IgG and IgM western blot assay for diagnosis of congenital toxoplasmosis

The aim of this study was to evaluate the utility of western blot (WB) analysis as a diagnostic tool for congenital toxoplasmosis in 215 newborn infants. The children were submitted to clinical examinations to assess macular, neurological and hearing signals. The WB results obtained were compared to the persistence of IgG antibodies at the end of 12 months, which is regarded as the "gold standard" diagnosis of congenital toxoplasmosis. Association between the WB results and the clinical signs presented by the infants was also assessed. Of the 215 children, 177 had a confirmed congenital toxoplasmosis diagnosis and 38 were uninfected. IgG-WB showed a sensitivity of 73.5% and a specificity of 97.4%. IgM-WB showed a sensitivity of 54.8% and a specificity of 94.7%. The IgG-WB and IgM-WB combination increased the sensitivity to 86.5%. The IgM-WB-positive children had a 1.4-fold greater risk of presenting active macular lesions than did those that were IgM-WB-negative. This study showed that the WB assay is a useful tool to confirm a diagnosis of congenital toxoplasmosis and that the IgM-WB-positive results can indicate active macular lesions in newborn infants.     

Clinical course of congenital toxoplasmosis in children]

Hundred eleven children with the congenital toxoplasmosis were treated at the Department of Infectious and Parasitic Diseases in Childhood in 1979-1988. Multi-symptomatic toxoplasmosis has been diagnosed in 35 cases, ocular form in 65, oligosymptomatic in 6, and asymptomatic in 5 cases. Clinical symptoms suggesting congenital toxoplasmosis was seen in the majority of children (63 cases) in the first year of life and the disease was diagnosed in 50% of cases (33 children) at this age. Congenital toxoplasmosis in the group of 78 children has been diagnosed later. The majority of cases was ocular form. Diagnosis of the oligo- and asymptomatic congenital toxoplasmosis is possible in the first year of life, only. A titre of antibodies is exclusively an indicator of the immunologic response, not a severity of infection and does not contribute to the prognosis. Antitoxoplasma drugs were administered to 102 children including 33 under the first year of life. Pyrimethamine, sulphonamides, and spiramycin were used in the treatment. Dosage, duration of therapy, and way of administration have been established individually in dependence of patients age and clinical form of the congenital toxoplasmosis. Two out of 35 children with multi-symptomatic congenital toxoplasmosis died whereas 13 demonstrate psychomotor retardation of significant degree despite the fact that 11 of them were treated in the first year of life.     

First Colombian multicentric newborn screening for congenital toxoplasmosis

Aims

To determine the incidence of congenital toxoplasmosis in Colombian newborns from 19 hospital or maternal child health services from seven different cities of five natural geographic regions (Caribbean, Central, Andean, Amazonia and Eastern).

Materials and Methods

We collected 15,333 samples from umbilical cord blood between the period of March 2009 to May 2010 in 19 different hospitals and maternal-child health services from seven different cities. We applied an IgM ELISA assay (Vircell, Spain) to determine the frequency of IgM anti Toxoplasma. The results in blood cord samples were confirmed either by western blot and repeated ELISA IgM assay. In a sub-sample of 1,613 children that were negative by the anti-Toxoplasma IgM assay, the frequency of specific anti-Toxoplasma IgA by the ISAGA assay was determined. All children with positive samples by IgM, IgA, clinical diagnosis or treatment during pregnancy were recalled for confirmatory tests after day 10 of life.

Results

61 positive samples for specific IgM (0.39%) and 9 positives for IgA (0.5%) were found. 143 questionnaires were positive for a clinical diagnosis or treatment for toxoplasmosis during pregnancy. 109 out of the 218 children that had some of the criteria for postnatal confirmatory tests were followed. Congenital toxoplasmosis infection was confirmed in 15 children: 7 were symptomatic, and three of them died before the first month of life (20% of lethality). A significant correlation was found between a high incidence of markers for congenital toxoplasmosis and higher mean annual rainfall for the city.

Conclusions

Incidence for congenital toxoplasmosis is significantly different between hospitals or maternal child health services from different cities in Colombia. Mean annual rainfall was correlated with incidence of congenital toxoplasmosis.     

