The size of the problem and the consequences of congenital CT

The estimated global incidence of CT is 190,100 annual cases; this corresponds to an incidence rate of approximately 1.5 cases in 1,000 live births [10]. In Brazil, the reported prevalence ranges from 0.1 cases to 3.4/1,000 live births and studies have estimated that approximately 35% of children had neurological diseases including hydrocephalus, microcephaly and mental retardation, 80% had ocular lesions and one report mentioned 40% of children with hearing loss [6,11].

Strategies to mitigate the impact of gestational and congenital toxoplasmosis

Prenatal treatment for Toxoplasma infection aims to prevent neurological or visual impairment by the reduction of mother-to-child transmission of infection or, once the fetal infection has occurred, by limiting cell damage caused by the parasite [12,13]. Information on the effectiveness of prenatal treatment is limited since it is challenging to carry out randomized studies with untreated versus treated groups [12]. However, it is possible to evaluate cases in which pregnant women were not treated as they arrived late to reference centers, missed appointments, or did not perform adequate prenatal care. The prenatal approach relies on the hypotheses that maternal treatment reduces the risk of mother-to-child transmission and congenital infection treated prenatally is associated with a lower risk of severe lesions [14,15]. Neonatal diagnosis can be performed by collecting anti-T. gondii serology in the maternity ward or through universal blood screening on filter paper. This neonatal screening was implemented in Denmark and is carried out in the states of Massachusetts and New Hampshire in the United States and Colombia [16–19]. The neonatal test using dried blood on filter paper became mandatory throughout Brazil in 2021 [20].

Brazil’s strategies

In Brazil, due to the high prevalence of toxoplasmosis, screening begins in the prenatal period to reduce the lifetime consequences of CT [21,22]. Since 2015, there has been a working group in the Brazilian Ministry of Health for the construction of integrated surveillance on gestational, congenital and acquired toxoplasmosis [23]. In 2016, gestational and CT became a disease of compulsory notification, which means an advance in the control of this pathogen once we understand better the impact that this disease has on the population [23]. Many advances have been achieved. The main purpose of screening is to identify susceptible pregnant women (without previous infection) for monitoring during pregnancy. Early detection aims to prevent fetal transmission and provide necessary gestational and postnatal treatment. All susceptible pregnant women (not infected) must perform at least three serology tests during pregnancy (in the first, second and third gestational trimesters) and another serology is recommended at the time of delivery or during the puerperium (on the maternity ward). The confirmed cases are referred for high-risk prenatal care. Ideally, serology for toxoplasmosis must be known in women before conception. Until 2018, there were no standardized guidelines for carrying out Brazil’s surveillance. However, some states of the union have developed protocols based on the local prevalence of the disease. The diagnosis is mainly based on indirect methods, such as serology (serology for the detection of IgG, IgM, IgA and the determination of IgG avidity anti-T. gondii), but also in methods of direct detection, such as molecular polymerase chain reaction techniques (real-time PCR and PCR), parasite isolation (cell cultures and inoculation in mice) and histological or immunohistological studies of the parasite [23]. The main methods used to confirm cases by the Brazilian Central Public Health Laboratories have been IgM, IgG serology and IgG avidity (until 16 gestational weeks). Eventually, they perform PCR according to laboratory capacity. Sometimes, it is necessary to combine different methods to achieve the proper assessment [23]. In 2021, the Ministry of Health included anti-T. gondii IgM serology in the heel prick test, which provides a greater chance of diagnosis in children despite maternal tests, given that it is performed universally (all children born in hospitals must have the heel prick test before discharge) [20].

Despite the advances, many challenges still need to be overcome, such as the implementation of public health services in the countryside, adequate training of health professionals to interpret laboratory results, guaranteeing the supply of drugs against gestational and congenital toxoplasmosis and, above all, providing evidence for the effectiveness of prenatal or postnatal treatment of toxoplasmosis.

In this study, we evaluated the benefit of drug treatment for pregnant women with acute toxoplasmosis as a protective factor in reducing the risk of congenital infection. Also, we evaluated the side effects of prenatal and postnatal drug treatment in children and assessed the risk of discontinuing the drug treatment in infants after two decreasing IgG anti-T. gondii serology tests. Finally, we describe clinical and radiological findings in children with congenital toxoplasmosis.

Ethics statement

The Ethics and Research Committee of the State University of Maringa approved this study (CAAE N° 09286918.5.0000.0104). The informed consent term was not obtained because the participants were kept anonymous.

Study design

The study was conducted from April 1, 2017, to December 31, 2021. This cohort study included all children born to women with confirmed acute toxoplasmosis (positive IgM and IgG anti-T. gondii) during prenatal care or diagnosed during delivery at the University Hospital of Maringá (HUM) University State of Maringá (UEM), South of Brazil. The HUM is certificated as a “Baby-friendly Hospital.” We used the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) to report this study.

Study population

The University Hospital of Maringá is located in the Northwest region of the state of Paraná. It is a teaching hospital that belongs to the State University of Maringá and provides care to 30 municipalities, which covers a population of approximately 800,000 inhabitants. Since 2005, the Hospital has offered specialized care to pregnant women suspected of having acute gestational toxoplasmosis through a high-risk outpatient clinic. In 2017, the hospital opened an exclusive outpatient clinic named "ToxoPed" for the follow-up of children born to these mothers.

Between 2017 and December 2021, 233 pregnant women were evaluated at the University Hospital of Maringá for suspected acute T. gondii infection. After analyzing and checking the exams (anti-T. gondii serologies, gestational ultrasounds) collected during the current pregnancy and evaluating the previous history of these women, we concluded that 130 had chronic toxoplasmosis (acquired before the current pregnancy), nine did not have the disease (they were referred due to an error in the interpretation of the serological exams) and 94 met the criteria for acute gestational toxoplasmosis.

