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Anaplasma phagocytophilum and human granulocytic anaplasmosis in Portugal
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Anaplasma phagocytophilum e anaplasmose granulocítica têm vindo a merecer um crescente interesse na comunidade científica, facto demonstrado pelo crescente número de publicações que surgiram nas duas últimas décadas, particularmente desde que foram documentados os primeiros casos de anaplasmose granulocítica humana (AGH). O reconhecimento da importância de A. phagocytophilum em termos de Saúde Pública na América do Norte e na Europa, associado à recente detecção do agente em Portugal, pôs em evidência a necessidade de estudos mais detalhados sobre a emergência deste Anaplasmataceae e de AGH no nosso país.
Inicialmente baseado num treino metodológico em Unidades de Investigação especializadas em Anaplasmataceae, o presente trabalho permitiu a transferência da tecnologia correntemente aplicada ao estudo de A. phagocytophilum e consequentemente o desenvolvimento desta linha de investigação no Centro de Estudos de Vectores e Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge (CEVDI/INSA). Ao estabelecer as bases para um estudo concertado em A. phagocytophilum e AGH, este trabalho tornou possível uma abordagem abrangente a aspectos essenciais da ecobiologia deste Anaplasmaceae e da doença que lhe está associada, focando-se na identificação de potenciais ixodídeos vectores, de mamíferos envolvidos no ciclo natural do agente, quer como reservatórios quer como hospedeiros susceptíveis, recolhendo evidências que apontam para a possibilidade de exposição do homem a este agente em Portugal.
Os estudos levados a cabo nos ixodídeos demonstram o envolvimento de duas espécies de Ixodes no ciclo de A. phagocytophilum, concretamente Ixodes ricinus na Ilha da Madeira e I. ventalloi no continente. A detecção de ADN do agente em I. ricinus vem reforçar estudos anteriores, sugerindo a sua persistência na Ilha da Madeira. Esta tese acrescenta ainda um novo dado ao conhecimento da história natural de A. phagocytophilum, com a primeira detecção de infecção em I. ventalloi. O facto de alguns dos artrópodes infectados terem sido obtidos a partir de gatos domésticos, não só acrescenta este mamífero à lista nacional de hospedeiros vertebrados parasitados por I. ventalloi mas sugere também o seu potencial envolvimento no ciclo de transmissão de A. phagocytophilum. A análise molecular dos dados obtidos evidencia ainda a existência de diferentes genótipos deste Anaplasmaceae nos ixodideos portugueses. As sequências nucleotídicas obtidas sugerem a proximidade genética entre A. phagocytophilum detectado em I. ricinus na Ilha da Madeira e estirpes isoladas de humanos na América do Norte, bem como de sequências detectadas na Europa Central e do Norte. No entanto estes genótipos divergem das sequências nucleotídicas obtidas a partir do I. ventalloi no continente, que representa um novo genótipo de patogenia não determinada.São também apresentadas evidências serológicas da exposição a A. phagocytophilum ou a outro agente antigenicamente semelhante em ratinhos Mus spretus, cavalos e canídeos domésticos no continente. A análise molecular das amostras biológicas obtidas a partir das populações em estudo permitiu a detecção da primeira infecção activa por A. phagocytophilum em vertebrados portugueses, com a identificação de ADN do agente num dos cavalos seropositivos. Sendo as sequências nucleotídicas obtidas semelhantes à das estirpes associadas aos casos de infecção em humanos, é reforçada uma vez mais a possibilidade de AGH ocorrer em Portugal. Esta tese refere ainda a identificação de outra Anaplasmataceae filogenicamente próxima de A. phagocytophilum - A. platys, detectada em canídeos seropositivos, evidenciando a possibilidade de ocorrerem reacções cruzadas entre estes dois agentes. Estes dados salientam a importância da vigilância em termos veterinários da ocorrência não só de anaplasmose granulocítica mas também de trombocitopénia cíclica infecciosa canina como causa de doença associada a ixodídeos em Portugal.
Para averiguar a possibilidade de exposição humana a A. phagocytophilum foram realizados estudos serológicos e moleculares retrospectivos e prospectivos, em amostras biológicas recebidas no CEVDI/INSA durante 2000-2006 para o diagnóstico laboratorial de pacientes com suspeita de doença associada a ixodídeos. Os resultados provam a existência de reacções serológicas positivas na população estudada, incluindo alguns casos que satisfazem os critérios serológicos para definição de AGH, embora sem evidência de infecção activa. Adicionalmente, alguns dos doentes seropositivos apresentaram evidência de infecção causada por outros agentes transmitidos por ixodídeos ou por bactérias relacionadas, nomeadamente borreliose de Lyme, febre Q e bartonelose. Embora estes resultados possam ser interpretados como falsos positivos resultantes de reacções cruzadas, a possibilidade de se tratarem de co-infecções ou exposições anteriores a vários agentes transmitidos por ixodídeos do género Ixodes também deverá ser considerada. Em suma, estes resultados apontam para a necessidade de desenvolver o diagnóstico de A. phagocytophilum, nomeadamente no que diz respeito à aplicação de técnicas directas de detecção do agente, bem como à integração dos dados do diagnóstico laboratorial em casos de suspeita de doença associada a Ixodes spp.