Clinical Features and Treatment of Ocular Toxoplasmosis

Ocular toxoplasmosis is a disease caused by the infection with Toxoplasma gondii through congenital or acquired routes. Once the parasite reaches the retina, it proliferates within host cells followed by rupture of the host cells and invasion into neighboring cells to make primary lesions. Sometimes the restricted parasite by the host immunity in the first scar is activated to infect another lesion nearby the scar. Blurred vision is the main complaint of ocular toxoplasmic patients and can be diagnosed by detection of antibodies or parasite DNA. Ocular toxoplasmosis needs therapy with several combinations of drugs to eliminate the parasite and accompanying inflammation; if not treated it sometimes leads to loss of vision. We describe here clinical features and currently available chemotherapy of ocular toxoplasmosis.     

A Survey of Toxoplasmosis Among Mentally Retarded Children

To determine what role, if any, toxoplasmosis plays in the mental retardation of children, sera from 345 mentally retarded children were tested for the presence of antibodies to Toxoplasma gondii. The serological tests employed were the complement-fixation, the Sabin-Feldman dye test and the immunofluorescence test. The donors were also skin-tested with toxoplasmin.Of 345 mentally retarded donors nine gave a positive skin reaction, 15 possessed complement-fixing antibodies, 21 had immunofluorescent antibodies and 45 had dye test antibodies to T. gondii.The incidence of antibodies to T. gondii in the mentally retarded group was approximately the same as in the normal control group of the same age, and less than in the group suspected of having toxoplasmosis. It is concluded that in the children in this study toxoplasmosis played little or no role as a predisposing factor in the occurrence of congenital mental deficiency.     

Factors of occurrence of ocular toxoplasmosis. A review

Acquired and congenital toxoplasmosis are frequently complicated by ocular toxoplasmosis. The diagnosis relies on clinical aspects, response to specific treatment and results of biological assays. The incidence and the prevalence of this complication are difficult to establish precisely and depend on the prevalence of the parasite infection in the general population, and are affected by factors such as type of exposure to the parasite, genetic backgrounds of the parasite and the host, and type of immune response elicited by the parasite.     

Age-dependent impairment of functional helper T cell responses to immunodominant epitopes of Toxoplasma gondii antigens in congenitally infected individuals

Human infection with Toxoplasma gondii is generally asymptomatic in immunocompetent adults while it causes significant morbidity in congenitally infected children. Cell mediated immunity plays the main role in host resistance to T. gondii infection and a Th1 cytokine profile is necessary for protection and control of infection. The present work focused on comparing the helper T cell response to the GRA1 antigen of the parasite between children with congenital toxoplasmosis and healthy adults with acquired infection. We demonstrated that in young children with congenital infection the specific T cell response to parasite antigens is impaired and that such hypo-responsiveness is restored during childhood. Also, we provided clear evidence that in individuals with congenital toxoplasmosis the acquisition of functional helper T cell responses is disease-unrelated and indistinguishable in terms of strength, epitope specificity, and cytokine profile from the corresponding responses in immunocompetent adults with asymptomatic acquired T. gondii infection.     

Diagnosis of toxoplasmosis in pregnancy: importance of immunoglobulin G avidity test

The immunoglobulin G (IgG) avidity test has proved to be a highly useful test in the diagnosis of toxoplasmosis during pregnancy, especially in combination with conventional serological assays. Acute infections at the time of gestation predispose the offspring to the risk of congenital toxoplasmosis. The IgG avidity test was developed to differentiate between recent and more distant infection; this method is valuable in the situation in which a single serum sample is obtained in the first trimester of pregnancy. This paper describes the utility of IgG avidity test during pregnancy, and its role in ruling out, by a high avidity, a recently acquired infection. Testing for specific IgG avidity has been reported to be useful for confirmatory testing in patients who have positive IgG and IgM antibodies.     

Ocular impairment of toxoplasmosis

The purpose of this review is to update the latest information about ocular toxoplasmosis. The infection can be congenital or acquired, but also depends about the immune condition of the patient and can affect the eye. Ocular symptoms are variable according to the age of the subject. Retinochoroiditis is the most common manifestation of toxoplasmic infection. Toxoplasmic retinochoroiditis typically affects the posterior pole, and the lesions can be solitary or multiple. Active lesions present as grey-white focus of retinal necrosis with adjacent choroiditis, vasculitis, hemorrhage and vitreitis. Anterior uveitis is a common finding. Atypical presentations include punctate outer retinitis, neuroretinitis and papillitis. Depending on the patient's age and the localization of the lesion, ocular symptoms vary usually presenting with reduced visual acuity or without symptoms. The laboratory diagnosis of toxoplasmosis is based on detection of antibodies and T. gondii DNA using polymerase chain reaction (PCR) which fulfillis clinical findings. Toxoplasmosis therapy includes antimicrobial drugs and corticosteroids. There are several regimens with different drug combinations including, among others, pyrimethamine, sulfadiazine, clindamycin, and trimethoprim-sulfamethoxazol.     

