17th International Congress on Toxoplasmosis

Professor

Professor Dr.

The Role of Age in Ocular Toxoplasmosis: Clinical Signs of Immunosenescence and Inflammageing – a perspective study

Epidemiology, Public Health and Clinical Aspects of Toxoplasmosis

Purpose: To investigate the association between age, immune response, and the clinical presentation in ocular toxoplasmosis (OT). Design: Retrospective, observational cohort study. Methods: A review of medical records of patients with active OT at the Uveitis Center, Charité Universitätsmedizin was conducted. Baseline parameters included age at presentation, primary manifestation or recurrence, visual acuity, intraocular pressure (IOP), size and location of active lesions, inflammatory activity, antibody index (AI), and complications of intraocular inflammation. The data are presented as mean ± standard deviation (SD). Results: Between 1998 and 2019, 290 patients with active OT were diagnosed at our tertiary reference center. The mean age was 37.7 ± 17.1 years, 53.8% were females, and 195 patients (70.9%) showed recurrent disease. Older age was associated with lower-baseline visual acuity (p = 0.043), poor visual outcome (p = 0.019), increased inflammatory activity (p &lt; 0.005), and larger retinal lesions (p &lt; 0.005). Older patients presented a lower AI (&lt;35 years: 45.1 ± 82.7, median: 12.1; ≥35 years: 18.6 ± 50.5, median: 5.8; p = 0.046), confirmed by a decrease of AI with increasing age (R² = 0.045; p = 0.024). Complications were observed more frequently in patients with primary ocular toxoplasmosis (p = 0.046) and were mostly accompanied by panuveitis (p = 0.018) and a longer duration of illness (p = 0.037). Furthermore, panuveitis (p = 0.025), ocular hypertension (p = 0.037), and improved visual acuity development (p = 0.035) were associated with the presence of primary ocular toxoplasmosis. Macular involvement (24.3% of patients) was positively correlated with complications (macular/peripapillary edema and retinal detachment, p &lt; 0.005) and poor visual outcome (p &lt; 0.005) and was negatively correlated with inflammatory activity (p &lt; 0.005). Conclusions: We found a strong and clinically-relevant impact of age on the clinical presentation and course of OT. While an unspecific inflammatory response increased with age, the specific, local humoral immune response declined. These findings are well in line with the concept of immunosenescence and inflammageing in uveitis. In addition, alongside patient age, the primary manifestation and the retinal lesion location were identified factors contributing to the severity of intraocular inflammation and the occurrence of ocular complications.

Oral presentation

CHRONIC LATENT TOXOPLASMA GONDII INFECTION PRECIPITATES COGNITIVE DECLINE IN AN INDUCIBLE ALZHEIMER'S DISEASE MODEL

Neuroimmune dysregulations strongly contribute to the pathophysiology of Alzheimer's Disease (AD), but the etiology of AD remains ill-defined and is certainly diverse. Because pathogens shape the immune system over lifetime, their association with AD have been suggested. Increasing evidence supports a role for the prevalent, brain-persisting parasite Toxoplasma gondii (Tg) in chronic neurological diseases. Yet, overall, our current knowledge about the impact of chronic Tg infection on AD pathology and clinical progression remains sparse. There have been limited attempts to describe the impact of Tg infection on neuropathological lesions in AD mouse models. Up to now, the consequences of latent Tg infection on the severity and progression of cognitive decline and on the neuroimmune landscape, as well as the underlying molecular mechanisms, remain ill-defined. Considering the temporal relationship between natural Tg infection and AD occurrence, it seems legitimate to evaluate the effects of a strain of Tg which is effectively controlled and result in latent infection, on the later development of AD. To tackle this question, we combined infection by transgenic Tg expressing a model antigen (Tg.GRA6-OVA) that is efficiently presented by MHC I in C57BL/6 mice and results in a CD8+ T cell-controlled persisting and latent infection, with an inducible mouse model of AD amyloidosis, the TetO-APPSweInd mouse (ihAPP), in which amyloid-β peptide production and deposition, can be induced upon removal of doxycycline treatment. Mice were kept on doxycycline-dosed chow until chronic latent Tg infection is established. Behavioral, immune and biochemical analysis were performed after doxycycline withdrawal. We found that Tg-infected ihAPP mice (Tg-ihAPP), displayed cognitive deficits earlier than non-infected AD mice (NI-AD), including impaired object recognition memory and spatial learning and memory deficits. NI-ihAPP mice are impaired in these tasks only 2 months later. Interestingly, Tg-ihAPP mice had less β-amyloid plaques in the cortex and hippocampus compared to NI-AD, confirming previous findings. Facs analyses of brain-isolated cells revealed unique changes in monocytes and CD8+ T cells in Tg-ihAPP mice compared to the other groups. Together, our data indicate that preexistent and in appearance silent chronic Tg infection may hasten the progression of cognitive decline in AD while contributing to Aβ plaques nibbling and involving neuroimmune mechanisms.

