Effect of prenatal treatment on mother to child transmission of Toxoplasma gondii: retrospective cohort study of 554 mother-child pairs in Lyon, France

RE Gilberta, L Grasa, M Wallonb, F Peyronb, AE Adesa and DT Dunna,c

a Centre for Paediatric Epidemiology and Biostatistics, Institute of Child Health, London, UK.
b Laboratoire de Parasitologie et de Pathologie Exotique, Hôpital de la Croix Rousse, Lyon, France.
c Medical Research Council Clinical Trials Unit, London, UK.

Dr Ruth Gilbert, Centre for Paediatric Epidemiology and Biostatistics, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Abstract

Background The aim of prenatal serological screening for toxoplasmosis is to identify and treat maternal infection as soon as possible in order to prevent transmission of the parasite to the fetus. However, despite widespread provision of prenatal toxoplasma screening across Europe, the effectiveness of prenatal treatment is uncertain. The study aimed to determine the effect of the timing and type of prenatal treatment on mother to child transmission of Toxoplasma gondii.

Method A cohort of 554 infected pregnant women were identified in Lyon, France between 1987 and 1995 and their children were followed to determine congenital infection status. We determined the effect of prenatal treatment on transmission by examining the effect of the delay between maternal seroconversion and start of treatment. We also compared the effect of the type of treatment and no treatment on the risk of mother to child transmission. Analyses were adjusted for gestation at maternal seroconversion.

Results Compared to treatment within 4 weeks from seroconversion, the adjusted odds ratios (OR) for mother to child transmission after a treatment delay of 4–7 weeks was 1.29 (95% CI : 0.61, 2.73) and after more than 8 weeks, 1.44 (95% CI : 0.60, 3.31). The adjusted OR associated with spiramycin alone compared with pyrimethamine-sulfadiazine treatment was 0.91 (95% CI : 0.45, 1.84) and the OR for no treatment compared with pyrimethamine-sulfadiazine treatment was 1.06 (95% CI : 0.37, 3.03).

Conclusions The authors hypothesize that the absence of an effect of prenatal treatment is due to transmission before the start of treatment.

Keywords Congenital toxoplasmosis, treatment, vertical transmission

Accepted 24 May 2001

Routine testing for toxoplasma infection during pregnancy is offered in many European countries.1 The aim is twofold. To treat infected pregnant women with antibiotics in order to reduce the risk of fetal infection and, if fetal infection has occurred, to reduce the risk of impairment in the child.2 In this report, we present results relating to the first aim of treatment, the reduction in mother to child transmission of infection.

Two possible mechanisms for the transmission of infection from mother to child have been suggested.3,4,5,p.156 First, infection is transmitted to the fetus via the placenta shortly after maternal infection and is most likely to occur in immunocompetent women when three factors occur ‘more or less concurrently': maternal parasitaemia in the presence of well-developed placental blood flow before the development of competent maternal cellular and humoral immunity to Toxoplasma gondii.5,p.156 This hypothesis underpins the rationale for the frequent re-testing of susceptible women in France in order to start treatment as soon as possible after detection of maternal infection.6

An alternative mechanism is that the parasite forms inflammatory foci in the placenta and tachyzoites are released into the fetal circulation at a later point in pregnancy.3,7 Wide acceptance of this hypothesis has resulted in the standard policy of continued treatment throughout pregnancy in infected women even following a negative diagnosis of fetal infection.8

Almost all women diagnosed with infection during pregnancy receive treatment. However, the delay between maternal seroconversion and start of treatment varies due to differences in the frequency of serological testing. We therefore examined the relationship between treatment delay and the risk of mother to child transmission in order to provide information on the effect of treatment. Further information on the timing of transmission was sought by investigating the association between the interval between maternal seroconversion and fetal diagnosis and the results of fetal diagnosis in children subsequently found to have congenital toxoplasmosis. Finally, we determined whether the risk of transmission differed according to whether women were prescribed spiramycin or the more potent regimen of pyrimethamine-sulfadiazine9–15 compared with those not treated.

