Avaliação de um novo kit (TF-TEST) nacional destinado ao diagnostico de enteroparasitoses em amostras fecais

Orientadores: Luiz Candido de Souza Dias e Sumie Hoshino ShimizuDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As enteroparasitoses humanas constituem atualmente um dos problemas de saúde pública tanto para países em desenvolvimento como para países desenvolvidos, em virtude do aumento no número de viajantes a regiões endêmicas e principalmente por causa de indivíduos imunocomprometidos. Devido aumentar a probabilidade do encontro de organismos pelo exame de amostras múltiplas, pelo menos três exames parasitológicos das fezes são necessários, a fim de obter resultados sensíveis. Desta forma, idealizamos e desenvolvemos um kit (TF-Test) prático e econômico, agora em vias de ser fabricado em escala industrial, por uma empresa (Immunoassay Ind. Com. Ltda., São Paulo, Brasil). Este kit possibilita, em dias alternados, a coleta de três amostras fecais, separadamente, em tubos contendo uma solução preservadora. No laboratório, as três amostras são processadas em uma só etapa, utilizando um conjunto, onde a mistura passa por duplo filtro, e é concentrada por uma centrifugação rápida. O presente trabalho trata sobre avaliações dos protótipos do kit realizadas em duas fases, sendo a primeira, intralaboratorial abrangendo um período de seis meses, e a segunda, interlaboratoriais de dois anos. Na primeira fase, a técnica de TF-Test foi avaliada no estudo de 30 pacientes com parasitoses intestinais e 30 sem infecção parasitária, obtendo uma sensibilidade de 96,6%, significativamente (p < 0,05) maior que as demais técnicas convencionais comerciais (Coprotest, StillQuick e Parasep) ou não (Lutz ou Hoffman, Pons & Janer). Ademais, a técnica em questão apresentou um elevado índice de concordância k (0,920), considerado como Quase Perfeito, diferindo das técnicas convencionais. Em vista do potencial uso do protótipo para finalidade diagnóstica, o trabalho teve continuidade na segunda fase, onde os protótipos aprimorados foram avaliados no estudo de 1.244 pacientes em seis diferentes laboratórios de setor público e privado, considerados como sendo de referência ou altamente conceituados. Os dados obtidos demonstram que a técnica de TF-Test é de fato melhor que as técnicas convencionais utilizadas (Coprotest, Lutz e associação de Lutz, Faust & Cols. e Rugai, Mattos & Brisola), por apresentar sensibilidades que variam de 82,6% a 100%, estatisticamente maiores (p < 0,05) que as técnicas convencionais. Em quatro laboratórios, o índice k de TF-Test teve a classificação de Quase Perfeito e em dois laboratórios, Substancial. A alta sensibilidade e os aspectos econômicos e práticos demonstram que a técnica de TF-Test é apropriada para diagnóstico individual, inquéritos populacionais, assim como, para avaliação de quimioterapia instituída em programas de controle das parasitoses intestinaisAbstract: Human intestinal parasitoses constitute a serious public health problem either in developing or developed countries. Those enteroparasitoses could be attributed to the increase of travelers around endemic areas and mainly due to immunocompromised patients. In order to obtain sensible results and to have a high chance to find organisms by multiple sample exams, it is necessary at least three serial parasitologycal faecal tests. For these reasons, a practice and non-expensive kit (TF-Test) was developed for being industrialized by a specialized company (Immunoassay Ind. Com. Ltda., Sao Paulo, Brazil). This kit makes possible to carry out three different faecal samples each other day. The three faecal samples are separately collected into tubes containing a preservative solution. Laboratory evaluation is done in just one step by using a pool of the three faecal samples. Mixture mass is passing by a double filter and then centrifuged for concentrating the mixture. Thus, the present study reports about evaluations of a prototype kit developed into two different phases: first phase was a six months stage inside developer laboratory and, the second one was a two years stage involving six different collaborative laboratories. In the first phase, parasitologycal exams using TF-Test was carried out in 30 patients with intestinal parasites and in 30 patients free of intestinal parasites and, it was significantly more sensible (96,6%, p<0,05) compared with conventional techniques as Coprotest, StillQuick e Parasep or with those frequently used as Lutz or Hoffman, Pons & Janer. Furthermore, TF-Test had a high agreement k index (0,920), which was considered as Almost Perfect, in addition to differ from conventional techniques. Thus, focusing in the potential use of TF-Test for diagnosing different intestinal parasites, this work had been continued for developing a better prototype kit of TF-Test. In the second phase, six different specialized laboratories from public or private service had evaluated faecal samples collected from 1.244 patients. Data from those laboratories attested that TF-Test was actually better than other known techniques (Coprotest, Lutz and Lutz, Faust & Cols. and Rugai, Mattos & Brisola), for presenting sensibility range between 82,6% and 100%, which was significantly higher than conventional methods (p < 0,05). In four of the six laboratories, k index of TF-Test was classified as Almost Perfect, and in the other two laboratories, the index was considered as Substancial. The high sensibility of TF-Test in addition to its practice and economic aspects shown that TF-Test method is very appropriated for individual diagnosis, population inquires as well as for evaluating therapy programs in the control of intestinal parasitosesMestradoParasitologiaMestre em Parasitologi

