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Prenatal Diagnosis Using Polymerase Chain Reaction on Amniotic Fluid for Congenital Toxoplasmosis

From the Laboratoire de la Toxoplasmose, Institut de Puériculture de Paris, Paris; the Laboratoire de Parasitologie-Mycologie, Hôpital de la Croix-Rousse, Lyon; and the Laboratoire de Parasitologie-Mycologie, Hôpital de la Timone, Marseille, France.

Address reprint requests to: Stephane Romand, MD Institut de Puericulture de Paris Laboratoire de la Toxoplasmose 26 Boulevard Brune Paris, 75014 France E-mail: romand{at}perinat.org

	Abstract

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Objective: To evaluate sensitivity, specificity, and predictive values of a prenatal amniotic fluid (AF) polymerase chain reaction (PCR) test for diagnosis of congenital toxoplasmosis.

Methods: A multicenter prospective study was done on 271 women with proved primary Toxoplasma infection during pregnancy and who had amniocentesis for prenatal diagnosis by PCR. Live-born infants were eligible for analysis only if a serologic follow-up could assess a definitive infection status.

Results: Of the 270 evaluable cases, 75 were congenitally infected, 48 of whom had a positive PCR at prenatal diagnosis. Overall sensitivity of PCR on AF was estimated at 64% (95% confidence interval [CI] 53.1%, 74.9%), negative predictive value of 87.8% (95% CI 83.5%, 92.1%), whereas specificity and positive predictive value were 100% (95% CIs 98%, 100% and 92.3%, 100%, respectively). Among cases with congenital toxoplasmosis, there were no significant differences between those with positive or negative PCR with regard to median gestational age at maternal infection, interval between maternal infection and amniocentesis, or duration of treatment before amniocentesis. However, sensitivity of PCR was found to be significantly higher for maternal infections that occurred between 17 and 21 weeks’ gestation (P < .02).

Conclusion: A negative PCR of AF cannot rule out congenital infection. In this case, continuation of treatment with spiramycin combined with ultrasonographic follow-up and postnatal follow-up are warranted. Our results also suggest presumptive treatment combining pyrimethamine and sulfonamides in case of maternal infection occurring late in pregnancy.

Since the introduction of polymerase chain reaction (PCR) applied to amniotic fluid (AF) for detecting Toxoplasma gondii, prenatal diagnosis of congenital toxoplasmosis has become a highly sensitive, simple, and safe procedure compared with previously used techniques requiring both amniocentesis and funicentesis.¹ A positive PCR on AF denotes fetal infection if no contamination has occurred during testing.

However, little is known about sensitivity and negative predictive value of prenatal diagnosis with PCR, because postnatal serologic follow-up of infants with a negative prenatal diagnosis is not always done. Yet, congenital infection can be ruled out only when there is complete clearance of maternally transmitted antibodies.

To address this issue, we carried out a prospective study in three centers (Paris, Lyon, and Marseille) that perform antenatal diagnosis for congenital toxoplasmosis in women who had primary Toxoplasma infection during pregnancy, had amniocentesis, and whose children had follow-up during the first year of life.

	Materials and Methods

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According to the French screening program for congenital toxoplasmosis, monthly repeated serologic testing of nonimmune pregnant women allowed us prospectively to collect consecutive cases of maternal primary Toxoplasma infection occurring during pregnancy. In each center, data from such cases were collected during a 2-year period (1996–1998), and women who had amniocentesis for prenatal diagnosis of toxoplasmosis and had serologic follow-up of their offspring in order to rule out or confirm congenital infection were included. In case of elective abortion, data were collected only if histopathologic examination was done.

A primary infection during pregnancy was defined by seroconversion (appearance of specific anti-Toxoplasma immunoglobulin IgG and IgM) on the basis that the last negative specimen was obtained after the beginning of pregnancy.² In each woman, the date of infection with regard to gestational age was estimated according to respective levels of anti-Toxoplasma IgG and IgM in the first positive sample.

