High prevalence of congenital toxoplasmosis in Brazil estimated in a 3-year prospective neonatal screening study

Eurico Camargo Netoa, Elaine Anelea, Rosélia Rubima, Adriana Britesa, Jaqueline Schultea, Daniela Beckera and Tamara Tuuminenb

a Centro de Triagem Neonatal and Laboratório Nobel RIE. Av. Ipiranga, 5.000, Porto Alegre, RS, 90.610–000, Brazil. E-mail: nobelrie{at}voyager.com.br or eneto{at}voyager.com.br
b Labsystems Research Laboratories, Sorvaajankatu 15, 00811, Helsinki, Finland.


    Abstract
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background A pilot neonatal screening programme revealed a high (approximately 1 per 4800 live births) prevalence of congenital toxoplasmosis (CT) in the State of Rio Grande do Sul, Brazil. The purpose of this paper was to estimate in a larger prospective study the prevalence of CT in the country.

Methods At the beginning of the study, an in-house indirect enzyme immunoassay (EIA) was used, to be later replaced with a commercial capture IgM fluorometric enzyme immunoassay (FEIA). Both methods detect specific anti-Toxoplasma gondii IgM-class antibodies eluted from dried blood spots.

Results Of the total of 140 914 samples received from all over the country, 47 cases were identified and confirmed as CT. This finding suggests a prevalence of 1 per 3000 live births. Of the 47 patients, only eight (17%) had clinical manifestations: two had intracranial calcifications, four had retinal scars, one had an intracranial calcification and retinal scars, and one had hepatosplenomegaly with lymphoadenopathy. The testing was paid for by the patients' families who volunteered for the study and gave their informed consent.

Conclusion The 3-year prospective study using sensitive detection methods, reliable confirmation, and feedback from clinicians showed that CT has an extraordinarily high prevalence in Brazil, in fact the highest ever reported in the world. Although the long-term efficacy of treatment of CT has not been well documented, in view of the availability of reliable diagnostics, confirmation and monitoring, functional logistics, and networking for screening, the insidious nature of the sequelae and the very high prevalence of the disease, neonatal screening for CT should be considered an alternative to no screening at all.

Keywords Congenital toxoplasmosis, neonatal screening, Brazil, prevalence

Accepted 17 April 2000


    Introduction
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Traditionally, screening for toxoplasmosis has been carried out in France1 and Austria2 as a mandatory part of prenatal care. Prenatal screening has also been carried out in pilot projects in Finland,3 Norway,4 some parts of Sweden5 and Germany.6 The screening programmes have revealed congenital toxoplasmosis (CT) prevalences varying from 1 per 10002 to 0.3 per 10003 live births. However, some programmes owing to their short-term nature and limited sample sizes hardly present a comprehensive picture of the spread of the disease. Furthermore, the benefits of prenatal programmes have been recently questioned.7 Thus, according to Eskild et al.7 ‘during almost two decades of experience, prenatal screening has not revealed either the natural history of toxoplasmosis or the efficacy of the antiparasite treatment during pregnancy’. More comprehensive results on the true prevalence of CT, approximately 1 per 10 000 live births, were recently published from an ongoing neonatal screening programme in Massachusetts.8,9 The experience of this programme prompted us to study the prevalence of CT in Brazil. Preliminary results from the neonatal screening programme have indicated a prevalence of approximately 1 per 4800 live births there.10

Since 1989, a private neonatal screening programme for congenital hypothyroidism and phenylketonuria has been implemented in Porto Alegre, the capital of the Rio Grande do Sul State, South Brazil. The programme offers a comprehensive panel of screening tests for the detection of genetic and infectious diseases (Table 1Go). The tests are paid for either by the patients' families or health insurance schemes. Generally, after the detection of the disease, the patients are referred to the local reference health centres for treatment and monitoring.


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Table 1 Tests offered by the Neonatal Screening Center of Porto Alegre
 
The present extended study was undertaken to confirm earlier results and to estimate the feasibility of routine neonatal screening. We describe our 3-year experience of testing 140 914 newborns from volunteer families for CT.


