Maternal infection was defined according to the Lebech’s classification system and case definition [4]. Seroconversion in pregnancy is coded as “1.1.1” case and presents a higher risk of vertical transmission. In selected cases, the diagnosis was confirmed by detecting Toxoplasma DNA using PCR technique on amniotic fluid. The extraction of Toxoplasma gondii DNA was performed with the Extrablood kit (ELITech group, Molecular Diagnostic s.p.a.) according to manufacturer’s instructions as previously reported [5]. The identification was carried out by Real Time PCR with the Toxoplasma g. Elite MGB kit (ELITech group, Molecular Diagnostic s.p.a.), which specifically detects a RE region of Toxoplasma gondii (a short sequence repeated 200 to 300 times in the microorganism genome).

Expectant mothers were then referred for a clinical and ultrasound follow up while on an appropriate treatment (either spiramycin or combination therapy with pyrimethamine, sulfadiazine, and folinic acid), according to specific guidelines [3]. Infants underwent an instrumental assessment of the central nervous system alongside an eye exam and auditory testing within the first month of life. Cranial Ultrasonography (US) was performed by an experienced neonatologist, who was blinded to the clinical data, using the Philips HD11 ultrasound imaging platform with 8.5–12.4 MHz transducers (Microconvex and Phased Array transducers). The fundus oculi examination was performed by a pediatric ophthalmologist skilled in congenital infections. Hearing function was evaluated by transitory evoked otoacoustic emissions (TEOAE) at birth and by automatic auditory brainstem response (ABR) after three months of life if the previous evaluation was not normal.

Paired serum samples from the mother and the neonate were collected for determination of IgG, IgA and IgM antibodies levels. The anti-Toxoplasma antibodies IgG and IgM, and IgG-Avidity in patients’ serum, were measured with an automated quantitative test using the VIDAS Instrument (Biomerieux SA). The assay principle combines a two-step enzyme immunoassay sandwich method with final fluorescent detection (Enzyme Linked Fluorescent Assay—ELFA). For the qualitative detection of anti-Toxoplasma IgA, the in vitro diagnostic kit PLATELIA™ TOXO IgA TMB (Bio-Rad) was used, it is a solid-phase immunoenzymatic double sandwich method, with capture of the IgA anti-Toxoplasma in the solid phase.

The results of IgG and IgM antibodies were expressed in International Units per ml (IU/ml) and were considered as negative when less than 8 IU/mL and 0.65 IU/mL, respectively. The IgG upper detection limit was 300 IU/mL. A titer equal to or greater than 300 IU/mL is generally considered high [4]. The results of the IgA antibodies were expressed by calculating the Simple Ratio according to the formula: Sample O.D.450/620 nm / mean O.D.450/620 nm Cut-Off. The sample was positive for Sample index ≥ 1.

Data was recorded on a standardized database.

Patients

This is a prospective collection of data gathered from January 2014 to December 2020 at the Perinatal Infection Unit of the University “Federico II” of Naples, a tertiary care hospital with a dedicated multidisciplinary team.

Study inclusion criterion was the presence of positive neonatal IgG and negative IgM and/or IgA serology. Antibody determination was scheduled at 2 and 3 months of age and then every two months until complete negativization. The latter was confirmed after 30 days and a final antibody determination was provided at one year of life [3]. A different schedule was followed in case of a non-reassuring titer decrease.

Exclusion criteria included: parental denial to participate; neonates with history of detection of Toxoplasma gondii DNA in amniotic fluid and/or a positive anti-Toxoplasma IgM and/or IgA serology who then received an early diagnosis of active CT [2, 3]; evidence of CT after complete clinical, radiologic, and laboratory evaluation; infants who did not complete the 12 months follow up.

Data analysis

Statistical analysis was performed using the R software environment for statistical computing.

Data is presented as a Median (I Quartile; III Quartile) for quantitative variables and as frequency (percentage) for categorical variables.

For categorical variables, the test for significant associations was performed using the chi-square test with continuity correction. Correlation between quantitative variables was analyzed using the Pearson correlation coefficient. Comparisons between groups for quantitative variables were performed using the Wilcoxon Test and Bonferroni method for multiple comparison correction.

P value less than 0.05 was considered as statistical significance.

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