Comparative performance of ISAGA IgM and ELISA assays for the diagnosis of maternal and congenital Toxoplasma infections: which technique could replace ISAGA IgM?

The ISAGA immunocapture test for the detection of anti-Toxoplasma immunoglobulin M is a manual technique known for its excellent sensitivity and specificity. The purpose of this retrospective, multicenter study was to compare the performances and agreement between ISAGA and other IgM detection techniques before cessation of ISAGA production. The analytic performance of the different tests was evaluated using 1,341 serum samples from adults with positive IgM and negative IgG to Toxoplasma gondii, and 1,206 sera from neonates born to mothers with seroconversion. The agreement between the tests was evaluated on 13,506 adult and 5,795 child serum samples. The sensitivity of Toxo-ISAGA IgM® (adults 98.7%, neonates 63.1%) was similar to that of Platelia Toxo IgM® (adults 94.4%, neonates 64.6%), and significantly higher than Liaison Toxo IgM® (adults 90.6%), Architect/Alinity Toxo IgM® (adults 95.7%, neonates 48.6%), and Vidas Toxo IgM® (adults 81.8%, neonates 17.5%). However, the specificities varied between 24.4% (Platelia Toxo IgM®) and 95.2% (Liaison Toxo IgM®) in adults and were >95% for all tests in neonates. An analysis of the kappa coefficients showed better agreement between ISAGA IgM® and the other tests in children (0.75-0.83%) than in adults (0.11-0.53%). We conclude that, in the absence of Toxo-ISAGA IgM®, the association of a very sensitive technique (Platelia Toxo IgM® or Architect/Alinity Toxo IgM®) and a very specific technique (Vidas Toxo IgM® or Liaison Toxo IgM®) is recommended for IgM detection in adult sera. For neonates, Platelia Toxo IgM® appeared to be the best alternative to replace Toxo-ISAGA IgM®.

Title: Performances comparatives des tests ISAGA IgM et ELISA pour le diagnostic des infections maternelles et congénitales à Toxoplasma : quelle technique pourrait remplacer ISAGA IgM ? Abstract: Le test d'immunocapture ISAGA pour la détection des immunoglobulines M anti-Toxoplasma est une technique manuelle connue pour son excellente sensibilité et spécificité. Le but de cette étude rétrospective et multicentrique était de comparer les performances et la concordance entre l'ISAGA et d'autres techniques de détection d'IgM avant l'arrêt de la commercialisation de l'ISAGA. Les performances analytiques des différents tests ont été évaluées à partir de 1 341 échantillons de sérum d'adultes présentant des IgM positives et des IgG négatives à Toxoplasma gondii, et de 1 206 sérums de nouveau-nés nés de mères présentant une séroconversion. La concordance entre les tests a été évaluée sur 13 506 échantillons de sérum d'adultes et 5 795 sérums d'enfants. La sensibilité de Toxo-ISAGA IgM® (adultes 98,7 %, nouveau-nés 63,1 %) était similaire à celle de Platelia Toxo IgM® (adultes 94,4 %, nouveau-nés 64,6 %) et significativement supérieure à celle de Liaison Toxo IgM® (adultes 90,6 %), Architect/Alinity Toxo IgM® (adultes 95,7 %, nouveau-nés 48,6 %) et Vidas Toxo IgM® (adultes 81,8 %, nouveau-nés 17,5 %). Cependant, les spécificités variaient entre 24,4 % (Platelia Toxo IgM®) et 95,2 % (Liaison Toxo IgM®) chez les adultes et étaient >95 % pour tous les tests chez les nouveau-nés. L'analyse des coefficients kappa a montré une meilleure concordance entre ISAGA IgM® et les autres tests chez les enfants (0,75­0,83%) que chez les adultes (0,11­0,53%). Nous concluons qu'en l'absence de Toxo-ISAGA IgM®, l'association d'une technique très sensible (Platelia Toxo IgM® ou Architect/Alinity Toxo IgM®) et d'une technique très spécifique (Vidas Toxo IgM® ou Liaison Toxo IgM®) est recommandée pour la détection des IgM dans les sérums adultes. Pour les nouveau-nés, Platelia Toxo IgM® apparaît comme la meilleure alternative en remplacement de Toxo-ISAGA IgM®.