    Subjects and Methods
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Clinical specimens
Blood samples from infants aged 3–15 days were spotted onto Schleicher and Schuell # 903 filter paper and dried overnight at room temperature. The samples were collected by heel prick and used for all routine neonatal screening tests and throughout the study. For confirmation, maternal and child serum samples were analysed.

Neonatal screening
The pilot screening programme was carried out from September 1995 to September 1996. During this time, 33 625 newborns were screened, and seven cases of CT were detected, indicating an initial prevalence of 1 per 4800.10 On the basis of these preliminary results, the screening programme was extended using, in the beginning, an in-house indirect enzyme immunoassay (EIA).10 In this EIA, Toxoplasma gondii tachyzoides purified and disrupted by sonication and cultivated in Eagle's medium with 10% fetal bovine serum were used as antigen. Polystyrene microplates were coated overnight in carbonate/bicarbonate buffer, pH 9.8. Dried blood spots, 6 mm in diameter, were placed into the wells of the microplates, and the blood was eluted with phosphate buffer saline, pH 7.4, for one hour at room temperature with shaking. After the first incubation, the plates were washed four times, and peroxidase-labelled sheep anti-human IgM conjugate (Genzyme Diagnostics, Cambridge, USA) was added. Thereafter, the plates were washed again as above and a substrate reaction with tetramethylbenzidine as chromogen was performed. The reaction was stopped with 2N hydrochloric acid, and optical densities (OD) were read at 450 nm. To calculate the cutoff value, 105 filter paper blood samples from healthy blood donors were screened and an average OD of 0.035 was obtained. Thus, the cutoff value was set as three-fold, 0.100. A sample was classified as normal if the OD was below 0.100; inconclusive if the OD varied between 0.100 and 0.200; and presumptively positive if the OD exceeded 0.200 when confirmed in duplicates from the same filter card. Internal quality control material on the filter paper was prepared from positive IgM anti-Toxoplasma gondii serum, which was mixed with human erythrocytes to achieve a haematocrit of 50%. These controls gave OD values of 0.100, 0.150 and 0.200. The sensitivity of the method was determined by testing of 39 artificially prepared positive dried blood samples. All samples were over the cutoff, to a preliminary analytical sensitivity of 100%. However, indirect methods are known to be less sensitive than capture IgM methods because of the competition of IgG with IgM for binding sites. Therefore, in 1997, a commercial capture Toxoplasma gondii IgM fluorometric enzyme immunoassay (FEIA) (Labsystems, Helsinki, Finland) method especially designed for newborn screening11 was adopted after evaluation in parallel with the in-house method. This method is a solid-phase capture EIA with fluorometric detection. Briefly, IgM class antibodies are eluted from single 3 mm dried blood spot disks and simultaneously captured by biotinylated sheep polyclonal anti-human antibody, which binds to streptavidin-coated microplates. After incubation and the first washing step, a mixture of Toxoplasma gondii disrupted tachyzoites as antigen (RH strain) and horseradish peroxidase-labelled monoclonal antibody derived against P30, a major membrane protein, is added and allowed to bind to immobilised IgM. After the second washing step, an enzymatic reaction with a fluorogenic substrate, 3-p-hydroxyphenylpropionic acid, is performed. The reaction is stopped by addition of glycine buffer, and fluorescence is measured using FluoroskanTM (Labsystems) at 405 nm, with 320 nm excitation. The reproducibility of the method measured by testing duplicate samples presented a calculated CV% below 13% from raw fluorescence. The samples were considered initially positive in the FEIA method when their fluorescence was above or below 20% of the fluorescence of the Borderline control supplied by the manufacturer. Initially, 3787 consecutive samples obtained from infants with a mean age of 18 days for the routine neonatal screening programme were tested using both methods. Thereafter, for the sake of economy, the FEIA was compared to the in-house method by doing them alternatively on consecutive days. Altogether, 10 320 samples were run, 6005 using the commercial Toxoplasma gondii IgM FEIA and 4315 using the in-house Toxoplasma gondii IgM EIA.

Confirmation, follow-up and treatment
The samples were analysed in duplicates from the same filter paper card when the results were between 0.100 and 0.200 of OD (EIA) and above or below the 20% of the Borderline control (FEIA) at the primary screen.

