Abstract
The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).
Highlights
Leishmaniasis is prevalent in 98 countries, with an incidence estimated at 1.5 to 2 million cases per year [1]
The human sera panel consisted of 65 samples from the cutaneous lesion (TL) patients infected with L. braziliensis and presenting cutaneous (CL, n = 45) or mucosal (ML, n = 20) clinical manifestations, from the Centro de Referencia em Leishmaniose (Januaria, Minas Gerais State, Brazil), and 55 samples from visceral leishmaniasis patients infected with L. infantum, from the University Hospital (Montes Claros, Minas Gerais State, Brazil)
Peroxidoxin of L. braziliensis has a homologous protein encoded in the genome of T. cruzi, H. sapiens and C. familiaris, polymorphic B-cell epitopes between these organisms may bind to specific antibodies discriminating samples from patients with leishmaniasis
Summary
Leishmaniasis is prevalent in 98 countries, with an incidence estimated at 1.5 to 2 million cases per year [1]. These methods have several limitations, such as variation in sensitivity because the parasite distribution in the tissue is not homogeneous and the reliance on invasive procedures and strict conditions for specimen collection that depends on complex structures and laboratory procedures—facts that hinders the employment of these methods in large-scale epidemiological studies [3] In this context, antigen- or antibody-based detection tests, such as enzyme-linked immunoassays (ELISA) have advantages, as they do not require special specimen-transport conditions and can be performed in local laboratories within 3–4 hours and can be used as important tools for the diagnosis and epidemiological study of leishmaniasis [4]. The ELISA has proved to be a sensitive method and suitable for epidemiological surveys; cross-reactivity with other infections such as American trypanosomiasis, as well as Leishmania vaccines, is often reported [6,7,8]
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