PRENATAL DIAGNOSIS OF CONGENITAL TOXOPLASMOSIS

500 SL.’BJE(:TS AND METHODS

Screening for Disease

Patients and Controls

PRENATAL DIAGNOSIS OF CONGENITAL

TOXOPLASMOSIS G. DESMONTS F. FORESTIER PH. THULLIEZ

F. DAFFOS M. CAPELLA-PAVLOVSKY M. CHARTIER

Laboratoire de Sérologie Néonatale et de Recherche

sur

la

Toxoplasmose, Institut de Puériculture de Paris; and Centre de Diagnostic Prénatal et de Foetologie, Hôpital Notre-Dame de Bon Secours, Paris,

France

Prenatal diagnosis of congenital toxoplasmosis was attempted by means of fetal blood sampling at 20 - 24 weeks’ gestation. It was possible to detect in fetal blood samples non-specific laboratory signs of fetal infection, specific antibodies of fetal origin (IgM), and parasitaemia by inoculation of the sample into mice. Amniotic-fluid samples were also inoculated into mice and parasites were often present when the fetus was infected. Ultrasound examination of the fetus was done repeatedly, mainly to detect any enlargement of the cerebral ventricles. Together the results of these examinations allowed a reliable diagnosis, which was confirmed by the presence of necrotic foci of toxoplasmic encephalitis in the fetus in every case. Only 1 case of congenital toxoplasmosis occurred among 209 cases with negative prenatal diagnoses.

Summary

INTRODUCTION .

WHEN Toxoplasma gondii infection occurs during pregnancy, the organism is often transmitted across the placenta to the fetus. If transmission occurs early in fetal life, it may cause stillbirth or fetal damage and delivery of a child with congenital toxoplasmosis.’ (Here, we use the term "toxoplasma infection" for cases with presence of parasites

proved by serology or parasitology, and "congenital toxoplasmosis" for cases in which signs of disease due to toxoplasma are present during life or specific lesions are seen at necropsy.) serological screening to detect and treat women toxoplasma infection during pregnancy is compulsory. Pregnant women are examined at the beginning of pregnancy for toxoplasma antibodies and tests are repeated monthly if necessary in order to detect seroconversion.l,2 Since this screening was introduced, the incidence of congenital toxoplasmosis has greatly decreased.But even some mothers treated during pregnancy give birth to infected infants. Thus, when a recently acquired toxoplasma infection is diagnosed in pregnancy (seroconversion from negative to positive tests; presence of IgM antibodies; rising titres; clinical signs when present)’ the pregnant woman and her physician face the dilemma of whether to treat the infection or to terminate the pregnancy.3,4 Inevitably, some pregnancies which would have ended in the delivery of normal, uninfected babies are needlessly terminated.5 In France, who acquire

The purpose of this study was to investigate the possibility of prenatal diagnosis of congenital toxoplasmosis, fetal toxoplasma infection, or both, in the hope of reducing the number of abortions of uninfected fetuses.

