Signatures of CD4+ T and B cells are associated with distinct stages of chronic chagasic cardiomyopathy.

Introduction: Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi. While most patients are asymptomatic, around 30% develop Chronic Chagasic Cardiomyopathy (CCC). Methods: Here, we employed high-dimensional flow cytometry to analyze CD4+ T and B cell compartments in patients during the chronic phase of Chagas disease, presenting the asymptomatic and mild or moderate/severe cardiac clinical forms. Results: Effector CD27-CD4+ T cells were expanded in both CCC groups, and only mild CCC patients showed higher frequencies of effector memory and T follicular helper (Tfh) cells than healthy donors (CTL) and asymptomatic patients. Unsupervised analysis confirmed these findings and further revealed the expansion of a specific subpopulation composed of Tfh, transitional, and central memory CD4+ T cells bearing a phenotype associated with strong activation, differentiation, and exhaustion in patients with mild but not moderate/severe CCC. In contrast, patients with mild and moderate/severe CCC had lower frequencies of CD4+ T cells expressing lower levels of activation markers, suggesting resting status, than CTL. Regarding the B cell compartment, no alterations were found in naïve CD21-, memory cells expressing IgM or IgD, marginal zone, and plasma cells in patients with Chagas disease. However, expansion of class-switched activated and atypical memory B cells was observed in all clinical forms, and more substantially in mild CCC patients. Discussion: Taken together, our results showed that T. cruzi infection triggers changes in CD4+ T and B cell compartments that are more pronounced in the mild CCC clinical form, suggesting an orchestrated cellular communication during Chagas disease. Conclusion: Overall, these findings reinforce the heterogeneity and complexity of the immune response in patients with chronic Chagas disease and may provide new insights into disease pathology and potential markers to guide clinical decisions.

The seasonality as a relevant aspect to be considered for differential diagnosis of Trypanosoma vivax infection and co-infections in female cattle.

Bovine Trypanosomiasis and other infectious diseases cause relevant loss for the livestock industry impacting productive/reproductive indices. This study intended to better understand the frequency, seasonality, and profile of infections associated with Bovine Trypanosomiasis. A total of 1443 serum samples were screened for T. vivax infection and other infectious diseases: Neosporosis, Leptospirosis, Bovine Leukosis Virus infection/(BLV), Infectious Bovine Rhinotracheitis/(IBR) or Bovine Viral Diarrhea/(BVD). Distinct methods were used for screening and diagnosis: immunofluorescence assay (Trypanosomiasis), ELISA (Neosporosis,BLV,IBR,BVD) and microscopic agglutination test (Leptospirosis). Our findings demonstrated that the seropositivity for Trypanosomiasis=57% was similar to Neosporosis=55%, higher than Leptospirosis=39% and BVL=34%, but lower than IBR=88% and BVD=71%. The seropositivity for Trypanosomiasis was higher in the autumn and lower in the winter. Regardless the season, the IBR seropositivity (min=73%;max=95%) was higher than Trypanosomiasis (min=48%;max=68%). Moreover, Neosporosis (min=71%;max=100%) and BVD (min=65%;max=76%) were more frequent than Trypanosomiasis in the summer, winter and spring. The diagnosis outcome revealed that Trypanosomiasis&IBR=43% and Trypanosomiasis&Neosporosis=35% were the most frequent co-infections with higher seropositivity in the autumn (58%) and summer (80%), respectively. Noteworthy, high seropositivity to Trypanosomiasis&BVD was registered in the autumn (46%). Together, our data re-enforce the relevance of differential diagnosis between Trypanosomiasis with other bovine infectious diseases and that differences in the seasonality profile is a relevant aspect to be considered while selecting the differential diagnosis to be applied.

Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Potential Biomarkers for Asymptomatic Visceral Leishmaniasis among Iraq-Deployed U.S. Military Personnel.

