Diagnosis of Congenital Toxoplasmosis: Challenges and Management Outcomes

Introduction Congenital toxoplasmosis (CT), despite being mostly subclinical at birth, can cause disabling disease in the fetus and lead to long-term sequelae. It is an important cause of chorioretinitis in infants and adolescents. Data on postnatal treatment are controversial, and there is a lack of universal guidelines. Methods A cross-sectional study of newborns with suspected CT was conducted between January 2007 and December 2021. Results Seventy-one patients with suspected CT were included. During pregnancy, 64 (90.1%) of the mothers underwent therapy, of which 59 (83.1%) with spiramycin. Amniocentesis identified one positive polymerase chain reaction assay. Most newborns were asymptomatic with normal laboratory, ophthalmological, and hearing screening. There was one case of hyperproteinorrachia. Fifty-seven patients (80.3%) started treatment: 42 (73.7%) with spiramycin, seven (12.3%) with pyrimethamine, sulfadiazine, and folinic acid (P+S+FA), and eight (14%) with P+S+FA intercalated with spiramycin. Adverse effects were found in 11 (19.3%) cases, mainly neutropenia. After investigation, we found three confirmed CT cases corresponding to 4.2% of suspected cases and an incidence of 0.4 per 10,000 births. All had normal clinical and laboratory exams in the neonatal period and started P+S+FA, fulfilling 12 months of therapy. During the follow-up, all presented normal psychomotor development without any long-term sequelae. Conclusion The lower incidence in our study, compared to the incidence in Europe, may be related to the decline in the prevalence of toxoplasmosis as well as the effectiveness of measures to prevent primary infection and a well-established program of antenatal screening, followed by the early initiation of treatment during pregnancy to prevent vertical transmission.


Introduction
Toxoplasmosis is caused by infection with Toxoplasma gondii, an intracellular protozoan parasite with a worldwide distribution and one of the most common parasitic infections in humans.T. gondii infection is most typically asymptomatic, but primary infection in pregnant women can lead to congenital toxoplasmosis (CT).CT results from the hematogenous transmission of the parasite across the placenta and can cause severe and disabling disease in the developing fetus and newborn [1][2][3].
The risk of fetal infection varies depending on whether the mother receives treatment in pregnancy and increases with gestational age.On the contrary, the risk of severe CT is inversely proportional to gestational age [2,4,5].Therefore, fetal infections that develop in the early stages of pregnancy may result in negative outcomes like spontaneous abortion or brain damage, whereas fetal infections that occur in the late stages of pregnancy are typically subclinical [6][7][8].
Although it is subclinical in roughly 75-90% of infected newborns, CT has a wide range of unspecific clinical manifestations.Only a small percentage presents with the complete classic triad (hydrocephalus, intracranial calcifications, and chorioretinitis) [2,3,9].The most characteristic ophthalmological lesion is focal chorioretinitis, and ocular lesions can develop or recur during childhood and adolescence or even later in life [3,10].
Data on the efficacy of prenatal and postnatal treatment are scarce, and there are no universal guidelines for postnatal treatment.Furthermore, the burden of the disease varies in different countries, and as a result, practices vary between centers [3,11].There is also no clear consensus on the management of asymptomatic infants in whom CT cannot be confirmed or ruled out during the initial evaluation [11].In the past, our protocol for treating asymptomatic doubtful cases of CT included treatment with cycles of pyrimethamine, sulfadiazine, and folinic acid (P+S+FA) for four weeks alternating with spiramycin for six weeks, which is no longer recommended because spiramycin is a parasitostatic drug and doesn't have benefits after the infection has occurred.Current treatment regimens include a combination of P+S+FA for one year and seem to be associated with significantly fewer and less severe sequelae [12,13].
This study aimed to characterize the suspected cases and determine the incidence of CT in a tertiary hospital.This research was previously presented as an abstract and e-poster at the 41st Annual Meeting of the European Society for Paediatric Infectious Diseases, held from May 8 to 12, 2023.

