RESUMO
Cestodes or tapeworms belong to a diverse group of helminths. The adult Taenia saginata and Taenia solium tapeworm can infest the human gut and the larval stage of Echinococcus spp. and T. solium can infect tissues of the human body, causing serious disease. Molecular diagnostics can be performed on proglottids, eggs and on cyst fluids taken by biopsy. Detection of cestodes when a helminthic infection is suspected is of vital importance and species determination is required for appropriate patient care. For routine diagnostics a single test that is able to detect and type a range of cestodes is preferable. We sought to improve our diagnostic procedure that used to rely on PCR and subsequent sequencing of the Cox1 and Nad1 genes. We have compared these PCRs with novel PCRs on the 12S rRNA and Nad5 gene and established the sensitivity and specificity. A single PCR on the 12S gene proved to be very suitable for detection and specification of Taenia sp. and Echinococcus sp. Both targets harbour enough polymorphic sites to determine the various Echinococcus species. The 12S PCR was most sensitive of all tested.
Assuntos
Infecções por Cestoides/diagnóstico , Echinococcus/classificação , Complexo I de Transporte de Elétrons/genética , Reação em Cadeia da Polimerase , RNA Ribossômico/genética , Taenia/classificação , Animais , Ciclo-Oxigenase 1/genética , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Diagnóstico Diferencial , Equinococose/diagnóstico , Echinococcus/genética , Marcadores Genéticos , Humanos , NAD/genética , Sensibilidade e Especificidade , Taenia/genética , Teníase/diagnósticoRESUMO
Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.
Assuntos
DNA de Protozoário/análise , Leishmania/classificação , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Medula Óssea/parasitologia , DNA de Protozoário/química , DNA Ribossômico/análise , DNA Ribossômico/química , Éxons/genética , Humanos , Leishmania/genética , Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Visceral/diagnóstico , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Estudos Prospectivos , RNA Ribossômico 18S/genética , RNA Ribossômico 5,8S/genética , Mapeamento por Restrição , Estudos Retrospectivos , Sensibilidade e Especificidade , Alinhamento de Sequência , Análise de Sequência de DNA , Pele/parasitologiaRESUMO
Surveillance data indicates that the number of cutaneous (CL), mucocutaneous (ML) and visceral leishmaniasis (VL) cases has increased globally and in Europe during the past decades. Leishmaniasis is only seen as an imported disease in the Netherlands. We investigated occurrence in the Netherlands through an analysis of Leishmania patients sent to our laboratory and to the nationwide network of the pathology departments between 1996 and 2007. The majority of patients suffered from CL, and an outbreak among military personnel stationed in endemic regions in 2005 was noted. ML was rarely found. However, the occurrence of VL has clearly increased. Physicians in non-endemic regions should be aware that leishmaniasis can be contracted as close as Southern Europe and that it is not limited to tropical and subtropical regions only.
