Assessing the risks for stillbirth in São Paulo, Brazil: protocol for a multidisciplinary case-control study - FetRisks.

Stillbirth is a fundamental component of childhood mortality, but its causes are still insufficiently understood. This study aims to explore stillbirth risk factors by using a multidisciplinary approach to stimulate public policies and protocols to prevent stillbirth, improve maternal care and support bereaved families. METHODS AND ANALYSIS: In this case-control study with stillbirths and live births in 14 public hospitals in São Paulo, mothers are interviewed at hospitals after delivery, and hospital records and prenatal care registries are reviewed. Maternal and umbilical cord blood samples and placentas are collected to analyse angiogenesis and infection biomarkers, and the placenta's anatomopathological exam. Air pollutant exposure is estimated through the participant's residence and work addresses. Traditional and non-invasive autopsies by image-guided histopathology are conducted in a subset of stillbirths. Subsample mothers of cases are interviewed at home 2 months after delivery on how they were dealing with grief. Information contained in the official prenatal care registries of cases and controls is being compiled. Hospital managers are interviewed about the care offered to stillbirth mothers. Data analysis will identify the main risk factors for stillbirth, investigate their interrelations, and evaluate health services care and support for bereaved families. We hope this project will contribute to the understanding of stillbirth's risk factors and related health services in Brazil, providing new knowledge about this central public health problem, contributing to the improvement of public policies and prenatal and puerperal care, helping to prevent stillbirths and improve the healthcare and support for bereaved families. ETHICS AND DISSEMINATION: This study protocol was approved by the Ethics Committee of the Municipal Health Secretary (process no 16509319.0.3012.5551) and of the Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (process no 16509319.0.0000.0068). Results will be communicated to the study participants, policy-makers and the scientific community.

Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis.

Nonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.

Evaluation of aversive behavior in Rattus norvegicus experimentally infected by two distinct strains of Toxoplasma gondii (ME49 and VEG): study of epigenetic markers.

BACKGROUND: Behavioral changes in Rattus norvegicus infected with two strains of Toxoplasma gondii (ME49 and VEG) were investigated. METHODS: Rats were evaluated for motor activity and aversion or attraction to cat urine 60 days after infection. After euthanasia, arginine-vasopressin gene methylation in the central nervous system was evaluated. RESULTS: A significant difference was observed in the methylation of the arginine-vasopressin promoter gene between rats infected with the ME49 and VEG strains. CONCLUSIONS: Although differences were not observed in many parameters, significant differences were observed in the methylation of the arginine-vasopressin promoter gene in rats infected with the two studied strains.

Isolation and characterization of Toxoplasma gondii isolates from human congenital toxoplasmosis cases reveal a new virulent genotype in São Paulo, Brazil.

Toxoplasma gondii causes severe disease in congenitally infected fetuses. The severity of fetal infection is related to the gestational stage at the time of maternal infection, parasite burden, and genotypic characteristics. South America has a high incidence of congenital toxoplasmosis and has the highest genotypic diversity of the parasite. In Brazil, clinical toxoplasmosis in children is notorious, however there are very limited data regarding the strains recovered from congenital infections. In this study, T. gondii strains from two cases of severe congenital toxoplasmosis from the São Paulo metropolitan area were isolated (TgHumIMTBr2 and TgHumIMTBr3) and biologically and molecularly characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and microsatellite analysis, revealing a new non-archetypal virulent genotype designated as #318. The other isolate, genotype #175, has already been described in domestic and wild animals in Brazil, but is now associated with acute toxoplasmosis in humans. These data reinforce the role of non-archetypal T. gondii genotypes in the severity of human congenital toxoplasmosis, highlighting the importance of studies focused on parasite isolation and genotyping for a better understanding of the virulence of isolates from human toxoplasmosis and contributing to the knowledge of the diversity of T. gondii in Brazil.

High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: Application of high pressure at pH 9.0.

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.

High level SARS-CoV-2 nucleocapsid refolding using mild condition for inclusion bodies solubilization: application of high pressure at pH 9.0

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.

Evaluation of aversive behavior in Rattus norvegicus experimentally infected by two distinct strains of Toxoplasma gondii (ME49 and VEG): study of epigenetic markers

ABSTRACT Background: Behavioral changes in Rattus norvegicus infected with two strains of Toxoplasma gondii (ME49 and VEG) were investigated. Methods: Rats were evaluated for motor activity and aversion or attraction to cat urine 60 days after infection. After euthanasia, arginine-vasopressin gene methylation in the central nervous system was evaluated. Results: A significant difference was observed in the methylation of the arginine-vasopressin promoter gene between rats infected with the ME49 and VEG strains. Conclusions: Although differences were not observed in many parameters, significant differences were observed in the methylation of the arginine-vasopressin promoter gene in rats infected with the two studied strains.