Clinical and diagnostic management of toxoplasmosis in the immunocompromised patient

With the advent of the highly active antiretroviral therapy (HAART), the natural course of HIV infection has markedly changed and opportunistic infections including toxoplasmosis have declined and modified in presentation, outcome and incidence. However, TE is a major cause of morbidity and mortality especially in resource-poor settings but also a common neurological complication in some countries despite the availability of HAART and effective prophylaxis. In most cases toxoplasmosis occurs in brain and toxoplasmic encephalitis (TE) is the most common presentation of toxoplasmosis in immunocompromised patients with or without AIDS. The need of a definitive diagnosis is substantial because other brain diseases could share similar findings. Rapid and specific diagnosis is thus crucial as early treatment may improve the clinical outcome. Classical serological diagnosis is often inconclusive as immunodeficient individuals fail to produce significant titres of specific antibodies. Polymerase chain reaction (PCR) has a high diagnostic value in the acute disease, but like many 'in-house' PCR assays, suffers from lack of standardization and variable performance according to the laboratory. Molecular diagnosis of toxoplasmosis can be improved by performing real-time PCR protocols. This article summarises the clinical manifestations, diagnostic procedures and management strategies for this condition.     

Early diagnosis of congenital toxoplasmosis in newborn infants using IgG subclasses against two Toxoplasma gondii recombinant proteins

The aim of this work was to evaluate the utility of ELISA-based testing of total IgG (IgGt) antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii for diagnosing congenital toxoplasmosis. Sera from 217 newborns initially testing positive for specific IgM in filter paper dried blood spots were tested for specific IgM and IgG by ELFA-VIDAS. Congenital toxoplasmosis was confirmed in 175 and ruled out in 42 infants. The validity of the ELISA tests was determined using the persistence of IgG antibodies (ELFA-VIDAS kit) at the end of 12 months, which is considered the reference test for the diagnosis of congenital toxoplasmosis. The frequency of positivity with IgGt against STAg, rSAG1 and rMIC3 was found in 97.2%, 96.3% and 80.2%, respectively, of the newborns with confirmed congenital toxoplasmosis. IgG1 reacted with all three antigens, while IgG3 and IgG4 reacted preferentially with rMIC3. Higher mean values of reactivity (sample optical density/cut-off) were found for all subclasses when using rMIC3. All of the antigens showed high sensitivity and low specificity in detecting anti-T. gondii IgGt and IgG1 and low sensitivity and high specificity in detecting IgG3 and IgG4. In conclusion, the combined detection of IgG antibody subclasses against recombinant toxoplasmic antigens may be useful for the early diagnosis of congenital toxoplasmosis.     

Congenital toxoplasmosis: systematic review of evidence of efficacy of treatment in pregnancy

ObjectiveTo summarise the evidence that treating toxoplasmosis in pregnancy reduces the risk of congenital toxoplasma infection and improves infant outcomes.DesignSystematic review of studies comparing at least two concurrent groups of pregnant women with proved or likely acute toxoplasma infection in which treatments were compared with no treatment and outcomes in the children were reported.SubjectsStudies were identified from Medline (1966-97), Pascal (1990-7), Embase (1993-7), and Biological abstracts (1993-5) plus contact with experts in the field, including the European Research Network on Congenital Toxoplasmosis.ResultsOut of 2591 papers identified, nine met the inclusion criteria. There were no randomised comparisons, and control groups were generally not directly comparable with the treatment groups. Congenital infection was common in treated groups. five studies showed that treatment was effective and four that it was not.ConclusionIt is unclear whether antenatal treatment in women with presumed toxoplasmosis reduces congenital transmission of Toxoplasma gondii. Screening is expensive, so the effects of treatment and impact of screening programmes need to be evaluated. In countries where screening or treatment is not routine, these technologies should not be introduced outside carefully controlled trials.

