Desenvolvimento e validação de um sistema de detecção simultâneo para dengue, zika e chikungunya empregando a metodologia de RT-PCR em tempo real com a utilização de sondas fluorogênicas

Abstract

Arboviruses are considered a serious threat to public health in several parts of the world as many of them are serious diseases to human beings, such as dengue, zika and chikungunya. Those are capable to cause encephalitis, persistent joint pain, hemorrhagic fevers and being linked to neurological diseases. Many diseases have advanced to different regions where their circulation had not been previously documented, causing new outbreaks. An important vector of arboviruses is the Aedes aegypti, which has, over the years, been found in regions not only tropical, in addition to being completely adapted to the urban environment. Dengue virus and Zika virus belong to the Flavivirus genus and have four distinct antigenic types (DENV1, DENV2, DENV3 and DENV4). Chikungunya virus, which cause the illness of the same name, belongs to Alphavirus genus. In Brazil and developing countries those diseases have been considered a serious public health problem and have become a huge challenge for health care systems. High demand for tests sent to central state laboratory and laborious detection methods create a huge obstruction to confirm positive cases. Ecovec is a Brazilian company and spin off from Federal University of Minas Gerais, focused on public healthy and has the intention to fill the inefficient gap in generate data for decision making by public managers. It has created and integrated monitoring systems of Aedes that provides weekly rates of vector infestation and viral circulation, pointing actions to combat the mosquito to avoid epidemics. The present work proposed to develop a unique assay for dengue, zika and chikungunya using fluorogenic probes for rt PCR, capable to detect virus in Aedes Aegypti and implement it in the Ecovec routine analysis. The first part of the study, designed to standardize singleplex reactions in control samples, showed satisfactory results with detection limits of 1 PFU in DENV assays and 10 PFU for the ZIKV and CHIKV assays. Triplicate analyses has shown reaction efficiency above 97% for all assays. It was verified the absence of cross reaction of the probes and primers and it was possible to evaluate the specificity of the assays when submitted to tests using controls of yellow fever virus and mayaro virus. 30 field samples containing Aedes aegypti collected in the field were used for the validation phase and all showed amplifications. RpL32 was used as an internal control. Three biplex reactions were setup to detect two targets in a single assay. All reactions have shown amplification curves in the positive controls, but it was not possible to observe amplification in the field samples. It is concluded that more tests are necessary to allow the detection of 3 targets in a single reaction but the standardization and singleplex validation has already made it possible to replace detection technique in our lab, which was previously performed by intercalating agents and nowadays the use of fluorogenic probes generate more reliable and and higher quality reducing the results delivery time.