Segurança biológica e ensaio in vitro de expressão gênica em PBMC de caninos vacinados experimentalmente contra Toxocara canis

Toxocariasis is a neglected zoonosis with a high prevalence and impact on the public health of impoverished populations in the world, its causative agent is the nematode of the genus Toxacara. Broad-spectrum antiparasitics are currently used to deal with the disease, however, there is no vaccine to prevent it. The objective of this research was to evaluate the biological safety of experimental clinical trials of reverse vaccinology; and also, to evaluate the in vitro response of PBMC of canines previously vaccinated with the recombinant proteins rTcVcan and rTcCad of Toxocara canis, through the gene expression assay measured by RT-qPCR of IL-5, IL-6, IL-17. , IL-10, TNF-α and INF-Ɣ cytokines. As result, once the clinical trials for the vaccination of the puppies were carried out, no differences were observed with respect to the reference ranges of the hematology analysis, as well as the analyzes of the liver and kidney functions in the puppies and; on the other hand, in the gene expression assays in PBMC, only the ACTB gene was successfully expressed, the cytokine genes did not present statistically significant results. In conclusion, the applied formulations were not toxic and had no adverse effects on the experimental animals and gene expression assays in PBMC of vaccinated puppies with the recombinant rTcVcan and rTcCad proteins against T. canis did not show conclusive results regarding the success of these procedures in the first phase of the experimental vaccine.La toxocariasis es una zoonosis desatendida de alta prevalencia e impacto en la salud pública de poblaciones empobrecidas del mundo, su agente causal es el nematodo del género Toxacara. Actualmente se utilizan antiparasitarios de amplio espectro para afrontar la enfermedad, sin embargo, no existe una vacuna para prevenirla. El objetivo de esta investigación fue evaluar la seguridad biológica de ensayos clínicos experimentales de vacunología inversa; y así mismo evaluar la respuesta in vitro de PBMC de caninos previamente vacunados con las proteínas recombinantes rTcVcan y rTcCad de Toxocara canis, a través del ensayo de expresión génica medida por RT-qPCR de citocinas IL-5, IL- 6, IL-17A, IL-10, TNF-α e INF-Ɣ. Como resultado, una vez realizados los ensayos clínicos para la vacunación de los cachorros, no se observaron diferencias con respecto a los rangos de referencia de los análisis de hematología, así como de los análisis de las funciones hepática y renal en los cachorros y; por otra parte, en los ensayos de expresión génica en PBMC, únicamente el gen ACTB se expresó con éxito, los genes referentes a las citocinas no presentaron resultados estadísticamente significativos. En conclusión, las formulaciones aplicadas no fueron tóxicas y no tuvieron efectos adversos en los animales experimentales y los ensayos de expresión génica en PBMC de los cachorros vacunados con las proteínas recombinantes rTcVcan y rTcCad contra T. canis no presentaron resultados concluyentes con respecto al éxito de estos procedimientos en la primera fase de la vacuna experimental.A toxocaríase é uma zoonose negligenciada de alta prevalência e impacto na saúde pública de populações empobrecidas no mundo, seu agente causal é o nematóide do gênero Toxacara. Atualmente, antiparasitários de amplo espectro são usados ​​para tratar a doença, mas não há vacina para preveni-la. O objetivo desta pesquisa foi avaliar a segurança biológica de ensaios clínicos experimentais de vacinologia reversa; e também avaliar a resposta in vitro de PBMC de caninos previamente vacinados com as proteínas recombinantes rTcVcan e rTcCad de Toxocara canis, através do ensaio de expressão gênica medida por RT-qPCR das citocinas IL-5, IL-6, IL-17. , IL-10, TNF-α e INF-Ɣ. Como resultado, uma vez realizados os ensaios clínicos para a vacinação dos filhotes, não foram observadas diferenças com relação aos intervalos de referência da análise hematológica, bem como das análises das funções hepática e renal nos filhotes e; por outro lado, nos ensaios de expressão gênica em PBMC, apenas o gene ACTB foi expresso com sucesso, os genes relacionados a citocinas não apresentaram resultados estatisticamente significativos. Em conclusão, as formulações aplicadas não foram tóxicas e não causaram efeitos adversos nos animais experimentais e os ensaios de expressão gênica em PBMC dos filhotes vacinados com as proteínas recombinantes rTcVcan e rTcCad contra T. canis não apresentaram resultados conclusivos quanto ao sucesso dos esses procedimentos na primeira fase da vacina experimental

