Specific Antibody Titer Decay in Neonates Prenatally Exposed to Toxoplasma Gondii and Their Mothers.

doi:10.21203/rs.3.rs-1312595/v1

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Specific Antibody Titer Decay in Neonates Prenatally Exposed to Toxoplasma Gondii and Their Mothers.

https://doi.org/10.21203/rs.3.rs-1312595/v1

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Backgrounds: For infants exposed in utero to Toxoplasma gondii, current guidelines recommend monitoring the specific antibody titer until 12 months of age. In this study, we investigated the antibody titer decay in the mother-infant dyad.

Methods: This is a single center, population-based cohort study of neonates referred for prenatal exposure to Toxoplasma gondii from January 2014 to December 2020. All infants underwent clinical, laboratory and instrumental investigation for at least 12 months.

Results: A total of 670 eligible neonates were referred to the Perinatal Infection Unit of the University Federico II of Naples. Six hundred thirty-six (95%) completed the serological follow up until 12 months. Specific IgG antibodies negativization occurred in 628 (98.7%) within 5 months. At 9 and 12 months, all patients had negative IgG. An initial neonatal IgG antibody titer ≥200 IU/ml was associated with a longer time to negativization (184 [177.5;256] days when above threshold vs. 139.5 [101;179] days when below it; p<0.001). Maternal IgG antibody titer ≥200 IU/ml at childbirth was also associated to delayed time to negativization in the infant (179 [163;184] days above the cut off vs 125 [96.8;178] days below it; p<0.001). Specific antibody negativization was irreversible in all patients.

Conclusions: Lower anti-Toxoplasma antibody titers detected at birth in the mother-infant-dyad lead to an earlier and irreversible negativization. This information allows to customize the infant follow up program and avoids invasive and expensive tests.

Congenital toxoplasmosis

antibody titer

follow-up

Congenital Toxoplasmosis (CT) is an important cause of chorioretinitis, intracranial calcification, hydrocephalus and potentially severe and permanent neurological deficits (psychomotor delay or other neurological deficiencies, seizures, and mental retardation) [1], though at birth most infants appear asymptomatic.

Soon after birth, infection is investigated clinically and instrumentally but the diagnosis mainly relies on positive IgM and/or IgA titers, detection of Toxoplasma gondii DNA in the amniotic fluid or cerebrospinal fluid (CSF), positive IgM/IgA in CSF [2, 3]. When diagnostic criteria are not present at birth, the persistence of specific antibodies beyond 12 months of age is as an accepted demonstration of congenital infection [2, 3]. For this purpose, asymptomatic infants undergo periodic blood drawings that are both invasive and costly. A shorter, customized laboratory follow up would be desirable but reliable predictors are currently lacking.

The present population-based, cohort study investigates the hypothesis that initial lower anti-Toxoplasma antibody titers detected in the mother-infant-dyad may lead to an earlier and irreversible negativization. This information would provide a solid base for saving pain to the infant and costs to the health system.

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.

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 detecting Toxoplasma DNA by PCR technique on amniotic fluid. The extraction of Toxoplasma gondii DNA was performed with Extrablood kit (ELITech group, Molecular Diagnostic s.p.a.) according to manufacturer’s instructions as previously reported [Pagliuca C et al., 2017]. The identification was carried out by Real Time PCR with 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 admitted to 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 central nervous system and eyes and hearing function 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 an 8.5–12.4 MHz transducers (Microconvex and Phased Array transducers). Fundus oculi examination was performed by a pediatric ophthalmologist skilled in congenital infections. The 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 on 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^TM TOXO IgA TMB (Bio-Rad) was used, it is a solid-phase immunoenzymatic double sandwich method, with capture of the IgA anti-Toxoplasma on 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._450/620nm / mean O.D._450/620nm Cut-Off. The sample was positive for Sample index ≥1.

Study inclusion criterion was the presence of a neonatal positive 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 was provided at one year of life [3]. A different schedule was followed in case of a non-reassuring titer decrease.

Exclusion criteria were: 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]; no evidence of CT after complete clinical, radiologic, and laboratory evaluation; infants who did not complete the 12 months follow up.

Data were recorded on a standardized database.

Data analysis

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

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

For categorical variables, test for significant associations were performed using the chi-square test with continuity correction. Correlation between quantitative variables was analyzed by 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.

Population characteristics (Figure 1)

During the study period 46 diagnosis of CT were made at the Perinatal Infection Unit because of prenatal diagnosis (positive amniocentesis) or positive IgM and/or IgA at birth or specific clinical signs and were not included in the study. Out of a total of 670 neonates responding to inclusion criteria, 636 (95%) completed the follow up until twelve months and were considered for the study.

Data on maternal history and neonatal characteristics are summarized in Table 1. In most cases infection occurred in the first trimester (467 cases, that is 73.4%). In 79 cases (12.4%) a seroconversion during pregnancy was demonstrated (Lebech’s classification “1.1.1”): 21 in the first trimester, 37 in second one and 21 in the third one. Unsafe eating habits were reported in 119 mothers (19%); 52 women (8%) were pet owners and had higher IgG titers at childbirth compared to those who did not have them (137 [78; 205.8] vs 101 [63.8-164.2], p=0.027). Maternal IgG titers at childbirth were not statistically associated with presence of symptoms in pregnancy and unsafe heating habits.

Table 1

Maternal and neonatal characteristics
Lebech’s classification of maternal infection 111 Others Not known	79 (12.4%) 541 (85.1%) 16 (2.5%)
Maternal country of birth Italy European Union Others	591 (92.9%) 36 (5.7%) 9 (1.4%)
Maternal age (years)	30.1±5.3 (15-45)
Unsafe maternal eating habits Yes No Not known	119 (18.7%) 143 (22.5%) 374 (58.8%)
Pet owners Yes No Not known	52 (8.2%) 253 (39.8%) 331 (52%)
Specific symptoms in pregnancy Yes No Not known	63 (9.9%) 408 (64.2%) 165 (25.9%)
Prenatal ultrasound scan Normal Pathological	630 (99.1%) 6 (0.9%)
Amniocentesis Negative for Toxoplasma DNA Positive for Toxoplasma DNA Not performed	151 (23.7%) 0 485 (76.3%)
Specific prenatal therapy Yes No	505 (79.4%) 131 (20.6%)
Time of maternal infection periconceptional period first trimester second trimester third trimester undetermined	28 (4.4%) 467 (73.4%) 78 (12.3%) 41 (6.4%) 22 (3.5%)
Neonatal gender Male Female	326 (51%) 310 (49%)
Neonatal gestational age at birth (weeks)	38.5±1.6 (29-42)
Neonatal birth weight (grams)	3127±489.3 (1310-4840)

This table shows the main characteristics of mother and children enrolled in the study.

Management and follow-up

The first blood sample collected (T0) showed an IgG titer higher than 300 IU/mL in 35 mothers (5.5%) and 10 of their neonates (1.6%). Among these 35 mothers, 5 (14.3%) were classified as “1.1.1” according to Lebech; none of the 10 neonates with an IgG titer higher than 300 IU/mL was born to a mother with a seroconversion in pregnancy. Both higher maternal and neonatal IgG titers were not related with a high-risk class according Lebech’s classification (p 0.595 and p 0.410 respectively). After one month (T1) and two months (T2) from birth, specific IgG were positive in 630 (99.1%) and 585 cases (92%), respectively. At three months (T3) 388 neonates (61%) had still specific IgG. At 5 months (T5) only 8 (1.3%) were still positive. At 7 (T7) and 12 (T12) months all neonates were negative (figure 1). Time to negativization was not related to a high-risk class according Lebech’s classification (p=0.108).

Considering the small number of mothers and neonates with antibody concentration over 300 UI/mL, we used a cut off of 200 UI/mL for our subsequent analysis.

The neonatal initial titer was related to maternal antibody concentration at birth (r=0.71; p<0.001). Time to negativization was directly related to maternal and neonatal initial IgG value (r=0.71, p<0.001 and r=0.60, p<0.001, respectively). Moreover, time to negativization was directly related to maternal titer at delivery and initial neonatal titer, categorizing the population of mothers and child in three groups based on intervals of IgG (8-100 UI/mL, 101-199 UI/mL, ≥200 IU/mL), as shown in figure 2. Considering maternal titer at delivery, time to negativization in child has a median of 105 days [84 ; 141] in the first group, 171 days [125 ; 182] in the second and 179 days [163 ; 184] in the last one. Considering neonatal initial titer, time to negativization in child has a median of 125 days [98 ; 178] in the first group, 179 days [172 ; 184] in the second and 184 days [177.5 ; 256] in the last one.

Positivity at 180 days of life was statistically associated to maternal antibody value at delivery above the threshold of 200 UI/mL (12.6% vs 35.6%, p<0.001).

It was also statistically significant if we considered the neonatal value at birth (1.2% vs 9.1%, p<0.001). In particular, the 518 babies whose mothers had an IgG titer at delivery <200 UI/mL negativized specific IgG earlier than the 118 babies whose mothers had with an IgG titer ≥200 UI/mL [125 days (96.8; 178) vs 179 days (163; 184) – p<0.001]. Moreover the 615 babies with an IgG titer <200 UI/mL at first check negativized specific IgG earlier than the 21 babies with an IgG titer ≥200 UI/mL [139.5 days (101; 179) vs 184 days (177.5; 256), p<0.001].

Negativization of specific IgG was irreversible in all tested babies.

CT can be diagnosed at birth in presence of a definite serological status and clinical manifestations. The overall incidence of seroconversion and suspected infection in pregnancy is 0.8 per 1,000 live births and the incidence of congenital toxoplasmosis 0.1 per 1,000 live births when applying a strict protocol of screening, follow-up, and treatment in pregnant women [5]. Assuming an overall mother to infant transmission rate of 25% [2], up to 75% of infants without congenital infection present with a positive serology at birth. Based on expert consensus, a congenital infection is definitely excluded if specific IgG are negative at twelve months of life. In order not to miss any late diagnosis and subsequent delayed treatment with negative effect on prognosis, these infants undergo a minimum of five laboratory evaluations [2, 3].

On a large cohort, we show a significant correlation between maternal and neonatal initial IgG titer and his/her age at antibody negativization suggesting that in children with rapidly undetectable antibodies, it could be considered to reduce the number of laboratory evaluations. In our population at 5 months only 8 (1.3%) were still positive but at 7 months were all negative. All of these babies with a later negativization had mothers with an IgG titer at delivery ≥200 UI/mL and had themselves an IgG titer ≥200 UI/mL at first check. Moreover, no child showed a recurrence of previously negative specific IgG.

Based on these data, whenever negativization happened, it seemed to be suggestable to repeat serological evaluation directly at one year of life with less effort for the patient, his/her family and the health care system. We calculate that, given a cost of 20 euro for each laboratory work up, the total expenditure sums up to 140 euro per patient. Reducing serological evaluation for an average number of 150 patients per year, we could have an average money saving of 5,500 euros per year.

We recommend the current follow up protocol for dyads with initial antibody titers above the 300 IU/ml threshold.

An implemented protocol for an early CT diagnosis may also be achieved with more specific blood tests. Capobiango JD et al [6] have described, an immunoblot test for the comparison of mother and infant IgG and IgM immunological profiles at birth or shortly after. Mahmoudi S et al characterized an interferon-gamma release assay (IGRA) where an early CT diagnosis was achieved stimulating T-cells from infected neonates with T. gondii antigens [7].

The latter test has shown promising results in our preliminary experience that is currently limited by the high costs of the laboratory procedure.

In conclusion, babies prenatally exposed to T. gondii with a specific titer at birth below 200 IU/ml may safely benefit of a follow up program consisting in serological evaluation till negativization and then directly at twelve months of life, with significant sparing of infant’s stress and valuable savings for the health care system.

Congenital Toxoplasmosis

CSF

cerebrospinal fluid

Cranial Ultrasonography

TEOAE

transitory evoked otoacoustic emissions

ABR

automatic auditory brainstem response

ELFA

Enzyme Linked Fluorescent Assay

IGRA

interferon-gamma release assay

Ethics approval and consent to partecipate

The Ethics Committee of our Institution (Comitato Etico “Carlo Romano”, Università Federico II di Napoli) approved this study (protocol number 98/17). Consent to participate was obtained from parents or legal guardians of children involved in the study.

Consent for publication

Consent for publication was obtained from parents or legal guardians of children involved in the study.

Availability of data and materials

The dataset generated during and analyzed during the current study are not publicly available because individual privacy could be compromised but are available from the corresponding author on reasonable request.

Competing interest

The authors declare that they have no competing interests.

Funding

The authors of this publication did not receive any research funding.

Authors’ contributions

SS designed the study, interpreted results and drafted the article. CG, PFB, EC and FC were involved in the acquisition of data. PD analyzed and interpreted the patient data. PS performed the serological analysis. LC and FG contributed to the revision of draft and interpretation of data FR conceived the study and revised the article critically for important intellectual content. All authors read and approved the final manuscript.

Acknowledgments

We thank all the children subjects and participating to the present study and their families.

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You are reading this latest preprint version

Specific Antibody Titer Decay in Neonates Prenatally Exposed to Toxoplasma Gondii and Their Mothers.

Status:

Version 1

Abstract

Figures

Background

Patients And Methods

Data analysis

Results

Population characteristics (Figure 1)

Management and follow-up

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Version 1