[Congenital toxoplasmosis: randomised comparison of strategies for retinochoroiditis prevention]. / Toxoplasmose congénitale : comparaison randomisée de stratégies de prévention des rétinochoroïdites.

In France, children with confirmed congenital toxoplasmosis receive a treatment for a period of 12 to 24 months. Such prolonged treatment may generate potentially severe risks, in particular hematologic and cutaneous. Our objective is to compare the effectiveness of two therapeutic strategies on the prevention of retinochoroiditis by a randomized, non-inferiority, open-label, parallel study including 486 children, 3 to 6 months of age with a non-severe form of congenital toxoplasmosis. Following randomization, pyrimethamine-sulphonamide treatment is initiated for a period of three months, followed by a treatment with Fansidar(®) for 9 months, or therapeutic abstention. Follow-up visits during a two-year period will include an examination of the eye, a blood test, and questionnaires to evaluate the children's quality of life and their parents' anxiety. Confirming the non-inferiority of the effectiveness of a short-term treatment will improve the quality of life of parents and children.

Development of a sensitive method for Toxoplasma gondii oocyst extraction in soil.

Toxoplasmosis is a world-wide infection caused by Toxoplasma gondii. Oocysts disseminated in the environment by infected cats provide a major source of infection for humans and intermediate hosts. The level of soil contamination and the dynamics of this contamination are mostly unknown due to the lack of sensitivity of detection method. Our aim was to improve the detection of T. gondii oocysts in soil samples by comparing three extraction protocols (A, B and C) on unsporulated and sporulated oocysts of different strains and ages, and by testing the effect of sporulation and soil characteristics on oocyst recovery using the most efficient method. The oocyst recovery obtained using protocol C, in which the flotation solution was placed under the sample solution after the dispersion step, was at least ten-fold higher than protocols A and B, in which the sample was just filtered before flotation. The efficiency of protocol C, tested on five artificial soil matrices and four natural soils inoculated with oocysts, was lowest in soils with high proportions of sand. We recommend the protocol C for field investigations, and we advise that results should be interpreted with caution, considering the effect of soil characteristics, especially sand content, on oocyst recovery.

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.

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.

Avaliação comparativa entre o desempenho dos reagentes paratoxoplasmose AxSYM, VIDAS e VIDIA em um país latinoamericano com alta soroprevalência / Comparative evaluation of AxSYM, VIDAS and VIDIA toxoplasmosis reagent performance in a high seroprevalence Latin American country

Aims: The purpose of this study was to compare the performance of three automated immunoassays for the detection of IgM and IgG Toxoplasma gondii antibodies using sera of pregnant women living in Colombia, a Latin American country with a high seroprevalence. Methods: A total of 905 sera were tested for IgM antibodies and 914 for IgG antibodies with AxSYM, VIDAS and VIDIA immunoassays. Discrepancies were resolved by using the dye test for IgG antibodies, and the ISAGA test for IgM. Results: The overall agreement between AxSYM, VIDAS and VIDIA assays was excellent for detection of IgG and IgM antibodies, and discrepancies were relatively rare (3.6% and 5.5% of sera for IgG and IgM antibodies, respectively). The performance of the three immunoassays was similar for the detection of IgG antibodies with high sensitivity (100.00% for VIDIA, 99.59% for VIDAS, 99.38% for AxSYM) and specificity (99.04% for VIDIA, 98.82% for AxSYM, 98.57% for VIDAS). The specificity for IgM antibodies was excellent for the three immunassays (99.88% for VIDIA, 99.76% for AxSYM and VIDAS). The sensitivity of the detection of IgM antibodies was higher with VIDIA (95.12%) than with VIDAS (76.74%) and AxSYM (61.90%) assays. The correlation between IgG titers was limited between AxSYM and VIDAS assays and between AxSYM and VIDIA assays, but was excellent between VIDIA and VIDAS assays. Conclusions: Our study performed with Latin American sera confirmed the excellent specificity of AxSYM, VIDAS and VIDIA assays for the detection of IgG and IgM antibodies already reported in other countries. The sensitivity of the detection of IgG antibodies was slightly higher with VIDIA than with VIDAS and AxSYM assays. The sensitivity of the detection of IgM antibodies was higher with VIDIA than with VIDAS and AxSYM assays.

Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings.

We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.

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.

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 mother's 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.

Spatial distribution of soil contamination by Toxoplasma gondii in relation to cat defecation behaviour in an urban area.