Contribution of serology in congenital toxoplasmosis diagnosis: results from a 10-year French retrospective study.

This study aimed to evaluate different serological strategies for the postnatal diagnosis of congenital toxoplasmosis (CT) and establish a biological algorithm for CT diagnosis. The study analyzed serological data of immunoglobulins M, A, and G (IgM, IgA, IgG) performed by immunoenzymatic and compared immunological profile (CIP) assays in 668 newborns with CT diagnosis across four testing periods: P1 (D0- D10), P2 (D11-D35), P3 (D36-D45), and P4 (>D45). Forty-nine percent of the 668 CT cases were diagnosed during P1 and 34%, 4%, and 12% during P2, P3, and P4, respectively. CIP assays detected neosynthetized IgMs/IgGs in 98% of CT cases diagnosed during P1, while IgMs and IgAs were detected in 90% and 57% of CT cases diagnosed during P2 and in 88% and 67% of diagnoses made during P3, respectively. Detection of neosynthesized IgMs/IgGs, IgMs, and IgAs by immunoassay contributed to CT diagnosis in 81%, 77%, and 60% of cases, respectively. In total, 46% of serum samples were positive for all three parameters, 27% for two, and 27% for one of the three. The study recommends using the CIP assay as standard during P1 for CT diagnosis and IgM and IgA immunoassays after P1. A clinical and biological follow-up in a specialized center with a close collaboration between biologists and clinicians is highly recommended to increase the chances of early diagnosis. Overall, this study provides useful information for the development of a biological algorithm for CT diagnosis, which can aid in early detection and appropriate treatment of this disease.

IgM triplet in neonatal diagnosis by immunoblotting and its potential use as a diagnostic marker for congenital toxoplasmosis.

Primary infection during pregnancy by the protozoan Toxoplasma gondii can be worrisome because transmission to the fetus may lead to congenital toxoplasmosis (CT). Neonatal diagnosis is usually performed by serological profile comparison of the mother and newborn. As previously reported in 2012 by C. L'Ollivier et al., three IgM bands at 75, 90 and 100 kDa called the "IgM triplet" has caught our attention and seems to be pathognomonic of CT. This retrospective multicenter study involved nine reference laboratories included in the French National Reference Center for Toxoplasmosis network and concerned determining the specificity and sensitivity of this IgM triplet. On this basis, we were able to propose a new read of the comparison of IgG and IgM immunoblot profiles of mother and infant to increase the sensitivity of this diagnostic marker. The effect of the trimester of pregnancy at the time of infection, but also of maternal treatment with pyrimethamine/sulfadiazine/folinic acid on the presence of this IgM triplet in the infant, could be studied. The presence of the triplet appears pathognomonic for the diagnosis of CT, and it increased the sensitivity of the immunoblot assay from 55.04% to 72.48%. As a result, it would be wise to enhance conventional immunoblot reading by adding the presence of the three IgM bands in the infant pattern for neonatal diagnosis of CT.

Title: La triplette IgM dans le diagnostic néonatal par immunoblot et son utilisation potentielle comme marqueur diagnostique de la toxoplasmose congénitale. Abstract: La primo-infection pendant la grossesse par le protozoaire Toxoplasma gondii peut se révéler préoccupante car la transmission au fœtus peut conduire à une toxoplasmose congénitale (TC). Un diagnostic néonatal est généralement réalisé par comparaison des profils sérologiques de la mère et du nouveau-né. Comme précédemment rapporté en 2012 par C. L'Ollivier et al., l'association de trois bandes d'IgM à 75, 90, et 100 kDa appelée la « triplette IgM ¼ a retenu notre attention et semble être pathognomonique de la TC. Cette étude rétrospective multicentrique impliquant neuf laboratoires de référence inclus dans le réseau du Centre National de Référence pour la Toxoplasmose a permis de déterminer la spécificité et la sensibilité de cette triplette IgM. Ainsi, cela a permis de proposer une nouvelle lecture de la comparaison des profils d'immunoblot IgG et IgM de la mère et du nourrisson pour augmenter la sensibilité de ce marqueur diagnostique. L'effet du trimestre de la grossesse au moment de l'infection mais aussi du traitement maternel par pyriméthamine/sulfadiazine/acide folinique sur la présence de la triplette IgM chez l'enfant a pu être analysé. La présence de cette triplette semble pathognomonique pour le diagnostic de TC et elle permet d'augmenter la sensibilité du test immunoblot de 55,04 % à 72,48 %. Ainsi, il pourrait être judicieux d'améliorer la lecture conventionnelle de l'immunoblot en ajoutant la présence des trois bandes IgM dans le schéma du nourrisson pour le diagnostic néonatal de TC.

