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Master's Dissertation
DOI
10.11606/D.17.2018.tde-15102018-115825
Document
Author
Full name
Marcilio Jorge Fumagalli
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Figueiredo, Luiz Tadeu Moraes (President)
Arruda Neto, Eurico de
Fonseca, Benedito Antonio Lopes da
Ribeiro, Bergmann Morais
Title in Portuguese
Desenvolvimento de métodos sorológicos para diagnóstico de infecções pelos vírus Chikungunya e Mayaro
Keywords in Portuguese
Arbovírus
Chikungunya
ELISA
Mayaro
Proteína de envelope e diagnóstico
Abstract in Portuguese
Devido a existência de 2 alphavírus artritogênicos no Brasil, os vírus Mayaro (MAYV) e Chikungunya (CHIKV) tornou-se importante desenvolver testes diagnósticos eficazes para discriminar suas infecções. No presente trabalho, desenvolvemos ELISAs indiretos para diagnóstico de CHIKV e MAYV utilizando proteínas de envelope viral E2 recombinantes, produzidas em Escherichia coli, as rE2-CHIKV e rE2-MAYV ELISAs. As proteínas E2 recombinantes tiveram suas antigenicidades verificadas nos ensaios utilizando anticorpos policlonais oriundos de camundongos hiperimunizados com CHIKV, MAYV e outros alphavírus. O rE2-CHIKV ELISA detectou anticorpos murinos de forma homotípica e não produziu reações cruzadas evidenciáveis utilizando anticorpos murinos específicos contra outros Alphavírus. O rE2-MAYV ELISA detectou anticorpos murinos homotípicos e também, reagiu cruzadamente com anticorpos murinos anti-CHIKV, mas não para outros Alphavírus. Esses ELISAs, também, foram usados na detecção de anticorpos em soros de pacientes com suspeita de infecção arboviral. Pelo o rE2-CHIKV ELISA, testaram-se 59 soros, resultando em 26 amostras IgG positivas. Resultados desse ELISA, quando comparados aos obtidos por teste de neutralização, demonstraram sensibilidade de 89,66% e especificidade de 100%. Soros humanos IgG positivos foram detectados em altas diluições pelo rE2-CHIKV ELISA. Quanto a detecção de IgM, o rE2- CHIKV ELISA apresentou moderada concordância com outros ensaios sorológicos. Com rE2- MAYV ELISA, testaram-se 68 soros resultando em 23 amostras IgG positivas, das quais 11 também mostraram-se positivas em teste de neutralização, demonstrando sensibilidade de 100% e especificidade de 78,95%. Portanto, os rE2-CHIKV e rE2 MAYV ELISAs, particularmente para detecção de IgG, mostraram-se adequadamente sensíveis e específicos para serem validados em estudos com maiores números de amostras e serem aplicados ao diagnóstico de pacientes infectados com CHIKV e MAYV.
Title in English
Development of methods for serological diagnose of Chikungunya and Mayaro infections
Keywords in English
Arbovirus
Chikungunya
ELISA
Envelope protein and diagnose
Mayaro
Abstract in English
Due the existence of 2 arthritogenic alphaviruses in Brasil, the viruses Mayaro (MAYV) and Chikungunya (CHIKV), it became important the development of efficient diagnose tests to discriminate their infections. In the present work, we developed indirect ELISAs for CHIKV and MAYV diagnosis using viral recombinant envelope proteins E2, produced in Escherichia coli, the rE2-CHIKV and rE2-MAYV. The recombinant E2 proteins had their antigenicity confirmed in the assay by using polyclonal antibodies produced in hyperimmunized mice with CHIKV, MAYV and other alphaviruses. The rE2-CHIKV ELISA detected homotypic murine antibodies and did not produced detectable cross-reactivity signal when using murine antibodies from other alphaviruses. The rE2-MAYV ELISA detected homotypic antibodies and also cross-reacted with murine anti-CHIKV antibodies, but not to other alphaviruses. These ELISAs were also tested for the detection of human antibodies, using patient sera suspected of arboviral infection. For rE2- CHIKV ELISA, it were tested 59 sera, resulting in 26 positive IgG samples. These ELISA results, when compared to those of a neutralizing assay, demonstrated a sensibility of 89.66% and specificity of 100%. The IgG positive human sera were detected in high dilutions by rE2-CHIKV ELISA. Regarding the detection of IgM, the rE2-CHIKV ELISA showed a moderate samples detection agreement when compared to other serologic assays. For rE2-MAYV ELISA, it were tested 68 sera, resulting in 23 positive IgG samples, of which 11 demonstrated to be positive by the neutralization assay, demonstrating a sensibility of 100% and specificity of 78.95%. Therefore, the rE2-CHIKV and rE2-MAYV ELISAs, especially for IgG detection, demonstrated to be properly sensitive and specific to be validated in studies using a greater number of samples, and also to be applied in the diagnosis of infected CHIKV and MAYV patients.
 
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Publishing Date
2018-10-18
 
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