A sensitive and reliable quantitative immunohistochemistry technique to evaluate the percentage of Trypanosoma cruzi-infected tissue area

Author(s)

Rente Ferreira-Filho, Julio Cesar ^[1] ; Almeida Braz, Lucia Maria ^{[1, 2]} ; Alves Andrino, Marcos Luiz ^[1] ; Yamamoto, Lidia ^[1] ; Yamashiro Kanashiro, Edite Hatsumi ^[1] ; Goncalves da Silva, Ana Maria ^[3] ; Kanunfre, Kelly Aparecida ^{[1, 4]} ; Okay, Thelma Suely ^{[5, 1]}

Affiliation

^[1] Univ Sao Paulo, Fac Med, Inst Med Trop, Lab Soroepidemiol & Imunobiol, Sao Paulo - Brazil

^[2] Inst Med Trop, Lab Soroepidemiol & Imunobiol, Predio 2, BR-05403000 Sao Paulo, SP - Brazil

^[3] Univ Sao Paulo, Fac Med, Inst Med Trop, Lab Imunopatol Doencas Infecciosas & Parasitarias, Sao Paulo - Brazil

^[4] Univ Sao Paulo, Fac Med, Dept Molestias Infecciosas, LIM48, Sao Paulo - Brazil

^[5] Univ Sao Paulo, Fac Med, Dept Pediat, Sao Paulo - Brazil

• Document type: Journal article
• Source: Parasitology International; v. 80, FEB 2021.
• Web of Science Citations: 0

Abstract

Quantification of parasites in the context of Chagas disease is required to monitor the treatment with benznidazole, disease-associated cardiomyopathies and graft rejection after heart transplantation. As parasitological exams lack sensitivity, Real Time Polymerase Chain Reaction (rt-PCR) has emerged to evaluate the parasite load in blood samples and cardiac biopsies. However, despite its higher sensitivity, rt-PCR does not provide information on the location and distribution of amastigote nests within infected tissues, the characterization of inflammatory infiltrates or changes to tissue architecture. On the contrary, a sensitive immunohistochemistry technique (IHC) could fill these gaps. In the present study, a quantitative IHC exam was standardized and validated by testing adipose and cardiac tissues of experimentally infected mice containing variable parasite load levels of T. cruzi assessed by a sensitive Sybr Green rt-PCR with kDNA primers. Tissues were divided into four groups according to the parasite load: group A100 parasites/50 ng of DNA; group B-10 parasites; group C - around 1 parasite and group D less than 1 parasite/50 ng/DNA. IHC was able to detect T. cruzi in the four groups, even in group D tissues containing fractions of a single parasite/50 ng of DNA sample according to rt-PCR. In conclusion, a highly sensitivity and reliable quantitative immunohistochemistry technique was developed and is proposed to estimate the percentage of T. cruzi-infected tissue area in chagasic patients presenting with cardiomyopathies, as a complementary test to rt-PCR. (AU)

• FAPESP's process: 17/26543-1 - Development of a multiplex-nested-RT-PCR for the simultaneous detection of RNA viruses associated with congenital infections.
• Grantee: Thelma Suely Okay
• Support Opportunities: Regular Research Grants