Study design, settings, and patients
A cross-sectional study was performed with a sample of newborns with suspected CT followed in the Neonatology Department of a tertiary center Data on the prenatal background (maternal gravidity and parity, maternal toxoplasmosis serology during pregnancy and trimester of seroconversion, amniocentesis and its PCR result, prenatal ultrasounds, and maternal therapy), birth data (type of delivery, sex, gestational age, and birth weight), postnatal period data (symptomatology and physical examination, including ophthalmological exam and hearing screening), postnatal investigation of CT (complete blood count, liver enzymes, cerebrospinal fluid (CSF) analysis, and transfontanellar ultrasound), treatment and its complications data, and follow-up were gathered from the electronic records.
A part of the investigation was carried out in a reference laboratory, involving the direct detection of the parasite through the inoculation of a newborn blood sample and placenta into mice.The results were observed at 10 days, three weeks, and six weeks after inoculation.Additionally, the investigation included a comparative analysis of mother/newborn IgG and IgM using Western blot, as well as a PCR assay conducted on newborn blood.

Ethical approval
This research complies with all the relevant national regulations and institutional policies and is in accordance with the tenets of the Helsinki Declaration.The study was approved by the Clinical Research Department and Ethical Committee of Centro Hospitalar Universitário de Santo António and Institute of Biomedical Sciences Abel Salazar (approval number: 2023.167).In line with recent normative, the requirement to obtain informed consent was waived for this study.

Statistical analysis
Data were collected, processed, and analyzed with IBM SPSS Statistics for Windows, Version 28.0 (Released 2021; IBM Corp., Armonk, New York, United States).Categorical variables were described using absolute and relative frequencies.

Results
During the study period, there were 49,025 births, and 80 (0.16%) of those had a suspicion of maternal toxoplasmosis during pregnancy.Fourteen cases were excluded, because suspicion was not supported (falsepositive cases, unconfirmed maternal seroconversion, and maternal infection prior to conception).Five newborns were referred from other hospitals, resulting in a final sample of 71 suspected CT.
The sample demographics and clinical characteristics of suspected CT cases are shown in Table  Most of the newborns (81.7%) were asymptomatic in the first days of life.Of the symptoms that can be linked to CT, jaundice was the only manifestation, and it was present in 13 cases (18.3%), six of which required phototherapy.One case of anemia and six cases of significant increase in liver enzymes were found on laboratory work-up that included a complete blood count and liver enzymes in 68 and 66 cases, respectively.CSF analysis was performed in 51 (71.8%) neonates and identified one case of hyperproteinorrachia.
Transfontanellar ultrasound was used for neuroimaging in 68 (95.8%) newborns, showing unspecific and non-typical findings for CT in nine cases.An ophthalmological exam was conducted in 64 (90.1%) and was altered in two cases, but these findings weren't related to CT.The hearing screening was performed in all neonates, being altered in one case.
The inoculation of a mouse with a placenta sample led to the detection of the parasite in two cases (one at 10 days and the other at six weeks after inoculation); the Western blot comparative analysis of mother/newborn IgG and IgM was performed in 37 (52.1%)cases, and it was positive in one; the PCR assay in newborn blood was done in 55 (77.5%) cases, and it was negative in all of them.
Three cases of positive serology for T. gondii were found at 12-month follow-up, all of them with positive IgG and one with positive IgM.The time for IgG to become negative was less than two months (n=5), between two and six months (n=25), between six and nine months (n=25), and between nine and 12 months (n=11), and two were lost to follow-up.
Fifty-seven patients (80.3%) started treatment for suspected CT: 42 (73.7%)with spiramycin, seven (12.3%) with P+S+FA, and eight (14%) with intercalated cycles of spiramycin and P+S+FA.The newborn with hyperproteinorrachia was also treated with corticosteroids.Neutropenia was the most frequent adverse effect and occurred in 10 cases (17.5%), all of which were under therapy with P+S+FA.The dose of FA was increased in all these patients, up to 20 mg three times per week, but due to sustained neutropenia, the treatment had to be suspended in one patient.Spiramycin was discontinued in one patient because of oral intolerance.Two negative serologies were obtained prior to treatment discontinuation, and the timing of suspension was less than one month (n=4), after one month (n=23), after two months (n=13), after three months (n=5), after four months (n=1), after five months (n=3), after seven months (n=1), after eight months (n=1), and after 12 months (n=3).Three patients were lost to follow-up.
In conclusion, there were three confirmed cases of CT (one of them was outborn), which corresponds to 4.2% of suspected cases and an incidence of 0.4 per 10,000 births with a vertical transmission rate of 3% in our unit.Table 2 summarizes the clinical presentation, investigation, and management of the three confirmed CT cases.[15].Some genotypes are more virulent and may contribute to justifying these geographical differences throughout the world, and in addition, some are also more correlated with chorioretinitis.
T. gondii can be transmitted to humans by consumption of raw and poorly cooked meat containing viable tissue cysts, ingestion of water or food contaminated by oocysts excreted in cat feces, or vertical transmission during pregnancy [12,16].The seroprevalence of T. gondii has been declining, particularly in developed countries, and this trend can be attributed to a number of factors, including increased frozen meat consumption, modern mass-produced meat production, improved hygiene, urbanization, etc. [9].In Portugal, according to the latest epidemiological data, the seroprevalence of T. gondii infection has been declining, from a rate of 47% in 1979 to 18% between 2009 and 2020.Therefore, approximately 80% of women of childbearing age are susceptible to toxoplasmosis infection during pregnancy, with a risk of CT in the newborn [17].It is essential to know the proportion of susceptible pregnant women in any given area to establish the best strategy for the local prevention of CT.