Semi-Automatic Data Annotation guided by Feature Space Projection

Data annotation using visual inspection (supervision) of each training sample can be laborious. Interactive solutions alleviate this by helping experts propagate labels from a few supervised samples to unlabeled ones based solely on the visual analysis of their feature space projection (with no further sample supervision). We present a semi-automatic data annotation approach based on suitable feature space projection and semi-supervised label estimation. We validate our method on the popular MNIST dataset and on images of human intestinal parasites with and without fecal impurities, a large and diverse dataset that makes classification very hard. We evaluate two approaches for semi-supervised learning from the latent and projection spaces, to choose the one that best reduces user annotation effort and also increases classification accuracy on unseen data. Our results demonstrate the added-value of visual analytics tools that combine complementary abilities of humans and machines for more effective machine learning.Comment: 28 pages, 10 figure

High prevalence of blastocystis Spp. infection in children and staff members attending public urban schools in São Paulo stare, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)After a gastroenteritis outbreak of unknown etiology in the municipality of Sebastiao da Grama, Sao Paulo, Brazil, we conducted a parasitological survey to establish the epidemiological profile of enteroparasitosis in children and staff members attending the public urban schools in operation in town. The cross-sectional study evaluated 172 children aged 11 months to 6 years old and 33 staff members aged 19 to 58 years old. Overall, 96 (55.81%) children and 20 (60.61%) staff members were mono-parasitized, while 58 (33.72%) children and 4 (12.12%) workers were poly-parasitized. Protozoa (88.37%; 72.73%) was more prevalent than helminthes (3.48%; 0%) in children and staff members respectively. Blastocystis spp. was the most prevalent parasite in children (86.63%) and staff members (66.67%). The age of 1 year old or less was found to be associated with increased prevalence of giardiasis [OR = 13.04; 95% CI 2.89-58.91; p = 0.00] and public garbage collection was identified as a protective factor against intestinal helminth infections [OR = 0.06; 95% CI 0.00-0.79; p = 0.03]. Although most of the children tested positive for Blastocystis spp. and also presented clinical signs/symptoms (62.2%), this association was not statistically significant [OR = 1.35; 95% CI 0.53-3.44; p = 0.51]. Intestinal parasites still represent a public health concern and this study underscores the importance of further investigations to better understand the pathogenic role of Blastocystis spp.After a gastroenteritis outbreak of unknown etiology in the municipality of Sebastiao da Grama, Sao Paulo, Brazil, we conducted a parasitological survey to establish the epidemiological profile of enteroparasitosis in children and staff members attending58CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)sem informaçã

EPIDEMIOLOGICAL ASPECTS OF FELINE TOXOPLASMOSIS

Toxoplasmosis is a zoonosis distributed worldwide, which is of great importance in human and veterinary medicine. Toxoplasmosis, caused by Toxoplasma gondii, can be transmitted through the ingestion of tissue cysts in infected meat, ingestion of food or water contaminated with oocysts and congenitally, by tachyzoites crossing the placenta from the infected mother to the fetus. Felines in particular (intermediate or definitive hosts for the parasites) have an important role in the epidemiology of this agent, as they can eliminate oocysts in the domestic environment. The aim of this review is to describe clinical manifestations of toxoplasmosis, to address the role of cats in the spread of the disease, as well as to discuss methods of diagnosis, therapeutic measures, prophylaxis and control of this disease.

