ROP39 is an Irgb10-specific parasite effector that modulates acute Toxoplasma gondii virulence.

Toxoplasma gondii (T. gondii) is a zoonotic apicomplexan parasite that is an important cause of clinical disability in humans. On a global scale, one third of the human population is infected with T. gondii. Mice and other small rodents are believed to be responsible for transmission of T. gondii to the domestic cat, its definitive host. Interferon-inducible Immunity-Related GTPases (IRG proteins) are important for control of murine T. gondii infections. Virulence differences between T. gondii strains are linked to polymorphic rhoptry proteins (ROPs) that cooperate to inactivate individual IRG family members. In particular, the pseudokinase ROP5 isoform B is critically important in laboratory strains of mice. We identified T. gondii ROP39 in complex with ROP5B and demonstrate its contribution to acute T. gondii virulence. ROP39 directly targets Irgb10 and inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuolar membrane (PVM). Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. This study identifies a novel T. gondii effector that is important for specific inactivation of the IRG resistance system. Our data reveal that yet unknown T. gondii effectors can emerge from identification of direct interaction partners of ROP5B.

Ocular Toxocariasis in Parasitology Consultation in Quindío, Colombia: Description of Cases and Contact Studies.

We report diagnosis, treatment and evolution of cases of ocular toxocariasis in specialized consultation in Quindío, Colombia. No cases were seen during the 2000-17 period, but five cases were confirmed from November 2017 to March 2019; two children resulted with definitive loss of vision on the affected eye. Studies in contacts found that 12 of 19 (63%) family members and 15 of 25 (60%) children <15 years of age living on the same street were positive for IgG Toxocara antibodies. Epidemiological studies are necessaries to establish the reasons for the increase in cases at this region.

Serotyping, host genes and cytokines response in human ocular toxoplasmosis.

In human ocular toxoplasmosis, serotype is related with greater severity. We analyzed Toxoplasma GRA6 serotype in 23 patients with ocular toxoplasmosis (13 confirmed, two co-infections- and eight unconfirmed cases) and 20 individuals chronically infected with Toxoplasma but without ocular involvement. In patients with ocular toxoplasmosis, we also studied host gene polymorphisms related to immune response (IL-1ß; IL-1α; IL-10; IFN-γ; TNF-α, IL-12), IL-17R, TLR-9, and P2RX7. Additionally, eight patients were studied for the production of TNFα, IL1-ß, IFN-γ and IL-10 by their peripheral leukocytes after ex vivo stimulation with soluble Toxoplasma antigens. There were no differences in the distribution of serotypes (GRA6-I versus GRA6 non-I) between infected individuals with- or without ocular involvement. Seropositivity for GRA6-I was associated with higher number of retinal lesions and higher levels of IL-1ß. Two polymorphisms were associated with specific clinical manifestations of ocular toxoplasmosis: IL-10 -819 C/T with bilateral lesions and IL-12 + 169,774 A/C with synechia. Higher levels of IL-10 were found in patients with the allele G/G at the polymorphic region IL-10 -1082. People with a GRA6 I serotype and possessing the allele G/G at the polymorphic region TNFα-857 suffered from an increased number of retinal lesions. We found a positive association between host cytokine genes polymorphisms and GRA6 serotypes correlated with specific clinical manifestations and immune response in ocular toxoplasmosis.

Human peripheral blood mononuclear cells as an ex vivo model to study the host parasite interaction in Toxoplasma gondii.

Toxoplasma gondii is a parasite that can invade any cell in the human body. Here, we implemented and described an ex vivo model with human peripheral blood mononuclear cells (PBMCs) without using culture supplements/antibiotics and without cryopreserved cells (EXMOWS) to study the interactions between T. gondii and human cells. To establish the EXMOWS, three independent tests were carried out. Firstly, blood samples from 5 individuals were included to assess the viability and adherence of PBMCs in plate culture. In a second trial, blood samples from three seropositive and two seronegative individuals for T. gondii were used to evaluate human PBMCs cells: parasites, multiplicity of infection (MOI) 1:1, 1:3 and 1:5 at different times post infection (1 h, 6 h and 24 h). The possible immunomodulatory effect of the infection for this EXMOWS were evaluated in a third trial where HFF cells were infected with T. gondii and co-cultured with PBMCs obtained from anti-Toxoplasma IgG positive and IgG negative individuals. One hour was enough time for T. gondii infection of human PBMCs and 2 h was the minimum incubation time to guarantee adherence before carrying out any infection assay. A minimum of 1:3 MOI was necessary to guarantee efficient infection in human PBMCs with T. gondii RH-GFP. All protocols, including PBMCs isolation and stimulation, should be conducted the same day. This EXMOWS can be adapted to study the early stages of interaction with other microorganisms of human interest, without need of using cryopreservation and supplements/antibiotics.

