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    • Early VL diagnosis makes it possible to

    2020-05-08

    Early VL diagnosis makes it possible to formulate a quicker and more effective treatment against the disease, which could increase the possibility of a cure for the patients, as well as reduce the toxicity of the drugs (Coelho et al., 2009); however, conventional and molecular parasitological methods presents variable sensitivity (Tavares et al., 2003, Srividya et al., 2012). Therefore, serological tests could be considered for the detection of antileishmanial antibodies, due to its simplicity, cost effectiveness, and less invasive procedure for sample collection (Coelho et al., 2015). A commercial kit, namely Kalazar Detect™ Test (InBios International®, Inc., Seattle, Wash, USA), was developed, which is a non-invasive immunochromatographic method to detect rK39-specific 69 7 in VL patients’ sera (Sundar et al., 2002, Grimaldi et al., 2012). However, problems, such as false-positive results in VL-related diseases patients, as well as false-negative results in subjects with low levels of antileishmanial antibodies, in immunocompromised subjects, or in subjects living in endemic areas, can hamper its diagnostic efficacy (Chappuis et al., 2007, Maia et al., 2012, Singh et al., 2012). Recent advances in genomics, proteomics, and bioinformatics are resulting in new strategies to identify antigenic markers for the diseases (Costa et al., 2011, Duarte et al., 2015). In one immunoproteomic study, enolase protein was identified in L. infantum amastigotes by antibodies in VL dog sera (Coelho et al., 2012). In another work, this antigen was recognized by antibodies in cutaneous and mucosal leishmaniosis patients’ sera (Duarte et al., 2015). In the present study, the diagnostic percentage of sensitivity and specificity values using recombinant enolase (rEnolase) protein were investigated in canine and human serum samples by an indirect ELISA method. Preliminary results showed that the detection of antibodies against recombinant protein can improve the serodiagnosis of human and canine visceral leishmaniosis.
    Materials and methods
    Results
    Discussion Visceral leishmaniosis is a fatal and relatively neglected disease, if acute and left untreated, of which dogs represent important reservoirs of the L. infantum parasite (Chávez-Fumagalli et al., 2013, Fonseca et al., 2014). Regarding its diagnosis, problems related to the occurrence of false-positive or false-negative results have been reported in the literature and, in this context, one could speculate that there is still a challenge to translate new knowledge into cost-effective and affordable diagnostic control tools. Despite the lack of pathognomonic manifestations, clinical signs in CVL include cutaneous alterations, local or generalized lymphoadenomegaly, loss of body weight, liver and spleen enlargement, glomerulopathy, ocular lesions, epistaxis, onycogryphosis, and lameness (Maia and Campino, 2008). The incidence of the canine disease is an important epidemiological parameter to consider in the prioritization of target control areas, considering that seropositive animals (including those asymptomatic) can infect phlebotomine sand flies (Coura-Vital et al., 2013). Regarding human VL, the disease is clinically diagnosed by the occurrence of anemia, leucopenia, fever, and hepatosplenomegaly; however, symptoms are unspecific, and parasitological tests are used to confirm this. These tests are based on the parasite detection in aspirates and are considered specific but present variable sensitivity. Molecular methods have been used and show an improved sensitivity, although there is a high risk of bleeding in splenic aspiration, and exams are dependent on technical expertise and sophisticated equipment (Lemrani et al., 2009, Santos et al., 2014). Recombinant Leishmania antigens have been evaluated as new diagnostic tools for VL. Fonseca et al. (2014) showed that the recombinant C9 protein presented an accuracy of 0.79, with specificity and sensitivity values of 80% and 70%, respectively, for the serodiagnosis of human VL. Oliveira et al. (2012) showed that an L. infantum hypothetical protein presented a sensitivity value of 36.4% for human VL diagnosis, although it has been well evaluated for CVL serodiagnosis. In our study, rEnolase was specifically recognized by antibodies in L. infantum-infected humans and dog sera, presenting a high percent diagnostic sensitivity and specificity values, when compared to those found when the rA2 protein (Farahmand et al., 2011, Costa et al., 2012, Akhoundi et al., 2013) or the crude soluble Leishmania extract were used. In addition, although all VL patients have shown positive PCR results for L. infantum kDNA, six of these were not detected by the rK39 immunochromatographic test. In contrast, rEnolase was recognized by all VL patient sera, suggesting that this antigen could become an interesting tool for VL serodiagnosis.