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Unraveling of Borrelia burgdorferi sensu lato genospecies diversity in Portugal towards the development of more efficient diagnostic tools for Lyme disease
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Ixodídeos (carraças de corpo-duro), são importantes vetores de agentes patogénicos, responsáveis por doenças emergentes como a doença de Lyme (DL). Esta zoonose é causada por espiroquetas do complexo Borrelia burgdorferi sensu lato (s.l.) transmitidas por carraças da espécie Ixodes ricinus, o principal vetor na Europa. A DL é uma doença multisistémica com diversas apresentações clínicas e diagnóstico complexo. Em Portugal é ainda pouco diagnosticada e a notificação, apesar de obrigatória, é escassa.
A presente investigação teve como principal objetivo desenvolver ferramentas moleculares, nomeadamente um algoritmo de PCR em tempo real e uma amplificação isotérmica, para identificar as espécies de B. burgdorferi s.l. mais prevalentes em Portugal. O desenvolvimento deste objetivo permitiu também avaliar as características bioecológicas da
ixodofauna presente em nove distritos do país (norte, centro e sul) nos quais o vetor I. ricinus havia sido anteriormente reportado, e ainda determinar a taxa de infeção por B. burgdorferi s.l. no vetor e hospedeiros. Os resultados obtidos são apresentados sob a forma de artigos científicos (dez), dos quais sete estão publicados, dois em submissão e um em preparação.
Do conjunto dos resultados alcançados, importa realçar as variações observadas na distribuição das carraças para possivelmente para novas regiões, provavelmente relacionadas com alterações ao nível da paisagem, clima e vegetação, às quais as carraças são muito sensíveis. Acresce o facto de várias espécies de B. burgdorferi s.l. terem sido detetadas em carraças que não aquelas até agora reconhecidas como vetores. A espécie B. lusitaniae foi a mais prevalente no vetor, estando este presente em seis dos nove distritos selecionados.
Surpreendentemente no decurso deste estudo, identificaram-se três espécies do complexo Borrelia recorrente em carraças da vegetação, nomeadamente, B. miyamotoi numa ninfa I. ricinus, e duas possíveis ‘novas’ espécies do complexo Borrelia recorrente em carraças da espécie Haemaphysalis punctata e Rhipicephalus sanguineus.
Paralelamente, foi identificado DNA de B. burgdorferi s.l. em amostras biológicas de animais de estimação (cães e gatos) e de animais silváticos (javalis), confirmando a importância destes animais, principalmente os de estimação, enquanto sentinela para uma deteção precoce da DL, ajudando na determinação do risco de transmissão das espiroquetas de B. burgdorferi s.l. a humanos e outros animais com importância económica (ex. bovinos), em áreas geográficas restritas.
Foi também possível otimizar dois protocolos moleculares para o diagnóstico laboratorial da DL, um dos quais, um algoritmo de PCR em tempo real que permite a identificação de quatro das espécies de B. burgdorferi s.l. mais prevalentes em Portugal, apresentando elevada
sensibilidade e especificidade e contribuindo para um diagnóstico mais preciso da DL.
Alguns dos aspetos introduzidos e explorados nesta tese necessitam ainda de uma investigação mais detalhada. No entanto, este trabalho alerta para a introdução de possíveis ‘novas’ espécies do complexo de Borrelia recorrente em carraças de corpo-duro da vegetação, cuja patogenicidade é ainda desconhecida, mas que poderão tornar-se um risco para a saúde pública; contribui para a atualização espacial de áreas importantes na ecoepidemiologia da DL em Portugal; e inova no diagnóstico molecular desta zoonose, constituindo um valioso suporte para os clínicos, permitindo uma terapêutica mais direcionada dos doentes.
