Developing a NGS panel for diagnosis of Lyme disease and its co-infections

• Autor(a) principal: Coelho, Eduardo Augusto
• 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
• topic: Coinfecções; Diagnóstico; Doença de Lyme; Ngs; Domínio/Área Científica::Ciências Médicas::Ciências Biomédicas

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.