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ANDRADE, F. B. Standardization and evaluation of multiplex PCR for diagnostic of typical and atypical enteroaggregative Escherichia coli. 2013. 85 p. Masters thesis (Biotechnology) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2013. The diarrheagenic Escherichia coli pathotype known as enteroaggregative E. coli (EAEC) is defined by the expression of the aggregative adherence pattern (AA) on cultured epithelial cells. The presence or absence of the aggR gene, located in the EAEC virulence plasmid, defines EAEC as typical or atypical, respectively. The gold standard diagnostic for EAEC is the observation of the AA pattern in adherence tests with cultured epithelial cells. However, this assay is expensive, time consuming and needs specific laboratory infrastructure. Molecular diagnostic assays have been developed as alternatives to the adherence tests. These tests detect plasmid markers not suitable for atypical EAEC diagnosis. The present study aimed the standardization of a multiplex PCR for the molecular diagnosis of typical and atypical EAEC. Initially the chromosomal genes aaiA, aaiC, aaiG and aaiU were evaluated for specificity and prevalence in EAEC. All of them were specific and aaiA/aaiG were the most prevalent in both groups of EAEC. Thus, the multiplex PCR was established, including these two genes together with the plasmid genes aatA and aggR. The test was successfully standardized using prototype EAEC strains and its detection limit was 125 ng of purified DNA. Then, the reaction was evaluated in a collection of 403 strains of fecal E. coli previously classified as belonging to diarrheagenic E. coli pathotypes or as non-pathogenic. The technique presented 94.8% of sensitivity, 94.5% of specificity, 74.3% of positive predictive value and 99.1% of negative predictive value. Moreover, there was an increase of 60% in detecting atypical EAEC. Therefore, the test developed is a sensitive and specific tool for the diagnostic of typical and atypical EAEC employing isolated colonies of E. coli from stool cultures.

Padronização e avaliação de PCR multiplex para o diagnóstico de Escherichia coli enteroagregativa típica e atípica