Análise comparativa de métodos moleculares de detecção e identificação de Leishmania spp. e desenvolvimento de metodologia para o diagnóstico de leishmanioses.

ZAMPIERI, R. A. Comparative analysis of molecular methods for detection and identification of Leishmania spp. and development of methodology for leishmaniasis diagnosis. 2013. 78 p. Masters thesis (Parasitology) Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2014. Leishmaniases are diseases that affect around 2 million people in 88 countries and represent a serious public health problem. In Brazil, seven Leishmania species are recognized as etiologic agents of these diseases. Therefore an accurate differential diagnosis is extremely relevant for the comprehension of epidemiological profiles and administration of therapeutic protocols. The employment of techniques using parasite DNA as target for detection and identification has been described as diagnostic strategy in several works. The present work aims are to evaluate sample collection and storage conditions for leishmaniasis diagnosis tests, evaluate DNA extraction and purification methods and develop precise protocols for detection, quantification and identification of Leishmania species responsible for the disease in Brazil. This way, were tested three solutions for sample conservation: PBS, NET and ethanol 70%; and three DNA extraction protocols: commercial kit of silica columns, saltingout protein precipitation and phenol-chloroform extraction. The samples from BALB/c mice experimentally infected with L. (L.) amazonensis and L. (V.) braziliensis, submitted to the described conservation treatments and extraction methods, were used in real time PCR assays for detection and quantification of parasite SSU rDNA and mammal gapd. The DNA of samples submitted to NET treatment produced higher amounts of target amplicons and the extraction method did not influence the test quality. Three different DNA targets were evaluated regarding sensitivity for conventional PCR detection: SSU rDNA, g6pd and kDNA. The higher sensitivity and specificity were reached when SSU rDNA was used as target. Besides, the use of nested PCR technique using the same target revealed to be sensitive enough to detect 10 ag of L. (L.) amazonensis DNA. In parasite/host mixture DNA samples, the simultaneous utilization of Leishmania SSU rDNA and g6pd, and mammalian gapd enabled parasite detection and subgenus discrimination, besides verification of amplifiable DNA presence. For the development of a new identification method, attempts to apply LCR technique were performed, but the obtained results did not provide accuracy for discrimination of L. (L.) amazonensis and L. (L.) chagasi SSU rDNA polymorphisms. Moreover, the application of HRM methodology exploring SSU rDNA polymorphisms proved to be quite accurate to discriminate L. (L.) chagasi among others tested species. HRM methodology also discriminated g6pd polymorphisms, allowing the L. (L.) amazonensis, L. (V.) braziliensis and other L. (Viannia) species discrimination by distinct amplicons dissociation profiles. So, the present work proposes optimized protocols for sample conservation, DNA extraction and identification assays of Leishmania, systematically tested in standardized samples.