Visceral leishmaniasis (VL) is a chronic infection caused by Leishmania (L.) donovani or L. infantum parasites. Despite having the infection, most individuals never develop the clinical disease and are able to control the parasite and remain asymptomatic. However, some progress to symptomatic VL, leading to death if untreated. The host immune response has a major role in determining the progression and severity of the clinical manifestations in VL; several immune biomarkers of symptomatic VL have been described with interferon-gamma release as a surrogate biomarker of host cellular immunity. However, new biomarkers to identify asymptomatic VL (AVL) are needed for the identification of people at risk for VL activation. In our study, levels of chemokine/cytokine in the supernatants of peripheral mononuclear blood cells (PBMC) from 35 AVL+ Iraq-deployed participants, stimulated in vitro with soluble Leishmania antigen for 72 h, were assessed by a bead-based assay that allows the measurement of multiple analytes. PBMC of AVL-negative military beneficiaries were used as controls. Monocyte Chemoattractant Protein-1, Monokine Induced by Gamma Interferon and Interleukin-8, were detected at high levels in AVL+ stimulated cultures from Iraq deployers compared to uninfected controls. Measurement of chemokine/cytokine levels can identify cellular immune responses in AVL+ asymptomatic individuals.

Serum biomarkers in patients with unilateral or bilateral active pulmonary tuberculosis: Immunological networks and promising diagnostic applications.

The present observational study was designed to characterize the integrative profile of serum soluble mediators to describe the immunological networks associated with clinical findings and identify putative biomarkers for diagnosis and prognosis of active tuberculosis. The study population comprises 163 volunteers, including 84 patients with active pulmonary tuberculosis/(TB), and 79 controls/(C). Soluble mediators were measured by multiplexed assay. Data analysis demonstrated that the levels of CCL3, CCL5, CXCL10, IL-1ß, IL-6, IFN-γ, IL-1Ra, IL-4, IL-10, PDGF, VEGF, G-CSF, IL-7 were increased in TB as compared to C. Patients with bilateral pulmonary involvement/(TB-BI) exhibited higher levels of CXCL8, IL-6 and TNF with distinct biomarker signatures (CCL11, CCL2, TNF and IL-10) as compared to patients with unilateral infiltrates/(TB-UNI). Analysis of biomarker networks based in correlation power graph demonstrated small number of strong connections in TB and TB-BI. The search for biomarkers with relevant implications to understand the pathogenetic mechanisms and useful as complementary diagnosis tool of active TB pointed out the excellent performance of single analysis of IL-6 or CXCL10 and the stepwise combination of IL-6 â†’ CXCL10 (Accuracy = 84 %; 80 % and 88 %, respectively). Together, our finding demonstrated that immunological networks of serum soluble biomarkers in TB patients differ according to the unilateral or bilateral pulmonary involvement and may have relevant implications to understand the pathogenetic mechanisms involved in the clinical outcome of Mtb infection.

Methods Applied to the Diagnosis of Cattle Trypanosoma vivax Infection: An Overview of the Current State of the Art.

Bovine trypanosomiasis caused by Trypanosoma vivax is a relevant disease in domestic ungulates in Latin America, causing different types of livestock losses, particularly in African and South American countries, leading to loss of millions of dollars/year related to dairy and meat production. In addition, T. vivax trypanosomiasis requires intensive veterinary care. While vector control is a feasible measure to manage disease spreading, the search for accurate diagnostic tools still represents a gap in routine veterinary practices and a challenge for the scientific community. The parasite is mechanically transmitted by fomites or by the saliva of haematophagous flies, such as Stomoxys sp. and Tabanus sp., infecting cattle as well as a number of animal hosts. The main symptoms of T. vivax bovine trypanosomiasis are apathy, fever, restricted growth, miscarriage, progressive weakness, neurological signs, pale mucous, loss of appetite, lethargy, and substantial weight loss. In most cases, the presence of animals with subclinical infections, nonspecific symptoms and without apparent parasitaemia presents a challenge when making a diagnosis, which requires accurate methods. Herein, we review state of the art concerning current methods available for the diagnosis of T. vivax bovine trypanosomiasis, focusing on clinical, parasitological, immunological and molecular approaches, highlighting the main features of each method, including "pros and cons". Overall, combining several diagnostic techniques is a better choice since it leads to fewer false negative results and contributes to better disease control.