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.

Early high avidity specific IgG production in experimental hamster visceral leishmaniasis.

Visceral leishmaniasis (VL) by Leishmania (Leishmania) infantum is epidemic in Brazil. Hypergammaglobulinemia appears early in patients with VL and is ineffective. Usually, high-affinity IgG B cells are selected during most infections, a critical step for an effective humoral response. The avidity of IgG antibodies in VL is unexplored due to the absence of temporal parameters in most patients, associated to low clinical significance. Experimental infection models overcome this fact, allowing the monitoring of the disease temporal evolution. In this study, the avidity of IgG antibodies was evaluated in experimental models, in infection in hamsters, and in immunization in rabbits. Specific IgG antibodies were detected by ELISA, using chaotropic solution to determine avidity, as reported for viral infections. The levels of IgG antibodies correlated with the progression of experimental infection in hamsters or antigenic stimulation in immunized rabbits. However, IgG avidity was high early in infected animals, even in early periods (> 80%), while in immunized rabbits, they had early antibodies of low avidity with progressive maturation, similar as other infections. These data suggest that the affinity maturation of the avidity of anti-Leishmania IgG antibodies promoted at an early stage, influencing the appropriate interaction between antigens and affecting the disease progression. This fact could be associated to monovalent immune complexes, as reported in human and experimental VL. This scenario may be related to an independent process of immune cell activation by the parasite but absent in antigen preparation used as immunogens.

Measles, rubella, mumps and Toxoplasma gondii antibodies in saliva of vaccinated students of schools and universities in São Paulo City, Brazil

ABSTRACT Introduction: Vaccines are well-established public health interventions with major impacton the prevalence of infectious diseases, but outbreaks are occurring frequently due to pri-mary and secondary failures, despite high coverage. Surveillance of efficacy and duration ofinduced immunity is a difficult task as it requires invasive blood sampling in children andteenagers. Saliva can be an acceptable alternative source of IgG to assess vaccine efficacyand toxoplasmosis incidence. We investigated IgG response for measles, mumps, rubella,and T. gondii in saliva samples of vaccinated young people. Methods: Saliva was collected from 249 public schools students from São Paulo, Brazil, aged7 to 13 years old, during an interactive exhibition on hygiene. We used S. aureus proteinA solid phase capture assay for IgG reactive to biotinylated purified proteins. Paired salivaand serum (47) were tested from young adults with serum evidence of T. gondii infectionand from negative children less than 12 month old for standardization. Reproducibility wasgreater than 98% and sensitivity and specificity of the saliva assays were greater than 95%,as well as the concordance of paired saliva and serum samples. Results: Saliva from high school students showed a prevalence of 8.5% (95% CI: 5.0-11.9%)for anti T. gondii IgG; 96.8% (94.6-99%) of anti-measles IgG; 59.1% (53-65%) of anti-rubella IgG,and 57.5% (51.3-63.6%) of anti-mumps IgG. Discussion: The prevalence of antibodies against mumps and rubella after 6-8 years of vaccination was lower than against measles among students. The findings of this study demonstrate the feasibility of saliva sampling for follow-up of vaccine immune status in teenagers. This useful approach allows for IgG detection for vaccine control or epidemio- logical studies.

Measles, rubella, mumps and Toxoplasma gondii antibodies in saliva of vaccinated students of schools and universities in São Paulo City, Brazil.

