Serological tests are preferentially used for the diagnosis of Chagas’ disease (CD) during the chronic phase because of the low parasitemia and high anti-
Trypanosoma cruzi
antibody titers. However, ...the current methods showed several disadvantages, as contradictory or inconclusive results, mainly related to the characteristics of the antigens used, in general, crude or whole parasites, but also due to antigen production protocol and the experimental conditions used in serological tests. Thus, better-quality serological assays are urgently needed. Here, we performed a wide immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted proteome based on genome sequence from
T
.
cruzi
strains to will be applied as synthetic peptides in the serodiagnosis of the chronic CD. Three B-cell epitopes derived from mucin-associated surface protein (MASP) family, expressed in both infective parasite stages, trypomastigote and amastigotes, conserved in
T
.
cruzi
strains, and highly divergent as compared with
Leishmania
spp. proteome, were selected for this study. The results demonstrated that synthetic peptide 2 and a mixture of peptides (Mix II: peptides 2 and 3) were able to identify all chronic CD cases, indeterminate or Chagas cardiomyopathy clinical presentation, and simultaneously able to discriminate infections caused by
Leishmania
parasites, with high accuracy (98.37 and 100.00%, respectively) and agreement (kappa index = 0.967 and 1.000, respectively) with direct methods as compared to current diagnostic pipeline performed by reference laboratories in Brazil. This study represents an interesting strategy for the discovery of new antigens applied to serologic diagnosis of infectious diseases and for the technological development of platforms for large-scale production of diagnostic tests.
Visceral leishmaniasis (VL) requires diagnostic assays to complement clinical suspicion. However, there is no standardization of a diagnostic flow using available assays. This study aimed to evaluate ...the performance of parasitological, molecular, and serological assays for diagnosing VL and propose a diagnostic flow based on performance, practicality, and invasiveness. We conducted a study of 10-year (2010–2020) routine diagnoses of VL at the Brazilian National Reference Laboratory. We propose a diagnostic flow where individuals suspected of VL are initially screened for antibodies using an immunochromatographic test (ICT) with rK39 antigen on the nitrocellulose membrane. This is followed by a blood polymerase chain reaction (PCR) for
Leishmania
sp. kDNA and direct parasitological exam and/or PCR in bone marrow aspirate. A positive result in any of these assays can define a VL case. If clinical suspicion persists in negative individuals, the diagnostic flow should be repeated. The proposed flow has the potential to standardize and improve the diagnosis of VL. It reduces the need for invasive tests without compromising diagnostic accuracy.
Leishmania braziliensis
is responsible for most cases of human tegumentary leishmaniasis (HTL) and has caused a wide range of clinical manifestations, including cutaneous (CL) and mucosal ...leishmaniasis (ML). The diagnosis is based on criteria that consider epidemiological data, clinical findings, and laboratory tests and is hard to establish. For laboratory tests, none of the assays available can be considered gold standards for disease detection. In addition, the Montenegro skin test, essential to supporting infectologists in the clinical management of the disease, is no longer available in Brazil. Thus, the aim of this study was to develop new targets to be used in diagnostic tests for HTL. In the first step, we carried out two-dimensional gel electrophoresis, followed by mass spectrometry, combined with heat map analysis and immunoproteomics approach, and disclosed eight proteins expressed in the amastigote stage specifically recognized by serum from CL and ML patients. A chimeric protein was designed based on the combination of thirteen linear B-cell epitopes, identified by immunoinformatics analysis, from
L
.
braziliensis
proteins. Our results showed that the strategy used in this work was successful in developing an antigen to be used in immunological assays (100.0% sensitivity and specificity) in the detection of HTL cases and in comparison with results obtained from an ELISA using soluble
L
.
braziliensis
antigen (SLb-Antigen) and immunofluorescence assay (Bio-Manguinhos/FIOCRUZ). The present technology opens the door for its use in field exams by means of an immunochromatographic test, which will be even more helpful in regions without laboratory structures.
Key points
• Rational strategy to develop antigens.
• Integration between immunoproteomic and immunoinformatics analysis.
• Chimeric protein shows high performance in HTL diagnosis.