Methods

Patients
This retrospective study was based on women in whom seroconversion to toxoplasma infection was confirmed by the Hôpital de la Croix Rousse, Lyon, France, between August 1987 and October 1995. The methods for ascertainment, diagnosis, treatment and follow-up of the cohort have been described elsewhere.16 In brief, women were included if they seroconverted from negative to positive specific IgG antibodies. We excluded women identified on the basis of detectable IgG antibodies as preconceptional infection cannot be excluded unless mother to child transmission occurs. In order to avoid selection bias, women referred for suspected fetal abnormalities were excluded. Dates of serology samples, prescription of anti toxoplasma drugs, detection of fetal abnormalities by ultrasound, and the results of fetal diagnosis were obtained from clinical records.

The standard treatment policy spiramycin (9 x 106 units/day) was prescribed immediately after confirmation of maternal infection. Exceptions to this regimen were women with a positive diagnosis of fetal infection and those infected after 32 weeks of gestation. Women in the latter two groups were prescribed pyrimethamine (50 mg/day) and sulfadiazine (3 g/day) alternating three weekly with spiramycin (subsequently referred to as pyrimethamine-sulfadiazine). Treatment was continued until delivery.

In children born to infected women, congenital toxoplasmosis was diagnosed if IgG persisted beyond 12 months, although in non live births positive findings by polymerase chain reaction (PCR) or mouse inoculation of amniotic fluid, fetal blood, or fetal tissue was accepted. The sole criterion for excluding congenital toxoplasmosis was a decline in specific IgG antibody below detectable levels before one year of age in the absence of treatment. Clinicians responsible for the clinical management of the mother routinely recorded the woman's compliance with treatment based on questioning after delivery.

Rationale for analyses
As toxoplasma infection is usually asymptomatic, seroconversion during pregnancy is used to identify maternal infection. However, as Figure 1Go illustrates, the date when seroconversion occurred is not known for certain, only the interval between the last negative and first positive test. We used a statistical model to take account of the uncertainty of the date of seroconversion and the strong effect of gestation at seroconversion on transmission by determining the probability of IgM seroconversion (change from negative to positive specific IgM antibodies) on all possible dates.16 Bayes' Theorem was used to calculate the probability of seroconversion based on information on the presence or absence of specific IgG antibodies at the first IgM positive test (using a previously derived function16), and whether the child was infected or not (Figure 2Go).



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Figure 1 Diagram to illustrate uncertainties about the timing of maternal infection and seroconversion

Maternal infection occurred during a period shortly before the last negative test or at any time up until a short but unknown period before the first positive IgM test. The woman had an antibody response and seroconverted at some point between the last negative IgM test and first positive IgM test. Intervals analysed are: (a) between seroconversion and the date treatment was prescribed or delivery (in untreated women); and (b) between seroconversion and the date of fetal diagnosis (not shown).

 


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Figure 2 Graphical illustration of the daily probability of seroconversion versus gestational age

The probability distributions (A–D) relate to women with an IgG and IgM negative test between 16 and 17 weeks and an IgM positive test between 23 and 24 weeks of gestation.

Curve A: the woman has an uninfected child and a negative IgG and positive IgM test at 24 weeks of gestation.

Curve B: the woman has an uninfected child and positive IgG and IgM tests at 23 weeks of gestation.

Curve C: the woman has an infected child, a negative IgG and positive IgM test at 23 weeks of gestation.

Curve D: the woman has an infected child and positive IgG and IgM tests at 24 weeks of gestation.

Modification of the probability of seroconversion is based on two characteristics. First, as specific IgM antibodies are detected before IgG antibodies, a positive IgM test combined with a negative IgG test indicates that seroconversion is most likely to have occurred shortly before the first positive IgM test. The probability distribution for seroconversion is therefore skewed to the right as shown in curves A and C. Second, as the risk of mother to child transmission increases steeply with gestation,16 women with an infected child are likely to have seroconverted nearer to the first positive test date. Conversely, those with an uninfected child are likely to have seroconverted closer to the date of the last negative test.

 
Effect of timing of treatment on transmission risk
If transmission generally occurs shortly after maternal infection, early treatment should be more effective than late treatment. We defined treatment delay as the interval between seroconversion and the date any type of anti toxoplasma treatment was first prescribed or, in untreated women, the date of delivery. We then compared the risk of mother to child transmission for a treatment delay of <4 weeks, 4–7 weeks, and >=8 weeks, taking account of gestation at seroconversion. The estimated number of women in each treatment category was calculated by summing the probabilities of seroconversion within each time interval.

Effect of type of treatment compared with no treatment on transmission risk
If there is a significant delay between maternal infection and transmission to the fetus, the risk of transmission in women prescribed the more potent regimen of pyrimethamine-sulfadiazine would be expected to be lower than in women prescribed spiramycin and those not treated.9–15 These three treatment groups were compared but, as pyrimethamine-sulfadiazine is routinely prescribed to women after a positive fetal diagnosis, we limited this analysis to women who did not undergo prenatal diagnosis.

Association between time to fetal diagnosis and result of fetal diagnosis
This analysis was limited to cases where fetal diagnosis was performed and where the infant had a definitive diagnosis of congenital toxoplasmosis. We examined whether the time interval between seroconversion and fetal diagnosis was associated with detection of fetal infection (defined as positive mouse inoculation of amniotic fluid or fetal blood, or PCR analysis of amniotic fluid). If delayed transmission was common, samples taken soon after seroconversion would be less likely to be positive than those taken later.

Analysis
In all the analyses, we took account of the confounding effects of gestational age at maternal seroconversion. Parameters were estimated by the method of maximum likelihood using a model that has been described previously.16 We assumed that the effects of treatment and gestation at seroconversion were additive on a logistic scale. Significance tests were based on the likelihood ratio test. Confidence intervals were obtained using the profile likelihood method.17

Results

Patients
Between 1987 and 1995, 788 mother-child pairs were identified. A total of 84 pairs were excluded from analysis due to: suspicion of referral for fetal infection (8); inadequate confirmation of maternal infection (9); incomplete data on prenatal treatment (7) and unknown congenital infection status (60) (Figure 3Go). Some 150 women identified through tests of recent infection were also excluded from analysis because pre conceptional infection could not be ruled out. In all, 554 women infected with T. gondii during pregnancy (identified through proven IgG serconversion) and their children were therefore analysed. Of these, 141/554 (25%) were IgM positive at their first prenatal test.



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Figure 3 Flowchart showing number of mother-child pairs identified, excluded and analysed

 
The cohort of 554 mother-child pairs included nine pregnancies which resulted in a non live birth: two stillbirths (one infected), two spontaneous abortions (both infected) and five planned terminations (performed after positive fetal diagnosis). All nine women received spiramycin and three, with planned terminations, received pyrimethamine-sulfadiazine. In total, including non live births, transmission of infection occurred in 161/554 mother-child pairs (29.1%, 95% CI : 25.3%, 33.0%). Of the 545 live births, 153 were infected.

Table 1Go shows the prenatal treatment prescribed by whether fetal diagnosis was performed. Treatment consisted of: spiramycin alone (416 women, 75%); pyrimethamine-sulfadiazine alternating with spiramycin (11 women, 2%); and spiramycin followed by pyrimethamine-sulfadiazine alternating with spiramycin (96 women, 17%). In 4 (0.8%) of the 523 treated women, compliance was recorded as possibly inadequate. Of the 31 women (6%) who were not treated, 25 had infection confirmed after delivery and no information is available for the remainder.