We studied 278 pregnant women at risk of giving birth to a child with congenital toxoplasmosis. referred to us by their physicians to discuss therapeutic abortion. All consented to sampling of fetal blood 5 patients desired immediate abortion and fetal blood was obtained before termination of pregnancy. The remaining 273 patients agreed to postpone the decision on termination of pregnancy until the results of serological and isolation studies were known. This group was treated with spiramycin according to the schedule usually followed when acquired toxoplasma infection is diagnosed in a pregnant woman (3 g daily, until the end of pregnancy). Between 20 and 24 weeks of pregnancy, a sample of fetal blood or samples of fetal blood and amniotic fluid were obtained. If the pregnancy was later terminated a second sample was taken just before termination. Fetal ultrasound examination was carried out every other week until delivery. If the pregnancy was terminated, or if fetal death or abortion occurred, the placenta and fetus were examined, and fetal and placental tissues were inoculated into mice to attempt isolation of toxoplasma. After delivery, placental tissues were inoculated into mice and the infant was repeatedly examined for clinical evidence of congenital toxoplasmosis, as well as for serological evidence of subclinical infection. Most of the children have been followed-up at the Institut de Puericulture. In a few cases (mothers who lived far from Paris and had been referred by other groups), results of postnatal examination were provided by other physicians. No information was provided in 5 cases and 58 women are not yet delivered. Sufficient information on the infants was obtained in 215 cases-prospective study group-to determine the incidence of congenital infection. Samples of fetal blood were obtained before termination of pregnancy from 3 additional cases; in 2 the referring physicians had presumptively diagnosed congenital toxoplasmosis because ultrasound had revealed hydrocephalus. These cases are not included in the prospective study group since they would have introduced a bias, but they are included in the prenatal diagnostic studies, as well 1 further case in which samples of fetal blood were sent to us by another group, for isolation and serological studies. It was not possible to arrange a deliberately selected group of we used two matched controls. As controls for the incidence of women for whom was no diagnosis groups prenatal attempted: those whose pregnancies were terminated and those whose pregnancies were allowed to proceed to spontaneous delivery. In both of the control populations the mother and the infant (or conceptus) were examined at the Institut de Puericulture. Data on these subjects have been publishedl-5 but the case-histories have been re-evaluated to constitute two categories which might be compared to the study population. For studies on incidence, the study population and the two control populations were classified into two categories: in category I toxoplasma infection was proved to have been acquired during the first trimester of pregnancy: in category II toxoplasma infection was recently acquired before or soon after conception.

studies,

Methods Fetal blood sczrrzpling.-15 -3 ml fetal blood was taken at 20-24 weeks’ gestation by direct puncture of the umbilical cord with a needle guided by ultrasoundThe Kleihauer-Betke staining procedureand isoelectric focusing of haemoglobin were carried out to detect maternal blood contamination.9 Histograms of white and red blood cells provided by a Coulter counter *S plus II’ were automatically recorded and compared to those for maternal blood. Isolatiorz stzzdies.-Fetal blood was centrifuged (2500Xg) and the sediment inoculated intraperitoneally into mice; the animals were observed as previously described.1 Serological studies.-IgM antibodies were sought bv the IgM immunosorbent assay and IgG antibodies by the dye testl1 1 and the agglutination test.12 The total level of IgG was determined by immunodifiusion assay m both maternal and fetal serum to calculate 1 and compare the antibody load.’ .

specific

501

eosinophilia and 6 thrombocytopenia, and the levels of IgM, y-glutamyl transpeptidase, and lactate dehydrogenase were raised in 6, 7, and 5 cases, respectively. Toxoplasma were isolated from amniotic-fluid samples in 7 of the 9 cases. IgM antibodies were not demonstrated in this fluid, nor any increase in the antibody load. On ultrasound an increase in the ventricle/hemisphere ratio was seen in 6 cases. In 2 (A and B; table n), this was the reason

TABLE I-MEANtSD LABORATORY MEASUREMENTS IN NORMAL AND TOXOPLASMA-INI’ECTEn FETUSES

*12

for referral to us. In 2 cases, the ratio was abnormal when the first sample of fetal blood was taken. In 2 cases, ultrasound examination was normal at first, but abnormal at the second examination before termination of pregnancy. In 3 cases with positive isolation studies, ultrasound examination was still normal when the pregnancy was terminated.

samples from 7 fetuses.

Fetal laboratorv data.-Infants tested during fetallifè who were proved to be free of toxoplasma infection were considered as a control group to define the normal range for laboratory measurements. Some measurements were retrospectively shown to be indicative of fetal infection; these include platelet count, eosinophil count, total IgM, y-glutamyl transpeptidase, and lactate

dehydrogenase. Aznniocentesis.-15-20 ml amniotic fluid

was

taken

at

the

same

time and by the same procedure as the fetal blood sample. The fluid

centrifuged (2500xg) and the sediment inoculated into mice. Serological tests for specific IgM and IgG toxoplasma antibodies were carried out in the supernatant fluid, and the antibody load of IgG was calculated.

was

Ultrasound examination. -Fetal ultrasound examination was done every 2 weeks from the time of the fetal blood sampling to the end of the pregnancy. Special attention was paid to the hemisphere/ ventricle ratio, the presence of ascites, hepatomegaly, the width of the placenta, and intracranial calcification.