Dogs are the main reservoirs in the domestic transmission cycle of visceral leishmaniasis, and the diagnosis is essential for the effectiveness of the control measures recommended by the Brazilian ...Ministry of Health. We assessed the diagnostic performance of the ELISA-Vetlisa/BIOCLIN prototype with serum samples from 200 dogs, in triplicate, including symptomatic, oligosymptomatic, asymptomatic, and healthy dogs, originated by two distinct panels (A and B) characterized by parasitological tests as the reference standard. In this study, the prototype kit showed a 99% sensitivity (95%CI: 94.5-100.0) and a 100% specificity (95%CI: 96.4-100.0). The sensitivity of the prototype kit did not vary significantly with the clinical status of the dogs. Considering the final result classification (positive or negative), agreement between the results of repeated tests was almost perfect (kappa = 0.99; 95%CI: 0.98-1.00). ELISA-Vetlisa/BIOCLIN is a promising option for the serological diagnosis of canine visceral leishmaniasis in Brazil.
•Tryparedoxin Peroxidase for the diagnosis of tegumentary leishmaniasis.•Integration between immunoproteomic and immunoinformatics analysis.•Flow cytometry analyses of protein levels in parasite ...evolutionary stages.•Prediction of linear B-cell epitopes to discover new diagnosis antigens.•MNEPAPP peptide shows high performance for tegumentary leishmaniasis diagnosis.
Diagnosis of tegumentary leishmaniasis (TL) is essential to avoid permanent damage and severe functional sequelae and there is an urgent need to discover new antigens. The present study aimed to comprehensively evaluate the potential use of the Tryparedoxin Peroxidase (TryP) as an antigen for serological tests. The proposal integrates data from immunoproteomics with immunoinformatics, in addition to a precise analysis of protein levels in the evolutionary stages of the parasite by flow cytometry. To evaluate the performance in the diagnosis of TL, Enzyme-Linked Immunosorbent Assay (ELISA) assays were performed using the recombinant protein and the respective B-cell epitope, followed by an analysis of the contribution of this peptide in the recognition of the protein by patients, evaluated by serum depletion assays. We showed that the TryP has a linear B-cell epitope with high divergence compared to orthologs from Trypanosoma cruzi and Homo sapiens. The results also show high expression and positive cells for TryP (TryP+) in the infective metacyclic promastigotes (MET) and intracellular (24 and 48 hours) stages. From the depletion assays, it was possible to confirm the contribution of the peptide in the specific recognition of the TryP protein by patients with TL (13.7-15.9%). ELISA using the peptide showed high performance in the diagnosis compared to the recombinant TryP (rTryP), Soluble Leishmania braziliensis Antigen (sLba) and Immunofluorescence Assay (IFA) with accuracy of 94.29, 89.29, 65.00 and 37.14%, respectively). We can conclude that the MNEPAPP peptide is a potential antigen for the diagnosis of TL.
Tegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and
Leishmania braziliensis
is its main etiological agent. The inefficiency of control measures, such as high ...toxicity and costs of current treatments and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4
+
/CD8
+
) epitope, derived from conserved proteins found in
Leishmania
species and associated with TL, to generate a chimeric protein (
r
MEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an
L. braziliensis
murine infection model:
r
MEP/TL in saline or associated with MPLA-PHAD
®
. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging,
r
MEP/TL and
r
MEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG
2a
, IgG
2a
/IgG
1
, NO, CD4
+
, and CD8
+
T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4
+
IL-10
+
and CD8
+
IL-10
+
T-cells. Vaccines also induced the development of central (CD44
high
CD62L
high
) and effector (CD44
high
CD62L
low
) memory of CD4
+
and CD8
+
T-cells. These findings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with
r
MEP/TL and, preferably, associated with an adjuvant, may be considered an effective tool to prevent TL.
Key points
•
Rational design approaches for vaccine development.
•
Central and effector memory of CD4+ and CD8+ T-cells.
•
Vaccine comprised of rMEP/TL plus MPLA as an effective tool to prevent TL.
