Extracellular Vesicles from Leishmania (Leishmania) infantum Contribute in Stimulating Immune Response and Immunosuppression in Hosts with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a chronic systemic disease. In Brazil this infection is caused by Leishmania (Leishmania) infantum. Extracellular vesicles (EVs) released by Leishmania species have different functions like the modulation of host immune systems and inflammatory responses, among others. This study evaluated the participation of EVs from L. (L.) infantum (Leish-EVs) in recognition of the humoral and cellular immune response of hosts with VL. Promastigotes were cultivated in 199 medium and, in the log phase of growth, they were centrifuged, washed, resus-pended in RPMI medium, and incubated for 2 to 24 h, at 25 °C or 37 °C to release Leish-EVs. This dynamic was evaluated using transmission (TEM) and scanning (SEM) electron microscopies, as well as nanoparticle tracking analysis (NTA). The results suggested that parasite penetration in mammal macrophages requires more Leish-EVs than those living in insect vectors, since promastigotes incubated at 37 °C released more Leish-EVs than those incubated at 25 °C. Infected THP-1 cells produced high EV concentration (THP-1 cells-EVs) when compared with those from the control group. The same results were obtained when THP-1 cells were treated with Leish-EVs or a crude Leishmania antigen. These data indicated that host-EV concentrations could be used to distinguish infected from uninfected hosts. THP-1 cells treated with Leish-EVs expressed more IL-12 than control THP-1 cells, but were unable to express IFN-γ. These same cells highly expressed IL-10, which inhibited TNF-α and IL-6. Equally, THP-1 cells treated with Leish-EVs up-expressed miR-21-5p and miR-146a-5p. In conclusion, THP-1 cells treated with Leish-EVs highly expressed miR-21-5p and miR-146a-5p and caused the dysregulation of IL-10. Indirectly, these results suggest that high expression of these miRNAs species is caused by Leish-EVs. Consequently, this molecular via can contribute to immunosuppression causing enhanced immunopathology in infected hosts.

Standardization of Semi-Quantitative Dot Blotting Assay-Application in the Diagnosis, Follow-Up, and Relapse of Paracoccidioidomycosis.

INTRODUCTION: This study standardized a semi-quantitative dot blotting assay (DB) and a quantitative real-time polymerase chain reaction (qPCR) to detect specific antibodies for Paracoccidioides brasiliensis and its DNA in PCM patients. METHODOLOGY: We evaluated 42 confirmed PCM patients upon admission using a serological double agar gel immunodiffusion test (DID), DB, and molecular tests (qPCR in total blood). The control groups included 42 healthy individuals and 37 patients with other infectious diseases. The serological progress during treatment was evaluated in eight patients, and there was a relapse diagnosis in ten patients using the Pb B.339 strain antigen. The cut-off points for the serological tests were determined by a receiver operator characteristic curve. RESULTS: The DB and DID tests showed similar accuracy, but the DB identified lower antibody concentrations. Cross-reactions were absent in the DB assay. In the relapse diagnoses, DB exhibited much higher sensitivity (90%) than DID (30%). CONCLUSIONS: A DB assay is easier and faster than a DID test to be performed; DB and DID tests show the same accuracy, while blood qPCR is not recommended in the diagnosis at the time of admission; cross-reactions were not observed with other systemic diseases; DB and DID tests are useful for treatment monitoring PCM patients; and a DB assay is the choice for diagnosing relapse. These findings support the introduction of semi-quantitative DB assays in clinical laboratories.

Exploring Peripheral Blood-Derived Extracellular Vesicles as Biomarkers: Implications for Chronic Chagas Disease with Viral Infection or Transplantation.

