serum samples with highavidity antibodies. Each of the 4 serum samples that were negative in the IgM ELISA and that had low IgG avidity titers had IgG titers of 512 (table 2); 3 had an acute pattern in the AC/HS test, and each was negative by IgA ELISA (table 2). Follow-up serum samples were available for 2 patients but did not reveal significant increases in antibody titers. For patients with a positive IgM test, the AC/HS test was the sole test that was most helpful in distinguishing recently acquired from more distant infection. Of the 125 women who were in their first trimester of pregnancy, 33 (26.4%) had an acute AC/HS test result, 53 (42.4%) had an equivocal result, and 39 (31.2%) had a nonacute result. Of the 33 women with an acute result in the AC/HS test, 17 (51.5%) had low avidity, 12 (36.4%) had equivocal avidity, and 4 (12.1%) had high-avidity test results (table 3). In all, 87 (69.6%) of the 125 women had either an acute or equivocal result in the AC/HS test (often interpreted as being suggestive of a recently acquired infection); IgG avidity results in these were low in 21 (24.1%), equivocal in 31 (35.6%), and high in 35 (40.2%; table 3). Of the 39 women who had a nonacute AC/HS test result, 94.9% had a highavidity test result; 2 (5.1%) were equivocal by avidity test, and none had a low-avidity test result. In 85 (68.0%) of the 125 cases, the overall interpretation of the TSP by TSL-PAMF physicians (without inclusion of the avidity test) was consistent with an infection acquired before conception. For 40 (32.0%) of the 125 women, interpretation of results in the TSP did not allow us to rule out infection acquired during pregnancy or near conception. In these women, spiramycin was recommended in an attempt to prevent transmission of the parasite from mother to fetus. Testing of IgG avidity in serum samples from these 40 women revealed high avidity in 7 (17.5%), borderline avidity in 17 (42.5%), and low avidity in 16 (40.0%). Figure 2. Correlation of IgM titers with avidity test results in 118 serum samples taken from 188 pregnant women during the first trimester. Two data points are superimposed, because results in 2 serum samples were identical (borderline avidity and negative IgM test results).

JID 2001;183 (15 April) Toxoplasma IgG Avidity 1251 Table 2. Serological test results in serum samples obtained from pregnant women during their first trimester who had negative results in the IgM ELISA and low IgG avidity. Specimen Gestation, weeks Avidity, % IgG a IgM IgA IgE ELISA IgE ISAGA AC/HS (int) 61642 b 10 6.3 2048 0.7 0.3 0 0 11600/13200 (A) 61681 c 6 7.1 4096 1.2 0.7 0.5 3 400/200 (A) 63081 b 8 1.6 2048 0.5 1.0 0.5 0 800/400 (A) 74108 12 7.1 512 1.2 1.0 0.8 0 100/400 (E) NOTE. A, acute pattern; AC/HS, differential agglutination test; E, equivocal pattern; int, interpretation; ISAGA, immunosorbent agglutination assay. For further explanation of the AC/HS test, see figure 1. a Reciprocal of serum dilution. b No significant increase in antibody titers was observed in follow-up serum samples obtained 4 weeks apart. c Serum sample obtained 8 weeks earlier revealed a negative result in the IgM ELISA and an equivocal pattern in the AC/HS test. Discussion Development of a serologic method to differentiate recent from more distant T. gondii infection in pregnant women by using a single serum sample has long been a goal of industry and academic investigators. Such a method would be of particular importance in the United States, where systematic serologic screening during pregnancy is not done. The lack of availability of serial serum samples from individual pregnant women in the United States has severely hampered research in this direction and has markedly compromised the ability of physicians to determine the risk for congenital infection. These problems led our group to develop a serologic test panel [11, 12] that assists in defining whether a positive IgM antibody test result for a given patient reflects recently acquired infection. Although the panel is useful, it does not provide definitive results in all cases. Our results reveal that the IgG avidity test is the first method that can differentiate between recent and more distant infection in a single serum sample if the sample is obtained in the first trimester of pregnancy. Kits in use in Europe extend this possibility to the fourth month of gestation [14, 18, 20]. Unfortunately, in many instances, the first serum sample is not submitted for testing until later in the pregnancy. The serum samples selected for this study had equivocal or positive results by IgM ELISA or AC/HS test. Additional serum samples from some of the same patients were tested by TSP, but the results did not allow clear-cut discrimination between recently acquired and distant infection. When IgG avidity and IgM ELISA results were compared in serum samples obtained during the first trimester of pregnancy, 51.9% of IgM ELISA positive serum samples (55.9% of IgM-positive and IgM-equivocal serum samples) had high IgG avidity test results that essentially ruled out recent infection. These results highlight the problem in interpretation of a positive test for IgM antibodies without confirmatory testing. In a recent study in which we used the TSP, a positive IgM test result did not indicate a recently acquired infection in 60% of pregnant women studied [24]. Of importance, IgM antibodies may persist for 1 year after acute T. gondii infection. The value of testing for IgG avidity, as shown in the present study, confirms and extends the results reported by Lappalainen et al. [14]. Initial findings with the IgG avidity method suggested that low-avidity antibodies indicated recently acquired infection [14]. Since then, studies have shown clearly that low-avidity antibodies can persist for many months beyond the acute infection and thus are not reliable for the diagnosis of recently acquired infection [20, 25]. In contrast, the consensus appears to be that the IgG avidity test is best used to rule out recently acquired infection. Depending on the method used, the presence of high-avidity IgG antibodies can be used to rule out the occurrence of acute infection within the past 3 5 months (table 4). Thus, its value is greatest when done in the first 3 4 months of gestation. A high-avidity result late in the second trimester or in the third trimester cannot be interpreted to mean that the infection was not acquired in the first 3 5 months of gestation. Of note, 20% of serum samples with a low-avidity result in the present study had negative results in the IgM ELISA; testing of the initial and follow-up serum samples in the TSP did not suggest recently acquired infection in any of these cases. Both the AC/HS test and the IgG avidity test were valuable in confirmatory testing of IgM-positive serum samples, and the 2 methods had excellent agreement (94.9% of serum samples with a nonacute pattern in the AC/HS test had high-avidity antibodies). Discrepant results between the AC/HS and IgG avidity tests only occurred in serum samples with high-avidity antibodies. The high numbers of equivocal results in the avidity and AC/ Table 3. Comparison of IgG avidity test and differential agglutination (AC/HS) test results in 125 serum samples taken from pregnant women during the first trimester. Avidity result Acute (n p 33) AC/HS test result Equivocal (n p 53) Nonacute (n p 39) Low 17 (51.5) 4 (7.5) 0 Borderline 12 (36.4) 18 (34.0) 2 (5.1) High 4 (12.1) 31 (58.5) 37 (94.9) NOTE. Data are no. (%) of serum samples. For further explanation of the AC/HS test, see figure 1.