Key messages

• Pregnant women in France and Austria are routinely screened for toxoplasmosis, and women negative for antibodies are followed up at regular intervals
• The value of antenatal toxoplasmosis screening programmes depends on safe treatments that reduce the risk of congenital disease
• This systematic review found no good comparative data measuring the potential harms and benefits of antiparasitic drugs used for presumed antenatal toxoplasma infection
• Most control groups were not comparable, and incidence of congenital infection was high in the intervention groups
• Countries considering introducing screening should do so only in the context of a controlled trial

     

Transplacental transmission of Toxoplasma gondii in minipigs infected with strains of different virulence

Infections with the zoonotic protozoan Toxoplasma gondii during pregnancy can result in severe fetal infections. To investigate the use of pigs as animal models for congenital toxoplasmosis, tachyzoites of 5 T. gondii strains, with low to intermediate virulence in mice, were intravenously inoculated into pregnant minipig gilts. Two strains caused abortions of uninfected fetuses following severe disease of the mothers. One strain caused no disease in the gilts but slightly elevated anti-T. gondii antibodies in 2 of 9 fetuses. One strain produced clinical disease with 4 mummified fetuses and 2 full-term, congenitally infected piglets in 1 gilt and no clinical disease but elevated specific fetal antibodies in both piglets of the other gilt. Infection with the fifth strain (SVS-O14), which was considered apathogenic to both pigs and mice based on the clinical course of this and previous experiments, resulted in significant numbers of congenitally infected piglets, as indicated by production of anti-T. gondii antibodies in all 12 fetuses; the parasite was identified in 3 of these fetuses. This pattern of infection indicates that pigs infected with SVS-O14 (or a similar strain) are relevant animal models for studies of transplacental transmission and pathogenesis of congenital toxoplasmosis.     

New prospects in immunology of toxoplasmosis: skin-tests for control of immunity and immuno-assays and agglutination tests for the detection of specific IgM antibodies

An excretory-secretory (ES) antigen was extracted from supernatants of cell cultures infected with Toxoplasma gondii, purified and controlled according to current standards. In 638 volunteers, the correlation with fluorescent antibody was 94.2% and no false positive skin tests were noted. The skin test did not transform an originally negative serological test into a positive one. For the prevention of congenital toxoplasmosis, this sensitive, specific and inexpensive skin test can be widely used for the detection of immunity to Toxoplasma in women before their first pregnancy. During pregnancy, the detection of specific IgM is very important for the diagnosis of a recently acquired toxoplasmosis and allows for an immediate treatment. For this detection and for the diagnosis of congenital toxoplasmosis, five different serological tests were compared: Indirect Fluorescent Antibody-test (IFA), ELISA test, ELISA test After Capture of IgM (ACCAs), Reverse Enzyme Immuno Assay R-EIA), Double-Sandwich Enzyme Linked ImmunoSorbent Assay (DS-ELISA) and ImmunoSorbent AGglutination Assay (ISAGA). For 37 sera of recently acquired toxoplasmosis, IgM were detected in 98.7% with ISAGA, in 89.5% with DS-ELISA and ELISA in 83% with R-EIA and in 59% with IFA test. The best specificity is obtained with ISAGA, DS-ELISA and R-EIA, from controls with non immune patients (99 cases), patients with chronic toxoplasmosis (77 sera), rheumatoid factors (35 sera) or anti-nuclear antibodies (7 sera). In 21 sera from infants with congenital toxoplasmosis, ISAGA was positive in 13 cases (62%), IFA in 5 cases (24%), ELISA and R-EIA in 2 cases (9.5%) and DS-ELISA in 9 cases (43%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Diagnosis of congenital toxoplasmosis: pre- and post-natal evaluation in Sicilian (Italy) epidemiological area. Preliminary data

To evaluate the usefulness of conventional serological methods with western blot assay (WB) in congenital toxoplasmosis diagnosis, we prospectively enrolled in a clinical and serological follow-up all pregnant women with Toxoplasma gondii infection and their offspring, referred to us from October 2004. Western blot and standard serological test were performed on sera collected from mother during pregnancy and from mother and child at birth, at postpartum month 1-3-6-9 and 12. At this point in time, 22 pregnant women and 14 infants have completed the follow-up. 4 newborns were infected and 2 had specific toxoplasmosis anomalies at the birth. In mothers without seroconversion, the WB performed during pregnancy demonstrates the highest accordance with postnatal follow-up whereas in 1 case the negative result of PCR analysis was not confirmed by postnatal observation. The detection of anti-T gondii IgG against 8 kDa accessory antigenic band and against the accessory band included between 35 and 40 kDa band in immunoblot assay was useful for diagnosis of acute phase but did not improve the evaluation of comparative postnatal profile. Althougth few infants have concluded the postnatal follow-up, the preliminary results showed a greater value of using a IgM and IgA WB test than other standard method for the early diagnosis of toxoplasmosis at birth also in child born to treated mothers. The comparative anti-T gondii IgG immunoblot profile of mother and child permitted us to reduce the time of ruling out infection in newborns born to mothers with probable or possible infection and/or when prenatal diagnosis is negative or not performed.     