Extracellular vesicles (EVs) are lipid bilayer envelopes that encapsulate cell-specific cargo, rendering them promising biomarkers for diverse diseases. Chagas disease, caused by the parasite Trypanosoma cruzi, poses a significant global health burden, transcending its initial epicenter in Latin America to affect individuals in Europe, Asia, and North America. In this study, we aimed to characterize circulating EVs derived from patients with chronic Chagas disease (CCD) experiencing a reactivation of acute symptoms. Blood samples collected in EDTA were processed to isolate plasma and subsequently subjected to ultracentrifugation for particle isolation and purification. The EVs were characterized using a nanoparticle tracking analysis and enzyme-linked immunosorbent assay (ELISA). Our findings revealed distinctive differences in the size, concentration, and composition of EVs between immunosuppressed patients and those with CCD. Importantly, these EVs play a critical role in the pathophysiology of Chagas disease and demonstrate significant potential as biomarkers in the chronic phase of the disease. Overall, our findings support the potential utility of the CL-ELISA assay as a specific sensitive tool for detecting circulating EVs in chronic Chagasic patients, particularly those with recurrent infection following an immunosuppressive treatment or with concurrent HIV and Chagas disease. Further investigations are warranted to identify and validate the specific antigens or biomarkers responsible for the observed reactivity in these patient groups, which may have implications for diagnosis, the monitoring of treatment, and prognosis.

Identification and validation of reference genes of circulating microRNAs for use as control in gestational toxoplasmosis.

Toxoplasmosis causes serious harm to the fetus, as tachyzoite dissemination, during pregnancy in women developing the primo-infection. The microRNAs (miRNAs) are small non-coding RNAs, which have regulatory roles in cells by silencing messenger RNA. Circulating miRNA are promising biomarkers for diagnosis and prognosis of numerous diseases. The miRNAs levels are estimated by quantitative real-time PCR (qPCR), however, the relative quantification of each miRNA expression requires proper normalization methods using endogenous miRNAs as control. This study analyzed the expression of three endogenous miRNAs (miR-484, miR -423-3p and miR-26b-5p) for use as normalizers in future studies of target miRNAs for gestational toxoplasmosis (GT). A total of 32 plasma samples were used in all assays divided in 21 from women with GT and 11 from healthy women. The stability of each endogenous miRNA was evaluated by the algorithm methods RefFinder that included GeNorm, Normfinder, BestKeeper and comparative delta-CT programs. The miR-484 was the most stably gene, and equivalently expressed in GT and NC groups. These results contribute to future studies of target miRNAs in clinical samples of women with gestational toxoplasmosis.

miRNA 511_5p is a potential biomarker for ocular toxoplasmosis.

BACKGROUND: Ocular toxoplasmosis (OT) is a frequent clinical manifestation due to infection by Toxoplasma gondii. It is characterized by an inflammatory process involving macrophages activated by pro-inflammatory cytokines. The expression of microRNAs takes place during the inflammatory process and, among them, miRNA 511 regulates the activation of macrophages. This study evaluated the expression of miRNA 511_5p in patients with OT and healthy controls. METHODS: A total of 361 patients from the Hospital de Base of Fundação Faculdade de Medicina de São José do Rio Preto were enrolled and divided into four groups: G1-patients with active ocular lesions and reagent serology for T. gondii; G2-patients with scars and reagent serology for T. gondii; G3-patients without ocular lesions or scars and reagent serology for T. gondii; G4-patients without ocular lesions or scars and non-reagent serology for T. gondii. All patients underwent clinical and laboratory evaluation to confirm the diagnosis of OT. Serology tests, RNA extraction and cDNA synthesis were performed. RESULTS: The miRNA 511_5p levels were compared among the groups. The G1 group showed a high blood plasma concentration of miRNA 511_5p (mean 22.34) compared with the G2 (4.65), G3 (8.91) and G4 (3.52) groups (p<0.0001). CONCLUSION: These data suggest that miRNA 511_5p has significant potential as a biomarker for OT.

Dogs with canine visceral leishmaniasis have a boost of extracellular vesicles and miR-21-5p up-expression.

