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Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis.

PloS one (2014-06-13)
Daniel Menezes-Souza, Tiago Antônio de Oliveira Mendes, Ronaldo Alves Pinto Nagem, Thaís Teodoro de Oliveira Santos, Ana Luíza Teixeira Silva, Marcelo Matos Santoro, Silvio Fernando Guimarães de Carvalho, Eduardo Antônio Ferraz Coelho, Daniella Castanheira Bartholomeu, Ricardo Toshio Fujiwara
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).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥99%
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Diisopropyl ether, puriss. p.a., ≥98.5% (GC)
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Diisopropyl ether, anhydrous, 99%, contains either BHT or hydroquinone as stabilizer
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3,3′,5,5′-Tetramethylbenzidine, ≥98% (TLC)
Supelco
Diisopropyl ether, analytical standard
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3,3′,5,5′-Tetramethylbenzidine, ≥98.0% (NT)
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3,3′,5,5′-Tetramethylbenzidine, tablet, 1 mg substrate per tablet
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Diisopropyl ether, ReagentPlus®, 99%, contains either BHT or hydroquinone as stabilizer
Sigma-Aldrich
Diisopropyl ether, contains either BHT or hydroquinone as stabilizer, ACS reagent, ≥99.0%
Supelco
3,3′,5,5′-Tetramethylbenzidine, standard for GC
Sigma-Aldrich
Diisopropyl ether, contains either BHT or hydroquinone as stabilizer, ACS reagent, ≥99.0%