When the samples were at repeat above or maximally 20% below the cutoff value, serum samples from the infant and the mother were requested. Specific anti-Toxoplasma gondii IgM and IgG class antibodies were measured in the mother's and the child's serum at the beginning of the programme using an indirect immunofluorescence (IF) method (Biolab-Merieux Diagnostica, Rio de Janeiro, Brazil) and later, owing to the inferior sensitivity of the IF IgM method, the MEIA IgM kit (Abbott Laboratories, Chicago, IL, USA) was also used.

When the initial laboratory findings indicated congenital infection, serum samples were collected for follow-up three times at 2-week intervals, and the detection of specific IgG and IgM antibodies in the serum was continued. The decision that the detected cases were true positives was based on a repeated positive screening test and one of the following criteria:

  1. positive confirmatory serum results for both IgM and IgG specific antibodies in the infant and the mother;
  2. a high IgG level in the infant and positive serum IgM and IgG in the mother;
  3. positive IgM and IgG results in the infant only;
  4. increase in specific IgG in the infants with titres of 1 per 256 to 1 per 256 000 during the follow-up period, sampling three times at 2-week intervals. An increase in the infant's specific antibodies excludes their maternal nature.

IgM IF was applied to 156 suspected case pairs, of which five were confirmed as CT in both the mother and the child, two cases in the child only and three in the mother only. Diagnosis was confirmed in the remainder of 366 suspected sample pairs using the MEIA, and 30 cases were then confirmed by the detection of IgM in the maternal serum and/or in the child's serum. The diagnosis of the remaining 12 cases was confirmed by an increase in IgG in the infants' samples at follow-up (Table 2Go). Two of the infants were adopted children, and four mothers were not tested for serum antibodies. It should be noted that the cutoff for the MEIA was used according to the manufacturer's instructions, without adjusting it for the infants' samples. In addition to laboratory testing, the infants underwent an ophthalmologic examination for retinal scars and cranial x-ray and/or tomography for cranial calcification by a local paediatrician. Infants diagnosed as having CT were referred to their local paediatricians for follow-up and treatment, except for six infants born in the Porto Alegre Metropolitan area who were treated in the capital. The treatment was given according to established protocols using pyrimethamine, sulphadiazine, and leucovorin during one year.


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Table 2 Demographic, laboratory and clinical data from detected cases with congenital toxoplasmosis
 

    Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
From September 1995 to December 1998, a total of 140 914 infants at the mean age of 18 days from various parts of the country were screened for Toxoplasma gondii IgM antibodies. Of these infants, 78 350 were tested using an in-house EIA and 62 564 using a commercial FEIA. Of the samples, 66 320 (47%) were from the State of Rio Grande do Sul and 74 684 (53%) from the rest of the country.

The screening programme detected 47 cases of CT. Thus, the calculated prevalence of CT in Brazil was found to be 1 per 3000 live births. Of the 47 detected cases, 29 (61.7%) were from the State of Rio Grande do Sul. It is of interest that of the 47 patients, 39 (83%) were asymptomatic during the period of observation ranging from 6 months to 3 years and 7 months. Only eight infants (17%) presented with clinical manifestations: one had intracranial calcifications, five had retinal scars, one had intracranial calcification and retinal scars, and one had hepatosplenomegaly with lymphoadenopathy. Table 2Go shows detailed demographic, laboratory, and clinical data on each infant. According to the criteria of confirmation (Subjects and Methods) the detected cases can be divided as follows: 20 cases, including four infants with symptoms; eight cases; seven cases, including one infant with symptoms and one who died of immunosuppression; 12 cases, including three infants with symptoms.

A total of 474 infants and their mothers were recalled for confirmatory serum tests. Negative results were obtained with 202 samples from these infants and their mothers (excluding four mothers not tested, and the two mothers of adopted infants). In the remaining 272 suspected cases, only IgG antibodies were found with decreasing levels without treatment at follow-ups at 2-week intervals. These antibodies were assumed to be of maternal origin as a result of past parasite exposure.

Specific IgG antibodies were detected in all pairs of patients and their mothers, whereas IgM antibodies were detected as follows: Of 32 mother-child serum pairs, IgM were present in 15 pairs, in five maternal samples and in eight infant samples. These results were obtained using the MEIA IgM (starting with patient No. 17, see Table 2Go). By contrast, in this group only 13 infants and/or their mothers were confirmed as having CT by IF. The discrepancy between the numbers of infants and those of mothers is due to the fact that two of the infants were adoptive and four mothers did not participate in sample collection.