RESULTS

Prenatal Diagnostic Studies Toxoplasma was isolated by inoculation of fetal blood into mice in 9 cases. Isolation was successful 6 times from the first sample; in 3 cases the first sample was negative but toxoplasma was isolated from the second. IgM antibodies were detected in 4 of the 9 parasitology-proved cases; no IgM antibodies were detected in the 272 cases with negative inoculation results. No increase in the antibody load was observed in any case. There were abnormalities in laboratory measurements for all of the 7 parasitology-proved cases examined (table 1). 6 had

Studies

after Termination of Pregnancy Pregnancy was terminated in the 9 cases in which the diagnosis of congenital toxoplasmosis was established. Table 11 summarises the main prenatal diagnosis data in these cases. In case C the fetus and placenta were not available for inoculations. Toxoplasma was isolated from the placentas of the other 8 cases. Foci of necrotic encephalitis were present in the 8 fetuses, including those in which ultrasound examination had been normal. Toxoplasma was seen in of brain tissues in every case and isolated after inoculation of brain tissues into mice. In 4 cases the mother requested termination of pregnancy despite the lack of evidence of congenital toxoplasmosis from examination of fetal blood. 1 other pregnancy was terminated after ultrasound showed.severe cardiac malformations. No lesions were seen in the brains of these 5 fetuses. Inoculation of placenta and fetal tissues into mice was negative in these 5 smears

cases.

Studies

after Spontaneous Abortion, Fetal or Neonatal Death There were 5 fetal losses in pregnancies with no prenatal evidence of congenital toxoplasmosis. The causes of death were polyhydramnios with diabetes mellitus; abruptio placentae with pre-eclampsia; twist of the middle of the cord, early and unexplained growth retardation, noted before fetal

TABLE 11-PRENATAL DIAGNOSIS OF CONGENITAL TOXOPLASMOSIS: SIGNIFICANT FINDINGS

0= negative or normal result; + = positive, abnormal, *No second sample of fetal blood taken.

or

significant result; ND = not

done.

502 TABLE

blood sampling; and severe growth retardation in 1 ofapair of twins probably related to interfetal transfusion, also noted before fetal blood sampling. No lesion suggesting congenital toxoplasmosis was present in the brains of these fetuses. Inoculation of the placentas into mice was negative in these 5 cases as was inoculation of fetal tissues, which were available in 3

V-FREQUENCY OF

TOXOPLASMOSIS,

FETAL LOSS, CONGENITAL

AND SUBCLINICAL CONGENITAL TOXOPLASMA

INFECTION AMONG THE OFFSPRING OF

468 CASES OF MATERNAL

TOXOPLASMA INFECTION

cases.

Studies

after Delivery

157 placentas were available after livebirths for inoculation into mice. Toxoplasma was isolated in 3 cases. 199 infants have been examined, most of them repeatedly. No sign of congenital toxoplasmosis was present in 198 of them. 1 infant, whose placenta had yielded toxoplasma, was symptom-free but intracranial calcifications were present on X-ray, ultrasound examination of the skull, and computerised tomography. Fundi, cerebrospinal fluid, and electroencephalogram were normal. This infant was considered as a of congenital toxoplasmosis and treated though case

TABLE VI-ATTEMPTS TO ISOLATE TOXOPLASMA FROM PLACENTA AND FETAL TISSUES AFTER TERMINATION OF PREGNANCY IN

177

CASES OF MATERNAL TOXOPLASMA INFECTION I

symptom-free. The correlation between results of prenatal investigation, inoculation of the placenta, and pathological or clinical examination of the infant is shown in table III.

was isolated in 2 cases from mixed tissues after curettage; in 4 from both placenta and fetal tissues inoculated separately; in 4 cases solely from the placenta.

