François Kieffer

Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

Background Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mothers genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite.

Why prevent, diagnose and treat congenital toxoplasmosis?

Evidence that prevention, diagnosis and treatment of toxoplasmosis is beneficial developed as follows: anti-parasitic agents abrogate Toxoplasma gondii tachyzoite growth, preventing destruction of infected, cultured, mammalian cells and cure active infections in experimental animals, including primates. They treat active infections in persons who are immune-compromised, limit destruction of retina by replicating parasites and thereby treat ocular toxoplasmosis and treat active infection in the fetus and infant. Outcomes of untreated congenital toxoplasmosis include adverse ocular and neurologic sequelae described in different countries and decades. Better outcomes are associated with treatment of infected infants throughout their first year of life. Shorter intervals between diagnosis and treatment in utero improve outcomes. A French approach for diagnosis and treatment of congenital toxoplasmosis in the fetus and infant can prevent toxoplasmosis and limit adverse sequelae. In addition, new data demonstrate that this French approach results in favorable outcomes with some early gestation infections. A standardized approach to diagnosis and treatment during gestation has not yet been applied generally in the USA. Nonetheless, a small, similar experience confirms that this French approach is feasible, safe, and results in favorable outcomes in the National Collaborative Chicago-based Congenital Toxoplasmosis Study cohort. Prompt diagnosis, prevention and treatment reduce adverse sequelae of congenital toxoplasmosis.

Risk Factors for Retinochoroiditis During the First 2 Years of Life in Infants With Treated Congenital Toxoplasmosis

Background: Retinochoroiditis is the main complication of congenital toxoplasmosis. Its risk factors have rarely been investigated and were the object of this study. Methods: A retrospective study was conducted on 300 infants with congenital toxoplasmosis born between July 1, 1996 and December 31, 2002 and treated with pyrimethamine and sulfonamide for at least 12 months. Results of eye tests were collected up to 24 months. Risk factors associated with first retinochoroiditis were identified by univariate then multivariate analyses (Cox model). Results: One hundred forty-nine boys and 151 girls were included. Maternal infection dated from the first trimester in 34 cases, the second in 97 cases, and the last in 169 cases. At birth, 22 infants had cerebral calcifications. During the first 2 years of life, first retinochoroiditis was diagnosed in 36 infants (12%). In multivariate analysis, 3 factors were significantly associated with first retinochoroiditis before the age of 2 years: a delay of >8 weeks between maternal seroconversion and first treatment [hazard ratio, 2.54; 95% confidence interval (CI), 1.14–5.65], female gender (hazard ratio, 2.02; 95% CI, 1.01–4.1), and cerebral calcifications at birth (hazard ratio, 4.3; 95% CI, 1.9–10). There was no correlation between gestational age at the time of maternal infection and risk for retinochoroiditis. Conclusions: A delay of >8 weeks between maternal seroconversion and the beginning of treatment, female gender, and especially cerebral calcifications are risk factors for retinochoroiditis during the first 2 years of life in infants treated for congenital toxoplasmosis.

Accuracy of real-time polymerase chain reaction for Toxoplasma gondii in amniotic fluid.

OBJECTIVE: To provide clinicians with information about the accuracy of real-time polymerase chain reaction (PCR) analysis of amniotic fluid for the prenatal diagnosis of congenital Toxoplasma infection. METHODS: This was a prospective cohort study of women with Toxoplasma infection identified by prenatal screening in three centers routinely carrying out real-time PCR for the detection of Toxoplasma gondii in amniotic fluid. The data available were gestational age at maternal infection, types and dates of maternal treatment, results of amniocentesis and neonatal work-up and definitive infectious status of the child. We estimated sensitivity, specificity and positive and negative predictive values both overall and per trimester of pregnancy at the time of maternal infection. RESULTS: Polymerase chain reaction analysis was carried out on amniotic fluid for 261 of the 377 patients included (69%). It was accurate with the exception of four negative results in children who were infected. Overall sensitivity and negative predictive value were 92.2% (95% confidence interval [CI] 81–98%) and 98.1% (95% CI 95–99.5%), respectively. There was no significant association with the trimester of pregnancy during which maternal infection occurred. Specificity and positive predictive values of 100% were obtained for all trimesters. CONCLUSION: Real-time PCR analysis significantly improves the detection of T. gondii on amniotic fluid. It provides an accurate tool to predict fetal infection and to decide on appropriate treatment and surveillance. However, postnatal follow-up remains necessary in the first year of life to fully exclude infection in children for whom PCR results were negative. LEVEL OF EVIDENCE: III

