Evaluation from a B-cell epitope-based chimeric protein for the serodiagnosis of tegumentary and visceral leishmaniasis.

The diagnosis of leishmaniasis presents problems due to the variable sensitivity and/or specificity of tests. In addition, high levels of anti-parasite antibodies can remain after treatment, making it difficult to conduct a prognostic follow-up of patients. In this context, it is necessary to identify new candidates to be examined for the sensitive and specific diagnosis of the disease. In the present study, four Leishmania proteins, previously shown as antigenic for tegumentary leishmaniasis (TL), were evaluated, and their linear specific B-cell epitopes were predicted and used to generate a new gene codifying chimeric protein called ChimB, which was cloned, and the recombinant version was expressed, purified, and evaluated in ELISA (Enzyme-Linked Immunosorbent Assay) to diagnose TL and visceral leishmaniasis (VL). A total of 220 human serum samples were used, and, when ChimB was used, results showed sensitivity, specificity, and positive and negative predictive values of 100% for the diagnosis of both diseases; however, when using peptides, the sensitivity values reached from 28.0% to 57.3% and specificity varied from 16.3% to 83.7%. A soluble Leishmania extract (SLA) showed sensitivity and specificity values of 30.7% and 45.9%, respectively. The area under the curve (AUC) value for ChimB was 1.0, while for synthetic peptides, this value reached between 0.502 and 0.635, whereas for SLA, the value was of 0.589. Serological assays using sera samples collected before and after treatment showed significant reductions in the anti-ChimB antibody levels after therapy, suggesting a prognostic role of this recombinant antigen. In conclusion, preliminary data suggest the use from ChimB as a potential candidate for the diagnosis and prognosis of leishmaniasis.

Diagnostic application of sensitive and specific phage-exposed epitopes for visceral leishmaniasis and human immunodeficiency virus coinfection.

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.

Recombinant Leishmania eukaryotic elongation factor-1 beta protein: A potential diagnostic antigen to detect tegumentary and visceral leishmaniasis in dogs and humans.

The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.

American tegumentary leishmaniasis in Brazil: a critical review of the current therapeutic approach with systemic meglumine antimoniate and short-term possibilities for an alternative treatment.

OBJECTIVES: Meglumine antimoniate (MA; Glucantime®), the 80-year-old first-line systemic treatment for all forms of American tegumentary leishmaniasis (ATL) caused by Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis and Leishmania (Leishmania) amazonensis, is highly toxic, presents adverse side-effects and may not attain clinical and parasitological cure. This critical review examines the necessity for intramuscular/intravenous administration of MA, the alternatives to this approach, and the possibilities of developing affordable, accessible and non-toxic drugs or new delivery methods. METHOD: PubMed searches were performed using the terms 'cutaneous leishmaniasis' or 'American tegumentary leishmaniasis' in combination with 'meglumine antimoniate' or 'N-methyl glucamine' or 'drug repositioning' or 'nanotechnology'. Searches covered a period of 20 years of peer reviewed journals and technical bulletins. We explored the mode of action, pharmacokinetics, toxicity and efficacy of MA, evaluated the progress of ATL therapy in Brazil, and examined the potential of drug repositioning and nanotechnology in accelerating the introduction and/or optimisation of an alternative treatment. RESULTS: The evidence suggests that ATL therapy will continue to rely on systemic MA in the foreseeable future even though an intralesional subcutaneous route has evolved over the last 10 years. The chances of developing a novel drug for ATL or a new mode of delivery of MA are low. While MA nanocarriers afford a promising approach, this technology is still in its infancy. A more immediate solution would be the production of a bioequivalent of miltefosine, an efficacious oral agent no longer protected by patent. CONCLUSION: Development of a contemporary treatment requires governmental commitment in bringing together private and public sectors.

OBJECTIFS: L'antimoniate de méglumine (AM; Glucantime®), le traitement systémique de première intention vieux de 80 ans pour toutes les formes de la leishmaniose tégumentaire américaine (LTA) causée par Leishmania (Viannia) braziliensis, L. (V.) guyanensis et L. (Leishmania) amazonensis, est hautement toxique, présente des effets secondaires indésirables et peut ne pas aboutir à une guérison clinique et parasitologique. Cette analyse critique examine la nécessité d'une administration intramusculaire/intraveineuse d'AM, les alternatives à cette approche et les possibilités de développement de médicaments abordables, accessibles et non toxiques ou de nouvelles méthodes d'administration. MÉTHODE: Des recherches sur PubMed ont été effectuées en utilisant les termes «leishmaniose cutanée¼ ou «leishmaniose tégumentaire américaine¼ en combinaison avec «antimoniate de méglumine ¼ ou «N-méthyl glucamine¼ ou «repositionnement de médicament¼ ou «nanotechnologie¼. Les recherches ont porté sur une période de 20 ans d'articles revue par des pairs et de bulletins techniques. Nous avons exploré le mode d'action, la pharmacocinétique, la toxicité et l'efficacité de l'AM, évalué les progrès du traitement de la LTA au Brésil et examiné le potentiel du repositionnement de médicaments et de la nanotechnologie pour accélérer l'introduction et/ou l'optimisation d'un traitement alternatif. RÉSULTATS: Les données suggèrent que le traitement de la LTA continuera à s'appuyer sur l'AM systémique dans un avenir proche, même si une voie sous-cutanée intralésionnelle a évolué au cours des 10 dernières années. Les chances de développer un nouveau médicament pour la LTA ou un nouveau mode d'administration d'AM sont faibles. Alors que les nanocarriers d'AM offrent une approche prometteuse, cette technologie en est encore à ses balbutiements. Une solution plus immédiate consisterait à produire un bioéquivalent de miltéfosine, un agent oral efficace, qui n'est plus protégé par un brevet. CONCLUSION: Le développement d'un traitement contemporain nécessite un engagement gouvernemental pour réunir les secteurs privés et publiques.

Myocardial fibrosis in chagas disease and molecules related to fibrosis.

Chronic Chagas cardiomyopathy (CCC) is responsible for the disease's greater morbidity and poor prognosis. Although understanding the pathophysiology of CCC and the fundamentals of its clinical management derives from research related to other cardiomyopathies, there are peculiarities that distinguish CCC from the others. CCC is the most fibrous heart disease, and its myocardial involvement is important as it disorganizes or disrupts the extracellular matrix, creating an environment conducive to the formation of arrhythmogenic foci. It is also considered the most arrhythmogenic of the known heart diseases, giving rise to complex arrhythmias, usually associated with varying degrees of stimulus conduction disorders. The central proposal of this review is to describe a possible association between the distribution and degree of myocardial fibrosis and cardiac arrhythmogenicity in patients with Chagas cardiomyopathy, drawing attention to the importance of noninvasive biomarkers for the quantification of myocardial fibrosis.

Screening diagnostic candidates from Leishmania infantum proteins for human visceral leishmaniasis using an immunoproteomics approach.

There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed in Leishmania infantum promastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.

Cytokines as biomarkers to monitoring the impact of multidrug therapy in immune response of leprosy patients.

Leprosy or Hansen's disease is a chronic infectious disease of the skin and nerves, caused by the intracellular bacilli Mycobacterium leprae. It is characterized by a spectrum of clinical forms depending on the host's immune response to M. leprae. Patients with tuberculoid (TT) leprosy have strong cell-mediated immunity (CMI) with elimination of the bacilli, whereas patients with lepromatous (LL) leprosy exhibit defective CMI to M. leprae. Despite advances in the understanding of the pathogenesis of leprosy and the development of new therapeutic strategies, there is a need for the identification of biomarkers which be used for early diagnosis and to discrimination between different forms of the disease, as prognostic markers. Here, we analyzed the serum levels of IL-1ß, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-γ and TNF in order to address the contribution of these cytokines in late phase of M. leprae infection, and the impact of multidrug therapy (MDT). Our results demonstrated that patients of LL group presented higher expression of serum levels of inflammatory cytokines before MDT, while TT patients presented a balance between inflammatory and regulatory cytokines. MDT changes the profile of serum cytokines in M. leprae infected patients, as evidenced by the cytokine network, especially in TT patients. LL patients displayed a multifaceted cytokine system characterized by strong connecting axes involving inflammatory/regulatory molecules, while TT patients showed low involvement of regulatory cytokines in network overall. Cytokines can be identified as good biomarkers of the impact of MDT on the immune system and the effectiveness of treatment.

High interleukin 17 expression is correlated with better cardiac function in human Chagas disease.

This study was designed to investigate whether the expression of interleukin 17 (IL-17) is associated with the indeterminate or cardiac clinical forms of Chagas disease and whether IL-17 expression can be correlated with patients' cardiac function. Our results demonstrated that cardiac Chagas patients have a lower intensity of expression of IL-17 by total lymphocytes and lower frequency of circulating T helper 17 cells. Correlative analysis showed that high IL-17 expression was associated with better cardiac function, as determined by left ventricular ejection fraction and left ventricular diastolic diameter values. Therefore, IL-17 expression can be a protective factor to prevent myocardial damage in human Chagas disease.

Agreement among four drug information sources for the occurrence of warfarin drug interactions in Brazilian heart disease patients with a high prevalence of Trypanosoma cruzi infection.

PURPOSE: The aim of this study was to assess the agreement of four renowned interaction lists on potentially severe warfarin drug interactions (DI) in outpatients at a university hospital in Brazil, specifically in subgroups of Trypanosoma cruzi-infected and non-infected patients and those with previous bleeding episodes. METHODS: This was a cross-sectional study in which adult outpatients with heart disease and indications for chronic warfarin use were enrolled. The occurrence of potentially severe warfarin DI was evaluated based on the lists provided by three compendia, i.e., Drug Interaction Facts (DIF), Drug Interactions: Analysis and Management (DIAM) and DRUG-REAX, and by the World Health Organization (WHO) Model Formulary. A kappa coefficient was used to calculate the agreement among the sources. RESULTS: A total of 280 patients were studied. Most patients were female (54.6 %) with an average age of 56.8 (standard deviation 13.1) years. The agreement among the four sources was fair (Fleiss' kappa coefficient = 0.295). T. cruzi-infected individuals were less likely to have severe warfarin DI than non-infected patients (p < 0.05 for DIAM, DRUG-REAX and the WHO Model Formulary). Potentially severe DI were more frequent in patients with previous bleeding episodes, based on the DIF compendia (p = 0.007). CONCLUSIONS: This evaluation of warfarin DI revealed that the disagreement between compendia is also observed in clinical practice. T. cruzi infection is associated with a lower prevalence of potentially severe warfarin DI, but with a wider variation in its detection. Our results suggest a wide spectrum of discrepancies in detecting heart disease patients at higher risk for severe warfarin DI and a possible heterogeneity in clinical guidance.

Warfarin drug interactions: a comparative evaluation of the lists provided by five information sources.

PURPOSE: Detecting potential drug interactions can lead to early interventions that protect patients from serious drug-related problems. The aim of this study was to evaluate the agreement among the lists of warfarin interactions provided by five information sources. METHODS: The lists of warfarin interactions and the corresponding severity ratings and documentation levels presented by the three compendia and by the World Health Organization (WHO) Model Formulary were all compared, and each list was compared to that provided on the package insert of Marevan, a brand of warfarin. The compendia used were: Drug Interaction Facts, Drug Interactions: Analysis and Management and DRUG-REAX. A kappa coefficient was used to calculate the agreement among the sources. RESULTS: A total of 537 interactions were listed. Only 13 (2.4%) were common to the five sources. The global Fleiss' kappa coefficient was -0.0080, which indicated poor agreement. Eleven warfarin interactions appeared only in the Marevan package insert. Importantly, 243 interactions (45.3% of the total) were deemed significant in at least one compendium. Only two warfarin interactions were reported as critical by all three compendia and by WHO. The most critical interactions cited by the compendia were missing from the package insert. CONCLUSIONS: Poor agreement was found among five sources listing warfarin interactions. Potentially severe clinical consequences might occur due to these discrepant recommendations. Finally, the lack of standard terminology and clinical guidance, as well as the possible inaccuracy of severity ratings and documentation might contribute to heterogeneous procedures in clinical practice.

Functional capacity and right ventricular function in patients with Chagas heart disease.