In urban areas, there may be a high local risk of zoonosis due to high densities of stray cat populations. In this study, soil contamination by oocysts of Toxoplasma gondii was searched for, and its spatial distribution was analysed in relation to defecation behaviour of cats living in a high-density population present in one area of Lyon (France). Sixteen defecation sites were first identified. Cats were then repeatedly fed with marked food and the marked faeces were searched for in the defecation sites. Of 260 markers, 72 were recovered from 24 different cats. Defecation sites were frequented by up to 15 individuals. Soil samples were also examined in order to detect the presence of T. gondii using real-time PCR. The entire study area was then sampled according to cat density and vegetation cover type. Only three of 55 samples were positive and all came from defecation sites. In a second series of observations, 16 defecation sites were sampled. Eight of 62 samples tested positive, originating in five defecation sites. Laboratory experiments using experimental seeding of soil showed that the inoculated dose that can be detected in 50% of assays equals 100-1000oocysts/g, depending on the strain. This study shows that high concentrations of oocysts can be detected in soil samples using molecular methods and suggests that spatial distribution of contamination areas is highly heterogeneous. Positive samples were only found in some of the defecation sites, signifying that at-risk points for human and animal infection may be very localised.

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.

Comparison of CD8+ T Cell responses to cytomegalovirus between human fetuses and their transmitter mothers.

BACKGROUND: The mechanisms responsible for the increased susceptibility of fetuses to cytomegalovirus (CMV) were studied by comparing CD8(+) T cell responses to the virus in susceptible fetuses to those in their comparatively more resistant mothers. METHODS: Included in the study were 16 transmitter mothers who underwent seroconversion during the first trimester of pregnancy as well as their fetuses, who were positive for CMV in amniotic fluid by polymerase chain reaction at 17-19 weeks of gestation. Fetal and maternal blood samples were collected between the 22nd and 39th week of gestation. Cytotoxic T lymphocytes (CTLs) that had activated (HLA-DR(+)), effector/memory (CD28(-)), and memory (CD18(high)) phenotypes; that stained with the HLA-A2/pp65 or the HLA-B7/pp65 multimer; and that secreted interferon (IFN)- gamma were enumerated by flow cytometry. Viral loads were determined simultaneously. RESULTS: The results showed (1) similar levels of activated, effector/memory, and memory CTLs in fetuses and mothers but a smaller pp65-specific CTL pool in fetuses (median, 0.015% vs. 0.99%; P=.003); (2) similar percentages of CTLs secreting IFN- gamma after stimulation with ionomycin/phorbol myristate acetate in fetuses and mothers but lower percentages of CTLs secreting IFN- gamma after stimulation with a CD3 monoclonal antibody in fetuses (median, 1% vs. 14%; P=.01); and (3) higher viral loads (mean, 17,290 vs. <250 genome equivalents/mL) in fetuses. CONCLUSION: Impaired viral clearance might be related to a defective expansion of the pp65-specific CTL pool and/or to the immaturity of IFN- gamma -secreting cells in fetuses.

Transmission of Toxoplasma gondii in an urban population of domestic cats (Felis catus).

Toxoplasma gondii is a protozoan parasite that infects humans and animal species worldwide. The relative importance of each potential transmission route in the complex life cycle of this coccidia is largely unknown, due to the lack of studies taking into account all routes simultaneously. In this study, we analyzed the transmission of T. gondii in an urban population of stray cats captured between 1993 and 2004. Analyzing prevalence, our aim was to determine which factors influence transmission in this population. Specific anti-T. gondii IgG antibodies were detected using the modified agglutination test. Firstly, we analyzed the kinetics of antibody titers in cats captured several times, using mixed linear models and correspondence analysis. We showed that antibody titers did not vary significantly with time and that titer 40 was the best threshold to separate individuals into two serological groups. Overall, prevalence was only 18.6%, thus transmission of T. gondii is infrequent in this population. As expected, a highly significant association was detected between age and presence of IgG antibodies. Prevalence was lowest in kittens aged 3-4 months, suggesting that newborn kittens may carry maternal antibodies and that vertical transmission is rare. After taking into account the effect of age, logistic regression showed that antibody carriage was related to factors that possibly related to the survival of oocysts: localization in the study site, origin of the cats, maximal temperatures and rain. Our results suggest that in this population, vertical transmission is rare, low predation limits prevalence, and oocyst survival is a determining factor in the risk of infection. We discuss the more general importance of conditions determining oocyst survival in the life cycle of T. gondii.

New Vidas assay for Toxoplasma-specific IgG avidity: evaluation on 603 sera.