Embora muitos dos aspectos focados neste trabalho requeiram uma investigação mais detalhada, esta tese contribui positivamente para a compreensão das questões fundamentais relacionadas com a ocorrência de A. phagocytophilum e o seu potencial como agente de doença em Portugal. Termina-se com a esperança de que estes primeiros passos ajudem a delinear novas linhas de investigação no âmbito do estudo da anaplasmose granulocítica e outras doenças emergentes associadas a espécies do género Ixodes.
Anaplasma phagocytophilum and granulocytic anaplasmosis are of increasing interest to the scientific community as indicated by the expanding number of reports published in the past two decades, and especially since the emergence of the first cases of human granulocytic anaplasmosis (HGA). The growing recognition of the Public Health importance of A. phagocytophilum in North America and in Europe, along with its recent detection in Portugal has signalled the need for more detailed studies that address the emergence of HGA and its causative agent in our country. Initially based on an methodological training in research units dedicated to Anaplasmataceae, this work enabled the transfer of technology as currently applied to A. phagocytophilum research and made possible the development of a new line of investigation at Centro de Estudos de Vectores e Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge (CEVDI/INSA). By establishing the foundations for concerted study on A. phagocytophilum and HGA, the work described herein has facilitated development of a broad approach toward fundamental issues in Anaplasmataceae ecobiology and disease by focusing attention on identification of potential ixodid vectors, mammals likely to be involved in the infectious agent’s life cycles either as reservoirs or affected hosts, and garnered the evidence indicating the potential for human exposure in Portugal. The studies conducted in ixodid ticks proves the involvement of two Ixodes species in A. phagocytophilum cycles, including Ixodes ricinus on Madeira Island and I. ventalloi on the mainland. The detection of A. phagocytophilum DNA in I. ricinus reinforces prior studies and suggests its persistence on Madeira Island. This thesis also adds new data to the understanding of the natural history of A. phagocytophilum by providing the first evidence of infections in I. ventalloi ticks. The fact that some infected arthropods infest domestic cats not only mandates the inclusion of these mammals on the national list of vertebrate hosts parasitized by I. ventalloi ticks, but also shows their potential complicity in A. phagocytophilum maintenance. Moreover, molecular data shows the existence of A. phagocytophilum variant genotypes in Portuguese ticks. Partial gene sequences from infected ticks demonstrates nucleotide polymorphisms that support a close relationship of A. phagocytophilum on Madeira Island I. ricinus to North American strains isolated from humans as well as genotypes detected in Central and Northern Europe. Yet, these variants diverge from those found in mainland I. ventalloi, which represents a new genotype of undetermined pathogenicity. Serological evidence of exposure to A. phagocytophilum or a close related agent is shown in Mus spretus mice, horses, and dogs in mainland. Molecular analysis of biological samples from these animal populations provides the first definitive evidence of A. phagocytophilum active infection in Portuguese vertebrates with detection of its DNA in one seropositive horse from mainland Portugal, where the A.phagocytophilum genotype found is closely related to strains isolated from humans, suggesting the potential for HGA in Portugal. This thesis further extends study to identification of another closely related Anaplasma species, and its potential for serological cross-reactions with A. phagocytophilum, as evident with the detection of A. platys DNA in seropositive dogs. These data also underscore the importance of veterinarians maintaining vigilance for detection of not only granulocytic anaplasmosis but also canine infectious cyclic thrombocytopenia as causes of tick-borne diseases in Portugal. Both prospective and retrospective serological and molecular investigations of human exposure to A. phagocytophilum were performed on samples received at CEVDI/INSA for the laboratory diagnosis of patients with suspected tick-borne diseases during 2000-2006. The results provide evidence for seropositive Portuguese patients, including cases that fulfil serological criteria for HGA, although active infections were not detected. Moreover some seropositive patients had additional evidence of other tick-borne agents or related bacteria infections, including Lyme borreliosis, Q fever and bartonellosis. Although possibly false positive cross-reactions to shared antigens, these reactions potentially could be the result of active dual infections, or past exposure to several agents transmitted by Ixodes species. Overall, these results argue for continued development of improved A. phagocytophilum diagnostics, especially direct detection techniques, and integrated analysis of diagnostic tests for patients with suspected Ixodes-borne disease. Although many aspects introduced and explored here will require expanded and more detailed investigations, this thesis contributes positively to a fundamental understanding of the extent to which A. phagocytophilum occurs in Portugal and its potential as a disease agent. It is hoped that these beginning studies will help to delineate new lines of research that more fully address granulocytic anaplasmosis and other emerging Ixodes-borne diseases.