This retrospective cohort study analysed extracellular vesicles (EVs) and microRNAs (miRNAs) excreted in canine sera from dogs with canine visceral leishmaniasis (CanVL). A total of 56 canine sera were divided into Group I (28, from healthy dogs) and Group II (28, from the same dogs, but already with CanVL). CanVL was determined by clinical and laboratory diagnoses. Canine sera were ultra-centrifuged to recover EVs (Can-EVs). Analyses by transmission electron microscopy, nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-poli-acrylammide gel eletroforesis (SDS-PAGE) and, Immunoblot confirmed the presence of (i) microvesicles/exosomes and (ii) the tetraspanins CD63 and CD9. EVs secreted by Leishmania (Leishmania) infantum-EVs were reactive against sera from dogs with CanVL (performed by ELISA and Immunoblot). NTA analyses exhibited that concentrations of Can-EVs from dogs with CanVL (7.78 × 1010 Can-EVs/mL) were higher (p < .0001) than the non-infected dogs (mean: 1.47 × 1010 Can-EVs/mL). These results suggested that concentrations of Can-EVs were able to distinguish dogs with CanVL from healthy dogs. The relative expressions of 11 miRNAs species (miR-21-5p, miR-146a-5p, miR-125b-5p, miR-144-3p, miR-194-5p, miR-346, miR-29c-3p, miR-155-5p, miR-24-3p, miR-181a-5p, and miR-9-5p) were estimated in purified miRNAs of 30 canine sera. Dogs with CanVL up-expressed miR-21-5p and miR-146a-5p when compared with healthy dogs. The other miRNA species were poorly or not expressed in canine sera. In conclusion, this study suggests that CanVL induces changes in size and concentration of Can-EVs, as well as, the up-expression of miR-21-5p and miR-146a-5p in infected dogs.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil.

BACKGROUND: Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES: This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS: T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS: It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS: The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Immunoanalysis of different antigenic preparations of Angiostrongylus cantonensis for neuroangiostrongyliasis diagnosis improvement.

BACKGROUND: Angiostrongylus cantonensis is the etiological agent of neuroangiostrongyliasis in humans, which is developed in gastropods and vertebrate species, mainly rodents. Human transmission occurs through consumption of molluscs and paratenic hosts infected with L3, and the migration of larvae to the central nervous system causes eosinophilic meningitis. Laboratory diagnosis is based on molecular and immunological tests, using young or adult females as a source of antigens. However, these tests give positive results only after several weeks of symptoms onset and also cross-reactions with others parasite infections may occur. OBJECTIVES: The purpose of this work was to study different antigenic preparations of distinct evolutionary phases of A. cantonensis, in order to improve serological techniques for disease immunodiagnosis. METHODS: For this purpose, antigenic fractions of different evolutionary forms were evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) and Western blot using serum samples. FINDINGS: All analysed fractions showed reactivity with serum samples from patients with neuroangiostrongyliasis, especially female membrane alkaline (FAM) and female soluble alkaline (FAS) fractions together with female soluble saline (FSS), improving the technique specificity. MAIN CONCLUSIONS: The results point to the possibility of use of raw female antigens in association with alkaline membrane antigens extracted from adult worms to aid in diagnosis and helps initiate neuroangiostrongyliasis surveillance and control actions.

Toxoplasmosis in Human and Animals Around the World. Diagnosis and Perspectives in the One Health Approach.

Toxoplasmosis is a unique health disease that significantly affects the health of humans, domestic animals, wildlife and is present in ecosystems, including water, soil and food. Toxoplasma gondii is one of the best-adapted parasites in the word. This parasite is able to persist for long periods in its hosts, in different geographic regions of the word. This review summarizes the current literature of these themes, focusing on: (1) toxoplasmosis, a zoonotic infection; (2) One health approach and toxoplasmosis; (3) human toxoplasmosis; (4) animal toxoplasmosis; (5) toxoplasmosis diagnosis, as immunological, parasitological and molecular diagnosis; (6) T. gondii outbreaks caused by infected meat, milk and dairy products, as well as, vegetables and water consume; (7) studies in experimental models; (8) genetic characterization of T. gondii strains; (9) extracellular vesicles and miRNA; and (10) future perspectives on T. gondii and toxoplasmosis. The vast prevalence of toxoplasmosis in both humans and animals and the dispersion and resistence of T. gondii parasites in environment highlight the importance of the one health approach in diagnostic and control of the disease. Here the different aspects of the one health approach are presented and discussed.