Coinfections and differential diagnosis in immunocompetent patients with uveitis of infectious origin.

BACKGROUND: Making a definite diagnosis of infectious uveitis is a challenging task because many other infectious, and non-infectious uveitis, may have similar non-specific symptoms and overlapping clinical appearances. Co-infections in immunocompetent patients are not frequently proved with traditional serologic-diagnostic tools. METHODS: Descriptive transversal study, in a Uveitis Service of an Ophthalmology Reference Center, in Bogotá, Colombia, from July 2014 to February 2016. Aqueous humor (AH) and/or vitreous fluid, blood and serum samples were collected from consecutive patients suspected of having infectious uveitis. The diagnosis of ocular toxoplasmosis (OT) was confirmed by the Goldmann-Witmer coefficient (GWC) and by polymerase chain reaction (PCR). Differential diagnosis by PCR in AH was done for viral origin such as Cytomegalovirus (CMV), Herpes simplex virus type 1 (HSV1), Herpes simplex virus type 2 (HSV2), Varicella zoster virus (VZV), Epstein-Barr virus (EBV) and Mycobacterium tuberculosis. RESULTS: In 66 Colombian patients with uveitis of presumed infectious origin: 22 (33.3%) were confirmed as OT, 16 (24.2%) as undetermined OT, five (7.5%) as co-infections and 23 (34.8%) as other uveitis. Toxoplasma coinfection with M. tuberculosis was identified in one case by PCR and in four cases with HSV by GWC. The initial clinical diagnosis changed, after laboratory examination, in 21 cases (31.8%). CONCLUSIONS: Clinical diagnosis can be changed by laboratory examination in a significant proportion of cases of uveitis. Diagnosis of OT should combine the use of PCR and GWC to reach the maximum of confirmation of cases. The use of multiple laboratory methods is necessary to identify co-infections and viral infections that can mimic OT in immunocompetent patients.

Food safety assessment and risk for toxoplasmosis in school restaurants in Armenia, Colombia.

We assessed the risk for toxoplasmosis in 10 school restaurants in Armenia (Quindio, Colombia). We analyzed the presence of Toxoplasma gondii DNA in the food, water, and living and inert surfaces of school restaurants, and we correlated these findings with the results of food safety inspection scores and with the prevalence of specific anti-T. gondii antibodies in children who ate at these restaurants. Of the 213 samples, 6.1% were positive using PCR to test for T. gondii DNA. Positive samples were found in meat, water, cucumber, guava juice, inert surfaces, and living surfaces. In 60% (6/10) of the public school restaurants, there was at least one PCR T. gondii-positive sample. In 311 serum samples from children who attended the restaurants, 101 (33%) were positive for IgG and 12 (3.9%) for IgM anti-T. gondii. The median of the compound score for the fulfillment of inspection for food safety conditions was of 60.7% (range 50-72). Higher T. gondii PCR positivity in surfaces, food, or water at each restaurant was correlated with lower inspection scores for water supply and water storage conditions. Lower scores in physical infrastructure and disinfection procedures and higher scores in furniture were correlated with a higher prevalence of IgG anti-T. gondii in children who ate at those restaurants. Inspection scores can identify restaurants with a higher risk for the presence of T. gondii.

Survey for Toxoplasma gondii by PCR detection in meat for human consumption in Colombia.

The overall risk for toxoplasmosis in meat produced in Colombia is unknown. We analyzed by PCR assay meat samples for human consumption in two types of plants in Colombia: 120 samples from class I plants (60 samples from chicken, 30 from swine and 30 from beef) and 60 from class II plants (30 samples from beef and 30 from swine). Presence of Toxoplasma DNA was established by targeted B1 nested PCR assay. We detected 79 (43%) samples that were positive by B1 nested PCR (33 from chicken, 22 from beef, and 24 from pork). No differences were found by region or species. Eleven positive samples were confirmed by sequencing of the B1 repeated region. Some polymorphisms were detected without relation with clonal groups nor meat species. Food animals are highly exposed to Toxoplasma in Colombia. Detailed studies are needed to establish the reasons for differences in Toxoplasma prevalence between farms, regarding practices of animal food production.

Striking Divergence in Toxoplasma ROP16 Nucleotide Sequences From Human and Meat Samples.

