An algorithm for visceral leishmaniasis diagnosis in Brazil from a 10-year analysis of National Reference Laboratory data.

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.

A multicenter comparative study of the performance of four rapid immunochromatographic tests for the detection of anti-Trypanosoma cruzi antibodies in Brazil.

Diagnosis of Trypanosoma cruzi (T. cruzi) 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 T. cruzi 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.

Chagas cardiomyopathy is associated with a high susceptibility to T. cruzi infection in monocyte-derived macrophages and a predominance of CD4+CD45RO+ T-cells with immunoregulatory patterns.

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.

Development of an immunogen containing CD4+/CD8+ T-cell epitopes for the prophylaxis of tegumentary leishmaniasis.

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 (rMEP/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: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, 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 (CD44highCD62Lhigh) and effector (CD44highCD62Llow) 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 rMEP/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.

Mapping linear B-cell epitopes of the Tryparedoxin Peroxidase and its implications in the serological diagnosis of tegumentary leishmaniasis.

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.

Development of a chimeric protein based on a proteomic approach for the serological diagnosis of human tegumentary leishmaniasis.

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.

Phase II validation study of the rK39 ELISA prototype for the diagnosis of canine visceral leishmaniasis in Brazil.

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.

Phase II validation study of the rK39 ELISA prototype for the diagnosis of canine visceral leishmaniasis in Brazil / Estudo de validação da fase II do teste ELISA rK39 para o diagnóstico da leishmaniose visceral canina no Brasil / Estudio de validación de la fase II del test ELISA rK39 para el diagnóstico de la leishmaniasis visceral canina en Brasil

Abstract: 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.

Resumo: Os cães são os principais reservatórios do ciclo de transmissão domiciliar da leishmaniose visceral, e o diagnóstico é essencial para a efetividade das medidas de controle recomendadas pelo Ministério da Saúde. Os autores avaliam o desempenho diagnóstico do protótipo da ELISA-Vetlisa/BIOCLIN em amostras sorológicas de 200 cães, em triplicata, incluindo cães sintomáticos, oligossintomáticos e saudáveis, com dois painéis distintos (A e B) caracterizados por testes parasitológicos enquanto referência. No estudo, o kit-protótipo mostrou sensibilidade de 99% (IC95%: 94,5-100,0) e especificidade de 100% (IC95%: 96,4-100,0). A sensibilidade do kit-protótipo não variou de maneira significativa de acordo com o estado clínico dos cães. Considerando a classificação final dos resultados (positivo ou negativo), a concordância entre os resultados dos testes em triplicata foi quase perfeita (kappa = 0,99; IC95%: 0,98-1,00). O protótipo ELISA-Vetlisa/BIOCLIN tem o potencial de ser utilizada para o diagnóstico sorológico da leishmaniose visceral canina no Brasil.

Resumen: Los perros son los principales reservorios en el ciclo de transmisión doméstica de la leishmaniasis visceral, por ello el diagnóstico es esencial para la efectividad de las medidas de control recomendadas por el Ministerio de Salud de Brasil. Evaluamos el desempeño diagnóstico de ELISA-Vetlisa/BIOCLIN prototipo con muestras de sérum de 200 perros, en triplicado, incluyendo sintomático, oligosintomático, asintomático y perros sanos, originadas por dos paneles distintos (A y B), caracterizados por test parasitológicos como referencia estándar. En este estudio, el kit prototipo mostró un 99% de sensibilidad (IC95%: 94,5-100,0) y un 100% de especificidad (IC95%: 96,4-100,0). La sensibilidad del kit prototipo no varió significativamente con el estatus clínico de los perros. Considerando la clasificación final del resultado (positiva o negativa), el acuerdo entre los resultados de los tests repetidos fue casi perfecto (kappa = 0,99; IC95%: 0,98-1,00). ELISA-Vetlisa/BIOCLIN tiene potencial para ser usado para el diagnóstico serológico de la leishmaniasis visceral canina en Brasil.

Comparative PCR-based diagnosis for the detection of Leishmania infantum in naturally infected dogs.

The applicability of molecular biology/PCR for canine visceral leishmaniasis diagnosis presents challenges, mainly due to the diversity of targets described. The objectives of this study were to compare the sensitivities and reliability of five targets (kDNA/120, kDNA/145, ITS1, hsp70/234 and hsp70/1300) in four different tissue samples (bone marrow, popliteal lymph node, skin and conjunctival swab). Sixty-five dogs (32 males and 33 females) naturally infected with Leishmania infantum and ten dogs without infection were examined. Dogs were characterized by serological and parasitological methods. The parasitological test was considered the gold standard for analysis. All tests presented high specificity 100% (95% CI 0.72-1), and variable sensitivity. The targets kDNA/145, ITS1, kDNA/120, hsp70/234 and hsp70/1300 detected 100% (65/65), 93.4% (61/65), 92.3% (60/65), 84.61% (55/65) and 72.3% (77/65) of positive animals respectively. The performance of PCR methods was analyzed in two different scenarios. The highest sensitivity value identified in all scenarios studied was kDNA/145. Our results suggest that popliteal lymph node and conjunctival swab samples, besides being less invasive collections, represent a good substratum for PCR-based diagnosis, and the target kDNA/145 is the best choice for detecting L. infantum DNA in naturally infected dogs.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis.

BACKGROUND: 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. OBJECTIVE: 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). METHODS: 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. FINDINGS: 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. MAIN CONCLUSIONS: 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.

Evaluation of three recombinant proteins for the development of ELISA and immunochromatographic tests for visceral leishmaniasis serodiagnosis

BACKGROUND 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. OBJECTIVE 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). METHODS 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. FINDINGS 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. MAIN CONCLUSIONS 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.

Immunogenomic screening approach to identify new antigens for the serological diagnosis of chronic Chagas' disease.

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.

Development and Validation of a PCR-ELISA for the Diagnosis of Symptomatic and Asymptomatic Infection by Leishmania (Leishmania) infantum.

A kDNA PCR enzyme-linked immunosorbent assay (kDNA PCR-ELISA) for the diagnosis of human visceral leishmaniasis (HVL) was developed. The detection limit of the reaction, precision measurements, and cut-off of the kDNA PCR-ELISA were defined in a proof-of-concept phase. A reference strain of Leishmania (Leishmania) infantum and a bank of 14 peripheral blood samples from immunocompetent patients with VL were characterized using techniques considered gold standards, and 11 blood samples obtained from healthy individuals of an endemic area were also assessed. Phase II evaluation determined the performance of the assay in peripheral blood samples from 105 patients with VL (adults and children), 25 patients with Leishmania/HIV coinfection, 40 healthy individuals, and 33 asymptomatic individuals living in endemic areas. The kDNA PCR-ELISA exhibited satisfactory precision, with a detection limit of 0.07 fg of DNA from L. (L.) infantum and 1 parasite/mL blood. The overall sensitivity of the assay for all groups studied was 100% (95% confidence interval [CI]: 97.1-100%), and the specificity was 95% (95% CI: 83.5-98.6%). The kDNA PCR-ELISA was shown to be a useful tool for VL symptomatic and asymptomatic individuals diagnosis and its use in endemic countries may help monitor control interventions.

Current situation and perspectives regarding human Chagas disease in midwestern of the state of Minas Gerais, Brazil.

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.