Novel paradigm enables accurate monthly gestational screening to prevent congenital toxoplasmosis and more.

BACKGROUND: Congenital toxoplasmosis is a treatable, preventable disease, but untreated causes death, prematurity, loss of sight, cognition and motor function, and substantial costs worldwide. OBJECTIVES: We asked whether high performance of an Immunochromatographic-test (ICT) could enable accurate, rapid diagnosis/treatment, establishing new, improved care-paradigms at point-of-care and clinical laboratory. METHODS: Data were obtained in 12 studies/analyses addressing: 1-feasibility/efficacy; 2-false-positives; 3-acceptability; 4-pink/black-line/all studies; 5-time/cost; 6-Quick-Information/Limit-of-detection; 7, 8-acute;-chronic; 9-epidemiology; 10-ADBio; 11,12-Commentary/Cases/Chronology. FINDINGS: ICT was compared with gold-standard or predicate-tests. Overall, ICT performance for 1093 blood/4967 sera was 99.2%/97.5% sensitive and 99.0%/99.7% specific. However, in clinical trial, FDA-cleared-predicate tests initially caused practical, costly problems due to false-positive-IgM results. For 58 persons, 3/43 seronegative and 2/15 chronically infected persons had false positive IgM predicate tests. This caused substantial anxiety, concerns, and required costly, delayed confirmation in reference centers. Absence of false positive ICT results contributes to solutions: Lyon and Paris France and USA Reference laboratories frequently receive sera with erroneously positive local laboratory IgM results impeding patient care. Therefore, thirty-two such sera referred to Lyon's Reference laboratory were ICT-tested. We collated these with other earlier/ongoing results: 132 of 137 USA or French persons had false-positive local laboratory IgM results identified correctly as negative by ICT. Five false positive ICT results in Tunisia and Marseille, France, emphasize need to confirm positive ICT results with Sabin-Feldman-Dye-test or western blot. Separate studies demonstrated high performance in detecting acute infections, meeting FDA, CLIA, WHO REASSURED, CEMark criteria and patient and physician satisfaction with monthly-gestational-ICT-screening. CONCLUSIONS/SIGNIFICANCE: This novel paradigm using ICT identifies likely false positives or raises suspicion that a result is truly positive, rapidly needing prompt follow up and treatment. Thus, ICT enables well-accepted gestational screening programs that facilitate rapid treatment saving lives, sight, cognition and motor function. This reduces anxiety, delays, work, and cost at point-of-care and clinical laboratories. TRIAL REGISTRATION: NCT04474132, https://clinicaltrials.gov/study/NCT04474132 ClinicalTrials.gov.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Genetic Variations in the Purinergic P2X7 Receptor Are Associated with the Immune Response to Ocular Toxoplasmosis in Colombia.

Ocular toxoplasmosis (OT) is characterized by inflammation within the eye and is the most recognized clinical manifestation of toxoplasmosis. The objective of this study was to identify new single-nucleotide polymorphisms (SNPs) in the P2RX7 gene that may have significance in the immune response to OT in Colombian patients. A case-control study was conducted to investigate the associations between SNPs (rs1718119 and rs2230912) in the P2RX7 gene and OT in 64 Colombian patients with OT and 64 controls. Capillary electrophoresis was used to analyze the amplification products, and in silico algorithms were employed to predict deleterious SNPs. Stability analysis of amino acid changes indicated that both mutations could lead to decreased protein structure stability. A nonsynonymous SNP, Gln460Arg, located in the long cytoplasmic tail of the receptor, showed a significant association with OT (Bonferroni correction (BONF) = 0.029; odds ratio OR = 3.46; confidence interval CI: 1.05 to 11.39), while no significant association between rs1718119 and OT risk was observed. Based on the 3D structure analysis of the P2RX7 protein trimer, it is hypothesized that an increase in the flexibility of the cytoplasmic domain of this receptor could alter its function. This SNP could potentially serve as a biomarker for identifying Colombian patients at risk of OT.

Performances of ICT Toxoplasma IgG-IgM test in comparison with Vidas® toxo competition to determine the immune status against Toxoplasma gondii.