BACKGROUND: ROP16 is a protein kinase of Toxoplasma gondii identified in the mouse model as a virulent marker, but it is unknown whether this finding is relevant in human toxoplasmosis. METHODS: We obtained the Toxoplasma ROP16 locus DNA sequence in samples from 12 patients with ocular toxoplasmosis, 1 sample from a patient with congenital toxoplasmosis, 22 samples from soldiers operating in the jungle, 2 samples from urban soldiers, and 10 samples from meat for human consumption. An enzyme-linked immunosorbent assay specific for antibodies against the ROP16 mouse-virulent peptide was performed in 46 serum specimens from patients with ocular toxoplasmosis and in 28 serum specimens from patients with chronic asymptomatic infection, of whom 19 had congenital infection and 11 had toxoplasmic lymphadenitis. RESULTS: We found a striking divergence of the ROP16 nucleotide sequences. Ten of 12 sequences (83.3%) from patients with ocular toxoplasmosis clustered with those of mouse-virulent strains, whereas 7 of 7 ROP16 sequences (100%) from meat were clustered with those of mouse-avirulent strains. Only 11 of 104 serum specimens (10.5%) had specific antibodies against the mouse-virulent peptide, and there was no association between clinical forms and positive results of serological assays. CONCLUSIONS: The majority of ROP16 nucleotide sequences from Colombian patients with ocular toxoplasmosis belonged to the group of mouse-virulent strains.

Th1 and Th2 immune response to P30 and ROP18 peptides in human toxoplasmosis.

We determined the specific lymphocyte proliferative response and cytokine profile production regarding Toxoplasma P30 (2017 from virulent and non-virulent strain) and ROP18 protein-derived peptides (from clonal lineages I, II and III) in 19 patients having ocular toxoplasmosis, five suffering chronic asymptomatic infection, nine with congenital toxoplasmosis and eight Toxoplasma negative people. A Beckman Coulter FC500 flow cytometer was used for determining antigen-specific T cells (CD3+ CD4+ or CD3+ CD8+ cells) in peripheral blood culture. IFN γ and IL10 levels were determined in culture supernatants. Specific CD4+ and CD8+ T cell response to total antigen and P30- and ROP18-derived peptides was observed in infected people. Ocular toxoplasmosis patients had a preferential Th2 response after antigenic stimulation. Non-virulent peptide 2017 was able to shift response toward Th1 in congenitally infected children and virulent peptide 2017 induced a Th2 response in chronically infected, asymptomatic people. An immune response in human toxoplasmosis after ex vivo antigenic stimulation was Th1- or Th2-skewed, depending on a patient's clinical condition. Colombian ocular toxoplasmosis patients' immune response was Th2-skewed, regardless of the nature of antigen stimulus.

Human T-cell activation with Toxoplasma gondii antigens loaded in maltodextrin nanoparticles.

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

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.

CD8+ T-cell Exhaustion Phenotype in Human Asymptomatic and Ocular Toxoplasmosis.

This work analyzed exhaustion markers in CD8+ T-cell subpopulations in 21 samples of peripheral blood mononuclear cells (PBMCs) from individuals with ocular toxoplasmosis (n = 9), chronic asymptomatic toxoplasmosis (n = 7), and non-infected people (n = 5) by using RT-qPCR and flow cytometry techniques. The study found that gene expression of PD-1 and CD244, but not LAG-3, was higher in individuals with ocular toxoplasmosis versus individuals with asymptomatic infection or uninfected. Expression of PD1 in CD8+ central memory (CM) cells was higher in nine individuals with toxoplasmosis versus five uninfected individuals (p = .003). After ex vivo stimulation, an inverse correlation was found between the exhaustion markers and quantitative clinical characteristics (lesion size, recurrence index, and number of lesions). A total exhaustion phenotype was found in 55.5% (5/9) of individuals with ocular toxoplasmosis. Our results suggest that the CD8+ exhaustion phenotype is involved in the pathogenesis of ocular toxoplasmosis.

Frequency of natural antibodies and concordance analysis for anti-TOXOPLASMA IgM tests in Colombian sera of pregnant women.