The results of the in-house EIA and those of the FEIA showed good agreement: the retest rates, i.e. the proportion of samples retested on the same dried blood spot for EIA and FEIA, were 2.2% and 0.5%, respectively. The recall rates, i.e. the proportion of initially positive samples requested for new testing on serum for EIA and FEIA, were 0.33% and 0.42%, respectively. With the 3787 samples, one case of CT was found by both methods and confirmed positive by serum findings, skull radiography, and ophthalmoscopy. The screening of 10 320 samples revealed two positive cases by both methods, one case positive by FEIA and a borderline case by EIA. Additional laboratory and clinical investigations confirmed these three cases as CT. At the beginning of FEIA use, one case was found positive by EIA but negative by FEIA. This case was confirmed as positive at follow-up. One case determined by EIA as disease-free was later found positive. However, acquired infection could not be ruled out in this case. Two infants proven positive on screening died without a clinical diagnosis. Table 3Go compares the performance of the in-house EIA against that of the commercial FEIA.


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Table 3 Comparison between the in-house indirect enzyme immunoassay (EIA) and the Toxoplasma gondii IgM fluoro-metric enzyme immunoassay (FEIA)
 

    Discussion
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Earlier epidemiological studies in Brazil have revealed an average seroprevalence of CT of approximately 80% (Camargo Neto, unpublished observation) in the general population, which speaks of a high risk of acquired acute infection in the mother. Unfortunately, no data are available on the seroprevalence of CT among women of childbearing age. Since many logistical problems, e.g. transport and storage of large quantities of serum samples, make prenatal screening impracticable, the only way to detect CT in infants is neonatal screening using specific IgM as a marker. Some authors12 have questioned the usefulness of the IgM method with the low observed detection rate of only 50%. However, recent findings9,13 suggest that the detection rate when IgM test is used alone approaches 80%. Several reasons may explain the low detection yield of the IgM screening. One is the use of an indirect method in which maternal IgG competes for antigen binding sites with newborn IgM present in the circulation in much lower quantities. The second reason is the use of the cutoff for the IgM method defined in adult population studies. The importance of the low cutoff for the efficacy of newborn testing has been repeatedly emphasized.810 The third reason may be that specific IgM is absent in some newborns12 as a result of the mother's treatment during pregnancy. Furthermore, the reliability of a positive IgM result as a marker of an acute primary infection has been questioned14 because indirect methods tend to produce false-positive results. It is well known that adult serum is abundant in IgM interfering naturally occurring antibodies15 that may non-specifically react with the antigen alone or with HRP-labelled conjugate preparations.16 Because newborn sera are devoid of these interfering factors, the cutoff can be set much lower to increase the analytical sensitivity at no expense of analytical specificity. Moreover, the use of the capture technology further increases the analytical sensitivity of the method, and thus the clinical sensitivity of the screening programme. In our study, the indirect in-house EIA was replaced with the commercial capture method, which proved reliable (Table 3Go) and confirmed earlier findings.11 To ensure the highest detection rate, we recalled for confirmation the samples that showed values 20% below the set cutoff at the primary screen. We took this precaution for two reasons: the imprecision of the method and the possible analytical variation of the sample at the cutoff level. Moreover, possible pre-analytical factors such as non-uniform impregnation of a filter paper with blood might have had untoward effects.

Our screening programme also detected one case of CT among the 55 tested samples (data not shown) that were submitted from Bolivia. This finding, the results of our study, and the similarity in economic and cultural conditions indicate that the very high prevalence of CT in Brazil can be extrapolated to other Latin American countries.

Our test samples were obtained from volunteer families. Selection bias may therefore have affected the results. Although the samples largely represented the demography of the Brazilian newborn population, the prevalence of CT is probably even higher among the poor that cannot afford testing.