1252 Liesenfeld et al. JID 2001;183 (15 April) Table 4. Study [reference] Reports in the literature on toxoplasma IgG avidity tests. Avidity test type No. and type of serum samples Exclusion period, months Cutoff for high avidity, % Positive predictive value of high avidity, % Comment Lappalainen et al. [14] Noncommercial titration 226 IgG /IgM, including 13 seroconverters 5 125 100 100% Positive predictive value for low avidity antibodies Holliman et al. [15] Noncommercial 25 Distant infected; 11 recently infected 3 150 96 80% Negative predictive value for high avidity antibodies Sensini et al. [16] Labsystems 485 Serum samples/308 patients, including 13 seroconverters ND 150 ND Delayed avidity maturation in patients receiving specific treatment Guttiérez et al. [17] ETI-Toxo-G Enzygnost 57 Distant infection 12 seroconverters ND 150 100 No signficiant differences between methods Jenum et al. [18] Titration (Platelia IgG); 107 recent infections 56 IgG /IgM 5 120 ND Unchanged avidity maturation in patients receiving specific treatment Ashburn et al. [19] Noncommercial titration 43, Including 1 seroconverter ND 130 ND Pelloux et al. [20] Vidas 330 26 Seroconverters 4 130 ND Avidity!30%, even 9 months after infection, in patients with immune disorders and in pregnant women Cozon et al. [25] Enzygnost 493 IgG /IgM 3 135 100 39.6% Negative predictive value of low avidity antibodies NOTE. ND, not determined. HS tests remain a drawback of these methods. In cases with equivocal results in either the AC/HS or avidity test (43.2% and 26.4%, respectively), only confirmatory testing by the TSP or testing of follow-up serum samples could help distinguish between recently acquired and distant infection. Interpretation of avidity test methods was based solely on results in serum samples obtained sequentially from pregnant women who seroconverted and not on outcome in their children. Follow-up data in only a small number of children ( n p 13) born to mothers who had high-avidity antibodies in the first trimester have been published elsewhere [26]. None of the 13 was infected with T. gondii, according to long-term serologic follow-up [26]. A question for consideration is whether a high-avidity test result in the first trimester can rule out the possibility of fetal infection in women who acquired the infection a few months before gestation. The likelihood of congenital transmission as a result of an infection acquired in the weeks before or near the time of conception is extremely low, approaching zero [27]. Congenital toxoplasmosis has been reported in only 6 children of women whose serologic test results revealed they probably were infected before pregnancy [1]. Each of these women was infected 1 3 months before becoming pregnant. Avidity testing does not overcome the limitations of serologic testing in this regard. Thus, the dictum that no serologic test is perfect can be extended to the avidity test. No investigators who have developed avidity tests have claimed that their methods can unequivocally rule out infection acquired a few weeks before the beginning of pregnancy. Unfortunately, commercial kits for IgG avidity are not licensed in the United States. This method would be well suited to rule out recently acquired infections in IgM-positive pregnant women who have high-avidity antibodies in their first months of gestation. An approach to testing for and management of T. gondii infection in pregnant women would be to screen for IgG and IgM antibodies in the first trimester of all women in whom the serologic status is not known at conception. For women with negative results in both tests, information and education would be of paramount importance to prevent infection [1]. In serum samples positive for both IgG and IgM antibodies, IgG avidity would be determined. A high-avidity test result would decrease significantly the number of additional confirmatory or follow-up serologic tests needed and the need for spiramycin and invasive techniques, including polymerase chain reaction (PCR) analysis of amniotic fluid. These findings were emphasized further by our experience in the present study: even in the setting of a reference laboratory in which TSP is used to distinguish between recently acquired and distant infection, high-avidity antibodies were detected in 17.5% of 40 women for whom spiramycin had been recommended, because recently acquired infection could not be ruled out in a single serum sample by the TSP. Since serum samples were chosen retrospectively, all decisions about spiramycin treatment were made without knowledge of avidity test results. Thus, a high-avidity test result in the first trimester decreases the need for follow-up serum samples and thereby reduces costs, rules out the need for PCR analysis of amniotic fluid and the need for treatment of the mother with spiramycin, removes the anxiety associated with further testing, and lessens the likelihood of misdiagnosis [24]. Whether the avidity test can replace any of the present tests in the TSP or should simply be added to that panel requires further evaluation of the avidity tests being marketed. At present no avidity test has been released by the Food and Drug Administration for US marketing. We now routinely use the avidity

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