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Table 1 Type of anti toxoplasma treatment prescribed to infected pregnant women
 
The median time between the first positive IgM test and the start of treatment was 6 days (range 0–146 days). However, because seroconversion inevitably occurred before it was detected, the proportion of women prescribed treatment within 2, 4 and 8 weeks of seroconversion were estimated to be 18%, 44% and 82%, respectively. The distribution of estimated treatment delay according to estimated gestation at seroconversion is shown for each individual woman in Figure 4Go.



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Figure 4 Treatment delay according to gestation at seroconversion

Treatment delay is plotted against gestation at seroconversion. Both measures are based on the estimated date for seroconversion (mean, weighted by the probability of seroconversion for all possible dates) for each individual woman.

White dots indicate mothers prescribed treatment with uninfected children. White squares indicate untreated mothers with uninfected children. Black dots indicate treated mothers with infected children. Black squares indicate untreated mothers with infected children. Ten women had an estimated treatment delay of more than 16 weeks.

 
Effect of timing of treatment on transmission risk
There was no significant difference in the risk of mother to child transmission after a treatment delay of 4–7 weeks and >=8 weeks compared with <4 weeks: adjusted odds ratios (OR) 1.29 (95% CI : 0.61, 2.73) and 1.44 (95% CI : 0.60, 3.31), respectively (Table 2Go). For women undergoing testing, the absolute risk difference is a more clinically relevant measure than OR. However, the magnitude of this difference depends on the gestation at seroconversion. For example, for a woman who seroconverts at 12 weeks gestation, the risk of transmission when given treatment after >=8 weeks compared with <4 weeks is increased by 1.5% (95% CI : -1.9%, 6.9%). If seroconversion occurs at 30 weeks gestation, this risk is increased by 8.8% (95% CI : -12.8%, 26.6%). Figure 5Go shows the transmission risk according to gestation at seroconversion together with the 95% CI for treatment within 4 weeks and for treatment >=8 weeks after seroconversion. There was no evidence that the effect of treatment delay differed between the first and second halves of pregnancy (P = 0.52). When we re-analysed the effect of treatment delay on mother to child transmission using the clinicians' own estimates of the date of maternal infection we obtained virtually identical results.


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Table 2 Effect of timing of treatment on odds ratio (OR) for mother to child transmission of toxoplasma infection
 


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Figure 5 Probability of mother to child transmission according to gestation at seroconversion and treatment delay

Solid lines indicate transmission risk for treatment delay within 4 weeks of seroconversion; dashed lines indicate transmission risk for a treatment delay of >=8 weeks after seroconversion. Faint lines represent 95% confidence limits (curves for treatment delay of 4–7 weeks not shown).

 
Effect of type of treatment compared with no treatment on transmission risk
This analysis was limited to the 273 women who did not undergo fetal diagnosis. Of this group, 55 (20%) were prescribed pyrimethamine-sulfadiazine, 16 (29%) before 28 weeks gestation. The women prescribed pyrimethamine-sulfadiazine were compared with 187 (68%) women prescribed only spiramycin and 31 (11%) who were not treated. The median interval between seroconversion and the date pyrimethamine-sulfadiazine was prescribed was 6 weeks (range 1–16 weeks). There was no evidence that the risk of mother to child transmission differed between the three groups. The adjusted OR associated with spiramycin treatment alone compared with pyrimethamine-sulfadiazine treatment was 0.91 (95% CI : 0.45, 1.84) and the OR for no treatment compared with pyrimethamine-sulfadiazine treatment was 1.06 (95% CI : 0.37, 3.03).

Association between time to fetal diagnosis and positive fetal diagnosis
Amniocentesis and/or cordocentesis were performed in 281 women (51%). In 274 (98%) of them mouse inoculation of amniotic fluid was performed and in 32 (11%) amniotic fluid was analysed with PCR. Of the 52 babies who had congenital toxoplasmosis confirmed postnatally, 39 (75%) had a positive fetal diagnosis and 13 were negative. The interval between seroconversion and fetal diagnosis was not associated with a positive diagnosis of fetal infection: adjusted OR for detection of fetal infection associated with a one-week increase in the time between seroconversion and fetal diagnosis was 0.86 (95% CI : 0.61, 1.19).

Discussion

We found no evidence that the risk of mother to child transmission was related to the timing of prenatal treatment despite reportedly good compliance. However, we cannot exclude a clinically important effect of treatment given within 4 weeks compared with >=4 weeks after maternal seroconversion. Although women were generally treated promptly (median 6 days) after the first positive IgM test, only an estimated 50% of women were prescribed treatment within one month of seroconversion. This interval is strongly determined by the frequency of antibody testing in susceptible women. In France, where monthly re-testing was made a legal requirement in 1992,18 the interval between seroconversion and start of treatment is probably the shortest achieved in any screening programme.

Possible explanations for findings
One possible explanation for our failure to detect an effect of the timing of treatment is that transmission occurs after a prolonged delay.3,7 However, other findings suggest that delayed transmission is unlikely. First, although pyrimethamine-sulfadiazine is more potent than spiramycin,9–15 we found no evidence that the effect of these two treatment regimens differed. Second, there was no association between the timing of fetal diagnosis and detection of fetal infection. Third, if delayed transmission was predominant it would be expected that fetuses exposed to maternal infection for longest would be most likely to be infected. Instead, the reverse pattern is observed as shown by the steep increase in the risk of transmission with gestation at seroconversion.16

Fourth, detection of infected placentas in uninfected children would provide evidence of delayed transmission, but such cases appear to be extremely rare.19–21 Finally, delayed transmission would be expected to result in an association between increasing interval between seroconversion and prenatal diagnosis and a positive diagnosis. However, we found no evidence for this.

An alternative explanation for our failure to detect an effect of the timing of treatment or an effect of more potent treatment, is that, even with monthly testing and very prompt treatment after a positive test result, transmission occurred before treatment started. Experimental studies in animals show that mother to fetus transmission of infection occurs during the phase of maternal parasitaemia.5,9,10,22,23,24,p.146 Less is known about the timing of transmission in humans, as it is rarely possible to accurately date the onset of maternal or fetal infection or the duration of parasitaemia. However, studies report a failure to detect parasitaemia in women identified by serological testing.5,p.147 This suggests that, in most women, parasitaemia ceases by the time seroconversion occurs. Information on the timing of seroconversion after infection is limited to an outbreak investigation of 14 infected individuals, which found that all had detectable antibodies to T. gondii within 2 weeks of infection.25

Findings of other studies
Our results on prenatal treatment support the findings of a study of 144 pregnant women referred to five European centres. The study found no evidence for an effect of prenatal treatment compared with no treatment, or the timing of treatment, on the risk of mother to child transmission after taking account of gestation at maternal infection.26 Two systematic reviews27,28 of the effect of prenatal treatment on mother to child transmission of infection found no controlled trials and none of the observational studies adequately accounted for the steep rise in the risk of mother to child transmission of infection with gestation at maternal infection.16 Without adjusting for this effect, apparently big treatment effects on transmission might be due to differences in timing of seroconversion between treatment groups.

Conclusions

We did not detect a significant effect of prenatal treatment on the risk of mother to child transmission of infection. Although this was the largest study reported to date, the sample size was not large enough to exclude a clinically important effect of treatment. We hypothesize that mother to child transmission of infection predominately occurs during maternal parasitaemia, which ceases when a serological response to toxoplasma infection is detectable. If our hypothesis is true, prenatal treatment of women identified by serological screening will not materially reduce the risk of mother to child transmission of infection.

Acknowledgments

The study was funded by The Wellcome Trust and by the European Commission BIOMED programme (BMH4-CT98-3927). We thank the staff of Laboratoire de Parasitologie et de Pathologie Exotique, Hôpital de la Croix Rousse, Lyon, for help with data collection, Catherine Peckham, for helpful comments on the paper and Eskild Petersen for commenting on drafts of the report and facilitating the collaboration.

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