Applied Microbiology and Biotechnology

, Volume 103, Issue 19, pp 8179–8190 | Cite as

Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis

  • Henrique Gama Ker
  • Wendel Coura-Vital
  • Diogo Garcia Valadares
  • Rodrigo Dian Oliveira Aguiar-Soares
  • Rory Cristiane Fortes de Brito
  • Patrícia Sampaio Tavares Veras
  • Deborah Bittencourt Mothé Fraga
  • Olindo Assis Martins-Filho
  • Andréa Teixeira-Carvalho
  • Alexandre Barbosa ReisEmail author
Methods and protocols


An accurate diagnosis of visceral leishmaniasis is an essential tool for control of the disease. While serologic methods are very useful, these conventional methodologies still present limitations in terms of sensitivity and specificity. The use of flow cytometry is a worldwide trend in the development of high-performance diagnostic methods. Herein, we describe a new flow cytometry serology test, characterized by the employment of the Cytometric Bead Array microspheres A4 and E4 coated with the recombinant antigens rLci1A and rLci2B respectively, to improve the serodiagnosis of canine visceral leishmaniasis. The tests were conducted in a wide variety of sera groups (n = 140), where the diagnostics development would be optimized accounting not just the ability to identify infected dogs with different clinical status, but also to exclude cross-reaction and differentiate vaccinated dogs from dogs infected. Serological testing of the antigenic system A4–rLci1A showed a sensitivity of 90.0% and specificity of 75%, while the E4–rLci2B testing demonstrated a sensitivity of 95.0% and specificity of 82.5%. The use of a multiplex assay of A4–rLci1A and E4–rLci2B, resulted in a diagnostic improvement, with a sensitivity of 95.0% and specificity of 91.2%. Our results show that this novel flow cytometry serology test is a viable tool for sensitive and specific serodiagnosis. Notably, the combination of distinct antigenic systems allows us to test for antibodies to multiple recombinant antigens from a single serum sample. This benefit emphasizes the importance of this methodology as an alternative in the serological diagnosis.

Key words

Leishmania infantum Canine visceral leishmaniasis Multiplexed diagnosis Serological methods Flow cytometry 



A.B.R., A.T.C., O.A.M.F., P.S.T.V, H.G.K., and R.C.F.B. are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and CAPES for PQ fellowships. We also thank Megan M. Keller of the Leishmania Research Laboratory, of Internal Medicine, University of Iowa for the critical reading and review of the English of our article.

Funding information

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, grants CBB - INV-00037-14 and CDS - APQ-03505-131), the Programa de Pesquisa para o SUS (PPSUS grant APQ-03505-13), and the Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais (INCT-DT).

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical statement

This study was approved by the Ethics Research Committee of the Universidade Federal de Ouro Preto (protocol number 083/2007). All procedures involving animals were performed in compliance with Brazilian federal law for animal experimentation (Law 11794/2008).


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Henrique Gama Ker
    • 1
  • Wendel Coura-Vital
    • 1
    • 2
  • Diogo Garcia Valadares
    • 1
  • Rodrigo Dian Oliveira Aguiar-Soares
    • 1
  • Rory Cristiane Fortes de Brito
    • 1
  • Patrícia Sampaio Tavares Veras
    • 3
    • 4
  • Deborah Bittencourt Mothé Fraga
    • 3
    • 4
  • Olindo Assis Martins-Filho
    • 5
  • Andréa Teixeira-Carvalho
    • 5
  • Alexandre Barbosa Reis
    • 1
    • 4
    Email author
  1. 1.Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas, Instituto de Ciências Exatas e BiológicasUniversidade Federal de Ouro Preto (NUPEB/UFOP)Ouro PretoBrazil
  2. 2.Pós-Graduação em Ciências Farmacêuticas, Departamento de Análises Clínicas, Escola de FarmáciaUniversidade Federal de Ouro Preto (NUPEB/UFOP)Ouro PretoBrazil
  3. 3.Laboratório Laboratório de Patologia e Biointervenção, Centro de Pesquisas Gonçalo MonizFundação Oswaldo Cruz (FIOCRUZ)SalvadorBrazil
  4. 4.Instituto de Ciência e Tecnologia de Doenças Tropicais (INCT-DT)SalvadorBrazil
  5. 5.Grupo Integrado de Pesquisas em Biomarcadores, Instituto René RachouFundação Oswaldo Cruz (FIOCRUZ)Belo HorizonteBrazil

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