Performance of a Real Time PCR for Pneumocystis jirovecii Identification in Induced Sputum of AIDS Patients: Differentiation between Pneumonia and Colonization.

Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and a diagnostic challenge. Conventional methods have low accuracy, hardly discriminating colonization from infection, while some new high-cost or broncho-alveolar lavage-based methods have limited usefulness in developing countries. Quantitative PCR (qPCR) tests may overcome these limitations due to their high accuracy, possibility of automation, and decreasing cost. We evaluated an in-house qPCR targeting the fungus mtSSU gene using induced sputum. Sensitivity of the assay (ten target gene copies/assay) was determined using recombinant plasmids. We prospectively studied 86 AIDS patients with subacute respiratory symptoms in whom PcP was suspected. qPCR results were determined as quantification cycles (Cq) and compared with a qualitative PCR performed in the same IS, serum 1,3-ß-D-Glucan assay, and a clinical/laboratory/radiology index for PcP. The qPCR clustered the patients in three groups: 32 with Cq ≤ 31 (qPCR+), 45 with Cq ≥ 33 (qPCR-), and nine with Cq between 31-33 (intermediary), which, combined with the other three analyses, enabled us to classify the groups as having PcP, not P. jirovecii-infected, and P. jirovecii-colonized, respectively. This molecular assay may contribute to improve PcP management, avoiding unnecessary treatments, and our knowledge of the natural history of this infection.

TNFα rs1799964 TT genotype may be a susceptibility factor for vertical transmission of Toxoplasma gondii and clinical signs in newborns from pregnant women with acute toxoplasmosis.

BACKGROUND: One of the main impacts of Toxoplasma gondii infection occurs during pregnancy and is related to the vertical transmission of the parasite (congenital toxoplasmosis), which can cause severe clinical outcomes and fetal death. During acute infection, in order to control the rapid replication of tachyzoites, different host immune response genes are activated, and these include cytokine-encoding genes. Considering that polymorphisms in cytokine genes may increase susceptibility to vertical transmission of T. gondii by determining the immune status of the pregnant woman, this study evaluated the influence of polymorphisms of tumor necrosis factor alpha (TNFα) rs1799964 (- 1031) and interleukin 1 beta (IL1ß) rs16944 (- 511) genes on gestational toxoplasmosis and on the vertical transmission of the parasite and verified the allele and genotype frequency of these polymorphisms in pregnant patients whose respective newborn did or did not present clinical abnormalities suggestive of congenital toxoplasmosis. METHODS AND RESULTS: A total of 204 pregnant patients with (n = 114) or without (n = 90) infection by T. gondii were enrolled. No associations were found involving the polymorphisms rs1799964 (- 1031) of the TNFα gene and rs16944 (- 511) of the IL1ß gene with the increased chance of T. gondii infection during pregnancy. However, it was observed that the maternal TT genotype referring to the polymorphism of the TNFα gene seems to influence the vertical transmission of the parasite (P = 0.01; χ2 = 6.05) and the presence of clinical manifestation in newborns from pregnancies with acute toxoplasmosis (P = 0.007; χ2 = 9.68). CONCLUSION: The TNFα rs1799964 TT genotype may act as a susceptibility factor for the vertical transmission of parasite and for the presence of clinical signs in newborns from pregnant women with acute toxoplasmosis.

Ocular Disease in Mice Inoculated with Pork Heart Samples Infected with Toxoplasma gondii.

PURPOSE: We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples. METHODS: Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis. RESULTS: Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed. CONCLUSION: Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.