Evaluación de técnicas coprodiagnósticas para Toxocara canis

The aim of this study was to evaluate the sensitivity of four coprodiagnostic techniques to quantitatively determine the presence of Toxocara canis eggs in fresh canine stool samples. For this, eggs were extracted from the uterus of adult female T. canis obtained from canine puppies. Eight concentrations of eggs were used (10, 50, 75, 100, 250, 500, 750 and 1 000 eggs per gram of faeces - hpg), being considered as the gold standard to determine the degree of precision of the Kato-Katz, McMaster, Improved Modified McMaster and Faust techniques. Three repetitions were used per technique and per concentration. The sensitivity of each test was performed by arithmetic calculation and ROC curve (95% CI), comparing them with the gold standard by means of a nonparametric test of the Wilcoxon signed ranges (p<0.05). The Faust technique detected T. canis eggs regardless of concentration, but in statistical inequality with the gold standard (p<0.001 to p<0.0001). The Kato-Katz technique did not show significant differences with the gold standard, but it was unable to detect the presence of T. canis eggs in 10 and 50 hpg concentration, while the improved modified McMaster technique showed similarity with the gold standard in 100 and 250 hpg. The McMaster method detected eggs from 100 hpg, obtaining hpg values below the gold standard (p<0.05 and p<0.001). Sensitivities of 74.72% (95% CI 72.93-80.22) were obtained for the Kato-Katz technique and between 30 and 40% (95% CI 26.71-48.29) for the other tests. It is concluded that the Kato-Katz technique obtained a better performance showing a constant quantitative diagnostic sensitivity when compared with other routine quantitative stool diagnosis techniques in veterinary laboratories for T. canis eggs in fresh dog faeces samples.El estudio tuvo como objetivo evaluar la sensibilidad de técnicas coprodiagnósticas para determinar cuantitativamente la presencia de huevos de Toxocara canis en muestras de heces frescas de caninos. Para ello, se extrajeron huevos del útero de hembras adultas de T. canis obtenidas de cachorros caninos. Se utilizaron ocho concentraciones de huevos (10, 50, 75, 100, 250, 500, 750 y 1 000 huevos por gramo de materia fecal – hpg), siendo considerados como el gold standard para determinar el grado de precisión de las técnicas Kato-Katz, McMaster, McMaster modificado mejorado y Faust. Se utilizaron tres repeticiones por técnica y por concentración. La sensibilidad de cada prueba se realizó mediante el cálculo aritmético y curva ROC (IC95%), comparándolas con el gold standard mediante una prueba no paramétrica de los rangos con signo de Wilcoxon (p<0.05). La técnica Faust detectó huevos de T. canis sin importar la concentración, pero en desigualdad estadística con el gold standard (p<0.001 a p<0.0001). La técnica Kato-Katz no presentó diferencias significativas con el gold standard, pero no detectó la presencia de huevos de T. canis en 10 y 50 hpg, mientras que la técnica McMaster modificado mejorado presentó similitud con el gold estandar en 100 y 250 hpg. El método de McMaster detectó huevos a partir de 100 hpg, obteniendo valores de hpg por debajo del gold standard (p<0.05 y p<0.001). Se obtuvieron sensibilidades de 74.72% (IC95% 72.93-80.22) para la técnica de Kato-Katz y entre 30 y 40% (IC95% 26.71-48.29) para las demás pruebas. Se concluye que la técnica Kato-Katz obtuvo un mejor desempeño en sensibilidad diagnóstica cuantitativa constante al comprarse con técnicas cotidianas cuantitativas de diagnóstico coprológico en laboratorios veterinarios para huevos de T. canis en muestras de heces frescas de canes

Biogeographical ancestry is associated with socioenvironmental conditions and infections in a Latin American urban population.

