Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis
Abstract
Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis Henrique Gama Ker a , b , Wendel Coura-Vital a , c , Rodrigo Dian de Oliveira Aguiar-Soares a , b , Bruno Mendes Roatt a , b , Nádia das Dores Moreira a , b , Cláudia Martins Carneiro a , b , Evandro Marques de Menezes Machado b , Andréa Teixeira-Carvalho d , Olindo Assis Martins-Filho d , Rodolfo Cordeiro Giunchetti e , Márcio Sobreira Silva Araújo d , Eduardo Antonio Ferraz Coelho f , Denise da Silveira-Lemos g and Alexandre Barbosa Reis a , b Laboratório de Pesquisas Clínicas, Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil a Laboratório de Imunopatologia, Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil b Pós-Graduação em Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil c Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil d Laboratório de Biomarcadores de Diagnóstico e Monitoração, Centro de Pesquisas Renè Rachou-FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil e Laboratório de Biotecnologia Aplicada ao Estudo das Leishmanioses, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil f Laboratório de Imunoparasitologia, Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil g ABSTRACT Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum -infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens ( Trypanosoma cruzi , Leishmania braziliensis , Ehrlichia canis , and Babesia canis ) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.