Anaplasma phagocytophilum and granulocytic anaplasmosis are of increasing interest to the scientific community as indicated by the expanding number of reports published in the past two decades, and especially since the emergence of the first cases of human granulocytic anaplasmosis (HGA). The growing recognition of the Public Health importance of A. phagocytophilum in North America and in Europe, along with its recent detection in Portugal has signalled the need for more detailed studies that address the emergence of HGA and its causative agent in our country. Initially based on an methodological training in research units dedicated to Anaplasmataceae, this work enabled the transfer of technology as currently applied to A. phagocytophilum research and made possible the development of a new line of investigation at Centro de Estudos de Vectores e Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge (CEVDI/INSA). By establishing the foundations for concerted study on A. phagocytophilum and HGA, the work described herein has facilitated development of a broad approach toward fundamental issues in Anaplasmataceae ecobiology and disease by focusing attention on identification of potential ixodid vectors, mammals likely to be involved in the infectious agent’s life cycles either as reservoirs or affected hosts, and garnered the evidence indicating the potential for human exposure in Portugal. The studies conducted in ixodid ticks proves the involvement of two Ixodes species in A. phagocytophilum cycles, including Ixodes ricinus on Madeira Island and I. ventalloi on the mainland. The detection of A. phagocytophilum DNA in I. ricinus reinforces prior studies and suggests its persistence on Madeira Island. This thesis also adds new data to the understanding of the natural history of A. phagocytophilum by providing the first evidence of infections in I. ventalloi ticks. The fact that some infected arthropods infest domestic cats not only mandates the inclusion of these mammals on the national list of vertebrate hosts parasitized by I. ventalloi ticks, but also shows their potential complicity in A. phagocytophilum maintenance. Moreover, molecular data shows the existence of A. phagocytophilum variant genotypes in Portuguese ticks. Partial gene sequences from infected ticks demonstrates nucleotide polymorphisms that support a close relationship of A. phagocytophilum on Madeira Island I. ricinus to North American strains isolated from humans as well as genotypes detected in Central and Northern Europe. Yet, these variants diverge from those found in mainland I. ventalloi, which represents a new genotype of undetermined pathogenicity. Serological evidence of exposure to A. phagocytophilum or a close related agent is shown in Mus spretus mice, horses, and dogs in mainland. Molecular analysis of biological samples from these animal populations provides the first definitive evidence of A. phagocytophilum active infection in Portuguese vertebrates with detection of its DNA in one seropositive horse from mainland Portugal, where the A.phagocytophilum genotype found is closely related to strains isolated from humans, suggesting the potential for HGA in Portugal. This thesis further extends study to identification of another closely related Anaplasma species, and its potential for serological cross-reactions with A. phagocytophilum, as evident with the detection of A. platys DNA in seropositive dogs. These data also underscore the importance of veterinarians maintaining vigilance for detection of not only granulocytic anaplasmosis but also canine infectious cyclic thrombocytopenia as causes of tick-borne diseases in Portugal. Both prospective and retrospective serological and molecular investigations of human exposure to A. phagocytophilum were performed on samples received at CEVDI/INSA for the laboratory diagnosis of patients with suspected tick-borne diseases during 2000-2006. The results provide evidence for seropositive Portuguese patients, including cases that fulfil serological criteria for HGA, although active infections were not detected. Moreover some seropositive patients had additional evidence of other tick-borne agents or related bacteria infections, including Lyme borreliosis, Q fever and bartonellosis. Although possibly false positive cross-reactions to shared antigens, these reactions potentially could be the result of active dual infections, or past exposure to several agents transmitted by Ixodes species. Overall, these results argue for continued development of improved A. phagocytophilum diagnostics, especially direct detection techniques, and integrated analysis of diagnostic tests for patients with suspected Ixodes-borne disease. Although many aspects introduced and explored here will require expanded and more detailed investigations, this thesis contributes positively to a fundamental understanding of the extent to which A. phagocytophilum occurs in Portugal and its potential as a disease agent. It is hoped that these beginning studies will help to delineate new lines of research that more fully address granulocytic anaplasmosis and other emerging Ixodes-borne diseases.
Descrição
Palavras-chave
Microbiologia médica Anaplasmataceae Anasplasma phagocytophilum Anaplasmoses granulíticas Portugal
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical