Adequação de técnicas moleculares para a detecção de Trypanosoma cruzi Chagas, 1909 em triatomíneos

Orientadora : Dra. Larissa ReifurCo-orientadora : Dra. Débora do Rocio KlisiowiczDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 11/03/2013Inclui referências : f. 48-55Área de concentração : ParasitologiaResumo: Em 2006, o Brasil recebeu a certificação internacional de eliminação da transmissão da doença de Chagas pelo Triatoma infestans, fornecida pela Organização Pan- Americana da Saúde, tendo o estado da Bahia como o último a receber a certificação. Apesar de o principal vetor ter sido eliminado, outras espécies assumiram o seu lugar e algumas delas apareceram infectadas com Trypanosoma cruzi. Atualmente, a Secretaria de Estado da Saúde da Bahia (SESAB) utiliza apenas o método direto a fresco para o diagnóstico de T. cruzi em conteúdo intestinal de triatomíneos. O método baseia-se na visualização de movimentação sugestiva do protozoário em fezes frescas, portanto, é um teste de baixa sensibilidade. A eficiência da detecção do parasito pode ser melhorada por técnicas moleculares. Neste trabalho foi realizada a adequação de duas técnicas, a amplificação em cadeia da polimerase (PCR) e a amplificação isotérmica mediada por alça (LAMP) para otimizar a detecção de T. cruzi em fezes e urina de triatomíneos do centro norte do estado da Bahia. As otimizações das técnicas de PCR e LAMP possibilitou detectar uma célula de T. cruzi (0,5 fg de DNA) e 1 ng de DNA da amostra controle, respectivamente. Para as amostras teste, 51 triatomíneos da espécie Triatoma brasiliensis e Triatoma pseudomaculata, foram utilizadas para a detecção do DNA de T. cruzi. A técnica de PCR foi capaz de detectar 31,36 % (16/51) de positividade em insetos, sendo mais sensível que o método direto a fresco, onde o mesmo detectou apenas uma amostra, correspondendo a 1,96 % (1/51). O uso de técnicas de detecção mais sensíveis e específicas é importante para apresentar a real situação de triatomíneos infectados por T. cruzi e a partir dos resultados, obter dados epidemiológicos mais acurados sobre a doença de Chagas. A presença de T. cruzi em triatomíneos com potencial transmissão vetorial é um indicativo que o parasito pode estar presente em uma carga parasitária suficiente para manter seu ciclo biológico na região. Palavras-chave: Trypanosoma cruzi, método direto a fresco, PCR, LAMP, triatomíneo.Abstract: In 2006, Brazil received the international certification of elimination of transmission of Chagas disease by Triatoma infestans, provided by the Pan American Health Organization. Bahia was the last Brazilian state to receive this certification. Although the Triatoma infestans has been eliminated from Brazil, other species proliferated and some of them have been found infected with Trypanosoma cruzi. Currently, the Secretaries of State for Health (SSH) adopt the direct fresh method for the diagnosis of T. cruzi in triatomine gut contents. This method is of low sensitivity and specificity as it is based on the visualization of movements suggestive of a tripanosomatid. Considering that parasite detection can be improved by molecular techniques, in this work, the polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) were used to optimize the detection of T. cruzi in feces and urine of triatomines. Using control DNA from an in vitro culture, it was possible to detect 0.5 fg of T. cruzi DNA, by PCR and 1 ng by LAMP. These reaction conditions were then used with DNA samples extracted from triatomines collected in the central north part of Bahia state. Out of the 51 triatomines collected, Triatoma pseudomaculata and Triatoma brasiliensis prevailed. Through PCR, 31,36 % (16/51) of the triatomines were positive for T. cruzi DNA, whereas the direct fresh method detected only one sample possibly positive, corresponding to 1,96 % (1/51). The LAMP technique probably needs more starting DNA in the sample to be able to amplify, thus it will be further optimized. In conclusion, the use of more sensitive and specific techniques is important to show the real situation of T. cruzi infected triatomines in Brazil. The presence of T. cruzi in triatomine species like T. brasiliensis and T. pseudomaculada, which are potential vectors for T. cruzi transmission, is an indication that the parasite may be present in sufficient amounts to maintain their life cycle in Bahia. Keywords: Trypanosoma cruzi, direct fresh method, PCR, LAMP, triatomine

