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

• Grant number: 17/26543-1
• Support Opportunities: Regular Research Grants
• Start date: July 01, 2018
• End date: June 30, 2020
• Field of knowledge: Health Sciences - Medicine - Maternal and Child Health
• Principal Investigator: Thelma Suely Okay
• Grantee: Thelma Suely Okay
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Kelly Aparecida Kanunfre

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. (AU)

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

YAMAMOTO, LIDIA; AMORIM FILHO, ANTONIO G.; QUEIROZ, JOELMA A.; DE CARVALHO, MARIO H. B.; RODRIGUES, JONATAS C.; KANUNFRE, KELLY A.; FRANCISCO, V, ROSSANA P.; OKAY, THELMA SUELY. Performance of a Multiplex Nested Polymerase Chain Reaction in Detecting 7 Pathogens Containing DNA in Their Genomes Associated With Congenital Infections. ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE, v. 144, n. 1, p. 99-106, JAN 2020. (17/26543-1)

RENTE FERREIRA-FILHO, JULIO CESAR; ALMEIDA BRAZ, LUCIA MARIA; ALVES ANDRINO, MARCOS LUIZ; YAMAMOTO, LIDIA; YAMASHIRO KANASHIRO, EDITE HATSUMI; GONCALVES DA SILVA, ANA MARIA; KANUNFRE, KELLY APARECIDA; OKAY, THELMA SUELY. A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area. Parasitology International, v. 80, FEB 2021. (17/26543-1)

DOS SANTOS, EMILLY HENRIQUE; YAMAMOTO, LIDIA; DOMINGUES, WILSON; DI SANTI, SILVIA MARIA; KANUNFRE, KELLY APARECIDA; OKAY, THELMA SUELY. A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene. Parasitology International, v. 76, JUN 2020. (18/20615-3, 17/26543-1)

RENTE FERREIRA FILHO, JULIO CESAR; ALMEIDA BRAZ, LUCIA MARIA; ALVES ANDRINO, MARCOS LUIZ; YAMAMOTOA, LIDIA; KANUNFREA, KELLY APARECIDA; OKAYA, THELMA SUELY. Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load. Experimental Parasitology, v. 200, p. 13-15, MAY 2019. (17/26543-1)