Trypanosoma cruzi antigen detection in blood to assess treatment efficacy and cure in mice models of Chagas disease.

Introduction: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi. Although endemic mainly in Latin America, CD has become a global public health problem due to migration of infected individuals to non-endemic regions. Despite progress made in drug development, preclinical assays for drug discovery are required to accelerate the development of new drugs with reduced side effects, which are much needed for human treatment. Methods: We used a cure model of infected mice treated with Fexinidazole (FZ) to further validate a novel Enzyme Linked Aptamer (ELA) assay that detects parasite biomarkers circulating in the blood of infected animals. Results: The ELA assay showed cure by FZ in ~71% and ~77% of mice infected with the VL-10 and Colombiana strains of T. cruzi, respectively. The ELA assay also revealed superior treatment efficacy of FZ compared to Benznidazole prior to immunosuppression treatment. Discussion: Our study supports the use of ELA assay as an alternative to traditional serology or blood PCR to assess the efficacy of antichagasic drugs during their preclinical phase of development. Further, the combination of high sensitivity and ease of use make this parasite antigen detection assay an attractive new tool to facilitate the development of much needed new therapies for CD.

Chagasic cardiomyopathy is marked by a unique signature of activated CD4+ T cells.

Chagas disease is a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic Chagas disease cardiomyopathy (CCC) is the most prominent clinical form and can lead to heart failure, thromboembolism, and sudden death. While previous reports have supported a role for CD4+ T lymphocytes in the pathogenesis of CCC a comprehensive analysis of these cells during different clinical forms is lacking. Here, we used high-dimensional flow cytometry to assess the diversity of circulating CD4+ T cells in patients with distinct clinical forms. We found increased frequencies of CD4+CD69+ T cells in patients compared to controls. CD39+ regulatory T cells, represented by mesocluster 6 were reduced in mild CCC patients compared to controls. Cytotoxic CD4+ T cells co-expressing granzyme B and perforin were expanded in patients with Chagas disease and were higher in patients with mild CCC compared to controls. Furthermore, patients with mild CCC displayed higher frequencies of multifunctional effector memory CD4+ T cells. Our results demonstrate an expansion in activated CD4+ T cells and a decrease in a functional subset of regulatory T cells associated with the onset of Chagas cardiomyopathy, suggesting their role in the establishment of cardiac lesions and as potential biomarkers for disease aggravation.

Chemokines in Leishmaniasis: Map of cell movements highlights the landscape of infection and pathogenesis.

Pathogen interactions with the host immune response components are critical for establishing protective immunity and pathological responses against Leishmania parasites. A predominant proinflammatory profile associated with enhanced phagocytosis trigger a cell-mediated immune response that is relevant to infection control. On the other hand, an anti-inflammatory phenotype, correlated with a predominant modulated/regulatory response, favors intracellular proliferation of Leishmania parasites and disease progression. In this context, chemokines play an important role in determining cellular composition at inflammatory sites. Leishmania infection induces the expression of various chemokines and chemokine receptors in the mammalian host, which can subvert the host immune responses. Indeed, the balance and dynamic changes in cytokines and chemokines may control or predict the disease outcome. In this review, we address our current knowledge regarding the chemokines and chemokines receptors' role in the immunopathogenesis of Tegumentary and Visceral Leishmaniasis.

A novel Trypanosoma cruzi secreted antigen as a potential biomarker of Chagas disease.

Chagas drug discovery has been hampered by a lack of validated assays to establish treatment efficacy in pre-clinical animal models and in patients infected with T. cruzi. Reduced levels of parasite secreted antigens in the blood of infected hosts could be used to demonstrate treatment efficacy. A published proteomic study of parasite secreted antigens identified the hypothetical protein Tc_5171 as a secreted antigen. In this report, we developed Tc_5171 specific antibodies and showed that the native protein was expressed by the three life cycle stages of the parasite. Anti-peptide antibodies were able to detect the parasite antigen in blood of infected mice during the acute and the chronic phase of infection. Benznidazole treatment of infected mice significantly reduced their blood antigen levels. Of clinical significance, patients diagnosed with Chagas disease, either asymptomatic or with cardiac clinical symptoms had significantly higher Tc_5171 antigen levels compared to endemic controls. Pair-wise analysis, before and after Benznidazole treatment, of patients with asymptomatic Chagas disease showed a significant reduction in antigen levels post treatment. Taken together, our results indicate that Tc_5171 could be used as a novel biomarker of Chagas disease for diagnosis and to assess treatment efficacy.

Human Chagas-Flow ATE-IgG1 for advanced universal and Trypanosoma cruzi Discrete Typing Units-specific serodiagnosis of Chagas disease.

The molecular and serological methods available for Discrete Typing Units (DTU)-specific diagnosis of Trypanosoma cruzi in chronic Chagas disease present limitations. The study evaluated the performance of Human Chagas-Flow ATE-IgG1 for universal and DTU-specific diagnosis of Chagas disease. A total of 102 sera from Chagas disease patients (CH) chronically infected with TcI, TcVI or TcII DTUs were tested for IgG1 reactivity to amastigote/(A), trypomastigote/(T) and epimastigote/(E) antigens along the titration curve (1:250-1:32,000). The results demonstrated that "AI 250/40%", "EVI 250/30%", "AII 250/40%", "TII 250/40%" and "EII 250/30%" have outstanding accuracy (100%) to segregate CH from non-infected controls. The attributes "TI 4,000/50%", "EI 2,000/50%", "AVI 8,000/60%" and "TVI 4,000/50%" were selected for DTU-specific serotyping of Chagas disease. The isolated use of "EI 2,000/50%" provided the highest co-positivity for TcI patients (91%). The combined decision tree algorithms using the pre-defined sets of attributes showed outstanding full accuracy (92% and 97%) to discriminate "TcI vs TcVI vs TcII" and "TcI vs TcII" prototypes, respectively. The elevated performance of Human Chagas-Flow ATE-IgG1 qualifies its use for universal and TcI/TcVI/TcII-specific diagnosis of Chagas disease. These findings further support the application of this method in epidemiological surveys, post-therapeutic monitoring and clinical outcome follow-ups for Chagas disease.

CXCL9 and CXCL10 display an age-dependent profile in Chagas patients: a cohort study of aging in Bambui, Brazil.

BACKGROUND: Chagas disease is endemic in Latin America and still represents an important public health problem in the region. Chronic cardiomyopathy is the most significant chronic form due to its association with morbidity and mortality. The last decade has seen increasing evidence that inflammatory cytokines and chemokines are responsible for the generation of inflammatory infiltrate and tissue damage, with chronic chagasic cardiomyopathy patients presenting a pro-inflammatory immune response. Although studies have evaluated the role of chemokines in experimental T. cruzi infection, few have addressed their systemic profile, especially for human infection and in aging populations. The present work aimed to use the data from a large population based study of older adults, conducted in an endemic area for Chagas disease, to examine the association between serum levels of cytokines and chemokines, T. cruzi infection and electrocardiogram (ECG) abnormality. METHODS: The present work evaluated serum levels of CCL2, CXCL9, CXCL10, CCL5, CXCL8, IL-1ß, IL-6, TNF, IL-12 and IL-10 by Flow Cytometric Bead Array assay (CBA) and the results expressed in pg/ml. The baseline survey started in January 1st 1997, with 1284 participants of an aged population-based cohort. Participants signed an informed consent at baseline and at each subsequent visit and authorized death certificate and medical records verification. RESULTS: Our results demonstrated that Chagas disease patients had higher serum levels of CXCL9, CXCL10 and IL-1ß and lower serum levels of CCL5 than non-infected subjects. Moreover, our data demonstrated that CXCL9 and CXCL10 increased in an age-dependent profile in Chagas disease patients. CONCLUSION: Together, this study provided evidences that serum biomarkers increase along the age continuum and may have potential implications for establishing clinical management protocols and therapeutic intervention in Chagas disease patients.

Laboratorial algorithm for serological diagnosis of visceral leishmaniasis using rK39-ICT, DAT-LPC and FC-Simplex IgG1.

The performance of distinct serological tests (rK39-ICT, IFAT, DAT-LPC, FC-Simplex IgG1) was assessed and a laboratorial algorithm was proposed for accurate diagnosis of VL. DAT-LPC and FC-Simplex IgG1 showed outstanding accuracy (AUC = 0.93) to identify VL patients. The use of a sequential serological algorithm (rK39-ICT screening followed by DAT-LPC or FC-Simplex IgG1) improved the global accuracy for VL (97.2%) diagnosis. An alternative approach for diagnosis of VL has been also assessed for interchangeable use of serum/whole blood lysate samples in DAT-LPC and FC-Simplex IgG1. Our data showed an outstanding agreement for the results obtained with whole blood lysate samples as compared to serum samples (DAT-LPC =100%; FC-Simplex IgG1 = 99%). Together, these findings provide insights to improve the current overall accuracy of VL diagnosis and present innovative laboratorial tests and alternative samples from use in public health services.

Distinct immune response profile during rhipicephalus (boophilus) microplus infestations of guzerat dairy herd according to the maternal lineage ancestry (mitochondrial DNA).

Rhipicephalus (Boophilus) microplus ticks cause major constraints to public and livestock health, and serious economic losses. It is well known that the immune response to infestations with cattle ticks is influenced by the host genetic background leading to distinct immunological profiles between bovine hosts genetically susceptible and resistant. The influence of Bos indicus (Bi) and Bos taurus (Bt) maternal lineage ancestry of mitochondrial DNA in the profile of the immune response of Zebu cattle to ticks remains unknown. The present work evaluated the hematological parameters and the immune response profile in the peripheral blood of a Guzerat dairy herd, further categorized into two maternal lineage ancestry subgroups (Bi-mtDNA and Bt-mtDNA) after experimental infestation with larvae of R. microplus. Our data demonstrated that although hematological and erythrogram analysis showed a similar profile throughout, some cell populations present a distinct profile between the groups. Especially MON, CD335+ and CD8+ T-cells are predominant in Bi-mtDNA. Moreover, an overall picture of R. microplus infestation demonstrated that Bi-mtDNA presented a more efficient and earlier innate immune response. Bi-mtDNA showed a greater number of connections with R. microplus counts and also with the CD25+ activation marker of the immune response. Bi-mtDNA showed greater number of connections, with an important participation of the innate immune while Bt-mtDNA showed a delay in the immune response. Elucidating the mechanisms by which resistant animals prevent heavy tick infestation is a crucial step in the development of predictive biomarkers for tick resistance for use in selective breeding programs, and is also potentially useful for the development of anti-tick vaccines.

Usefulness of FC-TRIPLEX Chagas/Leish IgG1 as confirmatory assay for non-negative results in blood bank screening of Chagas disease.

A relevant issue in Chagas disease serological diagnosis regards the requirement of using several confirmatory methods to elucidate the status of non-negative results from blood bank screening. The development of a single reliable method may potentially contribute to distinguish true and false positive results. Our aim was to evaluate the performance of the multiplexed flow-cytometry anti-T. cruzi/Leishmania IgG1 serology/(FC-TRIPLEX Chagas/Leish IgG1) with three conventional confirmatory criteria (ELISA-EIA, Immunofluorescence assay-IIF and EIA/IIF consensus criterion) to define the final status of samples with actual/previous non-negative results during anti-T. cruzi ELISA-screening in blood banks. Apart from inconclusive results, the FC-TRIPLEX presented a weak agreement index with EIA, while a strong agreement was observed when either IIF or EIA/IIF consensus criteria were applied. Discriminant analysis and Spearman's correlation further corroborates the agreement scores. ROC curve analysis showed that FC-TRIPLEX performance indexes were higher when IIF and EIA/IIF consensus were used as a confirmatory criterion. Logistic regression analysis further demonstrated that the probability of FC-TRIPLEX to yield positive results was higher for inconclusive results from IIF and EIA/IIF consensus. Machine learning tools illustrated the high level of categorical agreement between FC-TRIPLEX versus IIF or EIA/IIF consensus. Together, these findings demonstrated the usefulness of FC-TRIPLEX as a tool to elucidate the status of non-negative results in blood bank screening of Chagas disease.