Interpretation of IgM anti-Toxoplasma can be problematic given the phenomena of "natural" IgM. We analyzed 1,503 sera obtained during prenatal care program, and we established natural and false-positive results by doing follow-up. In 101 samples the concordance between enzyme linked immunosorbent assay (ELISA) and two semi-automatized systems: electro-chemiluminescence immunoassay (ELECSYS) and vitek immunodiagnostic assay system (VIDAS) was calculated. In 1,503 serum, 71 (4,7%) had ELISA IgG negative and ELISA IgM positive results and in 44 of these had a second sample 4 weeks later. In second samples, 27 (61,3 %) were IgM and IgG negative (false positive result in the first sample) and 13 (29,5%) were ELISA IgM positive and IgG negative (natural IgM). ELISA assay had a poor concordance with enzyme-linked fluorescent immunoassay as well as ELECSYS tests, contrarily, enzyme-linked fluorescent immunoassay and ELECSYS had optimal concordance, with 100 of 101 sera obtaining the same result by both tests. We recommended to use automatized assays to measure IgM anti-Toxoplasma.

Detection of Giardia duodenalis and Toxoplasma gondii in soil and water samples in the Quindío River basin, Colombia.

Two zoonotic protozoan pathogens, Giardia duodenalis and Toxoplasma gondii, are important causes of waterborne infections in the Quindío region in Colombia. No previous data exist on how contamination occurs at the source for drinking water consumed by the human population in this region. Our aim was to describe the frequency of G. duodenalis and T. gondii DNA in 11 sampling points during a five-month period in water and adjacent soil at the Quindío River basin (Andean region in the central western part of Colombia). The study employed nested PCR for T. gondii, using the B1 gene as the amplification target, and single-round PCR for G. duodenalis assemblage A and assemblage B, amplifying the gdh gene, followed by DNA sequencing. In 50 soil samples, 28% (14/50) were positive for T. gondii. For G. duodenalis, distribution was in equal parts for assemblage A (8%; 4/50) and assemblage B (8%, 4/50). Genotyping of T. gondii sequences showed two soil samples with type I strain, another two samples of soil with type III strain, but most samples were of unidentified strains. In water samples, T. gondii was detected in 9.1% (5/55), G. duodenalis assemblage A in 34.5% (19/55), and G. duodenalis assemblage B in 12.7% (7/55). T. gondii DNA positivity was associated with lower soil temperature (p = 0.0239). Presence of G. duodenalis and T. gondii was evidenced in soil and water samples in the Quindío River basin, indicating soil as the potential source of contamination for the river that it is destined for human consumption. Monitoring these protozoa in drinking water is necessary to prevent public health risks in human populations.

Evaluation of the impact of the first evidence-based guidelines for congenital toxoplasmosis in Armenia (Quindío) Colombia: An observational retrospective analysis.

Background: Colombia implemented the world's first evidence-based guidelines for congenital toxoplasmosis in 2013, no evaluation of its impact has been reported. Methods: We reviewed the clinical charts of cases referred to the specialized consultation of the health care centre at Universidad del Quindío during an 18-year period (2001-2019), where the diagnosis criteria and the correlation between prenatal treatment and symptoms at birth were analysed. Additionally, we described the diagnosis criteria and treatment for mothers during pregnancy at a primary prenatal care centre in the city of Armenia during 2018. Institutional consent was obtained to review clinical charts. Findings: At the referral centre, we found that before the implementation, 27.3% did not have prenatal diagnosis but after implementing the clinical practice guidelines, all mothers were diagnosed during pregnancy. In addition, we observed that prenatal treatment was associated with fewer symptoms and this improved significantly over time after implementing the guidelines. At the primary health care centre in 2018, we found that all mothers were diagnosed and treated, as recommended by the national guideline. Interpretation: The national guideline has had a positive impact by improving early diagnosis and treatment of prenatal toxoplasmosis and reducing severe forms, as observed at the referral centre. Funding: Colombian Ministry of Science.

Blastocystis Subtypes and Culture Characteristics of Isolates from Human Stools Related with the Presence of Gastrointestinal Symptoms: A Case-Control Study.

PURPOSE: To stablish if Blastocystis subtypes influences gastrointestinal symptoms. METHODS: Case-control study. We obtained sequencing for Blastocysts subtyping from 13 patients with gastrointestinal symptoms (diarrhea or abdominal pain) and 12 from individuals without symptoms. RESULTS: 12 sequences were from Subtype 2 and one from Subtype 3 in symptomatic individuals and nine samples were from Subtype 1, one from Subtype 2, and two from Subtype 3 in asymptomatic individuals. The prevalence of subtype 2 in symptomatic individuals was vastly different compared to the frequency in asymptomatic individuals (84.6% vs. 16.6%; OR 27.5 95% CI 3.2-233; Fisher exact test p = 0.0010201335). After in vitro culture, 22 isolates were obtained. Significant differences were observed for the 12 isolates from Subtype 2 that get a smaller number of total cells with dominant growth of vacuolar forms, compared with Subtypes 1 and 3, after eight days of culture. CONCLUSION: Our results suggest that gastrointestinal symptoms in Colombian individuals with Blastocystis infection depend on the infecting subtype with peculiar phenotypic characteristics in in vitro culture.