Chapter 112 – Congenital toxoplasmosis

Congenital toxoplasmosis results from the transplacental transmission of the parasite Toxoplasma gondii after a maternal infection acquired in pregnancy. Prevalence of congenital infection ranges from 0.1 to 0.3 per 1000 live births. The maternal-fetal transmission rate increases with gestational age at maternal seroconversion, from less than 15% at 13 weeks of gestation to over 70% at 36 weeks. Conversely, the later the maternal infection, the lower the risk of symptomatic congenital infection (infections acquired during the third trimester are most often asymptomatic at birth). Prenatal diagnosis is currently performed by PCR analysis in amniotic fluid. Antenatal management and treatment vary considerably among countries. In some European countries, maternal infections are detected through serological screening allowing a prompt treatment with spiramycin, which is expected to reduce the risk of vertical transmission. If PCR analysis in amniotic fluid is positive or if maternal infection was acquired in the third trimester of pregnancy, a combination with pyrimethamine and sulphonamide is given until delivery. Benefits of antenatal treatments remain controversial. Infected newborns are prescribed pyrimethamine and sulphonamide for 12 months. Despite antenatal and postnatal treatment, chorioretinitis can occur at any age (prevalence>20% at 10 years of age): long-term ophthalmological follow-up remains necessary.

Congenital Toxoplasmosis in France and the United States: One Parasite, Two Diverging Approaches

At 29 weeks of gestation, a pregnant woman in the United States was told that her fetal ultrasound had revealed the presence of hydrocephalus. She did not recall having any symptoms or risk factors during gestation. Serological testing at the national reference laboratory for toxoplasmosis in the US (http://www.pamf.org/serology/) confirmed an acute infection likely acquired around 17 weeks of gestation. She was started on pyrimethamine/sulfadiazine, at 31 weeks. Her amniotic fluid was positive for Toxoplasma gondii DNA by polymerase chain reaction (PCR). The infant was born with hydrocephalus, brain calcifications, and chorioretinitis. In France, a screening program has been in place since 1992, and pregnant women with negative serology are tested monthly until delivery. If this pregnant woman had been followed according to this program, she would have been started on spiramycin 14 weeks earlier than in the US, her amniotic fluid would have been tested for T. gondii by PCR at 18 weeks, and she would have been started on pyrimethamine/sulfadiazine 11 to 8 weeks earlier than in the US. In the US, such a program does not exist, and infection is usually diagnosed when clinical signs are present in the fetus or at birth. Severe disease is not an uncommon situation in the US [1], but it is apparently a rare event in France (Table 1) [2]. Why is a congenital infection caused by the same parasite approached so differently in France than in the US? Below, we will discuss several factors that could account for these diverging approaches. Table 1 Differences in T. gondii genetics, epidemiology, clinical manifestations, and approach to infection detection and management during pregnancy, between France and the US. France US Parasite Genetics Type II: >95%; other types are very rare Type II: 41.5%; non-type II (including atypicals): 58.5% IgG Seroprevalence in Women of Childbearing Age 37.0% 9.1% Incidence of Acute Infection among Toxoplasma-Seronegative Pregnant Women 2.1/1,000 0.2/1,000* Incidence of Congenital Toxoplasmosis 2.9/10,000 live births 0.5/10,000 live births* Clinical Signs of Congenital Toxoplasmosis in Newborns: Absent, Mild–Moderate, and Severe 85%, 10%, and 3% 12%, 11%, and 77% Screening of Toxoplasma-Seronegative Pregnant Women Yes. Systematic screening is performed every month No. However, systematic screening is performed in some obstetric practices Diagnosis of Acute T. gondii Infection by Seroconversion Yes, because sequential samples are available Rare Diagnosis of Acute T. gondii Infection by the Use of a Single Serum Rare Yes. Only a single serum is available. Positive Toxoplasma IgM samples require confirmatory testing at reference centers such as the Palo Alto Medical Foundation Toxoplasma Serology Laboratory** Recommendation of Treatment, Amniotic Fluid for T. gondii PCR Testing, and Serial Ultrasounds for Acutely Infected Women Yes Yes Indication of Infants Workup for Congenital Toxoplasmosis at Birth Yes, for each newborn born to a mother infected during gestation regardless of the presence of clinical signs or laboratory/radiological abnormalities Yes, for those with clinical signs and/or laboratory/radiological abnormalities suggestive of congenital infection. Seldom, for infected infants without clinical signs or laboratory/radiological abnormalities whose mothers were suspected of having or diagnosed with toxoplasmosis during gestation Postnatal Treatment of Congenitally Infected Infants Yes, for infected infants of acutely infected pregnant women (symptomatic or asymptomatic) diagnosed with congenital toxoplasmosis in utero or postnatally, regardless of the presence of clinical, laboratory, or radiological abnormalities Yes, for infants diagnosed with congenital toxoplasmosis because of the presence of clinical signs in utero or at birth. Seldom, for infected infants without clinical, laboratory, or radiological abnormalities in whom the diagnosis of congenital toxoplasmosis was made in utero or postnatally because of the presence of maternal illness, risk factors, or systematic screening by an obstetric practice Open in a separate window Ig, immunoglobulin.

