Long-term impact of congenital toxoplasmosis on phenotypic and functional features of circulating leukocytes from infants one year after treatment onset.

Changes in immune response of children with congenital toxoplasmosis (CT) regarding infection evolution and therapeutic intervention was addressed. Infants with CT presented increased counts of monocytes, CD3-CD16-CD56High, CD3+CD56+ and CD4+ T-cells 1-year after treatment onset (TOXO1-yearAT). Smaller numbers of CD3-CD16-CD56+ and TCRγδ+ T-cells were specifically observed in infants with retinochoroidal lesions (L(+)). When infants were classified based on the baseline status, expansion of CD3-CD16-CD56High and CD4+ T-cells were observed in L(+) who had active, active/cicatricial or cicatricial lesions. Infants who had active or active/cicatricial lesions display augmented numbers of monocytes, CD3-CD16+CD56+, CD3+CD56+, CD8+DR+ and TCRγδ+ T-cells and those with active/cicatricial or cicatricial at baseline displayed increase in CD14+CD64+ monocytes. Moreover, all L(+) had increased IFN-γ+ and IL-10+ CD4+ T-cells, while L(-) had increased ratios of TNF+, IFN-γ+ and IL-4+ NK-cells upon antigen-specific stimulation. Persistent alterations in leukocytes in TOXO1-yearAT suggest long-term sequels in the immune system of infants with CT.

Putative biomarkers for early diagnosis and prognosis of congenital ocular toxoplasmosis.

In the present study we have evaluated the performance of several immunological biomarkers for early diagnosis and prognosis of congenital toxoplasmosis. Our results showed that ex vivo serum levels of CXCL9, and the frequencies of circulating CD4+CD25+ T-cells and T. gondii-specific IFN-γ+CD4+ T-cells measured 30-45 days after birth presented high accuracy to distinguish T. gondii-infected infants from healthy age-matched controls (Global Accuracy/AUC = 0.9; 0.9 and 0.8, respectively). Of note was the enhanced performance (Accuracy = 96%) achieved by using a combined stepwise analysis of CD4+CD25+ T-cells and CXCL9. In addition, high global accuracy (AUC = 0.9) with elevated sensitivity (Se = 98%) was also reached by using the total frequency of in vitro IFN-γ-producing T. gondii-specific T-cells (∑ IFN-γ+ CD4+ & CD8+) as a biomarker of congenital toxoplasmosis. Furthermore, the analysis of in vitro T. gondii-specific IL5+CD4+ T-cells and IFN-γ+NK-cells displayed a high accuracy for early prognosis of ocular lesion in infant with congenital toxoplasmosis (Global Accuracy/AUC = 0.8 and 0.9, respectively). Together, these findings support the relevance of employing the elements of the cell-mediated immune response as biomarkers with potential to endorse early diagnosis and prognosis of congenital ocular toxoplasmosis to contribute for a precise clinical management and effective therapeutic intervention.

Establishing tools for early diagnosis of congenital toxoplasmosis: Flow cytometric IgG avidity assay as a confirmatory test for neonatal screening.

The aim of this study was to evaluate the performance of conventional serology (Q-Preven™ and ELFAVIDAS™) and flow cytometry-based serologic tools for early serologic diagnosis of congenital toxoplasmosis. The study groups included prospectively confirmed cases of congenital toxoplasmosis (TOXO=88) and age-matching non-infected controls (NI=15).The results demonstrated that all samples tested positive/indeterminate for anti-T. gondii IgM screening at birth using air-dried whole blood samples. Serum samples collected at 30-45days after birth tested positive for ELFAVIDAS™ IgG in both groups. While all NI tested negative for ELFAVIDAS™ IgM and IgA, only 78% and 36% of TOXO tested positive for IgM and IgA, respectively. Flow cytometry-based anti-T. gondii IgM, IgA and IgG reactivity displayed moderate performance with low sensitivity (47.6%, 72.6% and 75.0%, respectively). Regardless the remarkable specificity of IgG1, IgG2 and IgG3 subclasses for early diagnosis, weak or moderate specificity was observed (Se=73.9%, 60.2% and 83.0%, respectively). The analysis of IgG avidity indices (AI) demonstrated the highest performance among the flow cytometry-based methods (Se=96.6%; Sp=93.3%), underscoring the low avidity index (AI<60%) within TOXO (97.0%) in contrast with the high avidity index (AI>60%) in NI (93%). Analysis of anti-T. gondii IgG and IgG3 reactivity for mother:infant paired samples may represent a relevant complementary tests for early diagnosis. In conclusion, a feasible high-standard algorithm (Accuracy=97.1%) was proposed consisting of Q-Preven™ IgM screening at birth, followed by ELFAVIDAS™ IgM and flow cytometric IgG avidity analysis at 30-45days after birth as a high performance tool for early serological diagnosis of congenital toxoplasmosis.

Early serum biomarker networks in infants with distinct retinochoroidal lesion status of congenital toxoplasmosis.

The present study characterized the early changes in the serum chemokines/cytokine signatures and networks in infants with congenital-toxoplasmosis/(TOXO) as compared to non-infected-controls/(NI). TOXO were subgrouped according to the retinochoroidal lesion status as no-lesion/(NL), active-lesion/(ARL), active/cicatricial-lesion/(ACRL) and cicatricial-lesion/(CRL). The results showed that TOXO display prominent chemokine production mediated by IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10 and RANTES/CCL5. Additionally, TOXO is accompanied by mixed proinflammatory/regulatory cytokine pattern mediated by IL-6, IFN-γ, IL-4, IL-5 and IL-10. While TNF appears as a putative biomarker for NL and IFN-γ/IL-5 as immunological features for ARL, IL-10 emerges as a relevant mediator in ACRL/CRL. IL-8/CXCL8 and IP-10/CXCL10 are broad-spectrum indicators of ocular disease, whereas TNF is a NL biomarker, IFN-γ and MIG/CXCL9 point out to ARL; and IL-10 is highlighted as a genuine serum biomarker of ACRL/CRL. The network analysis demonstrated a broad chemokine/cytokine crosstalk with divergences in the molecular signatures in patients with different ocular lesions during congenital toxoplasmosis.

Cytokine Signatures Associated With Early Onset, Active Lesions and Late Cicatricial Events of Retinochoroidal Commitment in Infants With Congenital Toxoplasmosis.

BACKGROUND: Ocular toxoplasmosis is a prominent and severe condition of high incidence in Brazil. The current study provides new insights into the immunological events that can be associated with retinochoroiditis in the setting of congenital toxoplasmosis in human infants. METHODS: Flow cytometry of intracytoplasmic cytokines in leukocyte subsets following in vitro short-term antigenic recall in infants with congenital T. gondii infection. RESULTS: Our data demonstrates that whereas neutrophils and monocytes from T. gondii-infected infants display a combination of proinflammatory and regulatory cytokine profiles, natural killer cells showed a predominantly proinflammatory profile upon in vitro T. gondii stimulation. The proinflammatory response of CD4(+) and CD8(+) T cells, characterized by the production of interferon γ (IFN-γ) and interleukin 17 in patients with an active retinochoroidal lesion, revealed the presence of IFN-γ and tumor necrosis factor α during early and late immunological events. This specific proinflammatory pattern is associated with early events and active retinochoroidal lesion, whereas a robust monocyte-derived interleukin 10-mediated profile is observed in children with cicatricial ocular lesions. CONCLUSIONS: These findings support the existence of a progressive immunological environment concomitant with the initial, apical, and cicatricial phases in the process of retinochoroidal lesion formation in infants with congenital toxoplasmosis that may be relevant in the establishment of stage-specific clinical management.

IgA and IgG1 reactivities assessed by flow cytometry mirror clinical aspects of infants with ocular congenital toxoplasmosis.

This study intended to apply the flow cytometric analysis of IgA and IgG reactivity and intracytoplasmic cytokine analysis to understand and decode the clinical aspects of infants with ocular congenital toxoplasmosis. The Toxoplasma gondii-infected infants (TOXO) were subdivided according to their clinical aspects based on the absence (NRL), presence of active (ARL), active/cicatricial (ACRL) or cicatricial retinochoroidal lesions (CRL) and compared to non-infected controls (NI). The reactivity of anti-T. gondii IgG subclasses resembles the clinical aspects of ocular lesions. IgG and IgG1 discriminate infants with cicatricial lesions (ACRL and CRL) from both ARL and NLR. IgG2 and IgG3 are particularly higher in ACRL and CRL as compared to NLR. No differences were observed when IgG4 reactivity was evaluated. Thus, the results indicated that the reactivity patterns of IgA, IgG and IgG subclasses are able to discriminate ARL, ACRL and CRL from NLR or NI. IgA and IgG subclasses are relevant serological biomarkers with diagnostic and prognostic applicability, respectively. Moreover, IgA and IgG1 were closely related to cytokine production by innate/adaptive immunity cells. IgA reactivity was directly associated to TNF-α-derived from neutrophils, monocytes and CD8(+) T-cells, while IgG1 was inversely correlated with IFN-γ-producing CD4(+) and CD8(+) T-cells but positively correlated with IL-10(+) B-cells. These findings provide insights on the relationship between the cytokine production by innate/adaptive immunity and the antibody pattern of infants with ocular congenital toxoplasmosis. In addition, the present study supports the use of flow cytometric serology as a potential tool for the diagnosis and monitoring of ocular lesions in T. gondii-infected infants in the clinical setting.

Pathological changes in acute experimental toxoplasmosis with Toxoplasma gondii strains obtained from human cases of congenital disease.

There is a lack of studies using Toxoplasma gondii strains isolated from human patients. Here, we present a pathological study of three strains obtained from human cases of congenital toxoplasmosis in Brazil using inbred mice after oral infection with 10 tissue cysts. Multiplex-nested PCR-RFLP of eleven loci revealed atypical genotypes commonly found in Brazil: toxodb #8 for TgCTBr5 and TgCTBr16 strains and toxodb #11 for the TgCTBr9 strain. BALB/c and C57BL/6 mice were evaluated for survival and histological changes during the acute phase of the disease. All mice inoculated with the non-virulent TgCTBR5 strain survived after 30 days, although irreversible tissue damage was found. In contrast, no mice were resistant to infection with the highly virulent TgCTBR9 strain. The TgCTBr16 strain resulted in 80% survival in mice. However, this strain presented low infectivity, especially by the oral route of infection. Despite being identified with the same genotype, TgCTBr5 and TgCTBr16 strains showed biological differences. Histopathologic analysis revealed liver and lungs to be the most affected organs, and the pattern of tissue injury was similar to that found in mice inoculated perorally with strains belonging to clonal genotypes. However, there was a variation in the intensity of ileum lesions according to T. gondii strain and mouse lineage. C57BL/6 mice showed higher susceptibility than BALB/c for histological lesions. Taken together, these results revealed that the pathogenesis of T. gondii strains belonging to atypical genotypes can induce similar tissue damage to those from clonal genotypes, although intrinsic aspects of the strains seem critical to the induction of ileitis in the infected host.

Biomarker analysis revealed distinct profiles of innate and adaptive immunity in infants with ocular lesions of congenital toxoplasmosis.

Toxoplasma gondii is the main infectious cause of human posterior retinochoroiditis, the most frequent clinical manifestation of congenital toxoplasmosis. This investigation was performed after neonatal screening to identify biomarkers of immunity associated with immunopathological features of the disease by flow cytometry. The study included infected infants without NRL and with retinochoroidal lesions (ARL, ACRL, and CRL) as well as noninfected individuals (NI). Our data demonstrated that leukocytosis, with increased monocytes and lymphocytes, was a relevant hematological biomarker of ARL. Immunophenotypic analysis also revealed expansion of CD14(+)CD16(+)HLA-DR(high) monocytes and CD56(dim) cytotoxic NK-cells in ARL. Moreover, augmented TCRγ δ (+) and CD8(+) T-cell counts were apparently good biomarkers of morbidity. Biomarker network analysis revealed that complex and intricated networks underscored the negative correlation of monocytes with NK- and B-cells in NRL. The remarkable lack of connections involving B-cells and a relevant shift of NK-cell connections from B-cells toward T-cells observed in ARL were outstanding. A tightly connected biomarker network was observed in CRL, with relevant connections of NK- and CD8(+) T-cells with a broad range of cell subsets. Our findings add novel elements to the current knowledge on the innate and adaptive immune responses in congenital toxoplasmosis.

Real-time PCR as a prognostic tool for human congenital toxoplasmosis.

Real-time PCR (qPCR) was positive in 72/150 (48%) blood samples of newborns with congenital toxoplasmosis. Among infants with active retinochoroiditis, 68% had positive qPCR results, while positivity was 29% (P=0.009) in the absence of ocular involvement. Positive qPCR was associated with the presence of retinochoroidal lesions, with an odds ratio of 2.8.

Genetic characterization of Toxoplasma gondii revealed highly diverse genotypes for isolates from newborns with congenital toxoplasmosis in southeastern Brazil.

Recent studies of Toxoplasma gondii isolates from animals in Brazil have revealed high genetic diversity. Many of these isolates are virulent to mice. It is speculated that these isolates may also be virulent to humans. However, there is very limited data regarding T. gondii strains from human infection. Therefore, it is not clear whether there is any association between parasite genotypes and disease phenotypes. In this study, a total of 27 T. gondii strains were isolated from humans with congenital toxoplasmosis in Minas Gerais state, Brazil. The genetic variability was assessed by restricted fragment length polymorphism in 11 loci (SAG1, 5' plus 3' SAG2, alternative [alt.] SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Genetic analysis of 24 strains revealed 14 different genotypes, including 7 previously identified from animals and 7 new types. The widespread genotype BrII accounted for 29% (7/24) of the isolates and was the dominant genotype involved in this study. This is the first report of genotyping of T. gondii isolates obtained from blood samples from newborns with congenital toxoplasmosis. Genotypic characterization of these isolates suggests high genetic diversity of T. gondii in this human population in Brazil. Future studies are needed to determine the source of contamination of this human population.