Toxoplasmosis is a ubiquitous parasitic infection caused by Toxoplasma gondii (Tg). In immunocompetent people, the infection may be asymptomatic with the induction of an immune response that may prevent reinfection or transmission to the fetus in immune pregnant woman. In immunocompromised persons or seronegative pregnant woman with a primary infection during pregnancy, the infection may result in the loss of life, sight, cognition, and motor function in the immune-compromised person or immunologically immature fetus. The objective of this study was to evaluate a new immunochromatographic test Toxoplasma ICT IgG-IgM (ICT) that allows detection of specific anti-Tg immunoglobulins G (Ig G) and M (Ig M). We included 1145 prospectively obtained sera and 376 samples selected for specificity or sensitivity studies. The performance of ICT was compared using Vidas® Toxo Competition (TXC) and Toxoscreen®. In case of discrepancy, Vidas® Toxo Ig G or Ig M and LDBIO Toxo II IgG western blot were used to establish definitive results by additional methods. Sensitivity and specificity of ICT were respectively 99.3% and 100%. In comparison, Toxoscreen®'s sensitivity was 100% and the specificity was 99.8%. TXC had a sensitivity of 98.7% with a specificity of 99.1%. ICT has excellent performance even for low Ig G titers, especially in immunocompromised patients, and confirms the specificity of isolated Ig M. This ICT provides reliable results easily and quickly. This screening technique is not designed to differentiate the Ig M from Ig G. When positive, additional tests may be necessary.

CD4+ T Cell Responses to Toxoplasma gondii Are a Double-Edged Sword.

CD4+ T cells have been found to play critical roles in the control of both acute and chronic Toxoplasma infection. Previous studies identified a protective role for the Toxoplasma CD4+ T cell-eliciting peptide AS15 (AVEIHRPVPGTAPPS) in C57BL/6J mice. Herein, we found that immunizing mice with AS15 combined with GLA-SE, a TLR-4 agonist in emulsion adjuvant, can be either helpful in protecting male and female mice at early stages against Type I and Type II Toxoplasma parasites or harmful (lethal with intestinal, hepatic, and spleen pathology associated with a storm of IL6). Introducing the universal CD4+ T cell epitope PADRE abrogates the harmful phenotype of AS15. Our findings demonstrate quantitative and qualitative features of an effective Toxoplasma-specific CD4+ T cell response that should be considered in testing next-generation vaccines against toxoplasmosis. Our results also are cautionary that individual vaccine constituents can cause severe harm depending on the company they keep.

Evaluation of the acceptability of point of care diagnostic test for prenatal toxoplasmosis (translational research phase III).

BACKGROUND: A new point of care test (POC) was developed that is promising as a tool to enhance impact of prenatal care programs for toxoplasmosis, however, no reports exist about its use or acceptability for healthcare personnel and mothers in Colombia. METHODS: This was a translational research - phase III study of the acceptability of a new POC test (Toxoplasma ICT IgG-IgM, LDBio) for qualitative diagnosis of toxoplasmosis in 783 pregnant women and 30 health personnel in primary health care sites in the city of Armenia, Quindío (Colombia). Along with collection of the results of diagnostic POC and confirmatory test and demographic information, we evaluated acceptability through measure of the willingness, credibility, and satisfaction by using questionnaires with a Likert scale during routine prenatal care visits. RESULTS: POC positivity was 46.5% among pregnant participants and was significantly related to socioeconomic factors, including education level (p = 0.00000000) and insurance status (p = 0.00000015). A total of 93-97% of healthcare personnel indicated agreement to positive statements regarding total satisfaction and total credibility of the LDBio test, but qualitative questions identified "Difficulty in the test procedure" as the most common response about barriers to apply the test. Greater than 90% of pregnant participants agree that POC test should be routine for all pregnant woman and permanently implemented. CONCLUSIONS: The test had near complete acceptability. In future studies it is necessary to examine the effect of non-differentiation between IgG and IgM isotypes.

A fresh look at the role of spiramycin in preventing a neglected disease: meta-analyses of observational studies.

PURPOSE: We aimed to investigate the effect of antepartum treatment with spiramycin with or without subsequent pyrimethamine-sulfonamide-folinic acid, compared to no treatment, on the rate of mother-to-child transmission (MTCT) of Toxoplasma gondii (T. gondii) and incidence/severity of sequelae in the offspring. METHODS: Embase and PubMed were searched for literature on spiramycin in pregnant women suspected/diagnosed with T. gondii infection. Meta-analyses were performed using random-effects model. RESULTS: Thirty-three studies (32 cohorts and 1 cross-sectional study), with a total of 15,406 mothers and 15,250 offspring, were pooled for analyses. The MTCT rate for all treated patients was significantly lower than the untreated [19.5% (95% CI 14-25.5%) versus 50.7% (95% CI 31.2-70%), p < 0.001]. The transmission rate in patients on spiramycin monotherapy was also significantly lower than untreated [17.6% (95% CI 9.9-26.8%) versus 50.7% (95% CI 31.2-70%), p < 0.001]. CONCLUSION: Results indicate significant reduction in MTCT rates following spiramycin treatment of suspected/diagnosed maternal T. gondii infection.

