Philippe Thulliez

Prenatal Diagnosis of Congenital Toxoplasmosis with a Polymerase-Chain-Reaction Test on Amniotic Fluid

BACKGROUND Congenital infection with Toxoplasma gondii can produce serious sequelae. However, there is little consensus about screening during pregnancy, and the tests used to establish a prenatal diagnosis of toxoplasmosis are complex and slow. We evaluated a simpler approach that is based on a polymerase-chain-reaction (PCR) test. METHODS Prenatal diagnostic tests, including ultrasonography, amniocentesis, and fetal-blood sampling, were performed in 2632 women with T. gondii infection acquired during pregnancy. In 339 consecutive women, a competitive PCR test for T. gondii was performed on amniotic fluid, and its results were compared with those of conventional diagnostic tests. The PCR test targets the B1 gene of T. gondii, uses an internal control, and can be completed in a day. Positive tests were confirmed by serologic testing of newborns or by autopsy in terminated pregnancies. RESULTS Overall, the risk of fetal infection was 7.4 percent, but it increased sharply with gestational age. Congenital infection was demonstrated in 34 of 339 fetuses by conventional methods, and the PCR test was positive in all 34. In three other fetuses, only the PCR test gave positive results, and follow-up testing confirmed the presence of congenital toxoplasmosis. The PCR test gave one false negative result but no false positive results. The PCR test performed better than conventional parasitologic methods (sensitivity, 97.4 vs. 89.5 percent; negative predictive value, 99.7 vs. 98.7 percent). CONCLUSIONS For the prenatal diagnosis of congenital T. gondii infection, an approach based on a PCR test performed on amniotic fluid is rapid, safe, and accurate.

Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis

Abstract When infection with Toxoplasma gondii occurs during pregnancy, there is a risk that the parasite will cause severe congenital toxoplasmosis. We developed a method of diagnosing and treating congenital toxoplasmosis in utero. Diagnosis was based on the identification of maternal acute infection, followed by culture of fetal blood and amniotic fluid, testing of fetal blood for toxoplasma-specific IgM and nonspecific measures of infection, and ultrasound examination of the fetal brain. Treatment included the administration of antibiotics to all mothers with confirmed acute infection during pregnancy, with more intensive antibiotic treatment of those who had infected fetuses and who chose to continue the pregnancy. We report a prospective study of 746 documented cases of maternal toxoplasma infection, in which the infants were followed for at least three months. Infection was diagnosed antenatally in 39 of 42 fetuses. Twenty-four of the 39 pregnancies were terminated, and 15 were continued. All the m...

Genotype of 86 Toxoplasma gondii Isolates Associated with Human Congenital Toxoplasmosis, and Correlation with Clinical Findings

To study the influence of Toxoplasma gondii genotypes on the severity of human congenital toxoplasmosis (asymptomatic, benign, or severe infection or newborn or fetal death), 8 microsatellite markers were used to analyze 86 T. gondii isolates collected from patients with congenital toxoplasmosis. Seventy-four different genotypes were detected, some identical genotypes originating probably from the same source of contamination. The 3 less polymorphic microsatellite markers associated with 6 isoenzymatic markers allowed a classification of isolates into the 3 classical types and detected atypical genotypes. Whatever the clinical findings, type II isolates were largely predominant (84.88% in the whole collection and 96.49% in 57 consecutive cases). Type I and atypical isolates were not found in asymptomatic or benign congenital toxoplasmosis. However, in 4 cases in which children were not infected despite isolation of T. gondii from placenta, only type I isolates were found.

Fetal toxoplasmosis: outcome of pregnancy and infant follow-up after in utero treatment.

Eighty-nine cases of fetal Toxoplasma infection are reported in women treated with spiramycin during pregnancy. Thirty-four pregnancy terminations were performed (2.7% of the total number of acquired Toxoplasma infections during pregnancy). Fifty-two pregnancies were allowed to proceed (43 being additionally treated with pyrimethamine and sulfonamides), leading to the birth of 54 live infants. After a mean follow-up period of 19 months, 41 infants had evidence of subclinical Toxoplasma infection, 12 had a benign form, and one had severe congenital toxoplasmosis (this infant did not receive the additional treatment during pregnancy). Efficacy of the additional treatment with pyrimethamine and sulfonamides was demonstrated by a significant reduction of severe congenital toxoplasmosis and the relative decrease of the ratio of benign to subclinical forms. We recommend that spiramycin treatment be started as soon as possible once the diagnosis of maternal Toxoplasma infection during pregnancy is proved or strongly suspected, because a prolonged time interval between onset of infection and start of treatment seems to be associated with the presence of severe fetal lesions at the time of prenatal diagnosis.

