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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

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Abstract

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. rangeli was detected with 2 pa and genotyped (KP+/KP1-) with 25 pa. The present method more readily detected TcII 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.

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Funding

This work was funded by Conselho Nacional de Desenvolvimento Científico e Tecnólogico (CNPq; 472969/2012-9) and Fundação Araucária (37824/2013).

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Authors and Affiliations

  1. Departamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá (UEM), Avenida Colombo, 5790, Jardim Universitário, Maringá, Paraná, 87 020-900, Brazil

    Amanda Regina Nichi Sá, Karen Yuki Kimoto & Mônica Lúcia Gomes

  2. Docente do curso de Biomedicina, Centro Universitário Integrado, Rodovia BR 158, KM 207, Campo Mourão, Paraná, 87300-970, Brazil

    Amanda Regina Nichi Sá

  3. 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

    Mário Steindel & Edmundo Carlos Grisard

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  1. Amanda Regina Nichi Sá
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  2. Karen Yuki Kimoto
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  5. Mônica Lúcia Gomes
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Correspondence to Amanda Regina Nichi Sá or Mônica Lúcia Gomes.

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The experiments with human blood were approved by the Standing Committee on Ethics in Research with Humans (Comitê Permanente de Ética em Pesquisa com Seres Humanos [COPEP]) of the State University of Maringá (Universidade Estadual de Maringá [UEM]; protocol no. 012/2010). The experiments with animals were approved by the Committee of Ethical Conduct on the Use of Animals in Experimentation (Comitê de Conduta Ética no Uso de Animais em Experimentação [CEAE]) of UEM (protocol no. 076/2013).

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Sá, A.R.N., Kimoto, K.Y., Steindel, M. et al. 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. Parasitol Res 117, 2403–2410 (2018). https://doi.org/10.1007/s00436-018-5928-1

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