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SANTOS, D. C. The integration of molecular methods into the rapid laboratorial diagnostic of neurotropic viruses. 2009. 165p. Thesis (Microbiology PhD) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2009. Human herpesviruses (HHV), enteroviruses (HEV), and adenoviruses (HAdV) are causative agents of several respiratory, enteric, and systemic infections including those of the Central Nervous System. PCR and real-time PCR assays have been widely used in molecular diagnosis of viral infections, due to its sensitivity, rapid diagnosis, and practicality. In this study molecular techniques were used to detect HEV, HHV, and HAdV in cerebrospinal fluid samples (CSF). From January 2005 to March 2007, 295 CSF samples were collected from children and adults with suspected meningitis, meningoencephalitis, or unknown fever. Three methods for nucleic acid extraction were tested. Protocol C (DNA Qiablood kit Qiagen) was more sensitive and specific; and, therefore was the chosen method for this study. Nested PCR assays detected HEV in 28% of the samples, followed by HAdV (16%), HSV (4%), HHV-3 (1%), HHV-6 (0.7 %), HHV-4 (0.3%), and HHV-5 (0.3%). The real time PCR assay detected HEV in 23.3% of the samples and HSV in 5.1%. HEV positive samples were cultivated in RD cells and only 50% of the samples positive by PCR showed cytopathic effect. A clear serotype was identified for only two samples when a neutralization test was applied (HU 34 – serotype Echovirus 6 and serotype HU 102 Coxsackievirus B). Detected enteroviruses were sequenced to obtain the serotype. Echovirus 18 was the most frequent serotype identified (53%), followed by serotype Coxsackievirus B5 (26%). The less frequent serotypes were: Coxsackievirus B3, echovirus 3, 6, 11, 13, 16, and 20. Molecular biology techniques applied to the detection of HEV, HHV, and HAdV in CSF bring major benefits to the diagnosis of neural diseases due to their rapidity, high sensitivity, and specificity. These aspects are of great importance for effective diagnostic procedures and appropriate therapy.

Estabelecimento de métodos moleculares para aplicação no diagnóstico rápido de virus neurotrópicos.