Prenatal surveillance
In the literature, it is described that the risk of vertical transmission increases directly proportional to gestational age at the time of primary maternal infection and may be reduced by antenatal treatment [12].In our research, most (54.9%) of maternal seroconversion occurred in the first trimester, which is associated with a lower probability of placental transmission and can contribute to the lower number of cases of CT, with a vertical transmission rate of 3%.Spiramycin was the most used drug, and it was also the drug used by all three mothers of newborns with confirmed CT.However, currently, spiramycin is used for preventing fetal infection, and P+S+FA is reserved for the treatment of confirmed fetal infection or when fetal infection occurs in the third trimester and amniocentesis is not performed, due to the high fetal risk.

Postnatal period
The majority of CT cases, according to the literature, are asymptomatic, and in our study, the three confirmed cases were asymptomatic.
In CT, CSF analysis can reveal hyperproteinorrachia or mononuclear pleocytosis, and the detection of T. gondii by PCR can confirm the diagnosis.We found one case of hyperproteinorrachia, which corresponded to a CT-confirmed case, but PCR of CSF is not usually performed in our center.
In Europe, neuroimaging with transfontanellar ultrasound is more frequently used, which relates to the widespread availability and absence of radiation, despite computed tomography being described as having a higher sensitivity for the detection of calcifications [3].In our center, the neuroimaging exam used in all cases was transfontanellar ultrasound, and no calcifications or other typical findings of CT were found.

Specific investigation
A CT diagnosis can be reached through the combination of the following: newborn positive IgG and IgM for T. gondii; newborn IgG with a significantly higher value than maternal IgG at birth; positive PCR assay in the amniotic fluid; positive PCR assay in newborn blood or CSF; or the persistence of positive IgG after 12 months, which is the gold standard.However, interpretation of serologic testing can be challenging for the following reasons: positive newborn IgG can reflect past or current infection in the mother since it crosses the placenta; small amounts of maternal IgM can cross the placenta, resulting in low IgM levels in uninfected newborns if performed soon after birth; otherwise, if infection happens late in pregnancy, cases of false-negative IgM may occur, as IgM appears within 1-2 weeks of exposure; and maternal therapy can alter the serological profile of the newborn since it can delay IgG production [12].
The support of a reference laboratory is fundamental for an accurate diagnosis of CT, and in our three confirmed cases, all had positive findings in the investigation carried out in this laboratory: a positive result after inoculation in the mouse of a blood sample or placenta (n=2), a different pattern in Western blot (n=1), and detection of T. gondii in the amniotic fluid with a PCR assay (n=1).
After discontinuation of treatment, a rebound rise of IgM and/or IgG for T. gondii may be seen in CT cases, and this situation is thought to be due to a delayed serological response to infection rather than a relapse.Therefore, a positive IgG beyond 12 months of age is considered the gold standard and confirms a diagnosis of CT [12].In our study, the three cases of CT had positive IgG at 12 months, and one had concomitantly positive IgM.

