Toxoplasma gondii is an intracellular parasite that may cause illness in humans if acquired congenitally or after a decrease in immunity due to infection or to drug treatment.1,2 This parasitic infection is common in Mexico, being present in 15–65% of the population.3,4 Congenital toxoplasmosis is an obstetric problem in Mexico, although epidemiologic reports are not recent and provide only total immunoglobulin class antibody determination.5 In addition, there are no prenatal or post-natal screening programs in this country; thus, actual rate of the congenital infection is unknown. However, a study conducted in the State of Jalisco (occidental zone of the country) showed a frequency of IgM antibodies of 3% in a sample of women with normal pregnancies and a higher frequency among women in risk groups.6
In Mexico, there is a National Screening Program for neonatal disorders, but until recently, only non-infectious diseases were included.7 Taking advantage of the laboratory network in this program, we performed a pilot study to determine the frequency of IgM antibodies to T. gondii in an unbiased population of infants in Mexico City and herein report the results obtained.
This pilot study was reviewed and approved by the Research and Ethics Committees of the National Institute of Pediatrics of the Ministry of Health. The reference laboratory of the neonatal screening system for congenital metabolic disorders was chosen as a source of samples to determine the frequency of T. gondii infection in infants. Whole blood samples are normally obtained on filter paper attached to a small questionnaire that contains sociodemographic data to locate the newborn in case of positive or suspicious results, as well as the general clinical status of the newborn as determined by the pediatrician. To avoid contamination with blood from the mother, 1,003 samples obtained from the heels of children ranging in age from 1 to 139 days (mean ± SD = 18.3 ± 13.0 days) and weighing between 1,250 and 4,500 grams (mean ± SD = 3,116 ± 453.2 grams) were used. Fifty-one percent of the children were males and all were born in an area that covers one-eighth of the political division of the Federal District of Mexico City (Municipalities of Iztapalapa and Coyoacán). This region includes populations of low, medium, and high socioeconomic levels. The questionnaires chosen were from children consecutively born during March and April 2003.
The blood samples were tested for the presence of IgM antibodies to T. gondii by a commercial capture enzyme-linked immunosorbent assay (ELISA) kit that includes an elution step used while incubating the samples (Thermo Lab-systems, Helsinki, Finland). The manufacturer reports sensitivity and specificity values of 98%. Positive samples were re-assayed by the same test. Case-patients were then located and the parents were informed about this infection, as well as the problems for the baby that could be caused by the parasite if the result was confirmed. They signed an informed consent form and agreed to treatment of the infant with pyrimethamine, sulfadiazine, and folinic acid, following a recommended regimen.1 A blood sample from the child and the mother was taken to obtain serum and confirm infection by western blot for IgG antibodies according to a method previously reported.8 This procedure allows detection of antibodies uniquely produced by the newborn, i.e., neo-antibodies, which cannot be explained by mother to fetus transplacental transfer. Briefly, T. gondii tachyzoites (15 × 106, > 95% pure parasites per gel) were separated by electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred onto nitrocellulose membranes by standard techniques.9,10 The membranes were blocked with 5% defatted milk, cut into 0.3-cm strips, incubated with the serum samples diluted 1:200, developed by incubation with peroxidase-anti-human IgG conjugate diluted 1:2,000, washed with 0.01 M phosphate buffer, pH 7.2, 0.15 M NaCl, 0.05% Tween 20, and treated with substrate/chromogen solution containing H2O2 and 4-chloro-1-naphthol.
Positive cases are subjected to clinical follow-up at a third-level hospital in the Neurology, Ophthalmology and Infectious Diseases Services. Few risk factors could be tested for association with IgM antibodies; for this purpose, the odds ratio was determined and statistical significance was assessed by Fisher’s exact.11
One thousand three samples from newborns were tested. Six hundred sixty-two (66%) of the newborns were less than three weeks of age at the time of sampling. The corresponding group of mothers was young, with 90% less than 30 years old (mean ± SD = 24.3 ± 5.8 years). Five percent of the newborns were premature, 1.2% showed malformations, and 1.8% were diagnosed as ill by the pediatrician. Three cases were above the ELISA cut-off level in the screening technique (6.5, 18.2, and 152.9 IU/mL), but only two male children, 6 and 25 days old, were confirmed (Table 1), giving a positivity rate of 1.9/1,000 newborns (95% confidence interval = 0.24–7.18, P < 0.05). A second sample taken 44 days later from one of the two cases (25 days old) was negative by the IgM screening test, but was positive when tested by for IgG by Western blot. His mother was positive by both the screening test and Western blot, and also had IgG (Table 1 and Figure 1). The second case (six days old) remained positive for IgM 25 days after the first sample was obtained and was also shown positive for IgG. His mother also had IgM and IgG antibodies. In both cases, the Western blot pattern showed newborn-specific bands, supporting congenital infection (Figure 1). One of the two cases was born premature, even though he presented no clinical manifestations.
Neonatal screening of congenital toxoplasmosis is routinely performed in several countries and is claimed to control the disease.12 This type of program does not exist in Mexico; thus, the current frequency of neonatal infections is unknown. The results of this study suggest a frequency of infection with T. gondii of approximately 2/1,000 newborns in Mexico City. Thus, the main risk factors for T. gondii infection must be addressed in a larger population with a questionnaire specifically designed.
Approximately 2,000,000 children are born in Mexico each year; thus, 4,000 undetected infants with congenital infection might be born in Mexico annually. Since there is a nationwide network for detection of congenital metabolic disorders already in place, it would be relatively easy to incorporate the detection of T. gondii infection within it to study the actual prevalence and the risk factors for this infection in Mexico.
Laboratory and clinical data of the two positive cases*
|IgM capture ELISA (IU/mL)||IgG|
|Case||First test||Second test||ELISA (IU/mL)||(Western blot)||Birth data|
|* ELISA = enzyme-linked immunosorbent assay.|
|1 (25-day-old newborn)||18.5||1.4||320||Positive||Asymptomatic, premature|
|2 (6-day-old newborn)||152.9||178.2||5,988||Positive||Asymptomatic, born at term|
Authors’ addresses: Marcela Vela-Amieva, Martha Pérez-Andrade, Claudia González-Contreras, Joel Ortíz-Cortés, and Venancio Ortega-Velázquez. Servicio de Genética de la Nutrición, Instituto Nacional de Pediatría, Secretaría de Salud, Av. Insurgentes Sur 3700-C, Colonia Insurgentes Cuicuilco, Mexico City 04530, DF, Mexico, E-mail:
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