Chronic Toxoplasma gondii, Diabetes, and Retinopathy in Pregnancy

We carried out a prospective study of 690 Hispanic pregnant women (158 T.gondii (TG) positive, 532 TG negative) enrolled at an initial early prenatal visit and then followed for adverse events across the course of their pregnancies. After the initial study visit data were collected from the electronic health record (EHR). From the EHR, pre-pregnancy type 2 diabetes (T2D), and the occurrences of gestational diabetes (GDM) were significantly higher in the T. gondii positive women. GDM was diagnosed in 20% of the T. gondii positive women (χ2=4.4,df=1, p=0.04). compared to 11% in the T. gondii negative women (χ2=7.3, df=1, p=0.007). The frequency of an abnormal insulin glucose tolerance test (IGTT) without diagnosis of GDM was the same in both groups (about 16%). Additionally, from the initial enrollment, 285 women were recruited into a prospective study with 5 study visits in their prenatal clinics. There were 158 TG positive women who had a chronic, high avidity chronic infection and 127 T.gondii negative &quot;controls&quot;. Blood samples, health exams and retinal scannings were done at the clinic study visits. Serial retinal scanning was done only in the T. gondii positive women, as the aim of this analysis was to explore lesions and scars characteristic of toxoplasma disease. Only one scar was found in the population. Retinal images were scanned and graded by an independent retinal scanning service. We found diabetic retinopathy, mostly mild, non-proliferative in type, in 55 of the 130 women who had retinal images done, with 30 of these women having either GDM, positive IGTT, or T2D. The remaining 25 had no other related pathology. The relationship of chronic TG infection with retinopathy is unknown at this time. The increased prevalence of GDM and T2D in TG positive pregnant women may be important factors, as we have found differences in immune function in pregnant TG positive women (Prescott S, et al.,2023). Plasma levels of IFN- γ and IL-10 were significantly lower in GDM and IL-17 was lower in T2D. A comparison group of TG negative pregnant Hispanic women is necessary to determine this relationship. Prescott et al., Am J Reprod Immunol. 2023 Sep;90(3):e1376.

Serological Responses to Toxoplasma gondii and Matrix Antigen 1 Predict the Risk of Subsequent Toxoplasmic Encephalitis in People Living with HIV

Background: There is a lack of clinically useful predictors for fatal toxoplasmosis. We investigated the value of serological assays for antibodies to whole Toxoplasma antigens and to peptide antigens of the Toxoplasma cyst protein MAG1 for predicting incident toxoplasmic encephalitis (TE) in people living with HIV (PLWH). Methods: We performed a nested case-control study, conducted within the Multicenter AIDS Cohort Study (MACS), using serum samples obtained two years prior to diagnosis of TE from 28 cases and 37 HIV disease-matched Toxoplasma seropositive controls at matched time points. Sera were tested for Toxoplasma antibodies using a commercial assay and for antibodies to MAG1_4.2 and MAG1_5.2 peptides in ELISA. Results: Two years before clinical diagnosis, 68% of TE cases were MAG1_4.2 seropositive, as compared to 16% of controls (OR 25.0, 95% CI 3.14-199.18). The results for MAG1_5.2 seropositivity were 36% and 14% (OR 3.6, 95% CI 0.95-13.42). Higher levels of antibody to MAG1_4.2 (OR 18.5 per doubling of the OD value, 95% CI 1.41-242) and Toxoplasma (OR 2.91 for each OD unit increase, 95% CI 1.48-5.72) were also associated with the risk of TE. When seropositivity was defined as the presence of MAG1 antibody or relatively high levels of Toxoplasma antibody, the sensitivity was 89%, and specificity was 68% for subsequent TE. Conclusions: The presence of antibodies to MAG1 in PLWH can predict the occurrence of TE. The predictive performance of these antibodies is further improved by adding the levels of Toxoplasma antibodies. These measures could be clinically helpful in predicting subsequent diseases in multiple at-risk populations.