In vitro evaluation of new 4-thiazolidinones on invasion and growth of Toxoplasma gondii.

Treatments for toxoplasmosis such as pyrimethamine have shown numerous side effects. It has been reported that the likelihood of relapse associated with pyrimethamine-based therapy in patients with HIV and toxoplasmic encephalitis (TE) can have significant implications, even for patients who often develop new lesions in areas of the brain previously free of infection. This led us to research for new agents against Toxoplasma gondii. Recent findings have shown the potent biological activity of 4-thiazolidinones. We proposed to design and synthesize a new series of 2-hydrazono-4-thiazolidinones derivatives to evaluate the in vitro growth inhibition effect on T. gondii. The growth rates of T. gondii tachyzoites in Human Foreskin Fibroblast (HFF) cell culture were identified by two in vitro methodologies. The first one was by fluorescence in which green fluorescent RH parasites and cherry-red fluorescent ME49 parasites were used. The second one was a colorimetric methodology using ß-Gal parasites of the RH strain constitutively expressing the enzyme beta-galactosidase. The 4-thiazolidinone derivatives 1B, 2B and 3B showed growth inhibition at the same level of Pyrimethamine. These compounds showed IC50 values of 1B (0.468-0.952 µM), 2B (0.204-0.349 µM) and 3B (0.661-1.015 µM) against T. gondii. As a measure of cytotoxicity the compounds showed a TD50 values of: 1B (60 µM), 2B (206 µM) and 3B (125 µM). The in vitro assays and molecular modeling results suggest that these compounds could act as possible inhibitors of the Calcium-Dependent Protein Kinase 1 of T. gondii. Further, our results support the fact that of combining appropriate detection technologies, combinatorial chemistry and computational biology is a good strategy for efficient drug discovery. These compounds merit in vivo analysis for anti-parasitic drug detection.

High frequency of ocular toxoplasmosis in Quindío, Colombia and risk factors related to the infection.

OBJECTIVES: To determine the frequency of retinochoroidal lesions by ocular toxoplasmosis and their relationships with risk factors, in residents of two districts with high exposure to Toxoplasma, in Armenia-Quindío, Colombia. METHODS: Cross-sectional analyses of fundoscopy screening, serological tests, and questionnaires were performed to determine risk factors associated with ocular toxoplasmosis retinochoroidal lesions. Differences in proportions were analyzed using the chi-squared test. RESULTS: Of 161 individuals examined, 17 (10.5%) exhibited retinochoroidal scars suggestive of old inactive Toxoplasma gondii infection. All 17 individuals were seropositive for T. gondii antibodies. Consumption of bottled water was protective against T. gondii infection among individuals in this study. There were no specific epidemiological risk factors associated with ocular toxoplasmosis retinochoroidal lesions. CONCLUSION: Ocular toxoplasmosis is an important cause of visual impairment in Armenia-Quindío, Colombia. The consumption of boiled or bottled water is a major preventive public health measure to reduce infection by T. gondii and the subsequent onset of OT.

First Detection of Toxoplasma gondii DNA in a Wild Bat from Colombia.

INTRODUCTION: Toxoplasma gondii infections have been reported for many warm-blooded animals around the world including chiropterans. However, in Colombia, the country that holds the highest taxonomic richness of this order of mammals in the Neotropics, up to date there are no reports of T. gondii in bats (Carollia brevicauda). PURPOSE: The objective of the present study was to detect T. gondii DNA from internal bat organs from Quindío, Colombia. RESULTS: We report the first detection of T. gondii DNA from internal bat organs in the department of Quindio, Central Andes of Colombia. Out of three silky short tail bat (Carollia brevicauda) specimens collected at the natural reserve "La Montaña del Ocaso", organs were recovered (lungs, liver, heart, kidneys, small and large intestine) and tested for T. gondii through PCR for B1 sequence, with 1/3 (33.3%) positive result for the presence of T. gondii DNA in bat kidney tissues. CONCLUSION: Taking into consideration the high diversity of bat species in Colombia, and the complexity of the ecological and functional relationships that these organisms establish in the ecosystems they inhabit, we discuss on the urgent need for more detailed research and surveys for Toxoplansma in bats and other mammalian wild species.