ParasitologyA conserved Leishmania hypothetical protein evaluated for the serodiagnosis of canine and human visceral and tegumentary leishmaniasis, as well as a serological marker for the posttreatment patient follow-up
Introduction
Leishmaniasis is a disease complex caused by different species of protozoan parasites of the genus Leishmania. It includes clinical manifestations that vary from self-healing cutaneous lesions to the visceral form of the disease, which can be fatal if acute and untreated. Approximately 1.0 to 1.5 million tegumentary leishmaniasis (TL) cases and 0.2 to 0.5 million visceral leishmaniasis (VL) cases are registered annually (Alvar et al., 2012, World Health Organization (WHO), 2016). VL is caused by parasites of the Leishmania donovani complex, and in the Americas, the disease is caused by L. infantum species, where dogs are the main domestic reservoirs of parasites (Killick-Kendrick, 1999). TL is mainly caused by parasites of the Leishmania (Viannia) braziliensis, Leishmania (V.) guyanensis, and Leishmania (Leishmania) amazonensis species (Grimaldi Jr. and Tesh, 1993, Marzochi and Marzochi, 1994).
The outcome of human infection can vary from an asymptomatic disease to the acute form with symptoms, carrying a high risk of morbidity and mortality in the absence of adequate treatment (Ramos et al., 2017). Chemotherapy, based on the administration of pentavalent antimonials, has been used to treat the disease; however, toxicity, high cost, and/or parasite resistance are registered problems (Chávez-Fumagalli et al., 2015, Ponte-Sucre et al., 2017). In addition, infected dogs can develop the asymptomatic form of the disease, increasing the risks of parasite transmission to sand fly vectors and humans (Coura-Vital et al., 2013, Travi et al., 2018). In this context, the precise diagnosis of human and canine leishmaniasis can allow a faster treatment for human cases, in turn leading to a higher possibility of cure for the patients, as well as inducing a more adequate control of the canine disease (Coelho et al., 2016, Foglia-Manzillo et al., 2013, Fonseca et al., 2014).
The parasitological diagnosis is considered the gold standard for leishmaniasis (Cota et al., 2012). However, the technique presents low sensitivity and requires invasive collection procedures of the samples (Goto and Lindoso, 2010). The detection of Leishmania DNA, using the polymerase chain reaction (PCR) technique, is a specific method, but its sensitivity is also variable (Gomes et al., 2017, Rampazzo et al., 2017). Serological tests, including enzyme-linked immunosorbent assay (ELISA), indirect fluorescence antibody test (IFAT), direct agglutination test, Western blot, and immunochromatographic tests, are based on the detection of specific antibodies in patients' serum samples. These tools are also employed to diagnosis the disease; however, problems regarding the sensitivity and/or specificity of the tests are also registered (Georgiadou et al., 2015, Magalhães et al., 2017).
A number of recombinant proteins have been evaluated for the serodiagnosis of leishmaniasis, aiming to improve the sensitivity and specificity of the tests (Celeste et al., 2004, Mniouil et al., 2018, Wolf et al., 2014). However, a low number of molecules have shown a high efficacy to identify infected dogs and patients, and higher optimization is needed to reach more appropriate antigens to be employed in a safer diagnosis of disease (Leal et al., 2014, Salles et al., 2017). In addition, about 20% to 60% of the infected dogs develop asymptomatic VL and commonly go unidentified in the current serological analyses, hampering disease control measures (Coura-Vital et al., 2014, Faria et al., 2015). Regarding TL, most patients present low antileishmanial antibody levels, which are classified as false negative in the serological assays (Menezes-Souza et al., 2014, Souza et al., 2013). In addition, the maintenance of the parasite-specific humoral reactivity after the end of treatments in TL and VL patients is another problem that has been identified since their serology levels remain positive for months or even years after the patient has been treated, which make it extremely difficult to differentiate between those presenting past or active diseases (Lima et al., 2017, Portela et al., 2017).
It is known that antigens recognized by antibodies in both TL and VL patients´ sera, but not in those from treated patients, could be considered for the serodiagnosis of the disease (Magalhães et al., 2017). In a recent immunoproteomic approach performed in L. braziliensis species, some hypothetical proteins were recognized by antibodies in TL patients' sera (Duarte et al., 2015). In the present study, bioinformatics assays were used to functionally predict one of these recognized proteins, namely, LiHyE, as well as to evaluate this antigen as a recombinant protein by means of ELISA assays for the serodiagnosis of human and canine leishmaniasis, as well as the protein-specific humoral reactivity before and after the end of treatments in these patients.
Section snippets
Cloning, expression, and purification of recombinant LiHyE (rLiHyE) protein
The amino acid sequence of the LiHyE (XP_003392281.1) protein was cloned from genomic L. infantum DNA by using the following primers: 5′-GGATCCATGCCGCGTCCGTTCGGTGTTTG-3′ (forward) and 5′-AAGCTTAAGCGGGGATGTAGAACTTG-3′ (reverse), besides the BamHI and HindIII restriction enzymes. The DNA fragment was excised from a gel, which was purified and linked into the pGEM®-T vector (Promega, USA). The recombinant plasmid was transformed into Escherichia coli XL1-Blue (Phoneutria, Brazil) cells, and
Diagnostic evaluation in human leishmaniasis
The diagnostic evaluation of rLiHyE in human TL, represented by samples collected from patients developing CL or ML, as well as in VL subjects, was performed since antigen controls, rA2, and SLA were used. Results showed that rLiHyE was reactive with antibodies in CL, ML, and VL patients' sera samples but not with those present in cross-reactive diseases developing patients' sera, such as Chagas disease, paracoccidioidomycosis, leprosy, and aspergillosis (Fig. 1A). ROC curves were constructed
Discussion
A different variety of antigens has been tested for the serodiagnosis of leishmaniasis, such as recombinant proteins (Dias et al., 2017), synthetic peptides (Florez et al., 2017, Jamal et al., 2016), and/or polypeptide or polyprotein mixtures (Magalhães et al., 2017), since serology can be considered a useful diagnostic tool for leishmaniasis because the collection of the samples is less invasive than biopsies used for parasitological exams and high levels of antileishmanial antibodies can be
Competing interests
The authors declare to have no competing interests.
Funding
This work was supported by the Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT Nano-Biofar), 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 is a grant recipient of CNPq. MACF is a grant recipient of CAPES/FAPEMIG.
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