A new Leishmania hypothetical protein can be used for accurate serodiagnosis of canine and human visceral leishmaniasis and as a potential prognostic marker for human disease

Highlights

• A new Leishmania hypothetical protein was evaluated to diagnose VL.

• High sensitivity and specificity were found against disease in dogs and humans.

• The antigen showed better performance than other diagnostic tools.

• A low protein-specific humoral response was found after human treatment.

• The humoral immunity was based on protein-specific IgG2 antibody.

Abstract

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.

Introduction

Leishmaniases are diseases caused by protozoan parasites of the Leishmania genus, which are endemic in 98 countries in the world, with 380 million people exposed to the risks of infection (WHO, 2016). American visceral leishmaniasis (VL) is caused by L. infantum species, and the disease can be fatal if acute and left untreated (Alvar et al., 2012). In Brazil, dogs are considered the main domestic reservoirs of the parasites, and the incidence of canine VL is related to the human disease (Nogueira et al., 2019). The current control measures against disease have not been efficient in reducing the number of human VL cases Madeira et al., 2006; Sanches et al., 2016 Therefore, a rapid identification and control of the reservoir population, including infected domestic dogs, could be considered as one of the best ways to prevent human disease (Mohebali et al., 2018).

The diagnosis of VL is based on the clinical evaluation and laboratorial tests (Paiva-Cavalcanti et al., 2015). The infection is confirmed by parasitological methods, identifying parasites in organic aspirates of the infected hosts. However, although the specificity is high, the sensitivity is hampered, due to the need to detect the parasite or its content in the collected aspirates (Srividya et al., 2012). In addition, the sample collection is invasive. Molecular methods have shown higher sensitivity and specificity; however, they require sophisticated equipment and trained laboratory staff, which limit their use (Ruiter et al., 2014; Sakkas et al., 2016).

Immunological tests, such as the direct agglutination test (DAT), enzyme-linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT), and immunochromatographic strips, among others, have also been used for the diagnosis of VL (Elmahallawy et al., 2014; Farahmand and Nahrevanian, 2016). They present advantages, such as simplicity and low cost, as well as the fact that the sample collection is less invasive than parasitological methods (Travi et al., 2018). In addition, in the symptomatic VL, hosts usually develop high titers of anti-Leishmania antibodies and are thus easily detected in serological assays (Srivastava et al., 2011). However, most L. infantum-dogs and humans present the asymptomatic disease with a low antileishmanial serology. As consequence, they are not easily diagnosed by such tests (Echchakery et al., 2018). There are also problems with the antigenic cross-reactivity, when sera from dogs or humans developing VL-related diseases are evaluated (Coura-Vital et al., 2013).

DAT has been used for the diagnosis of canine and human VL (Lévêque et al., 2020). This test has been employed for decades in countries from South America, Iran, and Europe (Farahmand and Nahrevanian, 2016; Sarkari et al., 2018). It is based on the agglutination after incubation using patient serum sample and stained killed L. infantum or L. donovani promastigotes. DAT is a semi-quantitative assay, and results are based on antileishmanial antibody titers (Freire et al., 2019). This test presents high sensitivity and specificity to detect VL cases in immunocompetent patients and in VL/HIV co-infected patients (Cota et al., 2013; Oliveira et al., 2013). However, false-positive results can be found in malaria and Chagas Disease patients (Bangert et al., 2018; Maia et al., 2012). Regarding the canine VL, DAT presents high sensitivity and specificity to detect the infected animals (Mohebali et al., 2018) and is described as a suitable diagnostic method for large-scale screening for canine disease (Haddadzade et al., 2013).

Regarding the use of immunochromatographic strips, kinesin-derived proteins, such as k39, have also shown good performance for the diagnosis of canine and human VL (Bangert et al., 2018; Mohapatra et al., 2010). In spite of high sensitivity for the symptomatics, asymptomatic cases are less diagnosed due to the low antileishmanial serology. There are problems with the specificity of the tests, when cross-reactive samples or those from healthy hosts living in endemic regions of disease are evaluated (Salles et al., 2017; Srivastava et al., 2011). In the human VL, it is also difficult to serologically differentiate the active disease patients from those treated and cured, since specific serology can remain for months or even years after treatment (Portela et al., 2017).

Therefore, new antigens presenting better sensitivity and specificity should be identified. In a recent immunoproteomics study, parasite proteins were identified in L. infantum amastigotes and promastigotes through antibodies in VL dog sera (Coelho et al., 2012). One of these antigens, called LiHyG (XP_001467126.1), was identified in the amastigote stage through the asymptomatic sera. In this context, in the present study, the L. infantum LiHyG codifying gene was cloned and the recombinant protein (rLiHyG) was expressed, purified, and evaluated in ELISA experiments for the diagnosis of VL. In addition, a post-therapeutic serological follow-up was performed using sera samples of VL patients, which were collected before and six months after treatment, when the anti-protein and anti-parasite IgG, IgG1, and IgG2 antibody reactivity was investigated.