RESUMO
The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.
Assuntos
Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Humanos , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologia , Toxoplasmose Congênita/diagnóstico , Toxoplasmose Congênita/parasitologia , DNA , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , DNA de Protozoário/genética , DNA de Protozoário/análiseRESUMO
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®.
Assuntos
Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Criança , Adulto , Feminino , Recém-Nascido , Humanos , Toxoplasmose Congênita/diagnóstico , Toxoplasmose/diagnóstico , Estudos Retrospectivos , Imunoglobulina M , Ensaio de Imunoadsorção Enzimática , Anticorpos AntiprotozoáriosRESUMO
BACKGROUND: Experimental infection with Plasmodium falciparum results in malaria attack within a few days of exposure. However, we have regularly observed malaria attack within a short time after return, regardless of the time spent in an endemic area. We therefore aimed to assess whether the time before return and malaria attack varies according to length of stay. METHODS: We used anonymized data from the French National Reference Centre for Malaria between 2006 and 2016. We analyzed 11,823 cases aged at least 1 year and diagnosed with P. falciparum malaria 1 day to 1 year after returning to France, after a stay of 1 day to 1 year in an at-risk area. RESULTS: Trips had a median duration of 31 days [IQR: 19-56]. Median time between return from the endemic area and onset of malaria symptoms was 5 days [IQR: 0-10], and the median between return and malaria diagnosis was 9 days [IQR: 5-14]. Times to symptom onset or diagnosis were longer for stays of fewer than 15 days vs 15 days or more (for symptoms: 7 vs 4 days for longer stays, for diagnosis: 11 vs 9 days). For stays longer than 15 days, no variation was observed according to length of stay. CONCLUSIONS: Aside from at-risk stays of fewer than 15 days, the time between return and malaria attack is constant and rather short, even after long stays. The 2 weeks following return should be considered as a risk period whatever the length of stay in an at-risk area.
Assuntos
Antimaláricos , Malária Falciparum , Malária , Humanos , Antimaláricos/uso terapêutico , Tempo de Internação , Viagem , Malária/diagnóstico , Malária/tratamento farmacológico , Malária/epidemiologia , Malária Falciparum/diagnóstico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologiaRESUMO
Therapeutic strategies are shifting from a "one-size-fits-all" population-based approach to a stratified approach targeting groups with similar characteristics, or even individuals, tailoring treatments to the unique characteristics of each patient. Since such strategies rely on increasingly complex knowledge and healthcare technologies, along with an understanding of the tools of precision medicine, the appropriate dissemination and use of these strategies involves a number of challenges for the medical community. Having evaluation methodologies that have been jointly designed with the institutional, industrial, academic stakeholders, and also patients, like streamlining the processes and externally validating performances, could enhance the relevance of the "evaluation" aspect of precision medicine. Creating a network of expert precision-medicine centers and ensuring that precision-medicine procedures are reimbursed by social security would guarantee fair and sustainable access. Finally, training healthcare professionals, creating interfaces between precision-medicine expert centers and primary care professionals as well as patients, and integrating individual patient data into medical records are all key drivers that will enable information from precision-medicine to be made available and guarantee the proper use of these approaches.
Assuntos
Atenção à Saúde , Medicina de Precisão , Humanos , PacientesRESUMO
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.
Assuntos
Toxoplasma , Toxoplasmose Congênita , Recém-Nascido , Humanos , Toxoplasmose Congênita/diagnóstico , Estudos Retrospectivos , Anticorpos Antiprotozoários , Imunoglobulina M , Imunoglobulina G , Imunoglobulina ARESUMO
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 fÅtus 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.
Assuntos
Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Gravidez , Recém-Nascido , Lactente , Feminino , Humanos , Toxoplasmose Congênita/diagnóstico , Anticorpos Antiprotozoários , Immunoblotting , Toxoplasmose/diagnóstico , Imunoglobulina MRESUMO
BACKGROUND: IgG detection to determine immune status to Toxoplasma gondii infection and seroconversion mainly relies on ELISA techniques and, if necessary, on a confirmatory test, Western blot. This study evaluated the performance of the recomLine Toxoplasma IgG immunoblot (IB-recomLine) (Mikrogen) as a confirmatory test on a large number of sera. A total of 171 sera were selected (113 patients) and had previously been analyzed by two ELISA tests, ARCHITECT (Abbott) and VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). The sera were classified into three groups: group 1 included 50 sera without difficulty in interpreting the IgG results (patients with documented past infection or uninfected); group 2 included 47 sera with difficulty in interpreting the ELISA results; and group 3 included 74 sequential sera from 25 pregnant women with seroconversion. RESULTS: In group 1, overall IgG agreements were 94% and 90% with ARCHITECT and VIDAS, respectively. In group 2, low agreement was observed between IB-recomLine and WB-LDBIO, with eight false-positive and 13 false-negative results. In group 3, 4/13 seroconversions were detected earlier with IB-recomLine compared to other tests. CONCLUSIONS: IB-recomLine allowed for earlier diagnosis of toxoplasmic seroconversion compared to both ELISA tests and WB-LDBIO but led to insufficient performance to confirm the immune status when ELISA results were discordant or equivocal.
Title: Diagnostic sérologique de la toxoplasmose : évaluation du test commercial recomLine Toxoplasma IgG immunoblot (Mikrogen) basé sur des antigènes recombinants. Abstract: Contexte : La détection des IgG pour déterminer le statut immunitaire vis-à-vis de l'infection à Toxoplasma gondii et de la séroconversion repose principalement sur les techniques ELISA et, si nécessaire, sur un test de confirmation, le Western blot. Cette étude a évalué les performances de l'immunoblot recomLine Toxoplasma IgG (IB-recomLine) (Mikrogen) en tant que test de confirmation sur un grand nombre de sérums. Un total de 171 sérums ont été sélectionnés (113 patients) et ont été préalablement analysés par deux tests ELISA, ARCHITECT (Abbott) et VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). Les sérums ont été classés en trois groupes : le groupe 1 comprenait 50 sérums sans difficulté d'interprétation des résultats IgG (patients avec antécédents d'infection documentée ou non infectés); le groupe 2 comprenait 47 sérums avec des difficultés d'interprétation des résultats ELISA; le groupe 3 comprenait 74 sérums séquentiels de 25 femmes enceintes séroconverties. Résultats : Dans le groupe 1, les concordances globales des IgG étaient respectivement de 94 % et 90 % avec ARCHITECT et VIDAS. Dans le groupe 2, une faible concordance a été observée entre IB-recomLine et WB-LDBIO, avec huit faux positifs et 13 faux négatifs. Dans le groupe 3, 4/13 séroconversions ont été détectées plus tôt avec IB-recomLine par rapport aux autres tests. Conclusions : IB-recomLine a permis un diagnostic plus précoce de la séroconversion toxoplasmique par rapport aux tests ELISA et WB-LDBIO, mais a conduit à des performances insuffisantes pour confirmer le statut immunitaire lorsque les résultats ELISA étaient discordants ou équivoques.
Assuntos
Toxoplasma , Toxoplasmose , Humanos , Feminino , Gravidez , Anticorpos Antiprotozoários , Imunoglobulina G , Toxoplasmose/diagnóstico , Western Blotting , Imunoglobulina MRESUMO
The high sensitivity of the automated tests used for Toxoplasma gondii serology can yield false-positive IgM results due to aspecific reactions. On the other hand, specific therapy can delay IgG production and, therefore, the diagnosis of seroconversion. There is a need for confirmation tests to early detect seroconversions during pregnancy. We conducted a multicentre study to evaluate the diagnostic accuracy of the Toxo II IgG and a new, not yet commercialised Toxo II IgM western blot (WB) (LDBio diagnostics Lyon France) on 229 sera corresponding to 93 patients with seroconversions and 158 sera corresponding to 68 patients with nonspecific IgM. Sensitivity was 97.8% for IgM WB and 98.9% for IgG WB. Specificity was 89.7% and 100%, respectively. The concordance between IgM and IgG Toxo WB with the final diagnosis was very good, K = 0.89 and K = 0.99, respectively. In 5 cases (5.4%), the appearance of IgM, and in 55 cases (59.1%), the appearance of IgG was recorded by WB earlier than by traditional tests. In 10 cases (10.8%), IgM was detected after the traditional tests and in 2 cases (2.2%) for IgG. The association of IgG and IgM WB on the same sample not only detected all seroconversions but also correctly identified most of the false-positive results.