Treatment and adverse effects
It is fundamental to weigh the risks and benefits before starting therapy in suspected or inconclusive cases since therapy can be associated with adverse effects.There is no clear consensus on treating asymptomatic cases, and in some studies, clinicians consider monitoring serology while postponing treatment [12,18].
Complete blood counts and liver enzyme tests must be performed prior to initiating treatment, and subsequently, adverse effects must be monitored.Hematological toxicity is the principal adverse effect of pyrimethamine and sulfadiazine, and neutropenia is described as occurring in more than half the children treated with these drugs, but the addition of folinic acid reduces the likelihood of this adverse effect [3].In our study, of the 15 patients who did P+S+FA, neutropenia occurred in 10 (66.7%), and all but one resolved after increasing the dose of FA.Two cases of confirmed CT developed neutropenia after treatment with P+S+FA, and none had hepatotoxicity.

Follow-up
In our study, none of the three cases developed chorioretinitis during follow-up.However, patients with CT can be asymptomatic until the second or third decade of life, when lesions develop in the eye, presumably due to cyst rupture and the subsequent release of tachyzoites and bradyzoites [2].Nevertheless, regular follow-up in an ophthalmology consultation is recommended to be maintained due to the possibility of the occurrence or recurrence of chorioretinitis at least to an age when the child can identify significant visual changes and, according to some studies, annually until the second decade of life [2,10,19,20].

Strengths and limitations
The major strength of this study was the 15 years of data collection, which allowed for a comprehensive characterization of newborns with suspected CT at a tertiary pediatric hospital.However, this study has some limitations.Firstly, like any retrospective study, it has inherent limitations associated with data collection from medical records, including missing or incomplete information.Secondly, the low number of confirmed CT cases and the absence of a control group limit the ability to make direct comparisons or draw definitive conclusions regarding the positive cases.Despite these limitations, the study provides valuable insights into the clinical and investigation aspects as well as the therapeutics and complications associated with suspected CT.

Conclusions
The low incidence of CT in our study may be related to the decline in the prevalence of toxoplasmosis observed in our country, as well as the effectiveness of measures to prevent primary infection and a wellestablished program of neonatal screening, followed by the early initiation of treatment during pregnancy to prevent vertical transmission.New guidelines must be developed to avoid the high number of cases of unconfirmed CT that are submitted to therapy, whose adverse effects cannot be neglected, as documented in our study.Furthermore, the available evidence on CT is limited and relies essentially on observational studies.In the future, new studies are needed to determine the global impact of congenital toxoplasmosis, as well as the efficacy of postnatal treatment in preventing the emergence of late sequelae in asymptomatic cases.
(Centro Materno-Infantil do Norte, Centro Hospitalar Universitário de Santo António, Porto, Portugal) between January 2007 and December 2021.Inclusion criteria comprised neonates born in our hospital or referred from other hospitals with suspected CT: maternal serology for T. gondii with proven seroconversion during pregnancy; positive IgM and IgG with any degree of avidity or positive IgM with negative IgG; a positive polymerase chain reaction (PCR) assay for T. gondii in the amniotic fluid; and characteristic symptoms of CT.In contrast, exclusion criteria were cases of suspected CT where suspicion was not supported after an accurate investigation.