Prenatal diagnosis of congenital toxoplasmosis was done by PCR test of AF. Isolation of T gondii by mouse inoculation was attempted concomitantly in 238 of 271 cases. Amniocentesis was done about 4 weeks or more after the estimated date of infection and after 18 weeks’ gestation. The PCR methodology was identical at the three centers. Amplification targeted the same sequence of the repeated B1 gene of T gondii. In all reactions, PCR sensitivity was monitored by using an internal artificial DNA control and included a decontamination step with uracil-DNA-glycosylase to prevent carryover contaminations.¹

Definitive infectious status of infants was assessed postnatally by serologic follow-up. Congenital infection was confirmed in offspring by the persistence of specific IgG after 1 year of life. Conversely, a child was considered uninfected when disappearance of transmitted maternal IgG was documented by negative serology results in the absence of antiparasitic treatment.

For each case, the following data were analyzed: gestational age at which maternal infection occurred, time between maternal infection and amniocentesis, nature and duration of antiparasitic treatment before amniocentesis, result of prenatal diagnosis, and definitive status of infant.

Two hundred seventy-two offspring from 271 mothers (one twin pregnancy) met those inclusion criteria. Because of lack of independence, twins were not included and 270 cases were eligible for the study. Four cases resulted in elective abortion.

Discrete variables were analyzed with the ² test. For continuous variables, results were expressed as mean ± standard deviation, and analyses were performed by one-way analysis of variance. If data were not normally distributed, results were expressed as median (inter-quartile range) and analyses performed using a Kruskall-Wallis nonparametric test.

	Results

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Of the 270 evaluable cases, 75 were congenitally infected with T gondii. The maternal characteristics of cases are shown in Table 1. After the diagnosis of maternal infection, antiparasitic treatment with spiramycin was administered in 261 of 270 mothers (96.7%). The maternal-fetal transmission rate increased as time interval between maternal infection and amniocentesis decreased (P < .001) according to duration of gestation at maternal infection (Table 2).

In the 27 cases with congenital infection and negative results of PCR test on AF, transmission of infection was demonstrated by means of other specific markers antenatally (two cases in which parasite was detected in AF by mouse inoculation), immediately after birth (14 cases with presence of specific IgM in the child’s blood), between 15 days and 3 months of life (seven cases in which parasite was detected in the placenta or presence of IgM or IgA in the child’s blood), or after 3 months (three cases with increase in IgG titers during follow-up). In one other case, pregnancy was terminated after ventricular dilatation and cerebral calcifications were detected during ultrasonographic follow-up after amniocentesis. Fetopathologic examination demonstrated congenital toxoplasmosis.

Gestational age at maternal infection, interval between maternal infection and amniocentesis, and duration of treatment preceding amniocentesis did not differ significantly between congenitally infected fetuses with negative or positive prenatal diagnosis (Table 3). However, analysis of sensitivity and negative predictive value of prenatal diagnosis showed large variations according to gestational age at maternal infection (Figure 1). These variations showed that sensitivity of prenatal diagnosis with PCR was significantly higher when maternal infection occurred between 17 and 21 weeks compared with infection occurring before 17 or after 21 weeks’ gestation (² test [2 degrees of freedom] = 7.9; P < .02) (Table 4).

View larger version (38K): [in this window] [in a new window]   Figure 1. Prenatal diagnosis of congenital toxoplasmosis using polymerase chain reaction (PCR) on amniotic fluid (AF) according to gestational age at maternal infection. (unshaded bars = sensitivity of PCR on AF; shaded bars = negative predictive value of PCR on AF; CI = confidence interval.)

	Discussion

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Our results documented 27 congenitally infected infants for whom prenatal PCR on AF was negative, resulting in sensitivity and negative predictive values of 64% and 87.8%, respectively. Our results contrast with those of other studies evaluating PCR on AF that yielded much higher sensitivities for prenatal diagnosis. In those studies, a high proportion of infants with no or incomplete postnatal follow-up likely resulted in overestimation of sensitivity and negative predictive value.^1,6–8 Our reported sensitivity was even lower than that in two recent series with complete postnatal follow-up reported by Foulon et al⁴ and Gratzl et al,⁹ who obtained 81% and 100% sensitivity with PCR on AF among 65 and 49 cases, respectively. Our much larger series of 270 prospective consecutive cases with proved maternal infection during pregnancy might account for those differences. In a few cases, however, lack of sensitivity of PCR can be explained by purely technical pitfalls, because the parasite could be isolated by mouse inoculation of AF in two of 27 cases with a negative prenatal diagnosis by PCR. Thus, despite the lower sensitivity of mouse inoculation,¹ that method is recommended to detect some of the few false-negative results of PCR on AF. Nevertheless, a high proportion of cases could not be diagnosed antenatally although both methods were used.

We found that sensitivity of prenatal diagnosis and the risk of negative PCR depend on gestational age at which infection occurred. We found that PCR performed optimally when maternal infection occurred during the second trimester of pregnancy (between 17 and 21 weeks), compared with moderate performances for maternal infections occurring before 17 or after 21 weeks. Because all but one false-negative case were observed for infection acquired during the first or third trimester, the influence of gestational age at maternal infection was revealed only when maternal infections were considered separately for these three periods of gestation. A recent study identified a maximal risk for the development of severe clinical signs in congenitally infected children when maternal infections were acquired during the second trimester of pregnancy.¹⁰ Thus it appears that prenatal diagnosis is most accurate during that period. During the first trimester of pregnancy, the low rate of transplacental transmission of parasites counterbalances low sensitivity of prenatal diagnosis, resulting in a high negative predictive value. Conversely, the high rate of maternal-fetal transmission during the third trimester outweighs a mild sensitivity of prenatal diagnosis and results in a low negative predictive value.

A possible explanation for the false-negative antenatal diagnoses could be delayed transplacental transmission of parasites after amniocentesis. In our series, one such case illustrates this phenomenon. In this case, acute maternal infection was assessed at 15 weeks, and PCR on AF combined with mouse inoculation gave negative results. During subsequent ultrasonographic follow-up, development of ventricular dilatation at 31 weeks led to a second amniocentesis, which eventually demonstrated the presence of parasite in AF by PCR and mouse inoculation. Severe fetal abnormalities combined with congenital infection led to termination of the pregnancy. Of note, this case is similar to that previously reported by Hohlfeld et al¹ and to several others that have also been observed at our center. However, despite a continuous decrease in interval between maternal infection and amniocentesis, there was no significant difference between false-negative PCRs and positive prenatal diagnoses with regard to those intervals. Hence, other still-unexplained factors, including rapid modifications of placental permeability and perfusion, likely contribute to a high proportion of false-negative PCRs for maternal infections acquired late in pregnancy. Our findings did not support the hypothesis of an interaction between antiparasitic treatment with spiramycin and the result of prenatal diagnosis, as suggested by Foulon et al.⁴ However, parasitostatic treatment with spiramycin must be differentiated from other parasitocidal regimens combining pyrimethamine and sulfonamides, which have been shown to influence the recovery of Toxoplasma from AF.¹¹

Lack of a positive prenatal diagnosis in the 27 cases with false-negative PCR results did not result in more severe clinical outcomes at birth, compared with others with accurate prenatal diagnosis. With the notable exception of the case described above that had severe defects in utero despite initially negative results of PCR, only two of 26 infants had moderate findings at birth (cerebral calcifications). The 24 other children had subclinical infection until 1 year of life. However, long-term clinical outcome of congenital toxoplasmosis, if untreated, can result in visual impairments due to ocular infectious relapses.¹² Long-term positive prognosis depends on early initiation of therapy. This can be related to the sensitivity of the biologic prenatal diagnosis which may be the only prenatal marker of congenital infection.

Finally, our study showed that a negative result on prenatal diagnosis by PCR on AF cannot rule out congenital infection. For maternal infections occurring in late pregnancy, a high rate of maternal-fetal transmission combined with low sensitivity of prenatal diagnosis raises the question of a presumptive curative treatment with pyrimethamine and sulfadiazine, as suggested by others.¹³ For maternal infections occurring in early pregnancy, the possibility of delayed transplacental passage highlights the need to continue ultrasonographic monitoring and treatment with spiramycin throughout pregnancy. In all cases, congenital infection can be excluded in live-born infants only by postnatal serologic follow-up.

	Footnotes

 
The authors thank Dr François Kieffer for his substantial contribution in statistical analyses. Most of the women (223/271) were also part of a larger European Multicentre Study on Congenital Toxoplasmosis, funded by the European Commission BIOMED programme (BMH4-CT98-3927).

PII S0029-7844(00)01118-2

Received April 4, 2000. Received in revised form July 31, 2000. Accepted August 17, 2000.

	References

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