Type I and III interferons shape the retinal cytokine network and barrier function in an in vitro model of ocular toxoplasmosis.

Introduction: The particularities of the ocular immune environment and its barrier protection in the context of infection are not well elucidated. The apicomplexan parasite Toxoplasma gondii is one of the pathogens successfully crossing this barrier and establishing chronic infection in retinal cells. Methods: As a first approach, we studied the initial cytokine network in vitro in four human cell lines: Retinal pigmented epithelial (RPE), microglial, astrocytic and Müller cells. Furthermore, we looked at the consequences of retinal infection on the integrity of the outer blood-retina barrier (oBRB). We particularly focused on the roles of type I and type III interferons, (IFN-ß and IFN-λ). Especially IFN-λ is known for its significant role in barrier defense. However, its effect on the retinal barrier or T. gondii infection remains unexplored, unlike IFN-γ, which has been extensively studied in this context. Results and Discussion: Here, we show that stimulation with type I and III interferons did not limit parasite proliferation in retinal cells we tested. However, IFN-ß and IFN-γ strongly induced inflammatory or cell-attracting cytokine production, whereas IFN-λ1 showed less inflammatory activity. Concomitant T. gondii infection influenced these cytokine patterns, distinctly depending on the parasite strain. Interestingly, all these cells could be stimulated to produce IFN-λ1. Using an in vitro oBRB model based on RPE cells, we observed that interferon stimulation strengthened membrane localization of the tight junction protein ZO-1 and enhanced their barrier function, in a STAT1-independent manner. Conclusion: Together, our model shows how T. gondii infection shapes the retinal cytokine network and barrier function, and demonstrates the role of type I and type III interferons in these processes.

Dynamic Immune Profile in French Toxoplasmosis Patients.

BACKGROUND: Toxoplasma gondii infection is usually benign in Europe due to the strong predominance of type II strains. Few studies have been conducted to examine the immunological course of infection in humans and have yielded conflicting results, maybe influenced by heterogeneous parasite strains. METHODS: We measured 23 immune mediators in 39, 40, and 29 sera of French noninfected, acutely infected, and chronically infected immunocompetent pregnant women, respectively. RESULTS: Four different cytokine patterns were identified regarding their dynamics through infection phases. For 11 of the cytokines (IFN-ß, IFN-γ, IL-4, IL5, IL-6, IL-10, IL-12, IL-15, CXCL9, CCL2, and CSF2) the serum levels were significantly elevated during acute infection. The inflammatory mediators IL-1ß, IL-17A, IL-18, TNF-α, and CSF3 remained unchanged during acute infection, while they were significantly lower in chronically infected compared to noninfected patients. As for the anti-inflammatory cytokines TGF-ß and CCL5, their levels remained significantly elevated during chronic infection. We also observed a significant negative correlation of several cytokine concentrations with IgG levels, indicating a rapid decline of serum concentrations during the acute phase. CONCLUSIONS: These results indicate an anti-inflammatory pattern in chronically infected patients in a type II dominated setting and demonstrate the highly dynamic immune situation during acute infection.

Unusual presentation of toxoplasmosis with gastro-intestinal involvement in HLA non-identical stem cell transplantation.

Toxoplasmosis is a life-threatening infection in allogenic stem cell recipients usually involving the brain or retina and more rarely lungs or bone marrow. Digestive involvement has only been reported in AIDS patient. We report about a 38-year-old man who received a haploidentical allograft for acute myeloid leukemia and developed an unusual digestive presentation with severe protein-losing enteropathy following grade III acute digestive GvHD treated with steroids and ruxolitinib. Diagnosis was brought up because of concomitant brain involvement. Digestive symptoms fully recovered after treatment with sulfamethoxazole-trimethoprim. Digestive toxoplasmosis could be considered as a differential diagnosis or complication of severe digestive GvHD.