INTRODUCTION: Vaccines are well-established public health interventions with major impact on the prevalence of infectious diseases, but outbreaks are occurring frequently due to primary and secondary failures, despite high coverage. Surveillance of efficacy and duration of induced immunity is a difficult task as it requires invasive blood sampling in children and teenagers. Saliva can be an acceptable alternative source of IgG to assess vaccine efficacy and toxoplasmosis incidence. We investigated IgG response for measles, mumps, rubella, and T. gondii in saliva samples of vaccinated young people. METHODS: Saliva was collected from 249 public schools students from São Paulo, Brazil, aged 7 to 13 years old, during an interactive exhibition on hygiene. We used S. aureus protein A solid phase capture assay for IgG reactive to biotinylated purified proteins. Paired saliva and serum (47) were tested from young adults with serum evidence of T. gondii infection and from negative children less than 12 month old for standardization. Reproducibility was greater than 98% and sensitivity and specificity of the saliva assays were greater than 95%, as well as the concordance of paired saliva and serum samples. RESULTS: Saliva from high school students showed a prevalence of 8.5% (95% CI: 5.0-11.9%) for anti T. gondii IgG; 96.8% (94.6-99%) of anti-measles IgG; 59.1% (53-65%) of anti-rubella IgG, and 57.5% (51.3-63.6%) of anti-mumps IgG. DISCUSSION: The prevalence of antibodies against mumps and rubella after 6-8 years of vaccination was lower than against measles among students. The findings of this study demonstrate the feasibility of saliva sampling for follow-up of vaccine immune status in teenagers. This useful approach allows for IgG detection for vaccine control or epidemiological studies.

Natural versus recombinant viral antigens in SARS-COV-2 serology: challenges in optimizing laboratory diagnosis of COVID19

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.

Avaliação das reações de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) na detecção de anticorpos IgG anti-T. gondii em exsudatos cárneos bovinos / Evaluation of indirect Hemagglutination (HI) and Modified Agglutination (MAT) reactions in the detection of anti-T. gondii IgG antibodies in bovine meat exudates

Toxoplasmose é uma zoonose parasitária com ampla distribuição mundial provocada pelo Toxoplasma gondii, considerado um dos protozoários mais bem sucedidos do planeta, pois infecta cerca de um terço da população mundial. Dentre as formas de transmissão, o consumo de carne mal cozida, contendo cistos, tem sido considerado um fator de risco para aquisição desta zoonose. Uma abordagem alternativa para o controle da toxoplasmose pela ingestão de carne bovina seria a sorologia dos bovinos, já que animais soropositivos albergam cistos teciduais. Contudo, a obtenção de soro para esta avaliação, nem sempre é factível, dada a dificuldade de coleta de sangue durante a linha de abate e sua ausência em cortes comerciais. O exsudato cárneo é uma alternativa para detecção de anticorpos anti - T. gondii em cortes comerciais de carne, que foi a proposta deste estudo para avaliar o desempenho dos testes de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) quando comparados ao ELISA usando exsudato cárneo. Este estudo mostrou que a acurácia dos testes de aglutinação não foi viável devido aos baixos índices de sensibilidade e especificidade quando comparados ao ELISA. Estes dados demonstram a importância da escolha de testes eficientes como ELISA para aplicação no controle da qualidade e inocuidade de cortes comerciais de carne bovina.

Toxoplasmosis is a parasitic zoonosis with a wide worldwide distribution caused by Toxoplasma gondii, which is considered one of the most successful protozoa on the planet, since it can infect a third of the world population. Among the forms of transmission, consumption of undercooked meat has been considered as a risk factor for the acquisition of this zoonosis. An alternative approach to toxoplasmosis control by beef ingestion could be the serological diagnosis in cattle, since seropositives animals harbor tissue cysts. However, the use of serum for this evaluation is not always feasible due to the difficulty of blood collection during slaughter and its absence in commercial beef cuts. Meat exudate is an alternative for the detection of anti-T. gondii antibodies in commercial beef cuts, which was the propose of this study to evaluate the performance of Indirect Hemagglutination (HI) and Agglutination Modified (MAT) tests compared to ELISA using meat exudates. This study showed that the agglutination tests accuracy was not viable due to low sensitivity and specificity indexes when compared to ELISA. These data demonstrate the importance of choosing accurate tests such as ELISA for application in quality control and safety of commercial beef cuts.

Avaliação das reações de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) na detecção de anticorpos IgG anti-T. gondii em exsudatos cárneos bovinos / Evaluation of indirect Hemagglutination (HI) and Modified Agglutination (MAT) reactions in the detection of anti-T. gondiiIgG antibodies in bovine meat exudates