Disseminated congenital toxoplasma infection with a type II strain.

Disseminated congenital toxoplasmosis mimicking septic shock is unusual. We report a fatal case of disseminated congenital toxoplasmosis that was acquired after a third trimester maternal primary infection. The child had severe pneumonitis, purpura, and hepatitis. After 5 days of treatment, quantitative polymerase chain reaction analysis showed that parasite loads in the serum and in tracheal aspirates had decreased. The child died of refractory hypoxemia. Genotyping revealed a type II strain.

Use of IgG in Oral Fluid To Monitor Infants with Suspected Congenital Toxoplasmosis

ABSTRACT Infants born to mothers who seroconverted for toxoplasmosis during pregnancy are at risk of sequelae. In the case of a negative work-up at birth, congenital infection can be ruled out only by monitoring the disappearance of maternal immunoglobulin G (IgG) transmitted through the placenta, which can be achieved by regular blood sampling during the first year. To alleviate the discomfort of this follow-up, we developed an indirect enzyme-linked immunosorbent assay to detect specific IgG diffusing passively from the blood through the gingival epithelium by collecting oral fluid on microsponges. To assess the feasibility of the test, 212 patients were first enrolled. Levels of specific IgG in oral fluid were significantly higher in seropositive (n = 195) than in seronegative (n = 17) patients (mean optical densities, 1.145 ± 0.99 versus 0.092 ± 0.127; P < 0.0001). In a population of 93 patients <15 months of age born to mothers who displayed toxoplasmic infection during pregnancy, 70 were free of congenital infection and were followed up until their serology turned negative, and 23 were congenitally infected. The same patterns of IgG were observed in the oral fluid and sera in each group. Using a cutoff of 0.04 (optical density value), the sensitivity and specificity of the test were 67.9% and 80.3%, respectively, and the probability of not having a congenital infection when the test on oral fluid was negative was 99%. Although the performance of the test needs to be improved, oral fluid sampling appears to be a promising tool for monitoring infants with suspected congenital toxoplasmosis.

Prenatal therapy with pyrimethamine + sulfadiazine vs spiramycin to reduce placental transmission of toxoplasmosis: a multicenter, randomized trial