Recent Developments for Diagnosis of Toxoplasmosis

There are four groups of individuals in whom the diagnosis of toxoplasmosis is most critical: pregnant women who acquire their infection during gestation, fetuses and newborns who are congenitally infected, immunocompromised patients, and those with chorioretinitis (14, 20, 25). Although the diagnosis of patients in each of these four groups has been hampered by a number of problems, the most frequent challenge encountered by physicians the world over is how to determine if a pregnant woman acquired the acute infection during gestation. Women who acquired their infection prior to pregnancy are essentially not at risk for delivering an infected infant (unless the woman is immunosuppressed). In the United States there is no systematic screening of pregnant women to detect seroconversion during gestation. Much of the literature is based on studies from France, where such screening is performed monthly to detect recently acquired infection. Thus, in the United States, a single serum sample from each woman is submitted for evaluation, and from this sample the physician hopes to learn if the patient has recently been infected, thereby placing the fetus at risk. The high prevalence and lifelong persistence of toxoplasma immunoglobulin G (IgG) antibodies among healthy individuals in many geographical areas preclude the use of any titer in any serologic test as reflecting recent infection. Another problem is the frequent lack of reliability when IgM, IgA, or IgE toxoplasma antibody test results are used in an attempt to discriminate between recent and more distant infection. In addition there are the vagaries associated with lack of quality control, specificity, and/or sensitivity of many commercial serologic test kits on the market. In this setting, physicians and other health care workers responsible for the care of pregnant women are confused when faced with conflicting results and disagreements about interpretations of results. This often leads to incorrect information being provided by the laboratories to the physicians as well as by the physicians to their patients. In recent years, a major effort has been made toward improving our ability to diagnose recently acquired infection in the pregnant woman and congenital infection in the fetus and newborn. We now have a number of new methods that are proving to be of great value towards this end. Among these are the serum IgG avidity test, PCR with body fluids and tissues, and Western blots of serum from mother-baby pairs. At present, the first two methods are being widely used in Europe. Hopefully they will become more readily available in the United States as well. Although our focus here is on pregnant women and newborns, these methods are finding wide use in the other groups of patients alluded to above. Our purpose here is to review each of these methods, what is known about their proper interpretation, and their strengths and shortcomings.

Risk Factors for Toxoplasma Infection in Pregnancy: A Case-Control Study in France

Each year an estimated 4900 cases of primary Toxoplasma infection occur in pregnant women in France, a country with a high prevalence. Since 1992 all pregnant women at risk of Toxoplasma infection have been required to undergo monthly serological testing. This case-control study, the first of its kind in France, was undertaken to identify risk factors for Toxoplasma infection during pregnancy, with a view to improving primary prevention among non-immune pregnant women. A total of 80 pregnant women who seroconverted to Toxoplasma were matched with 80 pregnant women who had repeatedly negative tests. The women were interviewed by telephone, using a standardized questionnaire, to determine socio-demographic characteristics, exposure to possible risk factors and the type of information on prevention received during pregnancy. The risk factors for Toxoplasma infection included in a multivariate analysis were poor hand hygiene (OR = 9.9; 95%CI: 0.8-125), consumption of undercooked beef (OR = 5.5; 95%CI: 1.1-27), having a pet cat (OR =4.5; 95%CI: 1.0-19.9), frequent consumption of raw vegetables outside the home (OR = 3.1; 95%,CI: 1.2-7.7) and consumption of undercooked lamb (OR = 3.1; 95%CI: 0.85-14). Receipt of documentary advice on prevention was associated with a lower risk of infection. Prevention campaigns among pregnant women in France could be improved and should focus on eating habits, hand hygiene and cats.

Congenital Toxoplasmosis and Reinfection during Pregnancy: Case Report, Strain Characterization, Experimental Model of Reinfection, and Review

We present a case of disseminated congenital toxoplasmosis in a newborn born to a mother who had been immunized against toxoplasmosis before conception. The mother was reinfected, likely by ingestion of imported raw horse meat during pregnancy. This clinical presentation is exceptional in France and raised the possibility of infection by a highly virulent Toxoplasma strain. The strain responsible was isolated from the peripheral blood of the newborn, and when genotyped with microsatellite markers, it exhibited an atypical genotype, one which is very uncommon in Europe but had been described in South America. We tested the hypothesis of a reinfection with a different genotype by using an experimental mouse model, which confirmed that acquired immunity against European Toxoplasma strains may not protect against reinfection by atypical strains acquired during travel outside Europe or by eating imported meat.

Direct agglutination test for serologic diagnosis of Neospora caninum infection

Abstract A direct agglutination test was evaluated for the detection and quantitation of IgG antibodies to Neospora caninum in both experimental and natural infections in various animal species. As compared with results obtained by the indirect fluorescent antibody test, the direct agglutination test appeared reliable for the serologic diagnosis of neosporosis in a variety of animal species. The direct agglutination test should provide easily available and inexpensive tools for serologic testing for antibodies to N. caninum in many host species.

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.

DIAGNOSIS OF TOXOPLASMIC RETINOCHOROIDITIS WITH ATYPICAL CLINICAL FEATURES

PURPOSE To determine the value of aqueous humor analysis for confirming the diagnosis of ocular toxoplasmosis in patients who present with atypical clinical features and to relate the results of local antibody production and polymerase chain reaction (PCR) with the extent of active retinitis and the immune status of the patient. DESIGN Retrospective case series. METHODS Sixty-seven consecutive patients with retinitis or retinochoroiditis that was clinically consistent with atypical ocular toxoplasmosis underwent diagnostic anterior chamber paracentesis and serological studies. The aqueous humor was analyzed both by PCR to detect Toxoplasma gondii B1 gene and by the Goldman-Witmer coefficient to determine levels of local anti-T. gondii antibody production. RESULTS In nine of the 67 cases, PCR was positive for T. gondii; seven of these were negative for local antibody production. All nine patients had illnesses associated with immunosuppression or advanced old age and all had active retinitis with a mean area of 11.5 disk areas (DA). Twenty-five of the remaining 58 cases were positive for local antibody production. These 25 had a mean area of active retinitis measuring 2.6 DA, and 24 of these patients were immunocompetent. All 34 cases with laboratory evidence of ocular toxoplasmosis diagnosed by either method responded to anti-T. gondii agents. The remaining 33 were negative for T. gondii infection by these two methods; some had laboratory evidence of other infections. CONCLUSIONS Although in the present study, the sensitivity and specificity of the aqueous humor PCR and Goldman-Witmer coefficient could not be ascertained in the laboratory diagnosis of toxoplasmic retinochoroiditis, the PCR method appears to confirm the diagnosis in immunocompromised individuals with large atypical foci of retinitis. Conversely, determination of local antibody production may be appropriate for proper diagnosis in immunocompetent individuals presenting with small foci of retinitis.