Racial inequalities are observed for different diseases and are mainly caused by differences in socioeconomic status between ethnoracial groups. Genetic factors have also been implicated, and recently, several studies have investigated the association between biogeographical ancestry (BGA) and complex diseases. However, the role of BGA as a proxy for non-genetic health determinants has been little investigated. Similarly, studies comparing the association of BGA and self-reported skin colour with these determinants are scarce. Here, we report the association of BGA and self-reported skin colour with socioenvironmental conditions and infections. We studied 1246 children living in a Brazilian urban poor area. The BGA was estimated using 370,539 genome-wide autosomal markers. Standardised questionnaires were administered to the children's guardians to evaluate socioenvironmental conditions. Infection (or pathogen exposure) was defined by the presence of positive serologic test results for IgG to seven pathogens (Toxocara spp, Toxoplasma gondii, Helicobacter pylori, and hepatitis A, herpes simplex, herpes zoster and Epstein-Barr viruses) and the presence of intestinal helminth eggs in stool samples (Ascaris lumbricoides and Trichiuris trichiura). African ancestry was negatively associated with maternal education and household income and positively associated with infections and variables, indicating poorer housing and living conditions. The self-reported skin colour was associated with infections only. In stratified analyses, the proportion of African ancestry was associated with most of the outcomes investigated, particularly among admixed individuals. In conclusion, BGA was associated with socioenvironmental conditions and infections even in a low-income and highly admixed population, capturing differences that self-reported skin colour miss. Importantly, our findings suggest caution in interpreting significant associations between BGA and diseases as indicative of the genetic factors involved

Toxocara Seropositivity, Atopy and Wheezing in Children Living in Poor Neighbourhoods in Urban Latin American

Background Toxocara canis and T. cati are parasites of dogs and cats, respectively, that infect humans and cause human toxocariasis. Infection may cause asthma-like symptoms but is often asymptomatic and is associated with a marked eosinophilia. Previous epidemiological studies indicate that T. canis infection may be associated with the development of atopy and asthma. Objectives To investigate possible associations between Toxocara spp. seropositivity and atopy and childhood wheezing in a population of children living in non-affluent areas of a large Latin American city. Methods The study was conducted in the city of Salvador, Brazil. Data on wheezing symptoms were collected by questionnaire, and atopy was measured by the presence of aeroallergen-specific IgE (sIgE). Skin prick test (SPT), total IgE and peripheral eosinophilia were measured. Toxocara seropositivity was determined by the presence of anti-Toxocara IgG antibodies, and intestinal helminth infections were determined by stool microscopy. Findings Children aged 4 to 11 years were studied, of whom 47% were seropositive for anti-Toxocara IgG; eosinophilia >4% occurred in 74.2% and >10% in 25.4%; 59.6% had elevated levels of total IgE; 36.8% had sIgE≥0.70 kU/L and 30.4% had SPT for at least one aeroallergen; 22.4% had current wheezing symptoms. Anti-Toxocara IgG was positively associated with elevated eosinophils counts, total IgE and the presence of specific IgE to aeroallergens but was inversely associated with skin prick test reactivity. Conclusion The prevalence of Toxocara seropositivity was high in the studied population of children living in conditions of poverty in urban Brazil. Toxocara infection, although associated with total IgE, sIgE and eosinophilia, may prevent the development of skin hypersensitivity to aeroallergens, possibly through increased polyclonal IgE and the induction of a modified Th2 immune reaction

Efficient differentiation of Corynebacterium striatum, Corynebacterium amycolatum and Corynebacterium xerosis clinical isolates by multiplex PCR using novel species-specific primers

A multiplex-PCR (mPCR) assay was designed with species-specific primers which generate amplicons of 226 bp, 434 bp and 106 bp for differentiating the species C. striatum, C. amycolatum, and C. xerosis, respectively. mPCR results were 100% in agreement with identifications achieved by 16S rRNA and rpoB gene sequencing and by VITEK-MS.This work was supported by grants from FAPESB (JCB0031/2013) and CAPES (PROCAD 071/2013)

Reductions in the Prevalence and Incidence of Geohelminth Infections following a City-wide Sanitation Program in a Brazilian Urban Centre

In the city of Salvador, a large urban centre in Northeast Brazil, a city-wide sanitation intervention started in 1997, aiming to improve the sewerage coverage of households from 26% to 80%. Our aim was to study the impact of the intervention on the prevalence and incidence of geohelminths in the school-aged population. The longitudinal study comprised two cohorts: from the beginning of 1997 to 1998, where data was collected before the intervention, and at the end of 2003 to 2004, after the intervention. Copro-parasitological examinations were carried out on every individual from both cohorts, at the start and nine months later. Demographic, socio-economic, and environmental data were collected using semi-structured questionnaires. The variables utilized to demonstrate the effects of intervention, when utilized together in a multivariate model, accounted for a 100% observed reduction in the prevalence ratio (PR) and incidence ratio (IR). As well as proving that the variables associated with the effect of the program intervention were mediators in this reduction, the reduction in the PR and IR between these periods demonstrated that modifications to the urban environment, particularly those associated with sanitary sewage systems, affected the health of the population, significantly reducing the prevalence of geohelminths

