Developing a NGS panel for diagnosis of Lyme disease and its co-infections
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10400.6/9934 |
Resumo: | Lyme disease, also known as Lyme borreliosis, is the most common-tick borne disease in the northern hemisphere, with 300,000 cases estimated each year only in the United States. In addition to the transmission of Borrelia burgdorferi sensu lato (s.l.), the bacteria responsible for Lyme disease, ticks of the Ixodes ricinus complex are vectors for other infections, with the most common being babesiosis and human granulocytic anaplasmosis. The presence of co-infections may cause more severe clinical manifestations and their misdiagnosis may lead to an inappropriate treatment. The only accepted method to diagnose Lyme borreliosis, currently, is a serologic test based in a two-tier approach using an enzyme immunoassay (EIA) complemented with a Western immunoblot. This indirect method for Borrelia burgdorferi s.l. detection suffers from lack of sensitivity in the early stage of the disease, due to the time window needed for antibodies to be produced. Over the past few decades, many studies have been carried using direct methods, such as culture and PCR, in order to develop an alternative diagnostic method for this infectious disease, however this tests also suffer from a high rate of false negatives. In this study, a NGS panel was developed, for the Illumina's Miseq platform, which allows the simultaneous diagnose of Lyme disease and its most common co-infections. This panel includes seven specific primer pairs that target a gene fragment of each of the included pathogenic species, in regions that allow the genospecies distinction. The panel was tested in the library preparation for sequencing using samples of whole blood and samples of extracted DNA from the whole blood. Along with the developed panel, in order to evaluate its sensibility, both types of samples were also tested with specific primers that target the V3 and V4 regions of the 16S ribosomal RNA gene, widely used in microbiome analysis. Due to the difficulty to obtain samples from patients with Lyme disease and with the other infections covered by the panel, in this study, five samples of whole blood from patients diagnosed with babesiosis were tested, along with a positive and a negative controls. The condition that has shown better results was the one using extracted DNA from the blood combined with the panel, which detected Babesia microti DNA in all five samples of the infected patients. Despite the need to test this method in samples from patients with Lyme disease and the remaining infections included in the developed panel, the results obtained in this study are promising for the future use of this method as an alternative to the current tests, especially in the initial phase of infection. |
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Developing a NGS panel for diagnosis of Lyme disease and its co-infectionsCoinfecçõesDiagnósticoDoença de LymeNgsDomínio/Área Científica::Ciências Médicas::Ciências BiomédicasLyme disease, also known as Lyme borreliosis, is the most common-tick borne disease in the northern hemisphere, with 300,000 cases estimated each year only in the United States. In addition to the transmission of Borrelia burgdorferi sensu lato (s.l.), the bacteria responsible for Lyme disease, ticks of the Ixodes ricinus complex are vectors for other infections, with the most common being babesiosis and human granulocytic anaplasmosis. The presence of co-infections may cause more severe clinical manifestations and their misdiagnosis may lead to an inappropriate treatment. The only accepted method to diagnose Lyme borreliosis, currently, is a serologic test based in a two-tier approach using an enzyme immunoassay (EIA) complemented with a Western immunoblot. This indirect method for Borrelia burgdorferi s.l. detection suffers from lack of sensitivity in the early stage of the disease, due to the time window needed for antibodies to be produced. Over the past few decades, many studies have been carried using direct methods, such as culture and PCR, in order to develop an alternative diagnostic method for this infectious disease, however this tests also suffer from a high rate of false negatives. In this study, a NGS panel was developed, for the Illumina's Miseq platform, which allows the simultaneous diagnose of Lyme disease and its most common co-infections. This panel includes seven specific primer pairs that target a gene fragment of each of the included pathogenic species, in regions that allow the genospecies distinction. The panel was tested in the library preparation for sequencing using samples of whole blood and samples of extracted DNA from the whole blood. Along with the developed panel, in order to evaluate its sensibility, both types of samples were also tested with specific primers that target the V3 and V4 regions of the 16S ribosomal RNA gene, widely used in microbiome analysis. Due to the difficulty to obtain samples from patients with Lyme disease and with the other infections covered by the panel, in this study, five samples of whole blood from patients diagnosed with babesiosis were tested, along with a positive and a negative controls. The condition that has shown better results was the one using extracted DNA from the blood combined with the panel, which detected Babesia microti DNA in all five samples of the infected patients. Despite the need to test this method in samples from patients with Lyme disease and the remaining infections included in the developed panel, the results obtained in this study are promising for the future use of this method as an alternative to the current tests, especially in the initial phase of infection.A doença de Lyme, também conhecida como borreliose de Lyme, é a doença transmitida por carraças mais comum no hemisfério norte, com 300.000 novos casos estimados anualmente só nos Estados Unidos da América. Para além da transmissão de Borrelia burgdorferi sensu lato (s.l.), bactéria responsável pela doença de Lyme, as carraças do complexo Ixodes ricinus são vetores de outras infeções, sendo as mais comuns a babesiose e a anaplasmose granulocítica humana. A presença de coinfecções pode causar manifestações clínicas mais severas e o seu incorreto diagnóstico pode levar a um tratamento inapropriado. O único método aceite para o diagnóstico da borreliose de Lyme, atualmente, é um teste sorológico baseado numa abordagem de dois níveis no qual é realizado um imunoensaio enzimático complementado por um western blot. Este método indireto