Isosporose canina: uma abordagem clínica neonatal

Acute diarrhea in young dogs is among the main complaints described by tutors in veterinary services. Complications related to water and electrolyte imbalances due to intestinal alterations can compromise homeostasis in these pups, mainly due to dehydration. Consequently, they do not present a satisfactory clinical response and are exposed to the risk of early death. Among the different types of diarrhea, the infectious diarrhea due to viruses, bacteria, and parasites are particularly significant. Coccidia of the genus Isospora (Cvstoisospora spp.) is an important intestinal pathogen that causes severe clinical manifestations in puppies. Since high parasitic loads render these puppies highly susceptible to opportunistic infections, coccidia infection may cause impaired corporal and cognitive development and even lead to death. Studies regarding this issue, particularly concerning clinical knowledge in neonatology, are scarce in veterinary medicine. Thus, in this study, we intend to discuss the enteric alterations and the most commonly used current diagnostic techniques, as well as to analyze effective therapeutic and prophylactic proposals40637973803sem informaçãoAs diarreias agudas em cães jovens estão entre as principais queixas descritas pelos tutores nos serviços veterinários. As complicações relacionadas aos desequilíbrios hídrico e eletrolítico decorrentes das alterações intestinais comprometem a homeostase destes filhotes, principalmente devido a desidratação, não apresentam resposta clínica satisfatória e ficam expostos ao risco de morte precoce. Dentre os diferentes tipos de diarreia, sobressaem as de origem infecciosa. Os agentes podem ser vírus, bactérias, fungos e parasitos. Entre estes últimos agentes, os coccídios do Gênero Isospora (Cystoisospora spp.) se destacam como patógenos intestinais que ocasionam manifestações clínicas graves em filhotes, pois elevadas cargas parasitárias propiciam condição de maior susceptibilidade à infecções oportunistas, podendo também prejudicar o desenvolvimento corporal e cognitivo e/ou até mesmo levar a morte do cão. Diante de tal temática, na medicina veterinária os estudos são escassos, principalmente quanto ao conhecimento clínico em neonatologia de tal modo tencionamos discutir a respeito das alterações entéricas, métodos de diagnóstico mais utilizados e atuais, bem como analisar as propostas terapêuticas e profiláticas efetivassem informaçã

Caracterização molecular de Cryptosporidium em bezerros de assentamentos rurais da região Noroeste do estado de São Paulo, Brasil

The study was conducted on 25 properties of the settlements Sao Jose I and Salvador, located in the municipalities of Brejo Alegre and Birigui, in the State of Sao Paulo, Brazil. A record of variables was elaborated and included data such as gender, breed and age of the animals. A total of 231 stool samples were collected from bovines aged one to six months, 128 being females and 103 males, 131 crossbred and 100 Holstein. Among the 231 samples, 17 (7.36%) were positive for Crvptosporidium spp. both by malachite green negative staining and by nested-PCR. Of the 17 positive samples, 14 were sequenced in agarose gel. These sequences were detected between 99% and 100% of genetic similarity for the following species. One sequence was similar to C. parvum (AB513880.1), one to C. bovis (MF074602.1), two to C. ryanae (KT922233.1), one to C. felis (KM977642.1) and nine were similar for C. andersoni reference MF350628. C. andersoni was found in animals aged 2-6 months, an age group which is different from those described by several authors. The presence of C. parvum indicates that the calves in the studied region should be considered a potential source for zoonotic transmission. For the first time to our knowledge, C. felis was identified in cattle in America401491496FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2012/13733-3O estudo foi realizado num total de 25 propriedades localizadas nos assentamentos São José I e Salvador, situados nos municípios de Brejo Alegre e Birigui, no estado de São Paulo. Um registro de variáveis foi elaborado, incluindo dados como sexo, raça e idade dos animais. Foram colhidas 231 amostras de fezes de bovinos de um a seis meses de idade, sendo 128 fêmeas e 103 machos, 131 mestiços e 100 da raça Holandesa. Entre os 231 bovinos examinados, 17 (7,36%) foram positivos para Cryptosporidium spp. tanto pela coloração negativa de verde malaquita como pela nested-PCR. Das 17 amostras positivas, 14 apresentaram amplificação pela eletroforese em gel de agarose suficiente para fazer o sequenciamento de DNA. Essas sequências foram detectadas similaridade genética entre 99% e 100% com as seguintes espécies. Uma sequência foi semelhante com C. parvum (referência: AB513880.1), uma com C. bovis(MF074602.1), duas com C. ryanae (KT922233.1), uma com C. felis (KM977642.1) e nove foram semelhantes com C. andersoni (MF350628). O estudo caracteriza a presença do Cryptosporidiumspp. em bovinos oriundos de propriedades produtoras de leite na região Noroeste do estado de São Paulo, sendo o C. andersoni a espécie mais prevalente nesses animais, principalmente em uma faixa etária diferente das descritas por diversos autores. A presença de C. parvum indica que os bezerros da região estudada devem ser considerados como uma fonte potencial de oocistos de espécies zoonóticas.Identificamos com ineditismo o C. felis em bovinos na América, o que corrobora outros estudos realizados na Polônia e Espanha e evidencia a presença de espécies de Cryptosporidium em fezes em hospedeiros não naturais. Palavras-chave: Bezerros. Caracterização molecular. Criptosporidiose. Nested-PCR.Protozoan of the genus Cryptosporidium are obligate intracellular coccidia with a cosmopolitan distribution. Biological data and various molecular genotyping tools indicate that the genus Cryptosporidium comprises about 30 valid species and more than 70 genotypes that can infect fishes, amphibians, reptiles, birds, and mammals (NAKAMURA; MEIRELES, 2015).Among the named species, 13 have been described in cattle. Of these, nine present zoonotic potential, being more prevalent in humans: Cryptosporidium hominis, Cryptosporidium parvum, Cryptosporidium meleagridis, Cryptosporidium cuniculus and, occasionally,Cryptosporidium muris, Cryptosporidium suis, Cryptosporidium ubiquitum, Cryptosporidium felis(BORNAY-LLINARES et al. 1999; CARDONA et al., 2015) and Cryptosporidium canis (KVÁČ et al., 2016).Cattle are mainly infected with C. parvum, Cryptosporidium bovis, Cryptosporidium ryanaeand Cryptosporidium andersoni (XIAO, 2010). Of these, only the first is of zoonotic importance, being a concern for public health, since human cryptosporidiosis can be acquired from infected calves (LEARMONTH et al., 2004; SOPWITH et al., 2005; DEL COCO et al., 2014). These four species of Cryptosporidium are frequently diagnosed in cattle of different ages. Among these, C. parvumis the most prevalent in young pre-weaning calves (less than two months old) and shows low host specificity, and some genotypes are considered to have high zoonotic potential (DIXON et al., 2011).During the post-weaning phase (three to 11 months of age) a reduction in the prevalence of C. parvum is observed, however, in this stage an increase of C. bovis and C. ryanae infections is noticed. The latter two species are not considered zoonotic and show high specificity for bovine. C. andersoni is the species that mainly infects post-weaned calves and adult cattle. These parasitic species can infect other ruminants and rarely humans (DIXON et al., 2011).Due to the ability of this protozoan to infect different hosts and its constant presence in the environment, humans can acquire the infection in several ways, such as through ingestion of food and water contaminated with oocysts, direct contact with infected people (anthroponotic) or animals (zoonotic). Increases in environmental contamination and infected reservoirs assist in zoonotic transmission, as human cryptosporidiosis is also caused by certain species that also affect ruminants. The contact of humans with the feces of these animals that contains oocysts or contaminated water constitutes routes of transmission to humans (DEL COCO et al., 2014