Immunoanalysis of different antigenic preparations of Angiostrongylus cantonensis for neuroangiostrongyliasis diagnosis improvement

BACKGROUND Angiostrongylus cantonensis is the etiological agent of neuroangiostrongyliasis in humans, which is developed in gastropods and vertebrate species, mainly rodents. Human transmission occurs through consumption of molluscs and paratenic hosts infected with L3, and the migration of larvae to the central nervous system causes eosinophilic meningitis. Laboratory diagnosis is based on molecular and immunological tests, using young or adult females as a source of antigens. However, these tests give positive results only after several weeks of symptoms onset and also cross-reactions with others parasite infections may occur. OBJECTIVES The purpose of this work was to study different antigenic preparations of distinct evolutionary phases of A. cantonensis, in order to improve serological techniques for disease immunodiagnosis. METHODS For this purpose, antigenic fractions of different evolutionary forms were evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) and Western blot using serum samples. FINDINGS All analysed fractions showed reactivity with serum samples from patients with neuroangiostrongyliasis, especially female membrane alkaline (FAM) and female soluble alkaline (FAS) fractions together with female soluble saline (FSS), improving the technique specificity. MAIN CONCLUSIONS The results point to the possibility of use of raw female antigens in association with alkaline membrane antigens extracted from adult worms to aid in diagnosis and helps initiate neuroangiostrongyliasis surveillance and control actions.

Genetic diversity of Trypanosoma cruzi strains isolated from chronic chagasic patients and non-human hosts in the state of São Paulo, Brazil

BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.

Persistent cutaneous canine leishmaniasis caused by Leishmania (Viannia) braziliensis in an area with predominance of Nyssomyia neivai in the state of São Paulo, Brazil.

American cutaneous leishmaniasis (ACL) is a neglected zoonotic disease caused mainly by Leishmania (Viannia) braziliensis, which is endemic throughout Brazil. Canine ACL cases were investigated in a rural area of Monte Mor, São Paulo, where a human ACL case had been confirmed. Dogs were evaluated through clinical and laboratory diagnosis including serology, cytological tissue preparations and PCR on skin lesions, lymph node and bone marrow samples. Entomological investigations on sandflies trapped in the surroundings of the study area were performed for 14 months. Nyssomyia neivai was the predominant phlebotomine species, comprising 94.65% of the captured specimens (832 out of 879). This species was the most abundant in all trapping sites, including human homes and dog shelters. Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri and Expapilata firmatoi were also captured. Two of the three dogs examined were positive for anti-Leishmania IgG in ELISA using the antigen Fucose mannose ligand and skin samples were positive for L. (V.) braziliensis in PCR, but all the samples collected were negative for L. (L.) infantum. One of the dogs had a confirmed persistent infection for more than one year.

Combining urine antigen and blood polymerase chain reaction for the diagnosis of disseminated histoplasmosis in hospitalized patients with advanced HIV disease.

Disseminated histoplasmosis (DH) is endemic in Latin America and the Caribbean where diagnostic tools are restricted. We carried-out a 1-year prospective cohort study at a referral hospital in São Paulo, Brazil. Participants had > or =18 years old, were hospitalized due to any indication and had CD4+ < 200 cells/µl. A urine commercial monoclonal Histoplasma galactomannan enzyme-linked immunosorbent assay (IMMY, Norman, OK, USA) and 'in house' Histoplasma blood nested PCR were performed in all cases. Probable/proven DH cases were defined according to international guidelines. Conventional mycological methods were available in routine conditions to investigate suspected DH cases. Treatment of participants followed the institutional routine. One-hundred six participants were included. Median age (interquartile range [IQR]) was 39.5 years (30.0-47.3) and 80 individuals (75.5%) were males. Median (IQR) CD4 cell count was 26.5 (9.4-89.3) cells/mm3. DH was diagnosed in 8/106 patients (7.5%). Antigen assay and/or PCR were positive in 4.7% (5/106) of patients. The antigen assay and/or PCR identified 37.5% (3/8) of DH cases, which had not been diagnosed with conventional mycological methods, but had clinical manifestations compatible with HD. In conclusion, the use of Histoplasma urine antigen and Histoplasma blood PCR guided by CD4 status contributed to the diagnosis of DH in hospitalized individuals. These assays were complementary to conventional mycologic methods and are urgently needed in our setting. LAY SUMMARY: In this prospective cohort study carried-out in a referral center in São Paulo, Brazil, we found a high frequency of AIDS-related disseminated histoplasmosis (8/106, 7.5%). We used urine antigen test and blood PCR assay to improve the diagnosis of this opportunistic disease.

Characterization of murine extracellular vesicles and Toxoplasma gondii infection.

This study characterized extracellular vesicles (EVs) of sera from mice infected with Toxoplasma gondii or immunized with EVs derived T gondii. EVs were purified of sera from four groups (5 A/Sn mice/group). EV-IM: Mice immunized with T gondii-released EVs; ACT: mice in acute infection; CHR: mice in chronic infection; and NI: normal mice. EVs were purified by ultracentrifugation. Concentration of serum-derived EVs from NI group was smaller than EV-IM, ACT and CHR groups. Most of the EVs from ACT and CHR groups were microvesicles, and they were bigger than the NI group. The same results were shown by Transmission Electron Microscopy. The presence of exosomes was shown in immunoblotting by tetraspanin (CD63 and CD9) evidence. Splenocytes of EV-IM, CHR and NI groups were stimulated with T. gondii derived EVs. EV-IM and CHR groups up-expressed IFN-γ; TNF-α and IL-17, when compared with the NI group. IL-10 was up-expressed only in the EV-IM group. EV-IM, ACT and CHR groups expressed more miR-155-5p, miR-29c-3p and miR-125b-5p than the NI group. Host-T gondii interaction can occur, also, via EVs. miRNAs participate in the modulation of cellular immune response against T gondii. These data give subsidies to propose the differentiation between infect or noninfect hosts by concentration of EVs.

Characterization of extracellular vesicles isolated from types I, II and III strains of Toxoplasma gondii.

This study investigated the participation extracellular vesicles (EVs) in Toxoplasma gondii-host interaction. EVs of three T. gondii strains (RH, ME-49 and VEG) were purified by chromatography and ELISA. Results of "nanoparticle tracking analysis" and scanning electron microscopy showed that RH strain released more EVs than other strains. Images of transmission electron microscopy showed that in beginning of incubation (culture medium), EVs were inside of tachyzoites preparing to be released. After 24 hours, they were largely produced inside tachyzoites and were released through plasma membrane. The parasite burden of mice infected with RH strain plus EVs was increased and with early death of 1-2 days compared of those that received only parasites. EV proteins of ME-49 and VEG strains were poorly reactive to sera of infected patients in imunoblot. However, those from RH strain were reactive against sera of patients with cerebral toxoplasmosis. EVs stimulated murine splenocytes caused similar production of IFN-γ and IL-10 levels. RH strain derived EVs stimulated more TNF-α than those stimulated by other strains. T. gondii and infected hosts can express the same miRNAs (miR-155-5p, miR-125b-5p, miR-423-3p). In conclusion, T. gondii derived EVs promote host-parasite interactions, modulate host immune responses, carry virulent factors and cause an imbalance in cellular immune response.

Ready-to-use qPCR for detection of Cyclospora cayetanensis or Trypanosoma cruzi in food matrices.

Foodborne outbreaks caused by parasites have long been a public health issue. Among the available contamination detection methods, qPCR is one of the most sensitive and specific. However, it can be cumbersome and error-prone, if used by unexperienced users. Moreover, qPCR reagents usually require freezer temperatures for transportation and storage. We present a gelified reaction format that allows the reagents to be stored at 2-8 °C for up to 90 days without losing performance. The gelification process eliminates most operator mistakes during reaction setup, and renders the qPCR plates ready-to-use. The new reaction makeup was evaluated using artificially contaminated samples of distinct food matrices for sensitivity, specificity, repeatability, reproducibility, and stability. Samples consisted of cilantro leaves and raspberry fruits spiked with Cyclospora cayetanensis oocysts, as well as açai pulp and sugarcane juice tainted with Trypanosoma cruzi trypomastigotes. No significant difference between the gelified and the non-gelified qPCR was found. Our results suggest that gelifying the assay may help to achieve more reproducible qPCR data across laboratories, thus supporting surveillance actions. (170 words).

Immunization with extracellular vesicles excreted by Toxoplasma gondii confers protection in murine infection, activating cellular and humoral responses.

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.