*Toxoplasma

cases

Serology of Infant Cord or neonatal blood samples of the 198 symptom-free infants were examined. Specific IgM antibodies were not detected in any of these infants, except in a few cord-blood samples, due to placental leaks. The infants were repeatedly examined to detect the fall in passively transmitted maternal IgG antibodies. No serological evidence of subclinical toxoplasma infection has been seen up to now. TABLE 111-CORRELATION BETWEN PRENATAL

INVESTIGATION,

PRESENCE OF TOXOPLASMA IN PLACENTA ON DELIVERY, AND EVIDENCE FOR CONGENITAL TOXOPLASMOSIS IN FETUS OR INFANT

—

Inoculalions Positive

Toxoplasma isolated Evidence of congenital from placenta toxoplasmosis (Positive/no examined) (Positive/no examined)

during pregnancy

(n=9)

Negative (n = 272) Total (n=281)

8/8*

8/8*

3/157t

1/209

11/165

9/217

neither placenta nor fetus was available for examination. yet delivered. In 57 cases, placenta was not available for inoculation. *In 5 cases, no information on offspring was obtained. Congenital toxoplasmosis was excluded in 198 cases by the absence of clinical signs of congenital toxoplasmosis and in 10 cases by a negative result of the inoculation of tissues into mice. *In 1

t58

case

women are not

TABLE

IV-FREQUENCY

OF CONGENITAL TOXOPLASMOSIS AND

TOXOPLASMA INFECTION AMONG OFFSPRING OF

RISK

136 infants whose sera were examined routinely the Institut de Puériculture, antibodies have totally disappeared in 75. These children can therefore be considered to be free of subclinical congenital toxoplasma infection. Antibodies are decreasing normally in 61 infants. For these children the absence of subclinical infection is likely, but not definitely proved. This is also true of 2 cases in which toxoplasma was isolated from the placenta and in which no sign of congenital toxoplasmosis was present.

Among the

at

Incidence Studies shows the incidence of congenital toxoplasmosis congenital toxoplasma infection observed in our study-3% of cases. Table v shows the outcome in pregnancies continued in a group of 468 women with acquired toxoplasma infection. Table VI shows the results of attempts to isolate toxoplasma from the placenta and fetus after termination of pregnancy in 177 cases in which diagnosis of fetal toxoplasma infection was not attempted before termination. Table

IV

and

DISCUSSION

215MOTHERS AT

Comparing our prospective study group with the two control populations, it can be seen that when no pregnancies were terminated (table v), congenital toxoplasmosis was present in I - 5 - 6% of children, depending on the category of maternal infection. In the group where an attempt at prenatal diagnosis was made, once we exclude fetuses with other causes of death (5 cases) and those aborted by maternal decision (5 cases), 198 (96%) of the 205 pregnancies produced live-born infants free of congenital toxoplasmosis. If all the 215 women in the study population had decided on abortion, 198 babies would have been sacrificed unnecessarily. French law allows a pregnancy to be terminated at any time if two physicians, one of them being a "surveyor", agree

503

either that the life of the mother is in danger or that there is a high risk of severe damage in the child. These physicians have to take the responsibility for defining a "high risk". How high is the risk associated with toxoplasma infection acquired by a pregnant woman? If the infection is acquired early in pregnancy and is treated with spiramycin, transmission to the fetus occurs in only a small minority of cases. Ideally, termination of pregnancy should be limited to those cases which will result in severe damage and prenatal diagnostic techniques should be accurate enough to recognise all of these. For the cases reported in our diagnostic studies, the decision to terminate was taken in 5 cases after a positive result of inoculation into mice and in 4 cases after finding enlarged cerebral ventricles on ultrasound. In all of the 8 cases in which the fetus was available for examination, including those in which ultrasound examination was normal, necrotic foci or toxoplasmic encephalitis were present in the brain. Severe impairment, were the child born alive, was very likely and thus termination of pregnancy was