Epidemiological Aspects of Maternal and Congenital Toxoplasmosis in Panama.

In Panama, epidemiological data on congenital toxoplasmosis are limited, making it difficult to understand the scope of clinical manifestations in the population and factors that may increase the risk of infection. This study provides insight into the epidemiological situation of maternal and congenital toxoplasmosis in Panama and contributing information on the burden of this disease in Central America. Blood samples were collected from 2326 pregnant women and used for the detection of anti-T. gondii antibodies. A high seroprevalence (44.41%) was observed for T. gondii infection in pregnant women from different regions of Panama, with an estimated incidence rate of congenital toxoplasmosis of 3.8 cases per 1000 live births. The main risk factors associated with T. gondii infection using bivariate statistical analysis were an elementary level education and maternal age range of 34-45 years. Multivariate statistical analyses revealed that in some regions (San Miguelito, North and West regions), the number of positive cases correlated with the presence of pets, stray dogs and the consumption of poultry. In other regions (East and Metropolitan regions), the absence of pets was considered a protective factor associated with negative cases, while the presence of stray cats and the age range of 25-34 years did not represent any risk in these regions.

High performance of a novel point-of-care blood test for Toxoplasma infection in women from diverse regions of Morocco.

Point-of-care (POC) testing for Toxoplasma infection has the potential to revolutionize diagnosis and management of toxoplasmosis, especially in high-risk populations in areas with significant environmental contamination and poor health infrastructure precluding appropriate follow-up and preventing access to medical care. Toxoplasmosis is a significant public health challenge in Morocco, with a relatively heavy burden of infection and, to this point, minimal investment nationally to address this infection. Herein, we analyse the performance of a novel, low-cost rapid test using fingerstick-derived whole blood from 632 women (82 of whom were pregnant) from slums, educational centres, and from nomad groups across different geographical regions (i.e. oceanic, mountainous) of Morocco. The POC test was highly sensitive and specific from all settings. In the first group of 283 women, sera were tested by Platelia ELISA IgG and IgM along with fingerstick whole blood test. Then a matrix study with 349 women was performed in which fingerstick - POC test results and serum obtained by venipuncture contemporaneously were compared. These results show high POC test performance (Sensitivity: 96.4% [IC95 90.6-98.9%]; Specificity: 99.6% [IC95 97.3-99.9%]) and high prevalence of Toxoplasma infection among women living in rural and mountainous areas, and in urban areas with lower educational levels. The high performance of POC test confirms that it can reduce the need for venipuncture and clinical infrastructure in a low-resource setting. It can be used to efficiently perform seroprevalence determinations in large group settings across a range of demographics, and potentially expands healthcare access, thereby preventing human suffering.

Late Diagnosis of Congenital Toxoplasmosis with Macrocephaly in Dizygotic Twins after Incidental Detection of Leukocoria: A Case Report.

Untreated congenital toxoplasmosis remains an important cause of neurologic and ocular disease worldwide. However, congenitally infected infants may not have signs and symptoms their physicians recognize, leading to delayed diagnosis and missed opportunities for treatment. We describe a pair of twins diagnosed with congenital toxoplasmosis at 11 months of age following incidental detection of leukocoria in one twin.

Engineering and characterization of a novel Self Assembling Protein for Toxoplasma peptide vaccine in HLA-A*11:01, HLA-A*02:01 and HLA-B*07:02 transgenic mice.

Fighting smart diseases requires smart vaccines. Novel ways to present protective immunogenic peptide epitopes to human immune systems are needed. Herein, we focus on Self Assembling Protein Nanoparticles (SAPNs) as scaffolds/platforms for vaccine delivery that produce strong immune responses against Toxoplasma gondii in HLA supermotif, transgenic mice. Herein, we present a useful platform to present peptides that elicit CD4+, CD8+ T and B cell immune responses in a core architecture, formed by flagellin, administered in combination with TLR4 ligand-emulsion (GLA-SE) adjuvant. We demonstrate protection of HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02 mice against toxoplasmosis by (i) this novel chimeric polypeptide, containing epitopes that elicit CD8+ T cells, CD4+ T helper cells, and IgG2b antibodies, and (ii) adjuvant activation of innate immune TLR4 and TLR5 pathways. HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02q11 transgenic mouse splenocytes with peptides demonstrated predicted genetic restrictions. This creates a new paradigm-shifting vaccine approach to prevent toxoplasmosis, extendable to other diseases.

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites.

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Author Correction: New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Outcomes of hydrocephalus secondary to congenital toxoplasmosis.

OBJECTIVE: Hydrocephalus occurs in children with congenital toxoplasmosis and can lead to severe disability. In these cases, the decision to intervene is often influenced by the expectation of neurological recovery. In this study, clinical responses to neurosurgical intervention in children with hydrocephalus secondary to congenital toxoplasmosis are characterized. METHODS: Sixty-five participants with hydrocephalus due to congenital Toxoplasma gondii infection were evaluated as part of the National Collaborative Chicago-based Congenital Toxoplasmosis Study, and their neuroradiographic findings were reviewed. Clinical outcomes were scored on the basis of cognition and motor skills through the use of IQ scores and Gross Motor Function Classification System (GMFCS) level. Outcomes were then analyzed in relation to approach to management, anatomy of hydrocephalus, and time from diagnosis of hydrocephalus to surgical intervention. RESULTS: There was considerable variation in the outcomes of patients whose hydrocephalus was treated in early life, ranging from normal cognitive and motor function to profound developmental delay and functional limitation. Of the 65 participants included in the study, IQ and GMFCS level were available for 46 (70.8%). IQ and motor score were highly correlated (r = -0.82, p < 0.001). There were people with differing patterns of hydrocephalus or thickness of cortical mantle on initial presentation who had favorable outcomes. Time to neurosurgical intervention data were available for 31 patients who underwent ventriculoperitoneal (VP) shunt placement. Delayed shunt placement beyond 25 days after diagnosis of hydrocephalus was associated with greater cognitive impairment (p = 0.02). Motor impairment also appeared to be associated with shunt placement beyond 25 days but the difference did not achieve statistical significance (p = 0.13). Among those with shunt placement within 25 days after diagnosis (n = 19), the mean GMFCS level was 1.9 ± 1.6 (range 1-5). Five (29.4%) of 17 of these patients were too disabled to participate in formal cognitive testing, after excluding 2 patients with visual difficulties or language barriers that precluded IQ testing. Of the patients who had VP shunt placement 25 or more days after diagnosis (n = 12), the mean GMFCS level was 2.7 ± 1.4 (range 1-4). Of these, 1 could not participate in IQ testing due to severe visual difficulties and 8 (72.7%) of the remaining 11 due to cognitive disability. CONCLUSIONS: VP shunt placement in patients with hydrocephalus caused by congenital toxoplasmosis can contribute to favorable clinical outcomes, even in cases with severe hydrocephalus on neuroimaging. Shunt placement within 25 days of diagnosis was statistically associated with more favorable cognitive outcomes. Motor function appeared to follow the same pattern although it did not achieve statistical significance.

Correction to: The One Health Approach to Toxoplasmosis: Epidemiology, Control, and Prevention Strategies.

This article was originally published electronically on the publisher's internet portal (currently SpringerLink) on April 3, 2019 without open access.

Publisher Correction: Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The One Health Approach to Toxoplasmosis: Epidemiology, Control, and Prevention Strategies.

One Health is a collaborative, interdisciplinary effort that seeks optimal health for people, animals, plants, and the environment. Toxoplasmosis, caused by Toxoplasma gondii, is an intracellular protozoan infection distributed worldwide, with a heteroxenous life cycle that practically affects all homeotherms and in which felines act as definitive reservoirs. Herein, we review the natural history of T. gondii, its transmission and impacts in humans, domestic animals, wildlife both terrestrial and aquatic, and ecosystems. The epidemiology, prevention, and control strategies are reviewed, with the objective of facilitating awareness of this disease and promoting transdisciplinary collaborations, integrative research, and capacity building among universities, government agencies, NGOs, policy makers, practicing physicians, veterinarians, and the general public.

Evaluation of Three Point-of-Care Tests for Detection of Toxoplasma Immunoglobulin IgG and IgM in the United States: Proof of Concept and Challenges.

BACKGROUND: The cost of conventional serological testing for toxoplasmosis discourages universal adoption of prenatal monthly screening programs to prevent congenital toxoplasmosis. Point-of-care (POC) technology may constitute a cost-effective approach. METHODS: We evaluated the diagnostic accuracy of 3 Toxoplasma POC tests against gold-standard testing performed at Palo Alto Medical Foundation Toxoplasma Serology Laboratory (PAMF-TSL). The POC tests included the following: Toxo IgG/IgM Rapid Test (Biopanda) and the OnSite Toxo IgG/IgM Combo-Rapid-test that detect IgG and IgM separately, and the Toxoplasma ICT-IgG-IgM-bk (LDBIO) that detects either or both immunoglobulin IgG/IgM in combination. Samples were selected from PAMF-TSL biobank (n = 210) and Centers for Disease Control and Prevention Toxoplasma 1998 Human Serum Panel (n = 100). Based on PAMF-TSL testing, Toxoplasma-infection status was classified in 4 categories: acute infections (n = 85), chronic infections (n = 85), false-positive Toxoplasma IgM (n = 60), and seronegative (n = 80). The POC testing was performed in duplicate following manufacturer's instructions by investigators blinded to PAMF-TSL results. Sensitivity and specificity were calculated. RESULTS: A total of 1860 POC tests were performed. For detection of Toxoplasma IgG, sensitivity was 100% (170 of 170; 95% confidence interval [CI], 97.8%-100%) for all 3 POC kits; specificity was also comparable at 96.3% (77 of 80; 95% CI, 89.5%-98.9%), 97.5% (78 of 80; 95% CI, 91.3%-99.6%), and 98.8% (79 of 80; 95% CI, 93.2%-99.9%). However, sensitivity for detection of Toxoplasma IgM varied significantly across POC tests: Biopanda, 62.2% (51 of 82; 95% CI, 51.4%-71.9%); OnSite, 28% (23 of 82; 95% CI, 19.5%-38.6%); and LDBIO combined IgG/IgM, 100% (82 of 82; 95% CI, 95.5%-100%). Diagnostic accuracy was significantly higher for the LDBIO POC kit. The POC kits did not exhibit cross-reactivity for false-positive Toxoplasma-IgM sera. CONCLUSIONS: The 3 evaluated POC kits revealed optimal sensitivity for Toxoplasma-IgG antibodies. The LDBIO-POC test exhibited 100% sensitivity for the combined detection of IgG/IgM in acute and chronic Toxoplasma infection. Biopanda and Onsite POC tests exhibited poor sensitivity for Toxoplasma-IgM detection.

CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii.

Toxoplasma gondii, an Apicomplexan parasite, causes significant morbidity and mortality, including severe disease in immunocompromised hosts and devastating congenital disease, with no effective treatment for the bradyzoite stage. To address this, we used the Tropical Disease Research database, crystallography, molecular modeling, and antisense to identify and characterize a range of potential therapeutic targets for toxoplasmosis. Phosphoglycerate mutase II (PGMII), nucleoside diphosphate kinase (NDK), ribulose phosphate 3-epimerase (RPE), ribose-5-phosphate isomerase (RPI), and ornithine aminotransferase (OAT) were structurally characterized. Crystallography revealed insights into the overall structure, protein oligomeric states and molecular details of active sites important for ligand recognition. Literature and molecular modeling suggested potential inhibitors and druggability. The targets were further studied with vivoPMO to interrupt enzyme synthesis, identifying the targets as potentially important to parasitic replication and, therefore, of therapeutic interest. Targeted vivoPMO resulted in statistically significant perturbation of parasite replication without concomitant host cell toxicity, consistent with a previous CRISPR/Cas9 screen showing PGM, RPE, and RPI contribute to parasite fitness. PGM, RPE, and RPI have the greatest promise for affecting replication in tachyzoites. These targets are shared between other medically important parasites and may have wider therapeutic potential.

Global initiative for congenital toxoplasmosis: an observational and international comparative clinical analysis.

Globally, congenital toxoplasmosis remains a significant cause of morbidity and mortality, and outbreaks of infection with T. gondii represent a significant, emerging public health burden, especially in the developing world. This parasite is a threat to public health. Disease often is not recognized and is inadequately managed. Herein, we analyze the status of congenital toxoplasmosis in Morocco, Colombia, the United States, and France. We identify the unique challenges faced by each nation in the implementation of optimal approaches to congenital toxoplasmosis as a public health problem. We suggest that developed and developing countries use a multipronged approach, modeling their public health management protocols after those in France. We conclude that education, screening, appropriate treatment, and the development of novel modalities will be required to intervene successfully in caring for individuals with this infection. Gestational screening has been demonstrated to be cost-effective, morbidity-sparing, and life-saving. Recognition of the value and promise of public health interventions to prevent human suffering from this emerging infection will facilitate better patient and societal outcomes.