Toxoplasmose é uma zoonose parasitária com ampla distribuição mundial provocada pelo Toxoplasma gondii, considerado um dos protozoários mais bem sucedidos do planeta, pois infecta cerca de um terço da população mundial. Dentre as formas de transmissão, o consumo de carne mal cozida, contendo cistos, tem sido considerado um fator de risco para aquisição desta zoonose. Uma abordagem alternativa para o controle da toxoplasmose pela ingestão de carne bovina seria a sorologia dos bovinos, já que animais soropositivos albergam cistos teciduais. Contudo, a obtenção de soro para esta avaliação, nem sempre é factível, dada a dificuldade de coleta de sangue durante a linha de abate e sua ausência em cortes comerciais. O exsudato cárneo é uma alternativa para detecção de anticorpos anti - T. gondii em cortes comerciais de carne, que foi a proposta deste estudo para avaliar o desempenho dos testes de Hemaglutinação Indireta (HI) e Aglutinação Modificada (MAT) quando comparados ao ELISA usando exsudato cárneo. Este estudo mostrou que a acurácia dos testes de aglutinação não foi viável devido aos baixos índices de sensibilidade e especificidade quando comparados ao ELISA. Estes dados demonstram a importância da escolha de testes eficientes como ELISA para aplicação no controle da qualidade e inocuidade de cortes comerciais de carne bovina. (AU)

Toxoplasmosis is a parasitic zoonosis with a wide worldwide distribution caused by Toxoplasmagondii, which is considered one of the most successful protozoa on the planet, since it can infect a third of the world population. Among the forms of transmission, consumption of undercooked meat has been considered as a risk factor for the acquisition of this zoonosis. An alternative approach to toxoplasmosis control by beef ingestion could be the serological diagnosis in cattle, since seropositives animals harbor tissue cysts. However, the use of serum for this evaluation is not always feasible due to the difficulty of blood collection during slaughter and its absence in commercial beef cuts. Meat exudate is an alternative for the detection of anti-T. gondii antibodies in commercial beef cuts, which was the propose of this study to evaluate the performance of Indirect Hemagglutination (HI) and Agglutination Modified (MAT) tests compared to ELISA using meat exudates. This study showed that the agglutination tests accuracy was not viable due to low sensitivity and specificity indexes when compared to ELISA. These data demonstrate the importance of choosing accurate tests such as ELISA for application in quality control and safety of commercial beef cuts. (AU)

Saliva collection and detection of anti- T. gondii antibodies of low-income school-age children as a learning strategy on hygiene, prevention and transmission of toxoplasmosis.

School-age children are a social group in which blood collection for laboratory testing can be perceived as an invasive procedure, with low acceptance and tolerance of stakeholders. This problem could be circumvented by replacing serum samples with saliva. For this purpose, and to make the collection of saliva samples playful and instructive for children, educational activities on hygiene and toxoplasmosis transmission and prevention were performed using toys and audiovisual tools. The target audience consisted of 7-10 year-old children from low-income families who attended public schools in the city of São Paulo. Saliva samples were used in a previously described in-house Enzyme-Linked Immunosorbent Assays (ELISA) to detect anti- Toxoplasma gondii IgG antibodies and establish the immunological status of each of the participants. One year later, children's memory and fixation of concepts regarding hygiene habits, as well as transmission and prevention of toxoplasmosis were tested in the same schools, by means of a questionnaire application, using students who did not participate in the first intervention as controls. The prevalence of positive anti- T. gondii IgG among students was 50% (82/164). One year later, 45 children had more knowledge on toxoplasmosis (28/45 vs 29/147) and they drew the cat's involvement in the transmission of toxoplasmosis more often than controls (28/45 vs 29/147). Sorted according to the presence of specific IgG in saliva, recovered positive students presented worse memory of the above cited knowledge as did saliva-negative IgG students, but both groups had isolated higher frequency of fixed knowledge than non-intervened students. Our data show that there is a high prevalence of T. gondii infection in school-children from low-income areas; saliva is an alternative to blood for anti- T. gondii IgG detection; and a one-day educational intervention in school-children was effective in promoting knowledge fixation on hygiene and toxoplasmosis transmission and prevention after one year.

CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii.

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level. Bifidobacterium animalis subsp. lactis provided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probiotic B. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection.

Padronização da metodologia dot-ELISA para detecção de anticorpos IgG anti-Toxoplasma gondii em saliva / Standardization of dot-ELISA methodology for detection of IgG antibodies anti-Toxoplasma gondii in saliva

