Please cite this article in press as: Fonseca, A.M., et al., Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.04.028 ARTICLE IN PRESS G Model ACTROP 3365 1–6 Acta Tropica xxx (2014) xxx–xxx Contents lists available at ScienceDirect Acta Tropica jo ur nal home p age: www.elsevier.com/locate/actatropica Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis Aliani Moura Fonseca a,1 , Angélica Rosa Faria a,1 , Fernandes Tenório Gomes Rodrigues b , Q1 Ronaldo Alves Pinto Nagem b , Rubens Daniel Miserani Magalhães b , João Luís Reis Cunha a , Daniella Castanheira Bartholomeu a , Hélida Monteiro de Andrade a,∗ a Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Parasitologia, Avenida Antônio Carlos, 6627, Pampulha, CEP 31270-901 Belo Horizonte, Minas Gerais, Brazil b Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Bioquímica e Imunologia, Avenida Antônio Carlos, 6627, Pampulha, CEP 31270-901 Belo Horizonte, Minas Gerais, Brazil a r t i c l e i n f o Article history: Received 22 October 2013 Received in revised form 16 April 2014 Accepted 24 April 2014 Available online xxx Keywords: Visceral leishmaniasis Leishmania infantum Recombinant proteins Antigens Diagnosis a b s t r a c t Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. How- ever, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recom- binant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diag- nostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens’ sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leish- maniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance. © 2014 Published by Elsevier B.V. 1. Introduction Leishmaniasis is a serious public health problem that affects approximately 98 countries worldwide (Alvar et al., 2012). Vis- ceral leishmaniasis (VL), also known as kala-azar, is characterized by high fever, substantial weight loss, splenomegaly, hepatomegaly and anemia (Desjeux, 1996). In Brazil, where VL is caused by Leish- mania infantum (syn. L. chagasi), the disease has expanded from rural to urban areas (Margonari et al., 2006; Mauricio et al., 2000). ∗ Corresponding author. Tel.: +55 31 3409 3010. E-mail addresses: helida@icb.ufmg.br, helidandrade@gmail.com (H.M. de Andrade). 1 These authors have equally contributed to this article. Serological diagnosis is widely and frequently used to diagnose humans and dogs, the latter being identified as L. infantum reser- voirs (Alvar et al., 2004; Deane, 1961). Although specific humoral responses in human (Galvão-Castro et al., 1984) and canine vis- ceral leishmaniasis are generally very intense, with high levels of specific immunoglobulins, the rate of Leishmania infection is under- estimated in dogs from endemic areas (Alvar et al., 2004). According to the Brazilian Ministry of Health, canine visceral leishmaniasis (CVL) surveillance must be determined based on the DPP ® CVL rapid test and the enzyme linked immunosorbent assay (ELISA) produced by Bio-Manguinhos/FIOCRUZ, Brazil (Brasil, 2011). However, these tests have limitations such as low sensi- tivity, mainly in asymptomatic dogs (Grimaldi et al., 2012; Faria et al., 2011), and cross-reactivity (Porrozzi et al., 2007). Thus, new antigens have been studied to provide better assays. Recombinant http://dx.doi.org/10.1016/j.actatropica.2014.04.028 0001-706X/© 2014 Published by Elsevier B.V. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48