para a deteção de Borrelia burgdorferi s.l. carece de sensitividade na fase inicial da infeção, devido ao tempo necessário para os anticorpos serem produzidos. Ao longo das últimas décadas, vários estudos usando métodos diretos, tais como cultura e Polimerase Chain Reaction (PCR), foram realizados com o objetivo de desenvolver um método de diagnóstico alternativo para esta doença infeciosa. Contudo, estes testes demonstraram uma taxa elevada de falsos negativos. Neste estudo, foi criado um painel de Next Generation Sequencing (NGS), para a plataforma MiSeq da Illumina, que permite o diagnóstico simultâneo da doença de Lyme e das suas coinfecções mais frequentes. Este painel inclui sete pares de primers específicos para um fragmento de um gene de cada uma das espécies patogénicas incluídas, em regiões que permitem a distinção entre genoespécies. O painel foi testado na preparação das bibliotecas para sequenciação com amostras de sangue total e com amostras de ADN extraído do sangue total. A par do teste com o painel desenvolvido, com o intuito de avaliar a sensibilidade do mesmo, os dois tipos de amostras foram também testados com primers específicos para as regiões V3 e V4 do gene 16S do ARN ribossomal, amplamente usados na análise de microbiomas. Devido à dificuldade em obter amostras de pacientes com doença de Lyme e com as outras infeções abrangidas pelo painel, neste trabalho, foram testadas cinco amostras de sangue de pacientes diagnosticados com babesiose, juntamente com os controlos positivo e negativo. A condição que demonstrou melhores resultados foi aquela em que foi usado ADN extraído de sangue em combinação com o painel, com a qual foi possível identificar ADN de Babesia microti nas cinco amostras de pacientes infetados. Apesar da necessidade de testar o método em amostras de pacientes com doença de Lyme e com as restantes infeções incluídas no painel desenvolvido, os resultados obtidos neste trabalho demonstram-se promissores para a futura utilização deste método como alternativa aos exames atuais, especialmente na fase inicial da infeção.Gonçalves, Isabel Maria Theriaga Mendes VarandaDoria, GonçalouBibliorumCoelho, Eduardo Augusto2020-10-08T00:30:15Z2018-10-82018-11-022018-11-02T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.6/9934TID:202354083enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-12-15T09:51:18Zoai:ubibliorum.ubi.pt:10400.6/9934Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:50:01.144105Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
title |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
spellingShingle |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections Coelho, Eduardo Augusto Coinfecções Diagnóstico Doença de Lyme Ngs Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas |
title_short |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
title_full |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
title_fullStr |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
title_full_unstemmed |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
title_sort |
Developing a NGS panel for diagnosis of Lyme disease and its co-infections |
author |
Coelho, Eduardo Augusto |
author_facet |
Coelho, Eduardo Augusto |
author_role |
author |
dc.contributor.none.fl_str_mv |
Gonçalves, Isabel Maria Theriaga Mendes Varanda Doria, Gonçalo uBibliorum |
dc.contributor.author.fl_str_mv |
Coelho, Eduardo Augusto |
dc.subject.por.fl_str_mv |
Coinfecções Diagnóstico Doença de Lyme Ngs Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas |
topic |
Coinfecções Diagnóstico Doença de Lyme Ngs Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas |
description |
Lyme disease, also known as Lyme borreliosis, is the most common-tick borne disease in the northern hemisphere, with 300,000 cases estimated each year only in the United States. In addition to the transmission of Borrelia burgdorferi sensu lato (s.l.), the bacteria responsible for Lyme disease, ticks of the Ixodes ricinus complex are vectors for other infections, with the most common being babesiosis and human granulocytic anaplasmosis. The presence of co-infections may cause more severe clinical manifestations and their misdiagnosis may lead to an inappropriate treatment. The only accepted method to diagnose Lyme borreliosis, currently, is a serologic test based in a two-tier approach using an enzyme immunoassay (EIA) complemented with a Western immunoblot. This indirect method for Borrelia burgdorferi s.l. detection suffers from lack of sensitivity in the early stage of the disease, due to the time window needed for antibodies to be produced. Over the past few decades, many studies have been carried using direct methods, such as culture and PCR, in order to develop an alternative diagnostic method for this infectious disease, however this tests also suffer from a high rate of false negatives. In this study, a NGS panel was developed, for the Illumina's Miseq platform, which allows the simultaneous diagnose of Lyme disease and its most common co-infections. This panel includes seven specific primer pairs that target a gene fragment of each of the included pathogenic species, in regions that allow the genospecies distinction. The panel was tested in the library preparation for sequencing using samples of whole blood and samples of extracted DNA from the whole blood. Along with the developed panel, in order to evaluate its sensibility, both types of samples were also tested with specific primers that target the V3 and V4 regions of the 16S ribosomal RNA gene, widely used in microbiome analysis. Due to the difficulty to obtain samples from patients with Lyme disease and with the other infections covered by the panel, in this study, five samples of whole blood from patients diagnosed with babesiosis were tested, along with a positive and a negative controls. The condition that has shown better results was the one using extracted DNA from the blood combined with the panel, which detected Babesia microti DNA in all five samples of the infected patients. Despite the need to test this method in samples from patients with Lyme disease and the remaining infections included in the developed panel, the results obtained in this study are promising for the future use of this method as an alternative to the current tests, especially in the initial phase of infection. |
publishDate |
2018 |
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2018-10-8 2018-11-02 2018-11-02T00:00:00Z 2020-10-08T00:30:15Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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