After the development of the new version of the test Vidas Toxo IgG with antigens obtained from tachyzoites cultured on cells, a Vidas avidity test has been recently developed. The aim of this study was to assess the value of the determination of avidity on the new Vidas test. This avidity test was performed on 553 sera obtained from pregnant women whose dates of infection had been determined and on 50 sera obtained from immunosuppressed patients. In the group of infection occurring less than 4 months before sampling, the avidity index was <0.3 on 266 among 267 sera. In the group of infection occurring more than 15 months before sampling, the avidity index was >0.3 for 44/46 sera of pregnant women and for 47/47 sera of immunosuppressed patients. Thus, the new version of avidity test was helpful primarily to rule out that an infection had occurred within the prior 4 months.

European multicenter study of the LIAISON automated diagnostic system for determination of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM and the IgG avidity index.

The LIAISON system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles. The system allows fast and precise measurement of Toxoplasma-specific immunoglobulin G (IgG) and IgM antibody levels and measurement of the IgG avidity index even at low levels of Toxoplasma-specific IgG antibodies in a single step without manual interference. Seven European centers participated in a multicenter evaluation of the LIAISON system. The sensitivity and specificity of the LIAISON system compared to the Sabin-Feldman dye test were 99.3 and 96.8%, respectively. In a comparison of the LIAISON Toxoplasma-specific IgM assay with an immunosorbent agglutination assay, the LIAISON assay had a sensitivity of 96.7% and a specificity of 95.4%. The LIAISON IgG assay showed agreements of 91, 100, and 100% with the AXSYM IgG (Abbott), VIDAS IgG (bioMerieux), and Platelia IgG (Bio-Rad) assays, respectively. The LIAISON IgM assay showed agreements of 95% with the AXSYM IgM and Platelia IgM assays, 96% with the ISAGA IgM assay (bioMerieux), and 97% with the VIDAS IgM assay. The coefficient of correlation between the LIAISON system and the VIDAS Toxoplasma-specific IgG avidity index was 0.81. By use of the Toxoplasma-specific IgG avidity index assay with specific IgM-positive samples, the diagnosis of infection with Toxoplasma gondii in early pregnancy has been improved significantly. The LIAISON avidity assay is a valuable assay for the exclusion of recently acquired infection with T. gondii (less than 4 months) in pregnant women, and it decreases significantly the necessity for follow-up testing.

Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii.

OBJECTIVE: Our purpose was to evaluate Toxoplasma gondii concentration in amniotic fluid (AF) samples as a prognostic marker of congenital toxoplasmosis. STUDY DESIGN: A retrospective study was carried out in 88 consecutive AF samples from 86 pregnant women, which were found positive by prospective polymerase chain reaction (PCR) testing. Parasite AF concentrations were estimated by real-time quantitative PCR and analyzed in relation to the clinical outcome of infected fetuses during pregnancy and at birth, taking into account the gestational age at maternal infection. RESULTS: A significant negative linear regression was observed between gestational age at maternal infection and T gondii DNA loads in AF. After adjusting for time at maternal seroconversion by multivariate analysis, higher parasite concentrations were significantly associated with a severe outcome of congenital infection (odds ratio [OR]=15.38/log (parasites/mL AF) [95% CI=2.45-97.7]). CONCLUSION: PCR quantification of T gondii in AF can be highly contributive for early prognosis of congenital toxoplasmosis. Maternal infections acquired before 20 weeks with a parasite load greater than 100/mL of AF have the highest risk of severe fetal outcome.

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France.

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.

Ophthalmic outcomes after prenatal and postnatal treatment of congenital toxoplasmosis.

PURPOSE: To describe the ophthalmologic outcomes of cases of congenital toxoplasmosis treated prenatally and postnatally. DESIGN: Observational case series. METHOD: Follow-up ophthalmologic examinations of 18 children born to mothers who were infected before 25 weeks gestation were performed concurrently by two ophthalmologists. The infection in these children was first suspected when their mothers seroconverted during gestation. Toxoplasmic infection of the fetus was diagnosed by fetal blood or amniotic fluid analysis. Mothers were treated by a regimen of alternating pyrimethamine-sulfonamides and spiramycin during gestation. Pyrimethamine-sulfonamides treatment was continued from birth to 1 year of age. RESULTS: The median age of the children was 4.5 years (range 1-11), when the follow-up ophthalmologic examinations were performed. Visual acuity was decreased in only one child, who had extensive bilateral macular and peripheral lesions. A posterior pole scar was noted in four eyes (four children) for whom visual acuity remained normal. Peripheral lesions were observed in nine eyes (five children). Both eyes were normal in 11 of 18 (61%) of the children. CONCLUSIONS: In these children at a high risk for congenital toxoplasmic retinochoroiditis, a favorable visual outcome was observed in all but one case.

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.

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.