O diagnóstico da infecção pelo T. gondii é usualmente feito pelas técnicas sorológicas, mas a amostra (soro ou plasma) pode ser restrita em determinados grupos protegidos, em que a coleta de sangue é considerada agressiva e invasiva. Os anticorpos são encontrados em outros materiais biológicos, de coleta não invasiva, como a saliva. Os métodos de detecção de anticorpos no mercado estão padronizados para utilizar amostras de soro, e há metodologias alternativas de maior sensibilidade utilizando-se saliva, mas estas requerem equipamentos de difícil uso no campo. Dot-ELISA tem alta sensibilidade e leitura visual sem equipamentos, que facilita a execução de ensaio em campo utilizando-se técnica de triagem rápida e eficiente. Neste contexto, foi padronizado o dot-ELISA de alta sensibilidade para detecção de anticorpos anti-T. gondii em saliva e soro, utilizando-se amostras de 20 voluntários adultos. A sensibilidade e a especificidade do dot-ELISA padronizado foram semelhantes em soro e saliva, com exata distinção de amostras positivas e negativas, mesmo na ocorrência de baixas concentrações de anticorpos como na saliva. A saliva mostra ser material biológico adequado para detecção de anticorpos anti-T. gondii em estudos epidemiológicos da toxoplasmose em crianças ou outros grupos protegidos, em que a coleta de sangue é restrita...

HUMAN TOXOPLASMOSIS OUTBREAKS AND THE AGENT INFECTING FORM. FINDINGS FROM A SYSTEMATIC REVIEW.

Toxoplasmosis, a worldwide highly prevalent zoonotic infection, is transmitted either by the oocysts, from water and soil, or the tissue cysts, in raw or undercooked infected meat, of Toxoplasma gondii. An ongoing debate is whether there are differences between the clinical and epidemiological characteristics of the outbreaks due to one or the other infective form of the agent. We performed a systematic review, recovering 437 reported outbreaks of which 38 were selected. They were complete reports containing ascribed Toxoplasma infecting form, and clinical and demographic data. There was no gender or age group selection in the outbreaks, which were described more often in the Americas. A large number of individuals were affected when oocysts, associated with soil and water contaminated with cat feces, were considered the transmission source. Onset of symptoms occurred early when the infection was ascribed to meat tissue cysts (11.4 ± 6.7 days) with sharpened temporal distribution of cases, while a broader and prolonged appearance of new cases was observed when oocysts in water were the source of the infection (20 ± 7 days, p < 0.001). Such information may be useful in the design and implementation of control strategies.

HUMAN TOXOPLASMOSIS OUTBREAKS AND THE AGENT INFECTING FORM. FINDINGS FROM A SYSTEMATIC REVIEW / Surtos de toxoplasmose humana e a forma infectante do agente. Achados de revisão sistemática

SUMMARY Toxoplasmosis, a worldwide highly prevalent zoonotic infection, is transmitted either by the oocysts, from water and soil, or the tissue cysts, in raw or undercooked infected meat, of Toxoplasma gondii. An ongoing debate is whether there are differences between the clinical and epidemiological characteristics of the outbreaks due to one or the other infective form of the agent. We performed a systematic review, recovering 437 reported outbreaks of which 38 were selected. They were complete reports containing ascribed Toxoplasma infecting form, and clinical and demographic data. There was no gender or age group selection in the outbreaks, which were described more often in the Americas. A large number of individuals were affected when oocysts, associated with soil and water contaminated with cat feces, were considered the transmission source. Onset of symptoms occurred early when the infection was ascribed to meat tissue cysts (11.4 ± 6.7 days) with sharpened temporal distribution of cases, while a broader and prolonged appearance of new cases was observed when oocysts in water were the source of the infection (20 ± 7 days, p < 0.001). Such information may be useful in the design and implementation of control strategies.

RESUMO Toxoplasmose, infecção zoonótica altamente prevalente no mundo, é transmitida pela ingestão de oocistos em água e solo ou cistos teciduais em carne crua ou mal cozida. Um debate em andamento é se há diferenças nas características clínicas e epidemiológicas de surtos devido a uma ou outra forma infectante do agente. Realizamos revisão sistemática a partir de 437 relatos de surtos da doença, selecionando 38 artigos completos que descreveram a forma infectante do Toxoplasmacom dados clínicos e epidemiológicos. Não houve seleção por gênero ou faixa etária nos surtos, descritos mais frequentemente nas Américas. Quantidade maior de indivíduos foi afetada quando oocistos, associados com solo ou água contaminados com fezes de gato, foram considerados a fonte de transmissão. O início dos sintomas ocorreu mais precocemente quando a infecção foi atribuída a cistos na carne (11,4 ± 6,7 dias) com distribuição temporal nítida de casos, embora um aspecto mais amplo e prolongado de novos casos foi observado quando oocistos na água foram a fonte de infecção (20 ± 7 dias, p< 0.001). Essas informações podem ser úteis no desenvolvimento e implantação de estratégias de controle.