failures are the limited size of the available samples and the fact that parasitaemia may be intermittent. Thus, a negative isolation result does not definitely rule out infection of the fetus, and it may be necessary to obtain more than one sample of blood and amniotic fluid from fetuses in whom other features are abnormal. The main drawback of the inoculation procedure is that it takes 4 -weeks to prove the isolation of T gondii. Other parasitological methods might be helpful, such as determination of the presence of antigen. But at present, we strongly believe that the sole definitive proof of toxoplasma infection is the demonstration of parasites by inoculation into laboratory mice. We recommend that no group should undertake diagnosis of fetal toxoplasma infection without the help of a laboratory where the methods of isolation of parasites have been mastered. In this study, non-specific clinical laboratory data were not taken into account in making a decision to terminate pregnancy. It has been possible, retrospectively, to define the "normal" range of results in uninfected fetuses by gestational age. Some data appeared useful since one or more were

justified.

significantly abnormal in most positive cases (table I). The rises in y-glutamyl transpeptidase and lactate dehydrogenase might suggest liver damage. In future, these measurements will be helpful as early non-specific diagnostic markers. In the 1 child with congenital toxoplasmosis delivered the

These findings are in good agreement with those obtained in the two reference populations. In cases where pregnancies were terminated, gross lesions were consistently associated with the presence of parasites in the fetal tissues (table lB’). Transmission to the fetus of a maternal toxoplasma infection acquired during the first trimester of pregnancy resulted in clinical congenital toxoplasmosis more often than subclinical infection in the child (table B’). These facts suggest the following scheme of pathogenesis of congenital toxoplasmosis. If the placenta is not infected the fetus acquires no congenital infection. If transmission of parasites occurs early in fetal life (ie, before the 20th week of pregnancy) it results, in most cases, in fetal disease leading to severe congenital toxoplasmosis. If transmission is delayed or if toxoplasma infection is acquired by the mother during the third trimester, transmission occurs in the majority of cases but results in few cases of congenital disease and more subclinical infections. 1.5 The aim of prenatal diagnostic investigation is to recognise the cases of early transmission and thus of high fetal risk. In these cases, we consider termination of pregnancy justified if the parents so decide. 5 pregnancies were terminated despite the fact that no evidence of fetal infection was obtained. 4 of these occurred early in this study and the pregnancies were terminated at the request of the mothers. Every woman in the study population consulted us because she wanted to be certain there was no risk to her fetus; if there was she preferred an abortion. In these 4 cases, anxiety of both physicians and patients led to the decision to terminate. Our confidence in the possibilities of prenatal diagnosis has progressively increased with experience. Nevertheless, it should be noted that each one of the examinations carried out might have failed. The specific serological tests were the least useful. IgG antibody synthesis was not detected in any case. IgM specific antibodies were present in only 4 of 9 cases (4 of 14 samples of fetal blood), despite the high sensitivity of the method (it is positive in 75% of newborn infants with congenital toxoplasma infection). This failure of serological diagnosis is not surprising, since antibody synthesis is often delayed in infants with congenital toxoplasma infection and may not begin until some months after birth. Thus, the sensitivity of the immunological methods for recognition of the early fetal immune response needs to be improved. Isolation of parasites from fetal blood or amniotic fluid, was not successful in every sample. Among the reasons for the

results of the inoculation of fetal blood and amniotic fluid negative, and the other measurements were normal. The infant was and is still symptom-free. Intracranial calcifications were shown but no other sign of congenital toxoplasmosis was present. In this case, transmission may have occurred after the time of the fetal blood sampling. The technique we use for sampling fetal blood and amniotic fluid is rarely associated with untoward effects; fetal loss, growth retardation, and premature deliveries in this population were not significantly different to those in our current obstetric population. Prenatal diagnosis could therefore be attempted even in women who do not request termination of pregnancy, or if termination is not feasible for legal or ethical reasons. In these circumstances, a positive prenatal diagnosis might be an indication for treatment potentially more effective in the infected fetus than spiramycin alone-for instance, pyrimethamine plus sulphonamides. Prenatal diagnostic investigation might also be useful in maternal infection acquired after the 20th week of pregnancy. The demonstration of fetal infection without visible damage on ultrasound examination might be an indication for initiation of treatment with pyrimethamine plus sulphonamides before delivery, since clinical experience strongly suggests that the earlier treatment begins the less common will be delayed-onset disease and late sequelae in the were