Observations of children with chorioretinitis in congenital toxoplasmosis]

Hundred eleven children with congenital toxoplasmosis were followed up in the Department of Infectious and Parasitic Diseases in Childhood, Medical Academy in Warsaw, within 1979-1988. Ocular changes found in 91 children including chorioretinitis in 86 microphthalmia in 7, ophthalmic nerve atrophy in 15, vitreous body inflammation in 5, and cataract in 4. Only in 16 children the diagnosis was performed in the first year of life. In 15 children the recurrence of inflammatory process, most frequently in the time of puberty, was noted; twice in 5 of them. It was independent on the treatment which was previously administrated. Serological tests in ocular form of congenital toxoplasmosis do not indicate the dynamic changes in the inflammatory process. It is very important that small children are examined early and the treatment is started in the first year of life.     

Toxoplasmosis I. Studies by the Fluorescein-labelled Antibody Technique

The fluorescein-labelled antibody technique was investigated for the diagnosis of toxoplasmosis. The direct method, the inhibition and indirect modifications are suitable for the demonstration of Toxoplasma gondii in fluid and tissue-impression slides from animals in the acute phase of infection. The method was not applicable with the frozen tissue sections. The fluorescein-labelled antibody inhibition technique detected antibodies in immune sera from various species of animal. However the titres obtained were lower than with the complement-fixation test.     

Evaluation of laboratory diagnosis of taxoplasmosis by means of an ELISA-triple test

The combination of three parameters (IgG, IgM and circulating antigen) in a so-called ELISA triple test was suggested for advantageous diagnosing of human toxoplasmosis. A qualitative assay was used with the following arbitrary assumptions: IgG antibodies reflect an infection, IgM antibodies reflect a recent (primary) infection and circulating antigens reflect an active infection. The three assays were performed simultaneously in one microtiter plate. This approach was tested with 1091 patientsera submitted for routine diagnosis. In comparison with conventional indirect immunofluorescence and complement fixation test it was observed that combinations indicating a recently aquired infection (combinations with IgM and/or circulating antigen) mainly paralleled low or negligible conventional antibody titers. No strict association was seen between particular combinations and certain clinical symptoms suggestive for toxoplasmosis. In conclusion it was stated that the triple test for support of clinical diagnosis has some advantages but that a strong need exists to be able to demonstrate exacerbation or reinfections which are generally not characterised by IgM antibody formation of free circulating antigens but may be the reason for elevated conventional antibody titers.     

Clinical and Serological Assessment of Children Exposed in Utero to Confirmed Maternal Rubella

As a result of the work of the Public Health Laboratory Service Working Party on Rubella (1970) it was possible to examine the children of 60 susceptible women who were in contact with rubella during their pregnancy and who subsequently showed serological evidence of the infection, despite immunoglobulin prophylaxis. When the children were assessed between 8 months and 4 years 8 months of age a blood sample was also taken and tested for rubella antibodies so that a retrospective diagnosis of congenital rubella infection could be made. Only 19% of children exposed to subclinical maternal rubella showed evidence of intrauterine infection compared with 53% of those born to mothers who had clinical rubella. One of the 26 children exposed to subclinical maternal infection had a rubella defect, compared with 9 of the 34 children exposed to clinical maternal rubella.     

Use of IgG Avidity test in case definitions of toxoplasmosis in pregnancy

A survey network for congenital toxoplasmosis (TOXO-NET) was set up in December 1996 in Piedmont (Italy). Participants were asked to classify the infections in pregnant mothers and newborns by the criteria of the European Network on Congenital Toxoplasmosis published by Lebech in 1996. Because the IgG Avidity test is largely employed as a 2nd level test in toxoplasmosis diagnosis and it could be helpful to date infection, the co-ordinators of TOXO-NET suggested including it in the "case definition" of "probable" infection and "unlikely" infection. 117 cases of toxoplasmosis in pregnancy divided into the risk categories under Lebech's criteria were re-examined using the "new" case definitions. 77 out of 117 (65.8%) Toxoplasma gondii infections during pregnancy could be defined with only one serum sample using the IgG Avidity test. The IgG Avidity test proved a useful method to classify the Toxoplasma gondii infections in pregnancy, especially when we had only one serum sample.