Serological diagnosis and prognostic of tegumentary and visceral leishmaniasis using a conserved Leishmania hypothetical protein

https://doi.org/10.1016/j.parint.2018.02.001Get rights and content

Highlights

  • A conserved Leishmania protein, LiHyS, was evaluated in serological assays.

  • High sensitivity and specificity was reached when canine and human sera were tested.

  • The anti-LiHyS antibody presence was investigated before and after the treatment.

  • The specific-protein antibodies decrease after the end of the treatment.

  • The humoral response one year after was similar to this found in control subjects.

Abstract

New candidates for serological markers against leishmaniasis are required to be identified, since the presence of high titers of anti-Leishmania antibodies remain detected in sera of treated and cured patients, when current antigens have being employed. In this study, the diagnostic performance of a conserved Leishmania hypothetical protein was evaluated against a human and canine serological panel. The serological follow-up of the patients was also evaluated, using this recombinant antigen (rLiHyS) in ELISA assays. In the results, high sensitivity and specificity values were found when rLiHyS was used in the serological tests, while when the recombinant A2 (rA2) protein or an antigenic Leishmania preparation were used as controls, low sensitivity and specificity were found. Regarding the serological follow-up of the patients, significant reductions in the anti-rLiHyS antibody levels were found and, one year after the treatments, the anti-protein IgG production was similar to this found in the non-infected groups, reflecting a drop of the anti-rLiHyS antibody production. In conclusion, the present study shows for the first time a new recombinant antigen used to identify tegumentary and visceral leishmaniasis, as well as being able to serologically distinguish treated and cured patients from those developing active disease.

Introduction

Leishmaniasis is a disease complex caused by parasites from Leishmania genus, presenting an incidence of 0.2 to 0.4 million visceral leishmaniasis (VL) cases, and 0.7 to 1.2 million of tegumentary leishmaniasis (TL) cases [45]. Tegumentary leishmaniasis (TL) exhibits distinct clinical manifestations ranging from cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), to mucosal leishmaniasis (ML). Leishmania braziliensis is the main species responsible by the cases of the disease in the Americas, while L. infantum is the main responsible for the VL cases [34].

Dogs are considered as important reservoirs for visceral disease transmission, due to their close relationship with humans [10]. Regarding the canine disease, animals can develop from asymptomatic infection, when they are apparently healthy, to widespread chronic infections, which can lead to the death [25,40]. Regarding TL, of which dogs are accidental hosts, marsupials, rodents, and wild canids species have been found as reservoirs of the parasites [28].

Serological tests used to diagnose leishmaniasis present variable efficacy, indicating that there is a need for new studies to reach a safer diagnostic result [18]. In addition, distinct Leishmania species exhibit a distribution that overlap in many geographical regions, making it difficult to isolate the parasite species causing the disease [16]. Also, anti-Leishmania antibodies in cured and treated patients usually remain positive for months and years after the treatments, making it difficult to distinguish between past, current and cured infections [23,43].

Despite diagnostic methods available for VL, some problems persist and maximum sensitivity and specificity values are not reached [11]. In addition, most of the asymptomatic animals are not identified by the serological assays. In this context, an early and reliable diagnosis could refine the identification of these animals, as well as allow to control the spread of this disease in the world [25]. The serodiagnosis of TL also presents problems, since most of the patients present low levels of antileishmanial antibodies and, consequently, false-negative results are usually found in the serological assays [32,33]. As a consequence, the search for identifying new candidates to be employed in the improvement of the diagnosis of TL still remains, aiming to improve sensitivity and specific values.

Modern laboratorial techniques have allowed the identification of recombinant antigens to be evaluated in the serodiagnosis of canine and human leishmaniasis. Recombinant proteins, such as cytochrome c oxidase and IgE-dependent histamine-releasing factor [9], rLiHyD [22], rKLO8 [25], rLbHyM [23], rLiHyV [24], rHSP83 [6], rA2 [1,5], among others, have been studied with this purpose. However, although they present satisfactory results to identify the active disease, their optimization is still required to obtain maximum sensitivity and specificity values aiming to identify asymptomatic cases, as well as leishmaniasis-related diseases developing patients. In addition, no antigen is currently used to serologically follow-up the evolution of the patients treatment, since antibody titers remain positive for a long period of time after their parasitological and/or clinical cure [15,19].

In this context, the search for new Leishmania proteins, are able to stimulate the humoral response in the infected hosts, and allow distinguishing between treated and untreated patients, should help the development of more sophisticated tests to detect the disease [13]. In this context, in the present study, a conserved parasite protein, namely LiHyS (XP_001467126.1), which was recognized by VL dogs sera, but not by sera from healthy dogs or from those infected with Trypanosoma cruzi [8]; was cloned and its recombinant version (rLiHyS) was evaluated for the serodiagnosis of human and canine leishmaniasis. In our work, rLiHyS was also employed as a diagnostic antigen in ELISA assays to compare its reactivity in sera samples of untreated and treated patients.

Section snippets

Ethics statement

This study was approved by the Committee on the Ethical Handling of Research Animals of Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil (protocol number 0333/2015). The work was also approved by the Human Research Ethics Committee of UFMG (protocol number CAAE–32343114.9.0000.5149).

Canine sera

The sample used was composed by 113 domestic animals (Canis familiaris), and consisted of males (n = 67) and females (n = 46) of different breeds and ages. Healthy dogs (n = 21) were

Sequence evaluation and diagnostic performance of rLiHyS for canine leishmaniasis

Bioinformatics assays showed that LiHyS presents about 80% identity to its homolog in L. braziliensis, whereas 33% and 34% homology was found in two T. cruzi sequences (Fig. 1). In addition, the main B cell epitopes were found conserved in the Leishmania sequences, but not in the T. cruzi sequences, then showing a high conservation of this antigen in the Leishmania parasite. To evaluate the diagnostic application of rLiHyS for canine VL, ELISA assays using rA2 and L. infantum SLA as comparative

Discussion

The parasitological diagnosis based on Leishmania detection in lesion or mucosal fragments of TL patients remains as the gold standard to diagnose the disease. However, although the direct microscopic identification of the parasites is simpler, its sensitivity is hampered, mainly in the cases where parasitemia is low, such as in patients developing the chronic disease [21,35]. On the other hand, serological methods have been employed to diagnose VL, and they have been considered relevant to

Conflict of interest

The authors declare no commercial or financial conflict of interest.

Acknowledgments

This work was supported by grants from Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais (Edital 02/2017), Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT Nano-Biofar) (573924/2008-2), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014) and CNPq (APQ-482976/2012-8, APQ-488237/2013-0, and APQ-467640/2014-9). EAFC and RBC are grant recipient of CNPq. MACF is a grant recipient of CAPES/FAPEMIG.

References (45)

  • B.C. Salles et al.

    Leishmania infantum mimotopes and a phage-ELISA assay as tools for a sensitive and specific serodiagnosis of human visceral leishmaniasis

    Diagn. Microbiol. Infect. Dis.

    (2017)
  • W.W. Zhang et al.

    Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani

    Mol. Biochem. Parasitol.

    (1996)
  • L. Almeida-Silva

    Immunologic tests in patients after clinical cure of visceral leishmaniasis

    Am. J. Trop. Med. Hyg.

    (2006)
  • M.M. Arruda et al.

    Validity and reliability of enzyme immunoassays using Leishmania major or L. infantum antigens for the diagnosis of canine visceral leishmaniasis in Brazil

    PLoS One

    (2013)
  • M.M. Arruda et al.

    Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

    Mem. Inst. Oswaldo Cruz

    (2016)
  • B.J. Celeste et al.

    Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

    Braz. J. Med. Biol. Res.

    (2004)
  • E.A.F. Coelho et al.

    Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection

    Infect. Immun.

    (2003)
  • V.T.S. Coelho et al.

    Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach

    PLoS Negl. Trop. Dis.

    (2012)
  • W. Coura-Vital et al.

    Risk factors for seroconversion by Leishmania infantum in a cohort of dogs from an endemic area of Brazil

    PLoS One

    (2013)
  • W. Coura-Vital et al.

    Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and a new proposal for diagnosis

    PLoS One

    (2014)
  • D.S. Dias et al.

    Antigenicity, immunogenicity and protective efficacy of a conserved Leishmania hypothetical protein against visceral leishmaniasis

    Parasitology

    (2017)
  • M.C. Duarte et al.

    Proteins selected in Leishmania (Viannia) braziliensis by an immunoproteomic approach with potential serodiagnosis applications for tegumentary leishmaniasis

    Clin. Vaccine Immunol.

    (2015)
  • Cited by (21)

    • Evaluation from a B-cell epitope-based chimeric protein for the serodiagnosis of tegumentary and visceral leishmaniasis

      2022, Microbial Pathogenesis
      Citation Excerpt :

      Aiming to solve such a problem, Leishmania proteins, such as K39, A2, HSP83, heat shock proteins, LiHyp1, LiHyD, LiHyE, amongst others, have been used as recombinant antigens for the serodiagnosis of disease, and results have been promising [19–23]. However, few have been evaluated as prognostic markers for leishmaniasis [24,25]. In addition, the variability in the humoral response found for individual patients suggests that the combination of distinct antigens in a unique product could potentially improve the diagnosis efficacy [26,27].

    • Sensitive and specific serodiagnosis of tegumentary leishmaniasis using a new chimeric protein based on specific B-cell epitopes of Leishmania antigenic proteins

      2022, Microbial Pathogenesis
      Citation Excerpt :

      In addition, the specificity has been also variable by the presence of cross-reactive antigens in patients developing leprosy, malaria, tuberculosis, histoplasmosis, aspergillosis, systemic lupus erythematosus, among others [14–19]. There are also cases where treated patients maintain positive serology for months and/or years after treatment and clinic cure, being difficult to differentiate them from those with active disease [20,21]. In this context, the search continues to identify more sensitive and specific diagnostic targets for TL.

    • 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

      2021, Acta Tropica
      Citation Excerpt :

      In addition, we observed that B-cell epitopes were conserved between these parasite species. Thus, it is possible that antibodies in TL patients sera could react with rLiHyQ, even when levels of circulating anti-Leishmania antibodies in TL patients are low, such as found in other studies (Menezes-Souza et al., 2014, Carvalho et al., 2017, Dias et al., 2018). These facts suggest that there is a possible diagnostic role of rLiHyQ for tegumentary disease, although additional experiments will be certainly performed to confirm the diagnostic role.

    • Evaluation of Leishmania infantum pyridoxal kinase protein for the diagnosis of human and canine visceral leishmaniasis

      2020, Immunology Letters
      Citation Excerpt :

      The rA2 antigen is a Leishmania amastigote protein found in some parasite species [48]. Studies have shown the diagnostic role of this protein in symptomatic canine [49,50] and human [51,52] VL cases, although variable results have been found mainly when asymptomatic samples are evaluated [45,53–55]. Higher OD values were found when rA2 was tested against healthy dog samples, when compared to data obtained using the rPK protein or Peptide as antigens.

    View all citing articles on Scopus
    View full text