Development of a multiplex-nested-RT-PCR for the simultaneous detection of RNA viruses associated with congenital infections.

• Grant number: 17/26298-7
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: October 01, 2018
• End date: September 30, 2020
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Thelma Suely Okay
• Grantee: Lidia Yamamoto
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The aim of this proposal is to develop a multiplex-nested-RT-PCR capable of simultaneously detecting five RNA viruses associated with congenital infections: rubella virus, enterovirus (poliovirus, echovirus, coxckasie virus, other unclassified enterovirus) and three arboviruses (dengue, chikungunya and zika). Positive amplifications will be confirmed by Real Time PCR targeting only the pathogen evidenced in the combined test. Then, positive samples will be sequenced and compared with GenBank prototypes. In our laboratory, a multiplex-nested-PCR capable of detecting seven pathogens that contain DNA in their genomes and associated with congenital infections (Toxoplasma gondii, CMV, HSV, EBV, VZV, parvovirus B19 and adenovirus) was developed and after analysis of 357 clinical samples from suspected cases (n = 161) and unsuspected ones (n = 196), totaling 357 cases, the multiplex-nested-PCR detected 60 positive cases in 161 suspects (37.3%), contrasting with the 13 cases (8%) diagnosed by the available tests in the two participating obstetric centers, a 4.7-fold lower number of cases. The present proposal intends to take advantage of the experience acquired by the group over the last four years to develop a second, more complex molecular tool that should, together with the first one, enable the detection of 12 pathogens, significantly increasing prenatal diagnoses, providing treatment in some cases, and a more careful follow-up of all pregnancies in which infections were diagnosed.