RESUMO
Real-time PCR plays a crucial role in the diagnosis of toxoplasmosis. In this multicenter study, the Toxoplasma RealCycler Universal assay was assessed for the diagnosis of toxoplasmosis by eight reference laboratories. DNAs from diverse clinical samples were included: 141 characterized samples from patients with different clinical forms of proven toxoplasmosis and 27 from patients without toxoplasmosis were tested in duplicate with the commercial assay. Final diagnosis was affirmed by each center by analysis of clinical settings and biological follow-up. Calibrated Toxoplasma gondii standards and 11 external quality control samples were also included. Discrepant results observed after the first run of commercial PCR were controlled by both reference and commercial PCR assays. Using the commercial assay, the detection threshold varied from 0.01 to 1 tachyzoites/mL, depending on the center. The relationship between crossing point and DNA concentration was linear over 4 log units (r2 > 0.99), and PCR efficiencies were satisfactory (89% to 104%). The results of the 11 external quality control samples were concordant after one retesting, but those for 3 clinical samples remained discrepant. Sensitivity and specificity were calculated at 97.8% (95% CI, 97.8%-100%) and 100% (95% CI, 87.2%-100%), respectively. Provided that PCRs are performed at least in duplicate to detect low parasitic loads, Toxoplasma RealCycler Universal PCR showed suitable performances to diagnose the different forms of toxoplasmosis.
Assuntos
Toxoplasma , Toxoplasmose , DNA de Protozoário/análise , DNA de Protozoário/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Sensibilidade e Especificidade , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologiaRESUMO
Toxoplasmosis can be a life-threatening infection, particularly during pregnancy and in immunocompromised patients. The biological diagnosis of toxoplasmosis is challenging and has been revolutionized by molecular detection methods. This article summarizes the data of a multicenter study involving four centers to assess the performances of a commercial PCR assay as compared with four in-house PCR assays using Toxoplasma gondii standards, 20 external quality control specimens, and 133 clinical samples. This clinical cohort includes well-characterized clinical samples corresponding to different clinical situations: confirmed congenital toxoplasmosis (44 samples), toxoplasmosis in immunocompromised patients (25 samples), and chorioretinitis (5 samples). Furthermore, 59 samples from patients without toxoplasmosis were included as negative controls. The analytical sensitivities of the five methods tested were very similar; and the limit of Toxoplasma DNA detection was around 0.01 T. gondii genome per reaction for all the methods. The overall concordance between the commercial PCR and the four in-house PCR assays was 97.7% (130/133). The clinical sensitivity and specificity were >98% and could be increased for the commercial kit when PCR was performed in multiplicate to detect low parasitic loads. In conclusion, the commercial PCR assay shows suitable performances to diagnose the different clinical forms of toxoplasmosis.
Assuntos
Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Toxoplasmosis is a globally distributed parasitic infection that can be particularly severe when opportunistic or congenital. Its diagnosis requires accurate and rapid techniques that rely mainly on serology and molecular methods. AREAS COVERED: The aim of this review was to discuss the positioning of the molecular diagnosis of toxoplasmosis according to the different clinical situations possibly resulting from infection with T. gondii, and to detail recent developments in this technique. The English and French literature were searched with the following keywords: 'Toxoplasmosis', "Molecular diagnosis" and 'PCR'. EXPERT OPINION: Molecular techniques have revolutionized the diagnosis of toxoplasmosis, and practices have considerably evolved over the past decades. However, there is still a high degree of inter-laboratory heterogeneity which impairs comparisons between results and studies. Efforts to standardize practices are underway.
