Tesis
Tipificación de Borrelia burgdorferi en vector, roedor y muestras clínicas de la Republica Mexicana por secuenciación
Fecha
02/03/2010Registro en:
Covarrubias León, Yatzaret Yoselin. (2009). Tipificación de Borrelia burgdorferi en vector, roedor y muestras clínicas de la Republica Mexicana por secuenciación. (Maestría en Biomedicina y Biotecnología Molecular). Instituto Politécnico Nacional, Sección de Estudios de Posgrado e Investigación, Escuela Nacional de Ciencias Biológicas, México.
Autor
Covarrubias León, Yatzaret Yoselin
Institución
Resumen
ABSTRACT: Introduction. Lyme borreliosis is an emerging infectious disease caused by Borrelia burgdorferi, which is transmitted to man through of the Ixodes tick-bite. This disease is endemic in the northeastern U.S. with 10-17% and Europe with 15%. Infection in Mexico is 1.1% with higher prevalence in the states of the Northeast with 6.2% and Distrito Federal 3.3%. Recently cases have been confirmed in our country as well as vector and infected rodents detection. The identification of the spirochete in the vector is an epidemiological marker of high-risk areas for acquiring the disease and the identification in the patient is diagnostic. Different clinical manifestations have been associated to each genoespecie. For this raison, implementer different methods of molecular diagnostics are important to determine the infectious genoespecies of the patient that produce this clinical manifestation. Objective. Characterization of the species complex Borrelia burgdorferi sensu lato in clinical samples from patients with Lyme borreliosis acquired in Mexico and hosts the vector by sequencing. Material and methods. DNAs from ticks, mice tissues (ear, bladder, heart and joint) and clinical samples (skin biopsy, synovial and cerebrospinal fluids) were extracted with a commercial kit Qiagen ®. After confirm purity and integrity of DNAs were amplified by PCR for fla and ospA genes of B. burgdorferi. Positive samples were running on 1% agarose gels, the band cut and the amplified by PCR were extracted and purified. Since the amplified by PCR sequencing is done with the Applied Biosystem sequencer. These sequences were compared with the reference gene of B. burgdorferi sensu stricto by Chromas 223 program.
Results: Of 136 ticks DNA’s six (4.4%) were positives for the genes fla and 14 (10.3%) for ospA gen. In 27 Peromyscus sp. and Lyomis mice 7/27 (25 %) were positive for the genes fla and 6/27 (22 %) for ospA. DNA of clinical samples 7/84 (8.3 %) were positives, four skin biopsies, four CSF, five blood and two synovial fluid. The ospA gene sequences of DNA obtained of mouse joint (P. melanotis) collected at La Marquesa National park showed a point mutation adenine for thymine with a 99% homology with Borrelia burgdorferi strain B-31. This produce in amino acid change by the isoleucine. The fla gene sequences of sinovial fluid, blood DNAs show 96% homology to B. garinii, B. afzelii, B. spielmanii and B burgdorferi sensu stricto. Conclusion: Borrelia burgdorferi sensu stricto is the genoespecie that infects mice of La Marquesa National Park although that, in Mexican patients of diferents regions of Mexico with arthritis and neurological manifestations are infected with B. burgdorferi sensu lato similar to Europe strains. RESUMEN: La borreliosis de Lyme es una enfermedad infecciosa emergente causada por Borrelia burgdorferi, la cual es transmitida al hombre a través de la picadura de una garrapata del género Ixodes. Esta enfermedad es endémica en el noreste de E.U.A y Europa. En México la prevalencia nacional de la infección es de 1.1%, con mayor prevalencia en los estados del Noreste y el Distrito Federal, así que la detección de la espiroqueta tanto en vector, como en roedor, se considera como un marcador epidemiológico considerando zonas de alto riesgo para adquirir la enfermedad. La diferencia entre las manifestaciones clínicas que produce la borreliosis de Lyme esta relacionada con la genoespecie que esta infectando al paciente, por lo cual se considera importante realizar la tipificación.