An in-vitro method and predictive model to quantify Toxoplasma gondii inactivation by salting

Consumption of raw meat products is considered an important source of T. gondii infections. These products are usually processed with NaCl and other additives. Such additives affect the viability of T. gondii, but results described in literature are variable. Mouse bioassay is currently the standard method for T. gondii inactivation experiments, but ethically undesirable, costly and time consuming. Moreover, mouse bioassays provide very limited quantitative information on actual reductions in the concentration of viable parasites in tested meat products. Therefore it was our aim to validate and compare an in-vitro method as a quantitative alternative to the mouse bioassay for determining the effect of salting on T. gondii viability. Muscle tissue of sheep experimentally infected with T. gondii was used to prepare 50 g portions of minced meat supplemented with 0.6 - 2.7% NaCl and sodium lactate or sodium acetate. Portions were stored overnight at 4°C. Pepsin-HCl digestion was performed and digests were inoculated onto RK13 cell monolayers or into IFNγ-KO mice, to evaluate the presence of infective parasites. In addition, a four-fold dilution series ranging from 4 to 256 bradyzoites per ml of tissue digest or culture medium was tested to evaluate and compare the detection limit of these methods. Both, the in-vitro and the in-vivo method gave positive results for samples spiked with 4 or 16 bradyzoites and were consistently positive when a minimum of 64 bradyzoites was added. Viable parasites were detected in portions of experimental sheep tissue supplemented with up to 1.5% NaCl, but not in samples supplemented with both 1.2% NaCl and 1.4% sodium lactate. A statistical model was fitted, using actual and previous results with cell culture and, as an example, indicates a 5-log reduction in infective parasites after supplementation with 2.5% NaCl. In conclusion, the in-vitro method was successfully used to obtain data on T. gondii inactivation by salting. The model provides more insight in the possibilities to use salt for controlling the risk of T. gondii in raw meat products. Replacement of the mouse bioassay opens up possibilities to investigate other potential inactivation methods. In the future, we hope to also overcome the need for experimental infection to obtain positive muscle tissue and we look forward to discuss possibilities with in-vitro cultivated tissue cysts.

Usefulness of serotyping tools in Toxoplasma gondii infections in sheep and pigs

Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most relevant food-borne zoonotic diseases worldwide that causes important economic losses in the livestock sector related to reproductive failure, mainly in sheep and goats. To better understand the epidemiology of toxoplasmosis, predict the clinical consequences and design appropriate control strategies, it is crucial to determine the strain causing the infection. In this sense, serotyping methods offer a cost-effective, rapid, sensitive, and non-invasive alternative to DNA-dependent genotyping techniques, allowing the analysis of subclinical cases. This technique is based on the detection of strain-specific antibodies that react against segments of antigenic proteins presenting polymorphic variations. However, the prediction capability of promising peptides needs to be determined in each animal species, as differences in the strain-specific reactivity patterns have been described. In the present study we sought to evaluate the applicability in sheep and pigs of a panel of peptides previously characterized in mice and humans. To this end, we used 51 sheep serum samples obtained in previous experimental infections (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type I and 18 type III). Our ELISA test results show that a combination of GRA3, 5, 6 and 7 peptide homologous pairs can discriminate infections caused by type II and III strains in sheep and pigs. Namely, the best peptide pairs to discriminate infections in these two livestock species were GRA6-I/III-213 vs. GRA6-II-214 and GRA6-II-44 vs. GRA6-III-44, together with GRA3-I/III-38 vs. GRA3-II-38 in sheep and GRA7-II-224 vs. GRA7-III-224 in pigs. Notably, the GRA6-44 peptide pair, which were previously described to be inefficient in mice and humans, showed a high prediction capacity, especially in sheep, while GRA5-38 peptide versions failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. These results lay the grounds for future large-scale serotyping studies in sheep and pigs, as well as its potential extrapolation to other animal species in future studies.

Session VII: Epidemiology, Public Health & Clinical Aspects

Appointment

Chairs

Programme