infant. 13 Most of the patients in this study were in categoryI of maternal infection (definitely acquired in the first trimester) owing to our method of selection of cases. In category-11 patients (infection acquired at an unknown date, around the time of conception or before pregnancy) the risk of congenital toxoplasmosis was lower and would not have justified a potentially harmful examination for the fetus. Since the sampling procedure has proved so safe, its use might be extended to mothers for whom the risk of congenital toxoplasmosis is low, but who remain very anxious. Correspondence should be addressed to F. D., Department of Prenatal Diagnosis and Fetology, Hopitat Notre Dame de Bon Secours, 6 rue Giordano Bruno, 75014 Fans, France.

504

Occasional

Survey

titration gives variable estimates of infectivity, which can be altered to an unknown degree by the conditions of an experiment.3 Aggregation can lead to serious underestimates. For this reason in particular, interpretation of the limited data about the agent is controversial. All conventional viruses normally depend on protein coats for their protection and infectivity, so there was nothing intrinsically novel about the growing body of data suggesting that scrapie infectivity is protein-dependent.4 Prusiner5,6 has emphasised that protein is involved in the infectious process and that no nucleic acid has been detected by biochemical means so far. The apparent small size of the agent as measured by ionising radiation has been the source of much speculation over the years. However, a recent reanalysis of data shows that the target size, originally estimated to be equivalent to a nucleic acid of Mr 50 000-150 000,8 could be an order of magnitude larger. Another indication that the agent could be very small came from a gel-filtration study in which an Mr 60 000 was measured,s but this interpretation too has been seriously questioned.’ Nevertheless, Prusiner places great weight on these small estimates of size. Rohwer has shown that although scrapie shows high resistance to several inactivating procedures it is not very different from conventional viruses in its stability.9 Dickinson and colleagues have emphasised biological properties of the agent, whose determination is not dependent on the titration assay. In particular, they have shown that numerous strains of scrapie exist, some of which mutate. 10 Two simple hypotheses of the nature of the scrapie agent are based on present knowledge of biological systems, rather than requiring hypothetical or novel replication mechanisms. In the first a conventional virus, perhaps belonging to a new taxon, is proposed in which the viral genome codes for a protective coat protein.9 Alternatively, the virino hypothesis proposes that the agent consists of a small replicating informational molecule, presumably a nucleic acid, protected by protein of host origin.11,12 In this model the nucleic acid would have no protein coding capacity but might have regulatory functions analogous to those proposed for viroids. The virino hypothesis more adequately deals with the apparent small size of the agent, its resistance to inactivation, and its poor immunogenicity. By contrast, an unorthodox view of the agent has been embodied in the word "prion" invented by Prusiner from proteinaceous infectious particle. 5,6 This name was chosen to emphasise the involvement of protein in his concept of the agent. In addition, because of an erroneously small estimate of size for, the agent,’ he proposed that nucleic acid might be absent from "prions". The primary requirement of a protein-only hypothesis is that the molecular coding of the heritable ’.

ULTRASTRUCTURAL LINKS BETWEEN SCRAPIE AND ALZHEIMER’S DISEASE ROBERT A. SOMERVILLE AFRC and MRC Neuropathogenesis Edinburgh EH9

Unit, West Mains Road,

3JF

The discovery of abnormal fibrillar structures, scrapie-associated fibrils (SAF), in fractions with high infectivity from scrapie-infected brains has led to the proposal that SAF are a form of the infectious agent. On the basis of this proposal and on the congophilia shared by SAF and amyloid, it has been speculated elsewhere that the amyloid in Alzheimer’s disease is infectious. This speculation is not supported by available evidence and therefore a conventional origin for the amyloid in Alzheimer’s disease is favoured—that it originates by partial degradation of a host protein, as occurs in all other forms of amyloidosis characterised so far.

