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Desenvolvimento de métodos moleculares para a deteção de Trypanosoma spp em glossinas (Diptera: Glossinidae) da República da Guiné Bissau
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As moscas do género Glossina são vetores de patologias provocadas por várias espécies de parasita do género Trypanosoma, como a Tripanossomose Humana Africana e as Tripanossomoses Animais. A correta identificação destes tripanossomas é um ponto fulcral quando se procura determinar o risco de doença que determinadas populações de glossinas podem provocar.
No presente trabalho foram utilizadas e adaptadas diversas metodologias de diagnóstico molecular, nomeadamente o PCR em tempo real e o PCR-RFLP, que permitiram, através da utilização de primers genéricos, a determinação da carga parasitária e a correta identificação dos tripanossomas presentes no vetor. Os primers denominados Tryp18SF e Tryp18SR foram desenvolvidos especificamente para serem utilizados na identificação das espécies de tripanossoma utilizando PCR em tempo real com metodologia SYBR® Green I e PCR-RFLP. Os primers denominados Tryp18S2F, Tryp18S2R e sonda Tryp18S2P foram desenvolvidos para a quantificação dos parasitas presentes nas amostras utilizando PCR em tempo real com metodologia Taqman®.
Foi estudada uma amostra de 762 glossinas provenientes do território da República da Guiné-Bissau, zona atualmente considerada como estando livre de Tripanossomose Humana Africana, mas onde não são realizadas atividades de recenseamento de casos desde o final da ocupação portuguesa.
Das 762 glossinas utilizadas, 241 estavam infetadas com tripanossomas, o que representa uma percentagem de infeção geral de 31,6 %. Destas glossinas, 28.63 % encontravam-se infetadas com Trypanosoma grayi, 14.11 % com Trypanosoma congolense, 7.05 % com Trypanosoma vivax e 0.83 % com Trypanosoma brucei brucei, tendo as infeções mistas representado 1.66 %. Não foi possível a identificação conclusiva ao nível da espécie em 48.13 % das amostras positivas, tendo estas ficado consideradas como Trypanosoma spp. Não foram identificados vetores infetados com tripanossomas responsáveis pelas patologias humanas.
A utilização de primers genéricos para a identificação permitiu obviar o número de reações necessárias para identificar corretamente o parasita responsável pela infeção, e este trabalho é de resto a primeira descrição da utilização de primers genéricos que permitam a identificação de Trypanosoma grayi.
Esta é também a primeira descrição da utilização de PCR em tempo real com metodologia Taqman® para quantificação de tripanossomas, e a sua utilização com primers genéricos aumenta a aplicabilidade em estudos epidemiológicos de grande escala.
Palavras-chave: PCR em tempo real; PCR-RFLP; República da Guiné-Bissau; Glossina; Trypanosoma; Tripanossomose.
Flies of the genus Glossinaare vectors of Human African Trypanosomosis and Animal Trypanosomosis, diseases which are caused by various parasites of the genus Trypanosoma. Correct identification of trypanosomes is essential to assess the disease risk posed by certain Glossinapopulations.Several molecular diagnosis methodologies were appliedand adapted in the present work, namely real time PCR and PCR-RFLP, which allowed, through de use of generic primers, to determine the parasite loadand the correct identification of the trypanosomes present in the vector. One pair of primers, named Tryp18SF and Tryp18SR, were specifically designed for the identification of trypanosome species using real time PCR with SYBR® Green I methodology and PCR-RFLP. Another pair of primers, named Tryp18S2F, Tryp18S2R, along with the probe Tryp18S2P, wasdesigned for the quantification ofparasites present in the samples using real time PCR with Taqman® probemethodology.A sample consisting of 762 flies of the genus Glossinacaptured in the Republic of Guinea-Bissau was studied. This territory is currently listed as being free of Human African Trypanosomosis, but no large-scale epidemiologicsurveys regarding the disease have been made since the end of the portuguese occupation.Of the 762studied flies, 241 were infected with trypanosomes, which represents na overallinfection percentage of 31.6 %. Of these flies, 28.63 % were infected with Trypanosoma grayi, 14.11 % with Trypanosoma congolense, 7.05 % with Trypanosoma vivaxand 0.83 % with Trypanosoma brucei brucei, whereas mixed infections represented 1.66 %. No conclusive identification atthe species level was possible in48.13 % of the positive samples, and so these parasites wereconsidered to be Trypanosoma spp. Vectors infected with trypanossomes responsible for human diseases were not found.The use of generic primers for identification purposes allowedusto reduce the number of reactions needed to perform acorrect identification of the parasite responsible for the infection, and this work is the first to usegeneric primers that allow the identification of Trypanosoma grayi.This is also the first work to usereal time PCR with Taqman® probe methodology for the quantification of trypanosomes, and the use of generic primers will greatly improve its aplicability to large-scale epidemiological surveys.
Flies of the genus Glossinaare vectors of Human African Trypanosomosis and Animal Trypanosomosis, diseases which are caused by various parasites of the genus Trypanosoma. Correct identification of trypanosomes is essential to assess the disease risk posed by certain Glossinapopulations.Several molecular diagnosis methodologies were appliedand adapted in the present work, namely real time PCR and PCR-RFLP, which allowed, through de use of generic primers, to determine the parasite loadand the correct identification of the trypanosomes present in the vector. One pair of primers, named Tryp18SF and Tryp18SR, were specifically designed for the identification of trypanosome species using real time PCR with SYBR® Green I methodology and PCR-RFLP. Another pair of primers, named Tryp18S2F, Tryp18S2R, along with the probe Tryp18S2P, wasdesigned for the quantification ofparasites present in the samples using real time PCR with Taqman® probemethodology.A sample consisting of 762 flies of the genus Glossinacaptured in the Republic of Guinea-Bissau was studied. This territory is currently listed as being free of Human African Trypanosomosis, but no large-scale epidemiologicsurveys regarding the disease have been made since the end of the portuguese occupation.Of the 762studied flies, 241 were infected with trypanosomes, which represents na overallinfection percentage of 31.6 %. Of these flies, 28.63 % were infected with Trypanosoma grayi, 14.11 % with Trypanosoma congolense, 7.05 % with Trypanosoma vivaxand 0.83 % with Trypanosoma brucei brucei, whereas mixed infections represented 1.66 %. No conclusive identification atthe species level was possible in48.13 % of the positive samples, and so these parasites wereconsidered to be Trypanosoma spp. Vectors infected with trypanossomes responsible for human diseases were not found.The use of generic primers for identification purposes allowedusto reduce the number of reactions needed to perform acorrect identification of the parasite responsible for the infection, and this work is the first to usegeneric primers that allow the identification of Trypanosoma grayi.This is also the first work to usereal time PCR with Taqman® probe methodology for the quantification of trypanosomes, and the use of generic primers will greatly improve its aplicability to large-scale epidemiological surveys.
Descrição
Palavras-chave
PCR em tempo real PCR-RFLP República da Guiné-Bissau Glossina Trypanossoma Trypanossomose Biologia molecular Metodologia de análise e diagnóstico Tripanossoma spp Glossinas Diptera glossinidae
Contexto Educativo
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Instituto Higiene de Medicina Tropical