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Master's Dissertation
DOI
10.11606/D.87.2013.tde-06112013-111440
Document
Author
Full name
Fernanda Batista de Andrade
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2013
Supervisor
Committee
Elias Junior, Waldir Pereira (President)
Sircili, Marcelo Palma
Yamamoto, Denise
Title in Portuguese
Padronização e avaliação de PCR multiplex para o diagnóstico de Escherichia coli enteroagregativa típica e atípica
Keywords in Portuguese
Escherichia coli
Células epiteliais
Diarréia
Reação em cadeia por polimerase
Virulência
Abstract in Portuguese
O patótipo de Escherichia coli diarreiogênica denominado E. coli enteroagregativa (EAEC) é caracterizado pela expressão do padrão de adesão agregativo (AA) em células epiteliais cultivadas. Amostras de EAEC são classificadas como típicas ou atípicas, dependendo da presença ou não do gene aggR, respectivamente. O padrão AA no teste de adesão em células epiteliais é o diagnóstico padrão para a classificação de EAEC. Técnicas moleculares, como a PCR multiplex, são alternativas a esse teste. No presente estudo foi desenvolvida uma PCR multiplex para o diagnóstico molecular de EAEC típica e atípica, baseada na detecção dos genes aatA, aggR, aaiA e aaiG. A partir de colônias isoladas de E. coli de origem fecal o teste apresentou sensibilidade de 94,8%, especificidade de 94,7%, valor preditivo de teste positivo de 74,3% e valor preditivo de teste negativo de 99,1%. O teste desenvolvido mostrou-se uma alternativa diagnóstica sensível e específica para EAEC e permite um acréscimo significativo na detecção de EAEC atípica.
Title in English
Standardization and evaluation of multiplex PCR for the diagnostic of typical and atypical enteroaggregative Escherichia coli.
Keywords in English
Escherichia coli
Diarrhea
Epithelial cells
Polymerase chain reaction
Virulence
Abstract in English
The diarrheagenic Escherichia coli pathotype known as enteroaggregative E. coli (EAEC) is characterized by the expression of the aggregative adherence pattern (AA) on cultured epithelial cells. EAEC strains are classified as typical or atypical, depending on the presence or absence of the aggR gene, respectively. The AA pattern in the adherence assay with epithelial cells is the gold standard diagnostic for EAEC. Molecular techniques, such as multiplex PCR, are alternatives to this test. In the present study we developed a multiplex PCR for the molecular diagnostic of typical and atypical EAEC, based on the detection of aatA, aggR, aaiA and aaiG genes. The test presented 96.5% of sensitivity, 94.8% of specificity, 74.3% of positive predictive value and 99.1% of negative predictive value, when tested in isolated colonies of E. coli from feces. The test developed is a sensitive and specific diagnostic alternative for EAEC and allows a significant increase in atypical EAEC detection.
 
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Publishing Date
2014-01-17
 
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