Ixodids (hard-body ticks), are the most important vectors of pathogenic agents, responsible for emerging diseases like Lyme disease (LD). This zoonosis is caused by spirochetes of Borrelia burgdorferi sensu lato (s.l.) complex, transmitted by Ixodes ricinus tick, the main vector in Europe. LD is a multisystem disorder with different clinical presentations, and a complex diagnosis. In Portugal is still underdiagnosed and the notification, although mandatory, is scarce. The main goal of this research was to develop molecular protocols, namely a TaqMan realtime PCR algorithm and an isothermal amplification, for the identification of the most prevalent genospecies of B. burgdorferi s.l. in Portugal. The development of this goal, also allowed to evaluate the bio-ecological characteristics of the ixodofauna in nine districts of the country (north, center and south), where the presence of I. ricinus tick was previously reported, and also to determinate B. burgdorferi s.l. infection rate in the vector and hosts. The obtained results are presented in 10 scientific papers, from which seven are published, two in submission and one in preparation. Of all the achieved results, is important to highlight the variations in the distribution of ticks possible into new regions, probably related to changes in the landscape, climate and vegetation, to which ticks are very sensitive. Moreover, several B. burgdorferi s.l. species were detected in ticks, apart from those commonly recognized as vectors. B. lusitaniae species was the most prevalent species in the vector, which was collected in six of the nine selected districts. Unexpectedly, during this study, three species from Relapsing Fever Borrelia complex were identified in questing ticks, namely B. miyamotoi in an I. ricinus nymph, and two possible new Relapsing Fever like-Borrelia in Haemaphysalis punctata and Rhipicephalus sanguineus tick species. Alongside, B. burgdorferi s.l. DNA was identified in biological samples from pets (dogs and cats) and sylvatic animals (wild boars), confirming the importance of these animals, mostly pets, as sentinel for early detection of emerging LD, helping to access the risk of B. burgdorferi s.l. spirochetes transmission to humans and other animals with economic importance (e.g. cattle), in restricted geographical areas. It was also possible to optimized two molecular protocols for LD laboratory diagnosis, one of which, a TaqMan real-time PCR algorithm allowing the identification of four of the most prevalent species of B. burgdorferi s.l. in Portugal, presenting high sensitivity and specificity, and contributing for a more accurate diagnosis of LD. Some of the subjects introduced and developed in this thesis deserve more detailed investigation. However, this work alerts for the introduction of possible ‘new’ Relapsing Fever Borrelia species in questing hard-body ticks, whose pathogenicity is still unknown, but it may become a risk to public health; contributes to the spatial update of important areas for LD eco-epidemiology in Portugal; and innovates in the molecular diagnosis of this zoonosis, being a valuable tool to clinicians, allowing a more accurate therapy of the patients.
Ixodids (hard-body ticks), are the most important vectors of pathogenic agents, responsible for emerging diseases like Lyme disease (LD). This zoonosis is caused by spirochetes of Borrelia burgdorferi sensu lato (s.l.) complex, transmitted by Ixodes ricinus tick, the main vector in Europe. LD is a multisystem disorder with different clinical presentations, and a complex diagnosis. In Portugal is still underdiagnosed and the notification, although mandatory, is scarce. The main goal of this research was to develop molecular protocols, namely a TaqMan realtime PCR algorithm and an isothermal amplification, for the identification of the most prevalent genospecies of B. burgdorferi s.l. in Portugal. The development of this goal, also allowed to evaluate the bio-ecological characteristics of the ixodofauna in nine districts of the country (north, center and south), where the presence of I. ricinus tick was previously reported, and also to determinate B. burgdorferi s.l. infection rate in the vector and hosts. The obtained results are presented in 10 scientific papers, from which seven are published, two in submission and one in preparation. Of all the achieved results, is important to highlight the variations in the distribution of ticks possible into new regions, probably related to changes in the landscape, climate and vegetation, to which ticks are very sensitive. Moreover, several B. burgdorferi s.l. species were detected in ticks, apart from those commonly recognized as vectors. B. lusitaniae species was the most prevalent species in the vector, which was collected in six of the nine selected districts. Unexpectedly, during this study, three species from Relapsing Fever Borrelia complex were identified in questing ticks, namely B. miyamotoi in an I. ricinus nymph, and two possible new Relapsing Fever like-Borrelia in Haemaphysalis punctata and Rhipicephalus sanguineus tick species. Alongside, B. burgdorferi s.l. DNA was identified in biological samples from pets (dogs and cats) and sylvatic animals (wild boars), confirming the importance of these animals, mostly pets, as sentinel for early detection of emerging LD, helping to access the risk of B. burgdorferi s.l. spirochetes transmission to humans and other animals with economic importance (e.g. cattle), in restricted geographical areas. It was also possible to optimized two molecular protocols for LD laboratory diagnosis, one of which, a TaqMan real-time PCR algorithm allowing the identification of four of the most prevalent species of B. burgdorferi s.l. in Portugal, presenting high sensitivity and specificity, and contributing for a more accurate diagnosis of LD. Some of the subjects introduced and developed in this thesis deserve more detailed investigation. However, this work alerts for the introduction of possible ‘new’ Relapsing Fever Borrelia species in questing hard-body ticks, whose pathogenicity is still unknown, but it may become a risk to public health; contributes to the spatial update of important areas for LD eco-epidemiology in Portugal; and innovates in the molecular diagnosis of this zoonosis, being a valuable tool to clinicians, allowing a more accurate therapy of the patients.
Descrição
Palavras-chave
Biologia molecular Microbiologia médica Ixodídeos Carraças Borreliose de Lyme Zoonose Espiroquetas Borreliose burgdorferi Portugal
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical