Limit of detection of PCR/RFLP analysis of cytochrome oxidase II
for the identification of genetic groups of Trypanosoma cruzi
and Trypanosoma rangeli in biological material from vertebrate hosts
Amanda Regina Nichi Sá
Karen Yuki Kimoto
Edmundo Carlos Grisard
Mônica Lúcia Gomes
Received: 7 September 2017 /Accepted: 14 May 2018
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Mixed infections with Trypanosoma cruzi and Trypanosoma rangeli and their different genetic groups occur frequently
in vertebrate hosts and are difficult to detect by serology. In the present study, we evaluated the limit of detection of
polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of cytochrome oxidase II
(COII) for the identification of genetic groups of these two parasites in blood and tissue from vertebrate hosts.
Reconstitution experiments were performed using human blood (TcI/TcII and KP1+/KP1−) and mouse tissue (TcI/
TcII). We tested blood from patients who were in the chronic phase of Chagas disease and tissue from animals that
were experimentally infected with all possible combinations of six discrete typing units. In blood samples, T. cruzi and T.
rangeli were detected when 5 parasites (pa) were present in the sample, and genetic groups were identified when at least
50 pa were present in the sample. T. cruzi alone could be detected with 1 pa and genotyped (TcI/TcII) with 2 pa. T.
and KP1− in both admixtures and alone. In mouse tissue, TcI and TcII were detected with at least 25 pa. The analysis of
blood samples from patients and tissue from animals that were experimentally infected revealed low parasite loads in
these hosts, which were below the limit of detection of the present method and could not be genotyped. Our findings
indicate that the performance of PCR/RFLP analysis of COII is directly related to the amount and proportion of parasites
that are present in the sample and the genetic groups to which the parasites belong.
Keywords Trypanosoma cruzi
Infection with Trypanosoma cruzi and Trypanosoma rangeli
can occur in humans, animals, and vectors in the same geo-
graphical area, resulting in mixed infections in these hosts
(Coura et al. 1996; Chiurillo et al. 2003; Saldaña et al.
2012). T. cruzi is the etiological agent of Chagas disease,
and approximately seven million people are infected world-
wide (World Health Organization 2017). Although T. rangeli
is not considered pathogenic in vertebrate hosts, it was de-
scribed as an infectious agent in approximately 3000 people
in Central and South America (Stoco et al. 2017). Chagas
disease is usually diagnosed using serological methods.
However, T. cruzi and T. rangeli share similar antigens, and
cross reactions can occur, making specific diagnosis and
* Amanda Regina Nichi Sá
* Mônica Lúcia Gomes
Departamento de Ciências Básicas da Saúde, Universidade Estadual
de Maringá (UEM), Avenida Colombo, 5790, Jardim Universitário,
Maringá, Paraná 87 020-900, Brazil
Docente do curso de Biomedicina, Centro Universitário Integrado,
Rodovia BR 158, KM 207, Campo Mourão, Paraná 87300-970,
Departamento de Microbiologia, Imunologia e Parasitologia,
Universidade Federal de Santa Catarina (UFSC), R. Eng.
Agronômico Andrei Cristian Ferreira, Trindade, Florianópolis, Santa
Catarina s/n - 88040-900, Brazil