Fake news and covid-19: a concern due to the low vaccine coverage in Brazil / Fake News e covid-19: uma preocupação devido à baixa cobertura de vacinação no Brasil

Abstract This study comprised the application of a survey in São Paulo, Brazil, in 2 different periods of 2020: the beginning of the covid-19 pandemic and the disease's first peak (from March to April, 100 interviews) to the time of stability in case fatality rates (from May to July, 100 interviews); the questionnaire included was composed of 14 multiple-choice questions to evaluate the importance of mass communication channels, including social media, and the level of importance attributed to preventive measures at the beginning of the pandemic. The changes in people's behavior, even in a group with more schooling, which initially considered preventive measures to be very important (91%) but, in the second survey, was reduced to 82%. The reinforcement of preventive measures to reduce cases and deaths by covid-19 in Brazil is urgent, allied to recommendations with clear information on the importance of vaccination to avoid low rates as the current situation of vaccine coverage for preventable diseases.

Resumo Foi conduzida uma pesquisa em São Paulo, no Brasil, em 2 períodos distintos de 2020: sendo o primeiro no início da pandemia do covid-19 com um elevado pico de incidência da doença (de março a abril, foram realizadas 100 entrevistas) até o momento de estabilidade nas taxas de letalidade (de maio a julho, foram realizadas outras 100 entrevistas), composto por 14 questões de múltipla escolha para avaliar a importância dos canais de comunicação em massa (incluindo as redes sociais) e o nível de importância atribuído às medidas preventivas no início da pandemia. As mudanças no comportamento das pessoas, mesmo dentro de um grupo de nível educacional alto, que inicialmente considerava as medidas preventivas muito importantes (91%), apresenta considerável queda na segunda pesquisa realizada (redução para 82%). Há a necessidade urgente de reforço de medidas preventivas para redução de casos e óbitos por covid-19 no Brasil, aliadas a recomendações com informações claras como a importância da vacinação para evitar baixas taxas de cobertura vacinal que se apresentam em outras doenças preveníveis por vacinas.

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation.

BACKGROUND: Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. METHODS: Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. RESULTS: Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. CONCLUSION: Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.

Toxoplasma gondii isolated from a Brazilian patient with rare pulmonary toxoplasmosis has a novel genotype and is closely related to Amazonian isolates.

Pulmonary toxoplasmosis is rare in immunocompetent patients. Herein, a Toxoplasma gondii strain isolated in Brazil from an immunocompetent patient who had severe pulmonary involvement was biologically and molecularly characterized for the first time. The TgHumIMTBr1 isolate was bioassayed in mice showing a virulent phenotype. Restriction fragment length polymorphism (RFLP) genotyping using 11 markers [SAG1, SAG2 (5´3´SAG2 and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3] revealed a new non-archetypal genotype assigned as #312. Genotyping using ROP18/ROP5 markers exhibited the virulent combination of alleles 4 and 1. Microsatellite analysis using 15 markers (TUB2, W35, TgM-A, B18, B17, M33, IV.1, X1.1, N60, N82, AA, N61, N83, M48 and M102) revealed an atypical genotype with three unique alleles and a rare combination of alleles 246 (W35) and 203 (TgM-A) that is typical of the Amazon region. Non-archetypal genotypes with unique alleles may function in the occurrence of severe toxoplasmosis in immunocompetent patients in Brazil. Attempts to isolate or molecularly detect T. gondii for further genotyping studies would contribute to the understanding of causes related to the severity of toxoplasmosis in immunocompetent patients.

Natural versus Recombinant Viral Antigens in SARS-CoV-2 Serology: Challenges in Optimizing Laboratory Diagnosis of COVID-19.

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

Radiation effects on Toxoplasma antigens: different immune responses of irradiated intact tachyzoites or soluble antigens in experimental mice models.

Purpose: Purpose: Protein irradiation causes aggregation, chain breakage, and oxidation, enhancing its uptake by antigen-presenting cells. To evaluate if irradiated proteins participate on the protection, we studied the immune response induced in mice immunized with irradiated soluble extracts of T. gondii tachyzoites (STag) or irradiated intact T. gondii RH tachyzoites (RH0.25 kGy).Material and Methods: Soluble extracts of Toxoplasma gondii tachyzoites (STag) were irradiated at different dose by Cobalt-60 source. By polyacrylamide gel electrophoresis (SDS-Page) we evaluated the effects on primary structures of protein STags induced by irradiation. By Enzyme-linked Immunosorbent Assay (ELISA) we evaluated the difference between humoral immune response induced by irradiated STag or RH tachyzoites in immunized mice from the detection of specific immunoglobulin G (IgG) antibodies in the serum of immunized mice. From challenge with viable RH strain of T. gondii we evaluated the protection induced in the immunized animals. By cytometry we performed the phenotyping of T and B lymphocytes in the peripheral blood of the immunized animals.Results: Irradiation dose of 1.5 kGy induced minimal changes in most proteins, without affecting their antigenicity or immunogenicity. Immunization showed saturation at the dose of 10 µg/mice, with worst response at higher doses. STag irradiated at 1.5 kGy (STag1.5 kGy) induced higher survival and protection similar to T. gondii RH strain irradiated at 0.25 kGy (RH0.25 kGy), with higher serum levels of high affinity IgG compared to STag native. Blood immune memory cells of mice immunized with STag1.5 kGy had higher proportions of CD19+ (cluster of differentiation 19) and CD4+ (cluster of differentiation 14) cells, whereas mice RH0.25 kGy had high proportion of memory CD8+ (cluster of differentiation 8) cells.Conclusions: Our data suggest that major histocompatibility complex type I (MHCI) pathway, appears seem to be used by RH0.25 kGy to generate cytotoxic cells while STag1.5 kGy uses a major histocompatibility complex type II (MHCII) pathway for B-cell memory, but both induce sufficient immune response for protection in mice without any adjuvant. Irradiation of soluble protein extracts enhances their immune response, allowing similar protection against T. gondii in mice as compared to irradiated intact parasites.

Sertraline Delivered in Phosphatidylserine Liposomes Is Effective in an Experimental Model of Visceral Leishmaniasis.

Liposomes containing phosphatidylserine (PS) has been used for the delivery of drugs into the intramacrophage milieu. Leishmania (L.) infantum parasites live inside macrophages and cause a fatal and neglected viscerotropic disease, with a toxic treatment. Sertraline was studied as a free formulation (SERT) and also entrapped into phosphatidylserine liposomes (LP-SERT) against intracellular amastigotes and in a murine model of visceral leishmaniasis. LP-SERT showed a potent activity against intracellular amastigotes with an EC50 value of 2.5 µM. The in vivo efficacy of SERT demonstrated a therapeutic failure. However, when entrapped into negatively charged liposomes (-58 mV) of 125 nm, it significantly reduced the parasite burden in the mice liver by 89% at 1 mg/kg, reducing the serum levels of the cytokine IL-6 and upregulating the levels of the chemokine MCP-1. Histopathological studies demonstrated the presence of an inflammatory infiltrate with the development of granulomas in the liver, suggesting the resolution of the infection in the treated group. Delivery studies showed fluorescent-labeled LP-SERT in the liver and spleen of mice even after 48 h of administration. This study demonstrates the efficacy of PS liposomes containing sertraline in experimental VL. Considering the urgent need for VL treatments, the repurposing approach of SERT could be a promising alternative.

Gamma irradiation of Toxoplasma gondii protein extract improve immune response and protection in mice models.

Gamma radiation induces protein changes that enhance immunogenicity for venoms, used in antivenin production. Coccidian parasites exposed to gamma radiation elicit immune response with protection in mice and man, but without studies on the effect of gamma radiation in soluble acellular extracts or isolated proteins. Toxoplasmosis is a highly prevalent coccidian disease with only one vaccine for veterinary use but with remaining tissue cysts. Total parasite extracts or recombinant proteins used as immunogen induce usually low protection. Here, we study gamma radiation effect on T. gondii extracts proteins (STAG) and its induced immunity in experimental mice models. By SDS-PAGE, protein degradation is seen at high radiation doses, but at ideal dose (1500 Gy), there are preservation of the antigenicity and immunogenicity, detected by specific antibody recognition or production after mice immunization. Immunization with STAG irradiated at 1500 Gy induced significant protection in mice immunized and challenged with distinct T. gondii strains. In their blood, higher levels of specific CD19+, CD3+CD4+ and CD3+CD8+ activated cells were found when compared to mice immunized with STAG. Irradiated T. gondii tachyzoites extracts induce immune response and protection in mice in addition, could be a feasible alternative for Toxoplasma vaccine.

