Elsevier

Acta Tropica

Volume 224, December 2021, 106126
Acta Tropica

Potential of recombinant LiHyQ, a novel Leishmania infantum protein, for the diagnosis of canine visceral leishmaniasis and as a diagnostic and prognostic marker for human leishmaniasis and human immunodeficiency virus co-infection: A preliminary study

https://doi.org/10.1016/j.actatropica.2021.106126Get rights and content

Highlights

  • A new Leishmania protein classified as hypothetical was cloned, expressed and purified.

  • It was evaluated in ELISA experiments against 395 canine and human serum samples.

  • Sensitivity and specificity values of 100% were found for both serum classes.

  • Better performance than two distinct commercial kits was verified.

  • The protein was also well-evaluated as a prognostic marker for human leishmaniasis.

Abstract

Laboratory diagnosis of leishmaniasis shows variable efficacy in detecting infected mammalian hosts and there is a need to identify suitable antigens to improve the accuracy of diagnostic tests. In the present study, a L. infantum hypothetical protein called LiHyQ was evaluated for the diagnosis of tegumentary (TL) and visceral (VL) leishmaniasis using canine and human samples. A collection of dog sera (n=155) were tested and contained samples from asymptomatic (n=20) and symptomatic (n=25) VL animals, from healthy dogs living in endemic (n=25) or non-endemic (n=25) areas of disease, from Leish-Tec® vaccinated dogs (n=20) or from dogs infected with Ehrlichia canis (n=15), Babesia canis (n=10) and Trypanosoma cruzi (n=15). Sensitivity (Se), Specificity (Sp), Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of 100% were observed for rLiHyQ with these samples, whereas the Se, Sp, PPV and NPV values with L. infantum Soluble Leishmania Antigen (SLA) preparation were 60.0%, 99.0%, 96.0% and 86.0%, respectively. A collection of human sera (n=305) were tested and contained samples from TL (n=50) and VL (n=40) patients, from VL/HIV co-infected patients (n=35), from patients infected with HIV alone (n=30), Chagas Disease (n=30), malaria (n=10), tuberculosis (n=10), paracoccidioidomycosis (n=15), leprosy (n=30) or aspergillosis (n=15); and from healthy subjects (n=40). Se, Sp, PPV and NPV values of 100% were observed for rLiHyQ with these samples, whereas the Se, Sp, PPV and NPV values with SLA were 58.0%, 76.0%, 50.0% and 82.0%, respectively. The antibody reactivity against the protein was compared with commercial kits, and the kappa index varied from 0.95 to 1.00 for rLiHyQ, and of 0.55 to 0.82 for the kits. In addition, the serological follow-up of treated patients showed a significant reduction in rLiHyQ-specific IgG antibody levels. All canine and human samples were tested at the same time using the same reagents, in order to reduce experimental variation and interference in data interpretation. In conclusion, our preliminary data suggest a diagnostic and prognostic role for rLiHyQ against leishmaniasis.

Introduction

Leishmaniases are parasitic diseases that affect 12 million people in 98 countries worldwide; with 0.2 to 0.4 million cases of visceral leishmaniasis (VL) and 0.7 to 1.2 million cases of tegumentary leishmaniasis (TL) being reported annually (World Health Organization 2016). This tropical disease complex is caused by protozoan parasites of the genus Leishmania, and is found in the Americas, Europe, Africa and Asia (Alvar et al., 2012). The clinical manifestations depend on the parasite infecting species and the host immune status, and range from self-limiting cutaneous lesions to the fatal visceral disease (Torres-Guerrero et al., 2017). Dogs are important domestic reservoirs of Leishmania infantum parasites in the Americas, and have a significant role in the epidemiology of human disease (Marcondes and Day, 2019, Travi et al., 2018).

The diagnosis of leishmaniasis is based on clinical evaluation and laboratory data (Thakur et al., 2020). The gold standard is based on parasitological methods to detect parasites and/or their content in tissue fragments or organ aspirates. However, sample collection is invasive and requires trained professionals and/or sophisticated equipment (Srividya et al., 2012, Pessoa-E-Silva et al., 2019). The lack of precise diagnosis limits disease control and contributes to reservoir maintenance. Immunological methods have been used also to diagnose leishmaniasis. However, variable sensitivity and/or specificity have been reported, although such tests are desirable, since the collection of samples is less invasive and does not require trained professionals and/or sophisticated equipment (Das et al., 2020, Reimão et al., 2020).