Summary

INTRODUCTION

.

SOME heuropathological lesions in Alzheimer’s disease (AD) show close parallels with some of the lesions in experimental scrapie. In particular, amyloid and neuritic plaques are found in several scrapie models and resemble those found in AD .1,2 In addition there have been suggestions that AD may be caused by an infectious agent similar to that which causes scrapie and the related scrapielike diseases of man, Creutzfeldt-Jakob disease, Gerstmann Straussler syndrome, and kuru. However, the evidence for this suggestion is indirect and is based on extrapolations from the pathological similarities to scrapie and on some similarities in the mode of their pathogenesis.2

NATURE OF THE SCRAPIE AGENT

The nature of the scrapie agent is still not known, but many hypotheses have been proposed. A major difficulty in determining the nature of the agent or in assessing attempts at purification is the inadequacy of available methods for assaying infectivity, either LDso titration to a limiting dilution or a derivative incubation period assay. Even

G. DESMONTS AND OTHERS: REFERENCES 1

2.

3. 4. 5.

6.

7.

G. Toxoplasmosis. In: Remington JS, Klein JO, eds. Infectious diseases of the fetus and newborn infant. Philadelphia: WB Saunders, 1983: 143-263. Desmonts G Prévention de la toxoplasmose Remarques sur l’expérience poursuivie en France Conclusions of the Workshop on Infectious Diseases. Prevention of physical and mental congenital defects. New York- Alan R. Liss (in press). Foulon W, Naessens A, Volckaer M, et al. Congenital toxoplasmosis: a prospective survey in Brussels. Br J Obstet Gynaecol 1984; 91: 419-23. Beattie CP Congenital toxoplasmosis. Commentary. Br JObstet Gynaecol 1984; 91: 417-18. Desmonts G, Couvreur J. Congenital toxoplasmosis: a prospective study of 542 women who acquired toxoplasmosis during pregnancy. In: Thalammer O, Baumgarten K, Pollak A, eds Pathophysiology of congenital disease. Perinatal Medicine 6th European Congress Stuttgart: Georg Thieme, 1979: 51-60. Bessis R, Cherifi A, Vial M, et al. Diagnostic antenatal d’une toxoplasmose foetale par prélèvement sanguin échoguidé. Soirées échographiques gynéco-obstétricales Paris: Milupa Diététique 1984. 35-36. Daffos F, Capella-Pavlovsky M, Forestier F. Fetal blood sampling via the umbilical

Remington JS, Desmonts

cord using a needle guided by ultrasound. Report of 66 cases Prenatal Diag 1983, 3: 271-77. 8. Betke K Cytological differentiation of haemoglobin Bibl Haematol 1968; 29: 1085-93 9. Dubart A, Goosens M, Beuzard Y, et al. Prenatal diagnosis of hemoglobinopathies. comparison of the results obtained by isoelectric focusing and by chromatography

of

radioactive globin chains Blood 1980; 56: 1092-99. G, Naot Y, Remington JS An IgM immunosorbent agglutination assay for diagnosis of infectious diseases: diagnosis of acute congenital and acquired toxoplasma infections. J Clin Microbiol 1981; 14: 486-91. 11. Sabin AB, Feldman HA Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoon parasite (toxoplasma). Science 1948; 108: 660-63. 12. Desmonts G, Remington JS. Direct agglutination test for diagnosis of toxoplasma infection: method for increasing sensitivity and specificity. J Clin Microbiol 1980; 11: 562-68. 13 Couvreur J, Desmonts G, Aron-Rosa D. Pronostic oculaire de la toxoplasmose congénitale traitée Ann Pediat 1984; 31: 854-58. 10. Desmonts