Chronic virus infections supress atopy but not asthma in a set of children from a large latin american city: a cross-section study

<p>Abstract</p> <p>Background</p> <p>The prevalence of allergic diseases has increased over recent decades in affluent countries, but remains low in rural populations and some non-affluent countries. An explanation for these trends is that increased exposure to infections may provide protection against the development of allergy. In this work we investigated the association between exposure to viral infections in children living in urban Brazil and the prevalence of atopy and asthma.</p> <p>Methods</p> <p>School age children living in poor neighborhoods in the city of Salvador were studied. Data on asthma symptoms and relevant risk factors were obtained by questionnaire. Skin prick tests (SPTs) were performed to seven aeroallergens, and specific IgE was measured to four of these. Viral infections were determined by the presence of specific IgG in serum to Herpes simplex (HSV), Herpes zoster (HZV), Epstein-Barr (EBV), and Hepatitis A (HAV) viruses.</p> <p>Results</p> <p>A total of 644 (49.7%) children had at least one allergen-specific IgE> 0.35 kU/L and 489 (37.7%) had specific IgE> 0.70 kU/L. A total of 391 (30.2%) children were skin test positive (SPT+), and 295 (22.8%) children were asthmatic. The seroprevalence of viral infections was 88.9% for EBV, 55.4% for HSV, 45.5% for VZV and 17.5% for HAV. Negative associations were observed between SPT+ and HSV (OR = 0.64, CI = 0.51, 0.82) and EBV (OR = 0.63, CI = 0.44, 0.89) infections, but no associations were seen between viral infections and the presence of allergen-specific IgE or asthma.</p> <p>Conclusion</p> <p>These data do not support previous data showing a protective effect of HAV against atopy, but did show inverse associations between SPT+ (but not specific IgE+) and infections with HSV and EBV. These findings suggest that different viral infections may protect against SPT+ in different settings and may indicate an immunoregulatory role of such infections on immediate hypersensitivity responses. The data provide no support for a protective effect of viral infections against asthma in this population.</p

Dissociation between skin test reactivity and anti-aeroallergen IgE: Determinants among urban Brazilian children.

BACKGROUND: The dissociation between specific IgE and skin prick test reactivity to aeroallergens, a common finding in populations living in low and middle-income countries, has important implications for the diagnosis and treatment of allergic diseases. Few studies have investigated the determinants of this dissociation. In the present study, we explored potential factors explaining this dissociation in children living in an urban area of Northeast Brazil, focusing in particular on factors associated with poor hygiene. METHODS: Of 1445 children from low income communities, investigated for risk factors of allergies, we studied 481 with specific IgE antibodies to any of Blomia tropicalis, Dermatophagoides pteronyssinus, Periplaneta americana and Blatella germanica allergens. Data on demographic, environmental and social exposures were collected by questionnaire; serum IgG and stool examinations were done to detect current or past infections with viral, bacterial, protozoan and intestinal helminth pathogens. We measured atopy by skin prick testing (SPT) and specific IgE (sIgE) to aerollergens in serum (by ImmunoCAP). SIgE reactivity to B. tropicalis extract depleted of carbohydrates was measured by an in-house ELISA. Total IgE was measured by in house capture ELISA. SNPs were typed using Illumina Omni 2.5. RESULTS: Negative skin prick tests in the presence of specific IgE antibodies were frequent. Factors independently associated with a reduced frequency of positive skin prick tests were large number of siblings, the presence of IgG to herpes simplex virus, Ascaris lumbricoides and Trichuris trichiura infections, living in neighborhoods with infrequent garbage collection, presence of rodents and cats in the household and sIgE reactivity to glycosylated B. tropicalis allergens. Also, SNP on IGHE (rs61737468) was negatively associated with SPT reactivity. CONCLUSIONS: A variety of factors were found to be associated with decreased frequency of SPT such as unhygienic living conditions, infections, total IgE, IgE response to glycosylated allergens and genetic polymorphisms, indicating that multiple mechanisms may be involved. Our data, showing that exposures to an unhygienic environment and childhood infections modulate immediate allergen skin test reactivity, provide support for the "hygiene hypothesis"