Among the 94 acutely infected pregnant women, four children were born in particular hospitals and the mothers did no follow-up in our pediatric service. Twenty-six children were lost to follow-up during the COVID-19 pandemic and their outcome was not concluded.

There were three spontaneous abortions, where it was not possible to assess the presence of the parasite by the pathological anatomy. The family of one of the mothers who had an abortion was found to be infected (her three children and her husband); our study group evaluated the case, but we could not prove the source of infection.

Finally, we had the opportunity to follow-up 61 children in the “ToxoPed” outpatient clinic to conclude whether there was transplacental transmission of the parasite (Fig 1).

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Fig 1. Number of women and children evaluated for research.

https://doi.org/10.1371/journal.pntd.0011544.g001

Two groups of children were defined during follow-up. The first group was presumably infected children, called "exposed children". They presented negative serology for anti-T. gondii IgM, were IgG positive at birth and showed unaltered cerebrospinal fluid, physical examination, cranial computed tomography and retinal examinations. In this group, the IgG decreased and became negative during the first year of life and these children were considered free of toxoplasmosis. The second group was the children who were definitely infected during pregnancy. The following criteria were used to consider a child infected: i) positive anti-T. gondii IgM; ii) negative anti-T. gondii IgM but rising anti-T. gondii IgG; and iii) eye or brain lesions during follow-up.

Among the definitely infected children, we considered those who presented altered serology but unaltered brain tomography and no eye lesions to be asymptomatic.

Exclusion criteria were: children born to pregnant women with primary or acquired imunosupression, children with diseases or who used medications that could confuse the data analysis and children born to mothers who were chronically infected with T. gondii.

Laboratory procedure, follow-up and definitions

In the HUM/UEM high-risk prenatal care outpatient clinic, all of the pregnant women who confirmed acute toxoplasmosis received spiramycin when the infection was diagnosed in the first trimester and after 34 weeks of gestation, from 16 to 34 weeks of gestation, the women were treated with a combination of sulfadiazine, pyrimethamine and folinic acid. According to the institutional protocol (Table A in S1 File), all children started treatment at the maternity ward (rooming in) and were discharged using pyrimethamine, sulfadiazine and folinic acid. They had outpatient follow-ups every 45 days until they had completed 90 days. This way, the children had three serologies by the time they reached 90 days of life. The first serology was collected in the maternity ward after 24 hours of life (we did not use umbilical cord blood), the second when they were 30–35 days old and the third collected after 50 days. If the child presented a decrease in anti-T. gondii IgG serology at 90 days compared to the previous two readings, the medication was suspended and the follow-up continued every two months until one year. Children who were negative or had a zero value for anti-T. gondii IgG at 12 months old were defined as free of disease (the first group, “exposed”). Children considered infected received anti-Toxoplasma medication until 12 months of age. After reaching the age of one year, the medication was discontinued. All children can continue their follow-up at the "ToxoPed" outpatient clinic until they are 15 years-old. The intention is to follow these groups of children over time and study their neuropsychomotor development. Right now, our oldest patients are approximately 5 1/2 years old. Between 2017–2018, the amplified chemiluminescent method was used with Vitros ECiQ Toxoplasma IgG and IgM assays (Ortho-Clinical Diagnostics), according to the manufacturer’s specifications. Since December 2019, we have used microparticle immunoassay with ARCHITECT Toxoplasma IgG and IgM kits, according to the manufacturer’s specifications (Abbott, Wiesbaden, Germany).

In the group of infected children, after reaching the end of their first year of life, the medication was discontinued and the children continued with retinal assessments every six months in the first two years and were followed-up on psychomotor development. The social workers scheduled the child’s appointments in cases of absence. Neutropenia was considered when the number of neutrophils fell below 1,000/mm3.

Statistical analysis

The relative risk (RR) was calculated to assess whether prenatal treatment modifies the outcome in the child and to analyze the association between pediatric neutropenia and the condition “exposed” or “infected”. The significance of Fisher’s exact test level was p < 0.05 with a 95% confidence interval. Logistic regression was calculated to assess whether the use of pyrimethamine by the mother was associated with pediatric neutropenia (Tables B, C, and D in S1 File).

We examined the relationship between exposed children, infected children, the number of infants’ neutrophils, maternal treatment and postnatal treatment. We used factor analysis of mixed data (FAMD) to analyze the data set. FAMD is a principal components method to describe, summarizes and visualize multidimensional matrix with mixed data. As any principal components method, its aim is to study the similarities between individuals, the relationships between variables and to link the study of the individuals with the variables. Such methods reduce the dimensionality of the data and provide subspace that best represent the data. Briefly, this statistical method makes possible to analyze the similarity between individuals by considering mixed types of variables. We explore the association between all variables (Tables E and F in S1 File). In the multifactorial evaluation, the quantitative variables were grouped into dimensions 1 and 2 according to their contribution value (Figs A and B in S1 File).

After analyzing the variables, we built a decision tree to verify the safety of suspending the medication after three decreasing values of IgG anti-T. gondii; for this, we used the Rpart library. All analyses used R 4.0.2 software (R Foundation for Statistical Computing, Vienna, Austria). The principal libraries used were: factoextra, FactoMineR, ggplot2, dplyr and janitor. To correct the missing values, we used the algorithms contained in the input PCA of the R documentation [24,25]. The data is available on S1 Data.