Identification of Anti-Trypanosoma cruzi Lead Compounds with Putative Immunomodulatory Activity.

In seeking substitutions for the current Chagas disease treatment, which has several relevant side effects, new therapeutic candidates have been extensively investigated. In this context, a balanced interaction between mediators of the host immune response seems to be a key element for therapeutic success, as a proinflammatory microenvironment modulated by interleukin-10 (IL-10) is shown to be relevant to potentiate anti-Trypanosoma cruzi drug activity. This study aimed to identify the potential immunomodulatory activities of the anti-T. cruzi K777, pyronaridine (PYR), and furazolidone (FUR) compounds in peripheral blood mononuclear cells (PBMC) from noninfected (NI) subjects and chronic Chagas disease (CD) patients. Our results showed low cytotoxicity to PBMC populations, with 50% cytotoxic concentrations (CC50) of 71.0 µM (K777), 9.0 µM (PYR), and greater than 20 µM (FUR). In addition, K777 showed no impact on the exposure index (EI) of phytohemagglutinin-stimulated leukocytes (PHA), while PYR and FUR treatments induced increased EI of monocytes and T lymphocytes at late stages of apoptosis in NI subjects. Moreover, K777 induced a more prominent proinflammatory response (tumor necrosis factor alpha-positive [TNF-α+] CD8+/CD4+, gamma interferon-positive [IFN-γ+] CD4+/CD8+ modulated by interleukin-10-positive [IL-10+] CD4+ T/CD8+ T) than did PYR (TNF-α+ CD8+, IL-10+ CD8+) and FUR (TNF-α+ CD8+, IL-10+ CD8+). Signature analysis of intracytoplasmic cytokines corroborated the proinflammatory/modulated (K777) and proinflammatory (PYR and FUR) profiles previously found. In conclusion, the lead compound K777 may induce beneficial changes in the immunological profile of patients presenting the chronic phase of Chagas disease and may contribute to a more effective therapy against the disease.

Accomplishing the genotype-specific serodiagnosis of single and dual Trypanosoma cruzi infections by flow cytometry Chagas-Flow ATE-IgG2a.

The methods currently available for genotype-specific diagnosis of T. cruzi infection still present relevant limitations, especially to identify mixed infection. In the present investigation, we have evaluated the performance of Chagas-Flow ATE-IgG2a test for early and late differential diagnosis of single and dual genotype-specific T. cruzi infections. Serum samples from Swiss mice at early and late stages of T. cruzi infection were assayed in parallel batches for genotype-specific diagnosis of single (TcI, TcVI or TcII) and dual (TcI+TcVI, TcVI+TcII or TcII+TcI) infections. The intrinsic reactivity to TcI, TcVI and TcII target antigens, including amastigote (AI/AVI/AII), trypomastigote-(TI/TVI/TII) and epimastigote (EI/EVI/EII), at specific reverse of serum dilutions (500 to 64,000), was employed to provide reliable decision-trees for "early" vs "late", "single vs "dual" and "genotype-specific" serology. The results demonstrated that selective set of attributes "EII 500/EI 2,000/AII 500" were able to provide high-quality accuracy (81%) to segregate early and late stages of T. cruzi infection. The sets "TI 2,000/AI 1,000/EII 1,000" and "TI 8,000/AII 32,000" presented expressive scores to discriminate single from dual T. cruzi infections at early (85%) and late stages (84%), respectively. Moreover, the attributes "TI 4,000/TVI 500/TII 1,000", "TI 16,000/EI 2,000/EII 2,000/AI 500/TVI 500" showed good performance for genotype-specific diagnosis at early stage of single (72%) and dual (80%) T. cruzi infections, respectively. In addition, the attributes "TI 4,000/AII 1,000/EVI 1,000", "TI 64,000/AVI 500/AI 2,000/AII 1,000/EII 4,000" showed moderate performance for genotype-specific diagnosis at late stage of single (69%) and dual (76%) T. cruzi infections, respectively. The sets of decision-trees were assembled to construct a sequential algorithm with expressive accuracy (81%) for serological diagnosis of T. cruzi infection. These findings engender new perspectives for the application of Chagas-Flow ATE-IgG2a method for genotype-specific diagnosis in humans, with relevant contributions for epidemiological surveys as well as clinical and post-therapeutic monitoring of Chagas disease.