Assuntos
Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Humanos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose Congênita/diagnósticoRESUMO
BACKGROUND: Biomarker discovery remains a major challenge for predictive medicine, in particular, in the context of chronic diseases. This is true for the widespread protozoan Toxoplasma gondii which establishes long-lasting parasitism in metazoans, humans included. This microbe successively unfolds distinct genetic programs that direct the transition from high to low replicative potential inside host cells. As a slow-replicating cell, the T. gondii bradyzoite developmental stage persists enclosed in a cyst compartment within tissues including the nervous system, being held by a sustained immune equilibrium which accounts for the prolonged clinically silent phase of parasitism. Serological surveys indicate that nearly one third of the human population has been exposed to T. gondii and possibly host bradyzoites. Because any disruption of the immune balance drives the reverse transition from bradyzoite to fast replicating tachyzoite and uncontrolled growth of the latter, these people are at risk for life-threatening disease. While serological tests for discriminating recent from past infection are available, there is yet no immunogenic biomarker used in the serological test to allow ascertaining the presence of persistent bradyzoites. RESULTS: Capitalizing on genetically engineered parasites induced to produce mature bradyzoites in vitro, we have identified the BCLA/MAG2 protein being restricted to the bradyzoite and the cyst envelope. Using laboratory mice as relevant T. gondii host models, we demonstrated that BCLA/MAG2 drives the generation of antibodies that recognize bradyzoite and the enveloping cyst structure. We have designed an ELISA assay based on a bacterially produced BCLA recombinant polypeptide, which was validated using a large collection of sera from mice of different genetic backgrounds and infected with bcla+ or bcla-null cystogenic and non-cystogenic T. gondii strains. To refine the design of the ELISA assay, we applied high-resolution BCLA epitope mapping and identified a specific combination of peptides and accordingly set up a selective and sensitive ELISA assay which allowed the detection of anti-BCLA/MAG2 antibodies in the sera of human patients with various forms of toxoplasmosis. CONCLUSIONS: We brought proof of principle that anti-BCLA/MAG2 antibodies serve as specific and sensitive serological markers in the perspective of a combinatorial strategy for detection of persistent T. gondii parasitism.
Assuntos
Encéfalo/parasitologia , Toxoplasma/fisiologia , Toxoplasmose/diagnóstico , Animais , Biomarcadores/metabolismo , Doença Crônica , Camundongos , Testes Sorológicos , Toxoplasmose/parasitologia , Toxoplasmose/patologiaRESUMO
INTRODUCTION: Toxoplasma-PCR is essential to diagnose ocular, cerebral, disseminated and congenital toxoplasmosis. This multicenter study evaluated the impact of sample storage duration at +4°C on PCR assay performances in order to propose guidelines for the storage of samples during shipment or/and before PCR. MATERIALS AND METHODS: Five matrices, amniotic (AF), cerebrospinal (CSF), and bronchoalveolar lavage fluids (BALF), whole blood (WB) and buffy coat (BC), were artificially spiked with different amounts of Toxoplasma gondii (20, 100, 500 tachyzoites per mL of sample) or with previously infected THP1 cells. DNA extractions were performed at day 0 and after 2, 4 and 7 days of storage at +4°C. Each extract was amplified at least twice by real-time PCR. RESULTS: A total of 252 spiked samples was studied. No increase of crossing point was observed and all samples were positive for AF, BALF, BC and infected THP1-spiked WB after up to 7 days at 4°C. For CSF spiked with 20 parasites/mL, only 50% of PCR reactions were positive at D7 (p<0.05). For WB spiked with type II parasites, all reactions remained positive at D7 but amplifications were significantly delayed from D2; and for WB spiked with RH strain, the proportion of positive reactions decreased at D7. CONCLUSION: The storage of clinical samples at +4°C is compatible with the molecular detection of T. gondii parasites. Provided that PCR assays are performed in duplicate, storage of samples is possible up to 7 days. However, from the fifth day onwards, and for samples susceptible to contain low parasitic loads, we recommend to perform the PCR in multiplicate.
Assuntos
DNA de Protozoário , Preservação Biológica , Reação em Cadeia da Polimerase em Tempo Real , Manejo de Espécimes , Toxoplasma/genética , Toxoplasmose Congênita , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Feminino , Humanos , Masculino , Fatores de Tempo , Toxoplasmose Congênita/diagnóstico , Toxoplasmose Congênita/genéticaRESUMO
Toxoplasmosis represents one of the most common comorbidity factors in solid organ or hematopoietic stem cell transplant recipients as well as in other immunocompromised patients. In the past decades, availability and performance of molecular tools for the diagnosis or the exclusion of toxoplasmosis in these patients have greatly improved. However, if accurately used, serology remains a complementary and essential diagnostic tool for physicians and medical parasitologists for the prevention and management of toxoplasmosis in immunocompromised patients as well. It is required for determination of the immunological status of patients against Toxoplasma. It also helps diagnose and monitor complex cases of opportunistic Toxoplasma infection in immunocompromised patients. New perspectives are available to further enhance their yield and ease of use.
Assuntos
Hospedeiro Imunocomprometido , Testes Sorológicos/normas , Toxoplasmose/diagnóstico , Anticorpos Antiprotozoários/sangue , Humanos , Testes Sorológicos/tendências , Toxoplasma/imunologia , Toxoplasmose/sangueRESUMO
AIMS: We evaluate whether the serum and aqueous humour (AH) level of IgG anti-Hsp70.1 antibodies improved the biological diagnosis of ocular toxoplasmosis. METHODS AND RESULTS: In this prospective cross-sectional and multicentre study, serum and AH were collected at the time of active uveitis. Anti-Hsp70.1-antibody levels were determined by ELISA. Patients with confirmed (Group A1, n = 21) or suspected ocular toxoplasmosis (group A2, n = 30) were enrolled, as well as a control group of patients with cataract (group B, n = 42). Serum IgG anti-Hsp70.1 antibody levels were not significantly different within the group of uveitis patients (A1, n = 21 vs A2, n = 30, P = .8) and were significantly associated with the affected retinal zone (P = .006) and with the size of the retinal lesion (P = .03). Serum anti-Hsp70.1 antibody level was positive in 10 out of the 18 patients of group A2. Significant anti-Hsp-70.1 antibody level in AH was reported in only three patients (3 eyes) with confirmed ocular toxoplasmosis. CONCLUSION: While the level of IgG anti-Hsp-70.1 antibody in AH did not improve the laboratory diagnosis of ocular toxoplasmosis, its level in serum was of major significance for retinal damage diagnosis.
Assuntos
Anticorpos Antiprotozoários/análise , Humor Aquoso/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Imunoglobulina G/análise , Toxoplasmose Ocular/imunologia , Adulto , Anticorpos Antiprotozoários/imunologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Toxoplasma/imunologia , Toxoplasmose Ocular/diagnóstico , Uveíte/diagnóstico , Uveíte/imunologiaRESUMO
INTRODUCTION: Sleepiness is the main clinical expression of obstructive sleep apnea (OSA) syndrome resulting from upper airway collapse. Recent studies have discussed the fact that the presence of T. gondii cysts in the brain and the resulting biochemical and immunological mechanisms could be linked to neurobehavioral disorders. The aim of the present study was to explore the potential impact of chronic toxoplasmosis on sleepiness and on obstructive sleep apnea (OSA) severity in OSA obese patients. MATERIALS AND METHODS: A case control study on obese patients screened for OSA was performed. According to the sleep disorder and matched based on gender, age and body mass index (BMI), two groups of obese patients were selected from our sample collection database. All patients were tested for toxoplasmosis serological status measuring anti-Toxoplasma IgG and IgM levels. Univariable and multivariable logistic regression models were performed to assess the impact of chronic toxoplasmosis on sleepiness and OSA severity. RESULTS: 107 obese patients suffering from OSA were included in the study (median age: 53.3 years Interquartile range (IQR): [41.9-59.9]; median BMI: 39.4 kg/m2 IQR: [35.5-44.1], apnea-hypopnea index = 27.5 events/h [10.7-49.9]). Chronic toxoplasmosis was present in 63.4% and 70.7% of patients with or without sleepiness (p = 0.48), respectively and was not associated either to sleepiness (OR: 0.76, 95% CI: [0.52; 2.33], p = 0.64) or OSA severity (OR = 1.75, 95% CI: [0.51; 5.98] p = 0.37). CONCLUSION: Although chronic Toxoplasma infection in immunocompetent humans has been associated to several behavioral disorders or pathologies in recent literature, we demonstrate here that chronic toxoplasmosis is not associated to sleepiness and to sleep apnea syndrome severity in obese patients suspected of sleep apnea syndrome.
Assuntos
Doença Crônica/epidemiologia , Obesidade/epidemiologia , Apneia Obstrutiva do Sono/epidemiologia , Toxoplasmose Cerebral/epidemiologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Sonolência , Toxoplasma/imunologia , Toxoplasmose Cerebral/imunologiaRESUMO
Background Testing for anti-Toxoplasma immunoglobulin (Ig)M is of main importance in the context of pregnancy to promptly alert to an acute maternal infection prior to the detection of IgG and to identify infected newborns. Their absence helps exclude a recent maternal infection in the presence of IgG. Methods The performance of a Toxo IgM immunocapture prototype assay (bioMérieux, France) was compared with that of the VIDAS® Toxo IgM and the ARCHITECT® Toxo IgM (Abbott, Germany) assays at Grenoble and Lyon (France). A total of 1446 sera were sampled from (i) 1054 pregnant women found by routine workup to have no infection (n = 843), an acute infection (<4 months) (n = 28) or a chronic infection (>4 months) with residual (n = 120) or no IgM (n = 62); (ii) 50 three-serum panels sampled immediately after a maternal seroconversion; (iii) 242 samples taken in 41 children with a congenital toxoplasmosis (n = 122) and in 40 uninfected children (n = 120). Results In pregnant women, the overall agreement with the VIDAS® assay was 99.23% (CI: 99.16-99.27) and that with the ARCHITECT® assay was 99.14% (CI: 99.07-99.17). Sensitivity of the Toxo IgM prototype assay was 100% (CI: 87.66-100.00) and specificity was 99.64% (98.96-99.93). In acute maternal infections, IgM assays were detected as early with the prototype as with the other two. In the congenitally infected children, IgM were detected on their first sample in 25/40 with the prototype vs. 23/40 with the VIDAS® test. No uninfected child had positive IgM. Conclusion The prototype performed comparably to the ARCHITECT® and VIDAS® Toxo IgM assays for the diagnosis of maternal and congenital toxoplasmosis.
Assuntos
Anticorpos Antiprotozoários/sangue , Imunoglobulina M/sangue , Complicações Parasitárias na Gravidez/diagnóstico , Toxoplasmose Congênita/diagnóstico , Anticorpos Antiprotozoários/imunologia , Feminino , Humanos , Imunoglobulina M/imunologia , Testes Imunológicos/métodos , Gravidez , Complicações Parasitárias na Gravidez/sangue , Toxoplasma/imunologia , Toxoplasmose Congênita/sangueRESUMO
A difficulty when detecting both anti-Toxoplasma gondii-specific IgG and IgM in pregnant women is estimating the date of infection. The aim of this study was to compare the anti-Toxoplasma-specific immunoglobulin kinetics of 7 serological techniques to date the infection and to draw kinetic curves that are easy to use on a daily basis. IgG and IgM antibodies were measured on 691 sera samples. IgM appeared a few days, less than 1 week, after the beginning of the infection. Then, the levels of IgM reached a peak at approximately 3, 4, and 5â¯weeks with Toxo-ISAGA® IgM, IgM homemade indirect immunofluorescence assays, and Vidas Toxo® IgM, respectively. Furthermore, the Architect Toxo® IgG titers were higher than those of the Vidas Toxo® IgG results in recent infection (less than 6â¯months). This study provides new average IgM and IgG curves that can help to determine the approximate date of infection.