[Update on the diagnosis of parasitic and fungal infections]. / Actualités du diagnostic des infections parasitaires et fongiques.

The diagnosis of parasitic and fungal infections, historically based on the detection of these pathogens using direct diagnosis (macro/microscopic examination, culture) or serological methods, has considerably evolved in the last decades, especially with the development of molecular approaches and mass spectrometry. These techniques, as well as most analyses of parasitic and fungal serology, are mostly the preserve of Hospital University Centers Parasitology-Mycology laboratories. In 2016, the French association of medical parasitology and mycology teachers and hospital practitioners (Anofel) has provided a Catalogue of rare analyses, regularly updated and freely accessible on the Anofel website (https://anofel.net/). This tool, which hinges on 4 parts (parasitology, parasitic serology, mycology, and fungal serology), aims to provide information on all available analyses, and a list of hospital laboratories able to undertake them. It is complementary to the other reference works that were developed by our association, including the Guide of analyses and methods in parasitology and mycology, published in 2018, and the eANOFEL pictures and videos database, freely accessible online (http://www.eanofel.fr). In this article, we draw-up a state-of-the-art of the most specialized techniques available in the parasitology-mycology laboratories and presented in the Catalogue of rare analyses of the Anofel collegium, and their interest for the diagnosis of these infections.

Serological diagnosis of Toxoplasma gondii: analysis of false-positive IgG results and implications.

BACKGROUND: Primary infection by Toxoplasma gondii in pregnant women can result in serious outcomes for the foetus. A false-positive IgG result during pregnancy can lead to a misdiagnosis of past infection and to stopping preventive measures. We collected 189 sera with positive Architect® Toxo IgG assay (Abbott Laboratories) and negative IgG results with at least two other serological tests, in order to find an explanation for the suspected false-positive IgG results. We used the recomLine Toxoplasma IgG® immunoblot (Mikrogen Diagnostik) to search for specific antigenic reactivities of the sera, and the LDBio Toxo II IgG® immunoblot (LDBio Diagnostics) as a confirmatory test. RESULTS: The bands GRA8 and/or GRA7 were positive for 148 samples (78.3%). GRA8 was the most frequent band, appearing in 133 patterns (70.4%), whereas GRA7 was present for 49 samples (25.9%). Of the 81 samples tested with LDBio®, 23 (28.4%) turned out to be positive. Of the 58 negative LDBio® tests (71.6%) (real false-positive Architect® IgG), 23 samples (39.6%) did not show either a GRA8 or p30 band by recomLine®. Their false positivity with Architect® remains unexplained since Abbott uses these two recombinant antigens for their assay. CONCLUSIONS: The Architect® IgG false positivity for T. gondii seems to be due to reactivity against GRA8 for the majority of the sera and GRA7 to a lesser extent. The hypothesis of past contact with parasites genetically close to T. gondii such as Hammondia hammondi or Neospora caninum seems promising and should be assessed further.

TITLE: Diagnostic sérologique de Toxoplasma gondii : analyse des IgG faux positifs et implications. ABSTRACT: Contexte : La primo-infection à Toxoplasma gondii chez la femme enceinte peut avoir de graves conséquences pour le fœtus. Un résultat IgG faussement positif pendant la grossesse peut mener à un diagnostic erroné d'infection ancienne et à stopper les mesures préventives. Nous avons collecté 189 sérums présentant un résultat Architect® Toxo IgG (Abbott Laboratories) positif ainsi qu'un résultat IgG négatif par au moins deux autres tests sérologiques, dans le but de trouver une explication aux résultats IgG suspectés faux positifs. Nous avons utilisé l'immunoblot recomLine Toxoplasma IgG® (Mikrogen Diagnostik) pour chercher certaines réactivités antigéniques spécifiques des sérums et l'immunoblot LDBio Toxo II IgG® (LDBio Diagnostics) comme test de confirmation. Résultats : Les bandes GRA8 et/ou GRA7 étaient positives pour 148 (78,3 %) échantillons. GRA8 était la bande la plus fréquente, apparaissant dans 133 (70,4 %) profils alors que GRA7 était présente pour 49 (25,9 %) échantillons. Sur les 81 échantillons testés en LDBio®, 23 (28,4 %) se sont révélés positifs. Sur les 58 (71,6 %) tests LDBio® négatifs (réels faux positifs IgG Architect®), 23 (39,6 %) échantillons n'ont montré ni bande GRA8 ni bande p30 en recomLine® et leur fausse positivité reste donc inexpliquée puisque Abbott utilise ces deux antigènes recombinants dans son test. Conclusions : La fausse positivité IgG Architect® pour T. gondii semble être due à une réactivité envers la protéine GRA8 pour la majorité des sérums et envers GRA7 dans une moindre mesure. L'hypothèse d'un contact passé avec des parasites génétiquement proches de T. gondii comme Hammondia hammondi ou Neospora caninum semble prometteuse et devrait être approfondie.

Biological Diagnosis of Ocular Toxoplasmosis: a Nine-Year Retrospective Observational Study.

Ocular toxoplasmosis (OT), i.e., the ocular manifestation of Toxoplasma gondii infection, is one of the leading causes of posterior uveitis. While ocular lesions are often typical, atypical forms often require biological confirmation of the diagnosis. Our study sought to review the biological OT diagnoses made in our laboratory to further assess the role of each test in the diagnostic procedure. All ocular samples sent to our laboratory over the last 9 years for OT diagnosis were included. These samples were analyzed using T. gondii PCR and antibody detection by means of immunoblotting and Candolfi coefficient (CC) determinations, either alone or in combination. Since serum analysis is required to interpret both the CC and immunoblotting, blood serology for T. gondii was also performed in most cases. Of the 249 samples analyzed, 80 (32.1%; 95% confidence interval [95%CI], 26.3 to 37.9) were positive for OT. Of these 80 cases, 52/80 (65.0%; 54.6 to 74.5) displayed a positive PCR, 15/80 (18.8%; 10.2 to 27.3) a positive CC, and 33/80 (41.3%; 95%CI, 30.5 to 52.0) a positive immunoblot result. Overall, 63 of the 80 OT diagnoses (78.8%; 95%CI, 69.8 to 87.7) were made on the basis of a single positive test result. Our study results remind us that current biological diagnostic tools for OT must be employed in combination to obtain an optimal diagnosis based on the precious ocular fluids sampled by ophthalmologists. Clinicobiological studies that are focused on correlating the performances of the different tests with clinical features are critically needed to improve our understanding of the pathophysiology and diagnosis of OT.IMPORTANCE Ocular toxoplasmosis (OT), a parasitic infection of the eye, is considered to be the most important infectious cause of posterior uveitis worldwide. Its prevalence is particularly high in South America, where aggressive Toxoplasma gondii strains are responsible for more-severe presentations. The particular pathophysiology of this infection leads, from recurrence to recurrence, to potentially severe vision impairment. The diagnosis of this infection is usually exclusively based on the clinical examination. However, the symptoms may be misleading and are not always sufficient to confirm a diagnosis of OT. In such cases, biological tests performed by means of several techniques on blood and ocular samples may facilitate the diagnosis. In this study, we analyzed the tests that were performed in our laboratory over a 9-year period every time OT was suspected. Our report highlights that the quality of ocular sampling by ophthalmologists and combinations of several techniques are critical for a reliable biological OT diagnosis.

When biology supports clinical diagnosis: review of techniques to diagnose ocular toxoplasmosis.

Toxoplasmosis is a common infection whose worldwide prevalence is estimated at 30%, with large disparities across the world. Among infected subjects, the prevalence of ocular toxoplasmosis (OT) is, however, limited to about 2% in Europe and 17% in South America. In France, it is estimated that about 1 000 000 patients present either active OT or subsequent chorioretinal scars. Toxoplasmagondii is the first cause of posterior uveitis worldwide, responsible for retinochoroiditis, at times associated with anterior uveitis. To date, there is no consensus yet on how to diagnose OT, which is often based only on clinical presentation. Nevertheless, OT-associated symptoms are often atypical and misleading. Over the last 20 years, tremendous progress has been made in biological tools, enabling parasitologists to confirm the diagnosis in most suspected cases of OT. Using anterior chamber puncture, a safe and fast procedure, ophthalmologists sample aqueous humour for analysis using multiple techniques in order to reach high specificity and sensitivity in OT diagnosis. In this article, we present the different techniques available for the biological diagnosis of OT, along with their characteristics, and propose a diagnostic algorithm designed to select the best of these techniques if clinical examination is not sufficient to ascertain the diagnosis.

Contribution of neonatal amniotic fluid testing to diagnosis of congenital toxoplasmosis.

We evaluated the molecular diagnosis of congenital toxoplasmosis (CT) on neonatal amniotic fluid samples from 488 mother-child pairs. Maternal infection during pregnancy was diagnosed and dated or could not be ruled out. Forty-six cases of CT were defined according to the European Research Network on CT classification system and case definitions. Neonatal amniotic fluid testing had an overall sensitivity of 54% (95% confidence interval [95% CI], 39 to 69%) and a specificity of 100% (95% CI, 99 to 100%). Its sensitivity was 33% (95% CI, 13 to 59%) when antenatal diagnosis was positive and 68% (95% CI, 48 to 84%) when antenatal diagnosis was negative or lacking. This difference in sensitivity may have been due to treatment of antenatally diagnosed cases. Relative to postnatal serology, neonatal amniotic fluid testing allowed an earlier diagnosis to be made in 26% of the cases (95% CI, 9 to 51%).

Intraocular levels of interleukin 17A (IL-17A) and IL-10 as respective determinant markers of toxoplasmosis and viral uveitis.

Uveitis is a potentially blinding inflammatory disease. Thirty to 50% of uveitis cases are considered idiopathic. The present study sought to determine the intraocular cytokine patterns in the different etiological types of uveitis in order to better understand their immunological regulation and to determine whether the cytokine pattern may be a useful diagnostic tool. From a multicenter institutional prospective study, the clinical and biological data from patients with uveitis of various etiologies, determined after a complete workup, were compared with those from a control group of cataract patients. A multiplex assay was used to assess the profiles of 27 cytokines and chemokines in aqueous humor samples from these patients. In total, 62 patients with infectious or noninfectious uveitis and 88 controls were included. After a complete workup, the cause of uveitis remained unknown in 25 patients (40% idiopathic uveitis). Interleukin 1ß (IL-1ß) levels were markedly increased in viral uveitis, as were IL-10 levels, whereas IL-17A levels were augmented in toxoplasmic uveitis. Based on the cytokine pattern, the patients were reassigned to specific groups. At the end of the study, the diagnosis of idiopathic uveitis was still valid in only 11 patients (18%). The observation that some markers are specific to certain diseases enables a better understanding of the disease pathogenesis and paves the way for new diagnostic methods aimed to identify inflammatory markers, which may perhaps be targeted by therapy.

Ocular cytokinome is linked to clinical characteristics in ocular toxoplasmosis.

PURPOSE: To determine the cytokine levels in aqueous humor (AH) of Colombian patients with active ocular toxoplasmosis (OT), and to correlate them with their clinical characteristics. METHODS: 27 Cytokines/chemokines were assayed in 15 AH samples (nine patients with diagnosis of OT biologically-confirmed and six controls that underwent cataract surgery). Correlations were assessed between cytokine/chemokine levels, type of inflammatory response (Th1, Th2, Th17, Treg), and clinical characteristics. RESULTS: Th2 predominant response was related to more severe clinical features. The presence of VEGF and IL-5 was related to higher number of recurrences. Growth factors (VEGF, FGF, PDGF-ß), were related to higher number of lesions. Patients infected by type-I/III strains had a particular intraocular cytokine-pattern. CONCLUSIONS: Th2 response was related to more severe clinical characteristics in patients infected by Type I/III strains. IL-5 and VEGF were associated with recurrences. We correlate for the first time, specific cytokine-patterns with clinical characteristics and with the infecting Toxoplasma strain.

Severe South American ocular toxoplasmosis is associated with decreased Ifn-γ/Il-17a and increased Il-6/Il-13 intraocular levels.

In a cross sectional study, 19 French and 23 Colombian cases of confirmed active ocular toxoplasmosis (OT) were evaluated. The objective was to compare clinical, parasitological and immunological responses and relate them to the infecting strains. A complete ocular examination was performed in each patient. The infecting strain was characterized by genotyping when intraocular Toxoplasma DNA was detectable, as well as by peptide-specific serotyping for each patient. To characterize the immune response, we assessed Toxoplasma protein recognition patterns by intraocular antibodies and the intraocular profile of cytokines, chemokines and growth factors. Significant differences were found for size of active lesions, unilateral macular involvement, unilateral visual impairment, vitreous inflammation, synechiae, and vasculitis, with higher values observed throughout for Colombian patients. Multilocus PCR-DNA sequence genotyping was only successful in three Colombian patients revealing one type I and two atypical strains. The Colombian OT patients possessed heterogeneous atypical serotypes whereas the French were uniformly reactive to type II strain peptides. The protein patterns recognized by intraocular antibodies and the cytokine patterns were strikingly different between the two populations. Intraocular IFN-γ and IL-17 expression was lower, while higher levels of IL-13 and IL-6 were detected in aqueous humor of Colombian patients. Our results are consistent with the hypothesis that South American strains may cause more severe OT due to an inhibition of the protective effect of IFN-γ.

Interleukin 17A as an effective target for anti-inflammatory and antiparasitic treatment of toxoplasmic uveitis.

BACKGROUND: Toxoplasmosis is the most common cause of posterior uveitis in immunocompetent subjects. The requirement of limiting both parasite multiplication and tissue destruction suggests that the balance between T-helper (Th) 17 and T-regulatory cells is an important factor in toxoplasmosis-induced retinal damage. METHODS: In a prospective clinical study of acute ocular toxoplasmosis, we assessed the cytokine pattern in aqueous humors of 10 affected patients. To determine the immunological mechanisms, we evaluated intraocular inflammation, parasite load, and immunological responses using messenger RNA and protein levels in a mouse model. Anti-interleukin 17A (IL-17A) monoclonal antibodies (mAbs) were administered with the parasite to evaluate the role of IL-17A. RESULTS: Severe ocular inflammation and cytokine patterns comparable to human cases were observed, including IL-17A production. Neutralizing IL-17A decreased intraocular inflammation and parasite load in mice. Detailed studies revealed up-regulation of T-regulatory and Th1 pathways. When interferon γ (IFN-γ) was neutralized concomitantly, the parasite multiplication rate was partially restored. CONCLUSIONS: Local IL-17A production by resident cells plays a central role in the pathology of ocular toxoplasmosis. The balance between Th17 and Th1 responses (especially IFN-γ) is crucial for the outcome of infection. This data reveals new in vivo therapeutic approaches by repressing inflammatory pathways using intravitreal injection of IL-17A mAbs.

Evaluation of the usefulness of six commercial agglutination assays for serologic diagnosis of toxoplasmosis.

Six agglutination tests for detecting Toxoplasma gondii-specific antibodies (immunoglobulin G or M) in serum were performed and compared. In total, 599 sera were examined using direct and indirect agglutination assays. Sensitivity varied from 93.7% to 100% and specificity from 97.1% to 99.2%. In a selected population with interfering diseases, the percentage of false positives ranged from 4.3% to 10.9%. Although an overall agreement of 100% was found for chronic toxoplasmosis, sensitivity for the detection of confirmed acute toxoplasmosis ranged from 86.4% to 97.3%. Regarding the large variability in terms of the performance of the 6 assays, tests based on the hemagglutination principle were found to be better than the other agglutination tests for all the panels evaluated, meaning that they could be used as qualitative or semiquantitative low-cost screening assays.

Placental testing for Toxoplasma gondii is not useful to diagnose congenital toxoplasmosis.

We examined 785 placentas, including 51 from documented cases of congenital toxoplasmosis. Toxoplasma was detected in 16 placentas, including 1 in which congenital toxoplasmosis was ruled out. Placental screening had poor sensitivity (25%) but good specificity (99%), positive predictive value (93%), and negative predictive value (95%).

Cytokine profiles in toxoplasmic and viral uveitis.

BACKGROUND: Uveitis is a major cause of visual impairment throughout the world. Analysis of cytokine profiles in aqueous humor specimens may provide insight into the physiopathological processes that underly retinal damage in this context. METHODS: Using a multiplex assay, we determined the concentrations of 17 cytokines and chemokines in aqueous humor specimens obtained from patients with ocular toxoplasmosis or viral uveitis and compared these concentrations with those in specimens obtained from patients with noninfectious intermediate uveitis or cataract. RESULTS: Five mediators (interleukin [IL]-8, monocyte chemoattractant protein-1, tumor necrosis factor-alpha, IL-4, and IL-10) were detected in >50% of patients in all groups. In contrast, IL-5 and IL-12 were specific for ocular toxoplasmosis, and granulocyte monocyte colony-stimulating factor and IL-1 were specific for viral uveitis; these mediators could present specific markers for diagnostic purposes. Interferon-gamma, IL-6, and macrophage inflammatory protein-1beta were common markers of ocular toxoplasmosis and viral uveitis. IL-17 was a common marker of ocular toxoplasmosis and intermediate uveitis. CONCLUSIONS: We found specific cytokine profiles for each type of uveitis, with large interindividual variations and no etiological or clinical correlations. Ocular cytokine mapping contributes to a better understanding of the physiopathology of specific forms of uveitis and provides guidance for new targeted treatment.

IgG avidity assay firms up the diagnosis of acute toxoplasmosis on the first serum sample in immunocompetent pregnant women.

Rapid diagnosis of acute toxoplasmosis during pregnancy permits timely treatment and prevents or attenuates congenital toxoplasmosis. Specific IgM antibodies to Toxoplasma as marker of acute infection are often poorly informative, meaning that a complementary technique is needed to reach a diagnosis on the first sample. Here we evaluated 2 commercial kits designed to assist with the diagnosis of acute toxoplasmosis: Platelia Toxo IgG Avidity Complementary Reagents and Platelia Toxo IgA, both from BIO-RAD (Marnes La Coquette, France). We tested 2 groups of subjects: 36 patients with acute toxoplasmosis and 55 patients with chronic toxoplasmosis. The IgG avidity test had a sensitivity of 100% (36/36), a specificity of 92.7% (51/55), a positive predictive value of 90%, and a negative predictive value of 100%. Among the immunocompetent women population, the avidity test had perfect sensitivity and specificity, and positive and negative predictive values of 100%. The IgA test had a sensitivity of 88.8% (32/36) and a specificity of 85.4% (47/55), and positive and negative predictive values of 80% and 92.1%, respectively. When the 2 tests were combined, there was only 1 case in which the diagnosis of chronic toxoplasmosis could not be confirmed. The IgG avidity test can therefore be used to rapidly distinguish between chronic and acute infection on the first sample from a pregnant woman, provided there is no underlying immunodepression and no ongoing antitoxoplasmic treatment. In these 2 situations, the results must be interpreted with care, and other serologic markers, including IgA, should be tested. Determination of a pregnant woman's status on a first serum sample allows therapeutic and preventive management to be started without delay.

Regulation of Toxoplasma gondii multiplication in BeWo trophoblast cells: cross-regulation of nitric oxide production and polyamine biosynthesis.

Materno-foetal transmission causes one of the most severe forms of infection with the protozoan parasite Toxoplasma gondii. Several studies have shown T. gondii in placental trophoblast cells, which form the barrier between maternal blood circulation and foetal tissue. Parasite multiplication in trophoblast cells is thus a critical step leading to infection of the foetus. Here, we show that multiplication of T. gondii tachyzoites was slow in BeWo trophoblast cells, compared with MRC-5 fibroblast cells. However, unlike MRC-5 cells, even combined stimulation with interferon-gamma and tumor necrosis factor-alpha did not reduce T. gondii replication in BeWo cells. This was associated with a lack of indoleamine-2,3-dioxygenase induction by these cytokines. Neither low availability of iron salts, nor an immunosuppressive action of cyclooxygenase-2 could be attributed to the low T. gondii multiplication rate in BeWo cells. However, treatment with the nitric oxide synthesis inhibitor N(G)-methyl-l-arginine and addition of ornithine enhanced the proliferation rate of the intracellular pathogen. Despite detection of inducible nitric oxide synthase-II mRNA in BeWo cells, nitric oxide production could not be detected during cell culture. Thus, inhibition of arginase activity by nitric oxide synthesis may be partially responsible for the lower multiplication rate in BeWo cells.