Parasitological and immunological evaluation of cattle experimentally infected with Trypanosoma vivax.

Trypanosoma vivax infection causes relevant economical impact due to high morbidity and mortality leading to negative impact on local livestock. Despite parasitological and serological methods are used for the diagnosis of T. vivax infection, gaps regarding sensitivity and specificity of these methods still represent a challenge. The present study aimed to compare the kinetics of parasitological and serological parameters in cattle experimentally infected with T. vivax along with immunophenotypic analysis of whole blood leukocytes. Based on the parasitemia profile the analysis were performed in three distinct periods, referred as pre-patent, patent and post-treatment. Distinct kinetics of anti-T. vivax IgM and IgG were observed during the pre-patent, patent and post-treatment periods. Increased levels of WC1+ γδ T-cells were observed throughout the infection with strong correlations with other biomarkers observed during post-treatment period. Our findings demonstrated that there is a important participation of Monocytes:CD14+; NK-cells:CD335+ and WC1+ γδ T-cells that coincide with the peak of parasitemia and also with the adaptive immunity, specially CD4+ T-cells in T. vivax infection. The knowledge of the immune response is important not only for understanding the biology of the parasite in the host, but for the design of new treatment strategies for trypanosome infections.

Performance of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal and genotype-specific serodiagnosis of Trypanosoma cruzi infection.

Distinct Trypanosoma cruzi genotypes have been considered relevant for patient management and therapeutic response of Chagas disease. However, typing strategies for genotype-specific serodiagnosis of Chagas disease are still unavailable and requires standardization for practical application. In this study, an innovative TcI/TcVI/TcII Chagas Flow ATE-IgG2a technique was developed with applicability for universal and genotype-specific diagnosis of T. cruzi infection. For this purpose, the reactivity of serum samples (percentage of positive fluorescent parasites-PPFP) obtained from mice chronically infected with TcI/Colombiana, TcVI/CL or TcII/Y strain as well as non-infected controls were determined using amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI in parallel batches of TcI, TcVI and TcII target antigens. Data demonstrated that "α-TcII-TRYPO/1:500, cut-off/PPFP = 20%" presented an excellent performance for universal diagnosis of T. cruzi infection (AUC = 1.0, Se and Sp = 100%). The combined set of attributes "α-TcI-TRYPO/1:4,000, cut-off/PPFP = 50%", "α-TcII-AMA/1:1,000, cut-off/PPFP = 40%" and "α-TcVI-EPI/1:1,000, cut-off/PPFP = 45%" showed good performance to segregate infections with TcI/Colombiana, TcVI/CL or TcII/Y strain. Overall, hosts infected with TcI/Colombiana and TcII/Y strains displayed opposite patterns of reactivity with "α-TcI TRYPO" and "α-TcII AMA". Hosts infected with TcVI/CL strain showed a typical interweaved distribution pattern. The method presented a good performance for genotype-specific diagnosis, with global accuracy of 69% when the population/prototype scenario include TcI, TcVI and TcII infections and 94% when comprise only TcI and TcII infections. This study also proposes a receiver operating reactivity panel, providing a feasible tool to classify serum samples from hosts infected with distinct T. cruzi genotypes, supporting the potential of this method for universal and genotype-specific diagnosis of T. cruzi infection.

FC-TRIPLEX Chagas/Leish IgG1: a multiplexed flow cytometry method for differential serological diagnosis of chagas disease and leishmaniasis.

Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4 °C, and -20 °C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.