•Immunopathogenesis of chronic chagas cardiomyopathy.•Monocytes from CCC patients present high susceptibility to infection.•High IL-10 and low IFN-γ and IFN-γ/IL-10 levels its associated to ...susceptibility of phagocytes.•Chagas cardiomyopathy is associated with immunoregulatory CD4+CD45RO+ T-cells.
The pathogenesis of Chronic Chagas Cardiomyopathy (CCC) is still not fully understood, and the persistence of the parasite in tissues seems to be essential for the onset and progression of heart disease, tissue destruction, and chronic inflammation. It is clear that the polarity found between the asymptomatic (IND) and cardiac clinical forms refers mainly to the mechanisms involved in the regulation of the host's immune response. Thus, to elucidate aspects of the susceptibility of host phagocytes to T. cruzi infection, the present study explored novel aspects of innate immune response, integrating data on susceptibility to infection and intracellular replication, using monocyte-derived macrophages from CCC patients, together with memory CD4+ T-cells (CD45RO+). The isolation of PBMC was conducted by means of in vitro infection assay with T. cruzi trypomastigotes and flow cytometry analysis of the intracytoplasmic cytokine production by CD4+T-cells. Our findings indicated that monocytes derived from individuals with CCC are more susceptible to the infection and replication of intracellular amastigotes. Moreover, the stimulation of CD4+ T-cells from CCC patients, together with T. cruzi trypomastigotes, induces a predominance of a regulatory response over a type 1 response, demonstrated by an increase in IL-10 production and a reduction in the IFN-γ and IFN-γ/IL-10. Suppression of the function of monocyte-derived macrophages, from CCC patients, to control trypomastigote infection and intracellular replication sheds light on a potential susceptibility of these cells isolated from peripheral blood, which may reflect the ineffectiveness of parasite control by phagocytes in cardiac tissues, which can subsequently result in serious heart disease.
Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination ...programs; however, it has significant limitations due to low accuracy.
This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL).
A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection.
Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis.
The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.
Diagnosis of
(
) infection in the chronic phase of Chagas disease (CD) is performed by serologic testing. Conventional tests are currently used with very good results but require time, laboratory ...infrastructure, and expertise. Rapid diagnostic tests (RDTs) are an alternative as the results are immediate and do not require specialized knowledge, making them suitable for epidemiologic studies and promising as a screening tool. Nevertheless, few studies conducted comparative evaluations of RDTs to validate the results and assess their performance. In this study, we analyzed four trades of rapid tests (OnSite Chagas Ab Combo Rapid Test-United States, SD Bioline Chagas AB-United States, WL Check Chagas-Argentina, and TR Chagas Bio-Manguinhos-Brazil) using a panel of 190 samples, including sera from 111 infected individuals, most of whom had low
antibody levels. An additional 59 samples from uninfected individuals and 20 sera from individuals with other diseases, mainly visceral leishmaniasis, were included. All tests were performed by three independent laboratories in a blinded manner. Results showed differences in sensitivity from 92.8 to 100%, specificity from 78.5 to 92.4%, and accuracy from 90.5 to 95.3% among the four assays. The results presented here show that all four RDTs have high overall diagnostic ability. However, WL Check Chagas and TR Chagas Bio-Manguinhos were considered most suitable for use in screening studies due to their high sensitivity combined with good performance. Although these two RDTs have high sensitivity, a positive result should be confirmed with other tests to confirm or rule out reactivity/positivity, especially considering possible cross-reactivity with individuals with leishmaniasis or toxoplasmosis.
Recognising the importance of Chagas disease in Brazil, Bambuí set
up epidemiological surveillance for Chagas disease in 1974 and was the
first municipality to do so. To ascertain the current ...epidemiology of
Chagas disease in this municipality, 1.782 blood samples from the
general population were analysed; 7.7% of samples were found to be
seropositive for Chagas disease. A strong positive correlation between
increasing age and Chagas disease was evident in both genders, with the
highest prevalence in individuals aged over 60 years. Clinically, the
cardiodigestive form of Chagas disease was the most common in these
samples. These data confirm the interruption of Trypanosoma cruzi
transmission, in parallel with a still important residual morbidity of
Chagas disease in the county, thus supporting political decisions that
will prioritise epidemiological surveillance and medical treatment of
Chagas disease in the coming years.
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