Leishmania proteins have been evaluated as candidates for the diagnosis of leishmaniasis (Faria et al., 2015, Farahmand and Nahrevanian, 2016). In symptomatic VL, dogs and humans usually have an elevated antibody response directed against antigens, and assays based on recombinant antigens have showed high sensitivity and specificity (Dhom-Lemos et al., 2019, Pereira et al., 2020). However, in the asymptomatic disease, they tend to present lower antileishmanial serology, and tests show reduced sensitivity. There is also cross-reactivity with samples from other diseases, such as Chagas Disease, malaria and, tuberculosis, amongst others, which hampers the specificity of the tests (Daltro et al., 2019, Rodrigues et al., 2019). Most TL patients present low to moderate antileishmanial serology, and they are not detected with current serological assays (Souza et al., 2013, Lage et al., 2019). In addition, there are patients who remain serologically positive for months and/or years after treatment and clinic cure. Furthermore, patients co-infected with human immunodeficiency virus (HIV), usually develop lower serological responses to Leishmania antigens and the commercial tests show lower sensitivity, when compared to the serological responses of immunocompetent patients (Lindoso et al., 2016, Machado et al., 2020).

There is a need for suitable antigens for serological diagnosis of TL and VL in dogs and humans. In an immunoproteomic study, the reactivity of sera from asymptomatic and symptomatic VL patients was evaluated against L. infantum promastigote and amastigote protein extracts, and distinct proteins recognized by patients´ antibodies were identified (Lage et al., 2019). One of these antigens, called LiHyQ (LINJ_29_0870), was shown to be conserved between parasite species causing TL and VL. In the present study, the gene encoding LiHyQ was cloned and the recombinant protein was produced and evaluated in ELISA experiments for the diagnosis of canine and human leishmaniasis. In order to evaluate the possible prognostic role of LiHyQ antigen for leishmaniasis, antibody reactivity against the recombinant protein was investigated in samples collected from patients before treatment and six months after treatment with antimonials.

Section snippets

Bioinformatics assays

The L. infantum LiHyQ amino acid sequence was evaluated by BepiPred 2.0 program (http://www.cbs.dtu.dk/services/BepiPred/), with a cut-off of 0.65 for the prediction of B cell epitopes (Jespersen et al., 2017). Sequences containing six or more amino acid residues were selected. Homologous proteins expressed in other Leishmania and Trypanosoma species were aligned using the ClustalW program (https://www.genome.jp/tools-bin/clustalw) (Thompson et al., 1994). Regions with homology in the

Bioinformatic analyses of the LiHyQ sequence

The L. infantum LiHyQ protein was found conserved in other Leishmania species, such as L. donovani (LdCL_290013500), L. major (LmjF.29.0830) and L. braziliensis (LbrM.29.0860), with amino acid sequence similarity in the order of 99.79%, 91.32% and 72.14%, respectively (Fig. 1). A low homology with amino acid sequences of Trypanosoma cruzi-Esmo (TcCLB.504431.40; 29.83%) and T. cruzi-Non-Esmo (TcCLB.507951.90; 30.62%) proteins was observed. In addition, three linear B cell epitopes, composed of

Discussion

In the present study, we evaluated a hypothetical protein that was recently identified in L. infantum antigenic extracts by the reactivity of antibodies in sera from VL (Lage et al., 2019), as a diagnostic marker for canine and human VL and human TL. ELISA experiments showed that the rLiHyQ protein was sensitive and specific to detect both clinical forms of leishmaniasis in dogs and humans and to be detected by antibodies in sera from VL/HIV coinfected patients. The performance of the

Conflict of Interest

The authors have declared that no commercial or financial conflict of interest exist.

Financial support

This work was supported by grant MR/R005850/1 from the Medical Research Council (VAccine deveLopment for complex Intracellular neglecteD pAThogEns - VALIDATE), UK, and grant APQ-408675/2018-7 from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors also thank the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CNPq and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the student

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