Borreliosis de Lyme: estudio de posibles vectores ixódidos y evaluación de métodos de diagnóstico microbiológico.

by Navarro García, Amanda Estrella

SUMMARY Zoonosis caused by bacteria of the genre Borrelia. by the accidental transmission by infected ticks. The genre Borrelia. concerns to the family Spirochateaceae, and they are 4 the grounds of Lyme's borreliosis, associated in the complex broad Borrelia burgdorferi sensu (B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. Valaisiana) The adherence of 48 hours is the demanded minimum, for the inoculation of Borrelia burgdorferi sensu lato. Diagnostic of the Lyme borreliosis: Direcly diasnostic by detection of spirochaeta with the uses of the clinical specimens (skin biopsies, CSF semple, peripheral blood, etc.) of patients whith conventional laboratory tintions is relative easy to reconized. Culture of the organism in BSK medium, molecular characterization of Borrelia isolates by gene sequences from DNAs amplified (flagellin (flaB), and outer surface protein A (ospA)) from ticks or patiens confirmed the presence of several Borrelia species. In the indirect diagnosis use as methods of reference, the enzimoinmunoanálisis as test of screening and the inmunotransferencia for the confirmation Objetivos: 1.- Recolection and caracterization of ixodid ticks from diferent areas of Valencia countri. 2.- Investigated la presencia de Borrelia burgdorferi sensu lato in these ixode ticks recolected., 3.-To select clinical samples with result in the test of screening positive EIA. 4.-To analyze the above mentioned samples by means of other specific and confirmatory tests(proofs) as well as to evaluate the presence of cross-reactivity with other processes nosológicos. 5.-To study the clinical characteristics and evolution of the patients with serologic results suggestive of borreliosis of Lyme as well as to evaluate his significance. Materials and methods: Obtaining and characterization of ixodids, one proceeded to the detection of Borrelia burgdorferi by means of microscopic study, culture and PCR. The selection of clinical samples, it was realized by means of the method EIA, and the study of crossed reactivity and his confirmation by means of specific tests. And finally, there was realized the analysis of signs and clinical symptoms and his relation with results serológicos. There were realized softened and dissections those who a microscopic study was practised and culture in BSK II. In the 2º periode, we made a molecular characterization of B. Burgdorferi from ticks by gene sequences DNAs amplified (nexteed PCR using 2 primers of gen of outer surface protein A (OspA), by el Guttman metode et all that permeted the obtention in the 2ª amplification, a fragment of 345 pairs of bases. By the flagelina, gene, we using too 2 pairs of primers of the flagelin gene, by the Lebech metode et al. That allow to obtain in 2 ª amplification, a fragment of 275 bases pair. For the serologic study, there was in use first a method of sifted EIA IgM-IgG System VIDAS LYT (of bioMèrieux) and a complementary method IFI Lyme Spot (also of bioMèrieux) that determines antibodies of class IgM and IgG separately. As confirmatory method, there was in use the inmunotransferencia B. burgdorferi B31 (of MarDx Diagnostic) and B. garinii genogrupo 2 (of Gull Laboratories). The serologic cross reactivity, whith Treponema pallidum, Coxiella burnetii, Rickettsia conori and Brucella mellitensis, Epstein-Barr virus, Citomegalovirus virus and AIDS virus. The population of patients corresponded(fitted) with the attended ones in the area of the HGUV. Conclusiones: 1.-The collection and identification of ixodid ticks proceeding from different areas of the province of Valencia, it has revealed the existence of five genres, with seven different species. None of them, classified as Ixodes ricinus, recognized principal vector of Lyme's borreliosis. 3 2.-En none of the studied ticks has been identified an espiroqueta compatible with Borrelia burdorferi sl. or another Borrelia's species associated with borreliosis of Lyme disease. 3.-We considered that the method most adapted for the microbiological study in the ixodid, is the ticks dissection with extraction of its glandular structures and medium intestines. The use of specimens softened for detection for molecular methods is dissuaded by the probable inhibition of the amplification of the specific ADN 4.-The enzimoinmunoanálisis method of as screening test, a population of patients selects with clinical very changeable characteristics, but also a group of patients with typical manifestations and with a concrete clinical evolution, which they sustain the existence of probable cases of borreliosis of Lyme. 5.-La qualifications of the specific antibodies of class IgM and IgG by means of inmunofluorescencia and the criteria established for the interpretation of the confirmatory tests of inmunotransferencia with B. burgdorferi antigens and B. garinii, prove to be scantily profitable in our way, since they allow neither confirm nor to discard the existence of the disease in the group of studied patients. Allows to consider the need of a better epidemiological and clinical study of the suspicious cases of borreliosis of Lyme, and particularly the development of criteria of microbiological diagnosis us that they serve to the profile of the disease in our area.