Nanoliposomal Buparvaquone Immunomodulates Leishmania infantum-Infected Macrophages and Is Highly Effective in a Murine Model.

Visceral leishmaniasis is a fatal parasitic neglected disease affecting 1.5 million people worldwide. Based on a drug repositioning approach, the aim of this work was to investigate the in vitro immunomodulatory potential of buparvaquone (BPQ) and to establish a safe regimen to evaluate the in vivo efficacy of BPQ entrapped by negatively charged nanoliposomes (BPQ-LP) in Leishmania infantum-infected hamsters. Small-angle X-ray scattering, dynamic light scattering, and the ζ-potential were applied in order to study the influence of BPQ on the liposome structure. Our data revealed that BPQ was located in the polar-apolar interface, snorkeling the polar region, and protected against aggregation inside the lipophilic region. The presence of BPQ also decreased the Z-average hydrodynamic diameter and increased the surface charge. Compared to intravenous and intramuscular administration, a subcutaneous route was a more effective route for BPQ-LP; at 0.4 mg/kg, BPQ-LP reduced infection in the spleen and liver by 98 and 96%, respectively. Treatment for 5 days resulted in limited efficacy, but 10 days of treatment resulted in an efficacy similar to that of a 15-day regimen. The nanoliposomal drug was highly effective, with a mean 50% effective dose of 0.25 mg/kg, reducing the parasite load in bone marrow by 80%, as detected using quantitative PCR analysis. In addition, flow cytometry studies showed that BPQ upregulated cytokines as tumor necrosis factor, monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages, eliminating the parasites via a nitric oxide-independent mechanism. This new formulation proved to be a safe and effective treatment for murine leishmaniasis that could be a useful candidate against visceral leishmaniasis.

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum.

Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC50 values of 42.71µg/mL against promastigotes and 6.78µg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX(®) Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii.

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.

Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats.

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5 x 10(2) mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.

Toxoplasma gondii pneumonia in immunocompetent subjects: case report and review.

Pulmonary toxoplasmosis is rare in immunocompetent subjects. Here, we describe a 41-year-old previously healthy male patient who presented to the emergency department of a hospital with a life-threatening case of pneumonia due to Toxoplasma gondii infection, which responded to specific therapy. Clinical and image-based findings overlap with those for atypical pneumonias, and toxoplasmosis should be considered in the differential diagnosis--especially if immunoglobulin M-specific antibodies are detected.

200 Gy sterilised Toxoplasma gondii tachyzoites maintain metabolic functions and mammalian cell invasion, eliciting cellular immunity and cytokine response similar to natural infection in mice.

200Gy gamma-irradiated Toxoplasma gondii RH tachyzoites failed to reproduce in vitro and in vivo. In short-term cultures, these parasites maintained a respiratory response, the ability to invade cells and preserved protein and nucleic acid synthesis. ELISA and Western blotting techniques demonstrated the similarity in humoral response between mice infected with gamma-irradiated tachyzoites and animals infected with naive parasites and treated with sulfadoxine, higher than mice immunised with formaldehyde-killed tachyzoites. Splenocyte stimulation by T. gondii antigen produced lymphoproliferative response and cytokine profile (IL-10, IL-12, IFN-gamma and TNF-alpha) similar to those produced by chronic natural infection. Mice immunised with irradiated tachyzoites had extended survival times after subsequent tachyzoite challenge, and displayed minimal cerebral pathology after cyst challenge. Irradiated tachyzoites lose their reproductive ability whilst maintaining metabolic function and may provide a novel tool for the study of toxoplasmosis and vaccine development.