BACKGROUND: The efficacy of prophylaxis to prevent prenatal toxoplasmosis transmission is controversial, without any previous randomized clinical trial. In France, spiramycin is usually prescribed for maternal seroconversions. A more potent pyrimethamine + sulfadiazine regimen is used to treat congenital toxoplasmosis and is offered in some countries as prophylaxis. OBJECTIVE: We sought to compare the efficacy and tolerance of pyrimethamine + sulfadiazine vs spiramycin to reduce placental transmission. STUDY DESIGN: This was a randomized, open‐label trial in 36 French centers, comparing pyrimethamine (50 mg qd) + sulfadiazine (1 g tid) with folinic acid vs spiramycin (1 g tid) following toxoplasmosis seroconversion. RESULTS: In all, 143 women were randomized from November 2010 through January 2014. An amniocentesis was later performed in 131 cases, with a positive Toxoplasma gondii polymerase chain reaction in 7/67 (10.4%) in the pyrimethamine + sulfadiazine group vs 13/64 (20.3%) in the spiramycin group. Cerebral ultrasound anomalies appeared in 0/73 fetuses in the pyrimethamine + sulfadiazine group, vs 6/70 in the spiramycin group (P = .01). Two of these pregnancies were terminated. Transmission rates, excluding 18 children with undefined status, were 12/65 in the pyrimethamine + sulfadiazine group (18.5%), vs 18/60 in the spiramycin group (30%, P = .147), equivalent to an odds ratio of 0.53 (95% confidence interval, 0.23–1.22) and which after adjustment tended to be stronger (P = .03 for interaction) when treatment started within 3 weeks of seroconversion (95% confidence interval, 0.00–1.63). Two women had severe rashes, both with pyrimethamine + sulfadiazine. CONCLUSION: There was a trend toward lower transmission with pyrimethamine + sulfadiazine, but it did not reach statistical significance, possibly for lack of statistical power because enrollment was discontinued. There were also no fetal cerebral toxoplasmosis lesions in the pyrimethamine + sulfadiazine group. These promising results encourage further research on chemoprophylaxis to prevent congenital toxoplasmosis.

Cesarean delivery or induction of labor does not prevent vertical transmission of toxoplasmosis in late pregnancy

Toxoplasmosis during pregnancy can have deleterious effects on the fetus, ranging from severe neurologic or ophthalmologic lesions to fetal loss. In some cases, infection appears subclinical at birth but patients are at risk of developing chorioretinitis [1]. The stage of pregnancy at maternal infection is the main predictive factor for the risk of fetal toxoplasmosis and its severity [2]. For third trimester infections, the placental barrier is less efficient than at the beginning of pregnancy, and the risk of fetal infection reaches 58% [3]. Conversely, the severity of the disease is inversely related because the majority of newborns are asymptomatic. The management of maternal infections in the first and second trimesters is well codified, relying on amniocentesis to determine treatment with pyrimethamine and sulfonamides. The approach is less standardized for later maternal infections. Because of the high risk of congenital infection, obstetricians might consider performing cesarean delivery or induction of labor to prevent the risk of vertical transmission by reducing the length of fetal exposure to the parasite. The present study reports a series of 10 infants born tomothers who became infected with Toxoplasma gondii between 33 and 39 weeks of pregnancy (median, 36 weeks) and for whom a cesarean delivery or induction of laborwasperformedbetween 38 and40weeks (median, 39 weeks) to protect the newborn from toxoplasmosis. The study covered the period from October 1994 to July 2012. Date of maternal infection was serologically confirmed in the department. No amniocentesis was performed and five women were treated with spiramycin (Table 1). The median delay between the estimated date of maternal infection and delivery was three weeks. Nine newborns were diagnosed with congenital toxoplasmosis, subclinical at birth in eight cases, and one had chorioretinitis. In third trimester infections, vertical transmission occurs rapidly after maternal infection and cesarean delivery or induction of labor, even when performed shortly after the estimated date of infection, is inefficient at preventing congenital toxoplasmosis. Another approach comprises presumptive treatment with sulfadiazine and pyrimethamine; however, at the end of pregnancy, 30% of children would be treated unnecessarily [2]. Amniocentesis allows parasitic detection by polymerase chain reaction, with sensitivity and specificity of 73.5% and 100%, respectively, during the third trimester [3], and should be performed whenever possible, with treatment prescribed only for positive cases. Toxoplasmosis in late pregnancy should bemanaged first by amniocentesis whenever feasible. Induction of labor or cesarean delivery does not prevent congenital toxoplasmosis. Comparedwith termbirths, early-term deliveries are associated with greater morbidity [4]. http://dx.doi.org/10.1016/j.ijgo.2014.11.013 0020-7292/© 2014 Published by Elsevier Ireland Ltd. on behalf of International Federation of Gynecology and Obstetrics.