Vaccuuming method as a successful strategy in the diagnosis of active infestation by Pediculus humanus capitis

Most human epidemiological and clinical studies use visual inspection of the hair and scalp to diagnose Pediculus humanus capitis, however this method has low sensitivity to diagnose active infestations (presence of nymphs and adult lice). Vacuuming the hair and scalp has been used as a diagnostic method, but there are no previous data comparing its effectiveness with visual inspection. The aim of this study was to determine the prevalence of overall infestation (nits and trophic stages), of active infestation by Pediculus humanus capitis, and to evaluate the effectiveness of vacuuming in comparison with the visual inspection. Visual inspection was performed by three examiners and vacuuming of the scalp by one investigator, with an adapted vacuum cleaner. A total of 166 children aged 4 to 10 years old were randomly selected from public schools in Southern Brazil. Considering the positive results obtained by both methods, the prevalence of overall infestation was 63.3%, whereas active infestation was 18.7%. The visual inspection was more effective on diagnosing overall infestation, however, its effectiveness to detect active infestation was lower, ranging from 0.6% (RR=3%, p<0.001) to 6.6% (RR=35%, p=0.001), depending on the number of examiners. The effectiveness of vacuuming to diagnose active infestation was higher than the one of visual inspection, with a prevalence rate of 16.3% (RR=87%, p=0.332). As presented in our study, the vacuuming method was 2.74 to 7.87 times most likely to detect active infestation, thus it could be adopted as a more accurate method to diagnose active pediculosis

Adequação de técnicas moleculares para a detecção de Trypanosoma cruzi Chagas, 1909 em triatomíneos

Orientadora : Dra. Larissa ReifurCo-orientadora : Dra. Débora do Rocio KlisiowiczDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 11/03/2013Inclui referências : f. 48-55Área de concentração : ParasitologiaResumo: Em 2006, o Brasil recebeu a certificação internacional de eliminação da transmissão da doença de Chagas pelo Triatoma infestans, fornecida pela Organização Pan- Americana da Saúde, tendo o estado da Bahia como o último a receber a certificação. Apesar de o principal vetor ter sido eliminado, outras espécies assumiram o seu lugar e algumas delas apareceram infectadas com Trypanosoma cruzi. Atualmente, a Secretaria de Estado da Saúde da Bahia (SESAB) utiliza apenas o método direto a fresco para o diagnóstico de T. cruzi em conteúdo intestinal de triatomíneos. O método baseia-se na visualização de movimentação sugestiva do protozoário em fezes frescas, portanto, é um teste de baixa sensibilidade. A eficiência da detecção do parasito pode ser melhorada por técnicas moleculares. Neste trabalho foi realizada a adequação de duas técnicas, a amplificação em cadeia da polimerase (PCR) e a amplificação isotérmica mediada por alça (LAMP) para otimizar a detecção de T. cruzi em fezes e urina de triatomíneos do centro norte do estado da Bahia. As otimizações das técnicas de PCR e LAMP possibilitou detectar uma célula de T. cruzi (0,5 fg de DNA) e 1 ng de DNA da amostra controle, respectivamente. Para as amostras teste, 51 triatomíneos da espécie Triatoma brasiliensis e Triatoma pseudomaculata, foram utilizadas para a detecção do DNA de T. cruzi. A técnica de PCR foi capaz de detectar 31,36 % (16/51) de positividade em insetos, sendo mais sensível que o método direto a fresco, onde o mesmo detectou apenas uma amostra, correspondendo a 1,96 % (1/51). O uso de técnicas de detecção mais sensíveis e específicas é importante para apresentar a real situação de triatomíneos infectados por T. cruzi e a partir dos resultados, obter dados epidemiológicos mais acurados sobre a doença de Chagas. A presença de T. cruzi em triatomíneos com potencial transmissão vetorial é um indicativo que o parasito pode estar presente em uma carga parasitária suficiente para manter seu ciclo biológico na região. Palavras-chave: Trypanosoma cruzi, método direto a fresco, PCR, LAMP, triatomíneo.Abstract: In 2006, Brazil received the international certification of elimination of transmission of Chagas disease by Triatoma infestans, provided by the Pan American Health Organization. Bahia was the last Brazilian state to receive this certification. Although the Triatoma infestans has been eliminated from Brazil, other species proliferated and some of them have been found infected with Trypanosoma cruzi. Currently, the Secretaries of State for Health (SSH) adopt the direct fresh method for the diagnosis of T. cruzi in triatomine gut contents. This method is of low sensitivity and specificity as it is based on the visualization of movements suggestive of a tripanosomatid. Considering that parasite detection can be improved by molecular techniques, in this work, the polymerase chain reaction (PCR) and the loop-mediated isothermal amplification (LAMP) were used to optimize the detection of T. cruzi in feces and urine of triatomines. Using control DNA from an in vitro culture, it was possible to detect 0.5 fg of T. cruzi DNA, by PCR and 1 ng by LAMP. These reaction conditions were then used with DNA samples extracted from triatomines collected in the central north part of Bahia state. Out of the 51 triatomines collected, Triatoma pseudomaculata and Triatoma brasiliensis prevailed. Through PCR, 31,36 % (16/51) of the triatomines were positive for T. cruzi DNA, whereas the direct fresh method detected only one sample possibly positive, corresponding to 1,96 % (1/51). The LAMP technique probably needs more starting DNA in the sample to be able to amplify, thus it will be further optimized. In conclusion, the use of more sensitive and specific techniques is important to show the real situation of T. cruzi infected triatomines in Brazil. The presence of T. cruzi in triatomine species like T. brasiliensis and T. pseudomaculada, which are potential vectors for T. cruzi transmission, is an indication that the parasite may be present in sufficient amounts to maintain their life cycle in Bahia. Keywords: Trypanosoma cruzi, direct fresh method, PCR, LAMP, triatomine

Aspectos de susceptibilidade à pediculose

Orientadora : Drª Débora do Rocio KlisiowiczCoorientador : Drª. Vânia Aparecida VicenteTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 30/11/2017Inclui referênciasResumo: A pediculose, doença causada pelo artrópode Pediculus humanus capitis é considerada um problema de saúde pública, pois afeta milhões de pessoas em todo o mundo, no entanto, crianças em idade escolar são as mais acometidas. É de conhecimento popular que alguns indivíduos são mais suscetíveis à parasitose que outros, porém, fatores inerentes ao hospedeiro, que podem influenciar na suscetibilidade à essa doença não estão totalmente esclarecidos. Assim, o objetivo do presente estudo foi isolar e identificar a microbiota do couro cabeludo e analisar se determinadas caracteríscas da morfologia do cabelo (tipo, cor, comprimento, escamas e diâmetro) podem interferir na predisposição à doença. Para a análise da microbiota do couro cabeludo, 10 crianças com pediculose (grupo A) e 10 crianças sem pediculose (grupo B) foram analisadas. As amostras da microbiota do couro cabeludo foram coletadas através de swabs que foram friccionados na cabeça e acondicionados em meios de transporte Stuart. O isolamento da microbiota fúngica foi feito em Ágar Sabouraud (SBA) com tetraciclina e o isolamento bacteriano foi feito em ágar sangue. A identificação molecular foi realizada através de sequenciamento da região 16S e ITS do DNA ribossomal bacteriano e fúngico, respectivamente. Um total de 186 isolados foram obtidos sendo 35 bactérias e 40 fungos (grupo A) e 47 bactérias e 64 fungos (grupo B). Os resultados sugerem que a microbiota isolada pode estar envolvida na suscetibilidade à pediculose. Em relação à microbiota bacteriana, no grupo A, Staphylococcus captis foi significativamente diferente (P 0,05). No entanto, quando comparado os grupos etários, meninos com idades de 2-3 anos estavam mais infestados que as meninas. Palavras-chave: Piolho; cabelo; bactérias; fungos e prevalência.Abstract:Pediculosis, a disease caused by the arthropod Pediculus humanus capitis is considered a public health problem as it affects millions of people around the world. However, school children are the most affected. It is popular knowledge that some individuals are more susceptible to parasitosis than others, but factors inherent to the host that may influence the susceptibility to this disease are not well understood. In this context, the objective of the present study was to isolate and identify the scalp microbiota and to analyze if certain characteristics of hair morphology (scales, type, color, length and diameter) may interfere with predisposition to the disease. For analysis of the scalp microbiota, 10 children with pediculosis (group A) with 10 children without pediculosis (group B) were analyzed. Samples of the scalp microbiota were collected through swabs that were rubbed on the children's heads and packed into Stuart transport medium. Isolation of the fungal microbiota was done in Sabouraud Agar (SBA) with tetracycline and bacterial isolation was done on blood agar. Sequencing was done for the 16S and 18S region of the bacterial and fungal ribosomal DNA, respectively. A total of 186 isolates were obtained from group A, from these 35 bacteria and 40 fungi and for group B, 47 bacteria and 64 fungi were isolated. The results suggest that the isolated microbiota may be involved in pediculosis susceptibility. In relation to the bacterial microbiota, in group A, Staphylococcus capitis was significantly different (p 0.05). However, when compared to age groups, boys aged 2-3 years were more infested than girls. Keywords: Head lice; hair; bacterium; fungi and prevalenc

Pediculosis capitis risk factors in schoolchildren: hair thickness and hair length

The human head lice is a cosmopolitan ectoparasite that causes pediculosis. The main way of spreading lice is through direct head-to-head contact. It is popular knowledge that some individuals are more susceptible to contracting head lice than others. Reports of individuals who have never been affected by the disease are common, even living in the same environment and under the same conditions as people who regularly have lice infestations. Previous research has been carried out on the risk of this infection associated with different human factors like gender or age. However, studies on the influence of the individual hair characteristics are scarce. The objective of the study was to analyze the pediculosis risk using geographical location, gender, age and individual hair characteristics as variables. Pediculosis was diagnosed through the detection of living lice in the hair. This cross-sectional school-based epidemiological study was conducted in 310 schoolchildren aged 1 to 13 years of schools in 4 municipalities situated in the State of Paraná, Brazil. The prevalence of head louse infection in primary school students was 49.35 %. The Odds Ratio of presence of pediculosis (OR) was estimated using multivariate logistic regression analysis. The results obtained indicate that hair length and thickness increase the risk of infection. Furthermore, the inclusion of hair color, hair shape, kind of hair-scale as covariates increases the risk of pediculosis, indicating that these variables partly explain this susceptibility and that pediculosis is independent of gender. A smaller hair diameter may favor insect fixation to the hair in the nymphal phases. These results may explain why girls are a greater risk as they let their hair grow for cultural reasons, i.e., being of female gender is an agglutinating variable. The conclusions drawn may explain the discrepancies obtained in previous analyses

High occurrence of Calodium hepaticum (syn. Capillaria hepatica) spurious infection in a village in the Atlantic Forest of southern Brazil

Calodium hepaticum (syn. Capillaria hepatica) is a nematode of the Capillariidae family that infects rodents and other mammals. In Brazil, human spurious infections of C. hepaticum have been detected in indigenous or rural communities from the Amazon Basin, but not in the southern states of the country. Here, we report the highest occurrence (13.5% of 37 residents) of C. hepaticum human spurious infection detected in Brazil and the first record in a southern region, Guaraqueçaba. The finding is explained by the area being located in the Atlantic Forest of the state of Paraná, surrounded by preserved forests and because the inhabitants consume the meat of wild mammals