In conclusion, the reliable methods of detection and confirmation of our prospective study revealed a prevalence of CT of 1 per 3000 live births, which is the highest rate ever reported in the world. Although treatment efficacy has not been documented in recent study better than in previous studies of prenatal screening, this should not be used as an argument for abstaining from screening, especially in areas with a high CT prevalence. When facilities are available for the detection, confirmation, follow-up, and treatment of CT, no screening means not caring for children and is thus unethical. Moreover, our findings should help national health care authorities to implement preventive measures.


    Acknowledgments
 
We express our gratitude to Dr Roger Eaton and Dr Ho-Wen Hsu from the New England Regional Newborn Screening Programme, Massachusetts State Laboratory Institute, Boston, MA, USA, for their valuable comments.


    References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
1 Jeannel D, Niel G, Costagliola D, Danis M, Traore BM, Gentilini M. Epidemiology of toxoplasmosis among pregnant women in the Paris area. Int J Epidemiol 1988;17:595–602.[Abstract]

2 Aspöck H, Pollak A. Prevention of prenatal toxoplasmosis by serological screening of pregnant women in Austria. Scand J Infect Dis Suppl. 1992;84:32–38.[Medline]

3 Lappalainen M, Koskela P, Hedman K et al. Incidence of primary toxoplasma infection during pregnancy in Southern Finland: a prospective cohort study. Scand J Infect Dis 1992;24:97–104.[ISI][Medline]

4 Stray-Pedersen B, Lorentzen-Styr AM. The prevalence of toxoplasma antibodies among 11 736 pregnant women in Norway. Scand J Infect Dis 1979;128:716–21.

5 Ahlfors K, Börjeson M, Huldt G, Forsberg E. Incidence of toxoplasmosis in pregnant women in the city of Malmö, Sweden. Scand J Infect Dis 1989;21:315–21.[ISI][Medline]

6 Krausse T, Straube W, Wiersbitzky S, Hitz V, Kewitsch A. Toxoplasmose screening in der Schangerschaft—ein Pilotprogram in Nordosten Deutschlands. Geburtsshilfe Frauenheilkd 1993;53:613–18.

7 Eskild A, Oxman A, Magnus P, Bjorndal A, Bakketeig LS. Screening for toxoplasmosis in pregnancy: what is the evidence of reducing a health problem? J Med Screening 1996;3(4):188–94.[Medline]

8 Guerina NG, Hsu H-W, Meissner HC et al. Neonatal serological screening and early treatment for congenital Toxoplasma gondii infection. N Engl J Med 1994;330:1858–63.[Abstract/Free Full Text]

9 Eaton RB, Hsu H-W, Grady GF. Newborn screening for congenital toxoplasmosma infection. Oral presentation at the 4th International Neonatal Screening Syposium, 13–16 June, 1999, Stockholm, Sweden.

10 Neto EC. One year experience on neonatal screening for congenital toxoplasmosis in South Brazil. Proceedings of the 3rd International Neonatal Screening Symposium, 21–24 October, 1996, Boston, Massachusetts, USA, pp. 68–70.

11 Eaton BR, Petersen E, Seppänen H, Tuuminen T. Multicenter evaluation of a fluorometric enzyme immunocapture assay to detect Toxoplasma-specific immunoglobulin M in dried blood filter paper specimens from newborns. J Clin Microbiol 1996;34:3147–50.[Abstract]

12 Pratlong F, Boulot P, Issert E et al. Fetal diagnosis of toxoplasmosis in 190 women infected during pregnancy. Prenatal Diag 1994;14: 191–98.[ISI][Medline]

13 Lynfield R, Guerina NG. Toxoplasmosis. Pediatrics in Review 1997; 18:75–83.[Medline]

14 Liesenfeld O, Press C, Montoya JG et al. False-positive results in immunoglobulin M (IgM) Toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test. J Clin Microbiol 1997;35:174–78.[Abstract]

15 Potasman I, Araujo FG, Remington J. Toxoplasma antigens recognised by naturally occurring human antibodies. J Clin Microbiol 1986;24: 1050–54.[ISI][Medline]

16 Tuuminen T, Seppänen H, Pitkänen E-M, Palomäki P, Käpyaho K. Improvement of immunoglobulin M capture immunoassay specificity: Toxoplasma antibody detection method as a model. J Clin Microbiol 1999;37:270–73.[Abstract/Free Full Text]