AIMS: Right ventricular (RV) dysfunction is an important factor on effort tolerance in cardiopulmonary diseases. Nevertheless, the role of RV function in predicting exercise capacity in patients with Chagas disease has not been reported. This study aims to evaluate whether RV function assessed by tissue Doppler can predict functional capacity in patients with Chagas heart disease. METHODS AND RESULTS: We evaluated 65 patients (48.6 +/- 9.1 years, 60% men) with Chagas heart disease. Standard and tissue Doppler echocardiography were performed before maximal exercise testing. Tissue Doppler imaging (TDI) was used to measure RV peak annular systolic and diastolic velocities. Exercise testing was performed using a standard Bruce protocol. Linear regression analysis was used to determine multivariate peak oxygen consumption (VO(2)) predictors. All patients were in NYHA functional class I or II. Mean peak VO(2) was 32.4 +/- 10.2 mL/kg/min and mean LV ejection fraction was 43 +/- 11%. There was correlation between TDI RV peak systolic velocity and LV ejection fraction (r = 0.5; P < 0.001). In a multivariate analysis, after adjustment for age and gender, RV function emerged as an independent predictor of functional capacity, as demonstrated in the model: peak VO(2) (r = 0.71) was: 42.22-(9.77 x female gender)-(0.29 x age) + (1.54 x RV systolic velocity). CONCLUSION: In this cross-sectional study, RV function was an important, independent determinant of exercise capacity in patients with Chagas heart disease. TDI RV systolic annular velocity was most closely associated with peak VO(2), regardless of the influence of age, gender, and other echocardiographic parameters.

Left ventricular diastolic function and exercise capacity in patients with Chagas cardiomyopathy.

UNLABELLED: Parameters of diastolic function have been shown to correlate with exercise capacity (EC) in individuals with impaired left ventricular (LV) systolic function. However, the role of LV diastolic function in predicting EC in Chagas cardiomyopathy has not been reported. OBJECTIVES: This study aimed to determine the relationship between LV diastolic parameters assessed by echocardiography and EC in patients with Chagas cardiomyopathy. METHODS: We studied 40 patients (23 men; 49 + or - 8 years), with diagnosis of Chagas disease and dilated cardiomyopathy. Medical therapy was individually adjusted according to standardized guidelines. Methods of acquiring two-dimensional Doppler, tissue Doppler imaging (TDI), and their measurements were described. Exercise testing was performed by a Bruce protocol. Brain natriuretic peptide (BNP) levels were also determined. RESULTS: Most patients (63%) were in NYHA functional class I. Mean peak oxygen consumption estimated (peakVO(2)) was 31.7 + or - 10.2 mL/kg per minute, and mean left ventricular ejection fraction (LVEF) was 36.3 + or - 7.8%. Univariate analysis showed that various echocardiographic parameters of diastolic function were correlated with peakVO(2). There was no correlation between BNP levels or LVEF and EC. Multivariate analysis, after adjustment for age and gender, revealed that E/E' ratio and left atrial volume (LAV), emerged as independent predictors of EC, as demonstrated in the model: peakVO(2)= 60.825 + (0.439 x LAV) - (1.620 x E/E' ratio) - (0.483 x age) - (4.821 x female gender). The R(2) of this model was 0.52. CONCLUSIONS: Functional capacity assessed by peakVO(2) was related to increase LV filling pressures, independently on systolic function in patients with Chagas cardiomyopathy. (Echocardiography 2010;27:519-524).

Feasibility and Safety of Laparoscopic-Guided Epicardial Access for Ventricular Tachycardia Ablation.

Background The usual approach to epicardial access in patients with Chagas cardiomyopathy and megacolon is surgical access to avoid bowel injury. However, there are concerns regarding its safety in cases of Chagas cardiomyopathy with reports of prolonged mechanical ventilation and high mortality in this clinical setting. The aim of this study was to examine feasibility and complication rates for ventricular tachycardia ablation performed with laparoscopic-guided epicardial access. Methods and Results This single center study examined complication rates of the first 11 cases of ventricular tachycardia ablation in patients with Chagas cardiomyopathy, using laparoscopic guidance to access epicardial space. All 11 patients underwent epicardial VT ablation using laparoscopic-guided epicardial access, and the complication rates were compared with historical medical reports. The main demographic features of our population were age, 63±13 years; men, 82%; and median ejection fraction, 31% (Q1=30% and Q3=46%). All patients were sent for ventricular tachycardia ablation because of medical therapy failure. The reason for laparoscopy was megacolon in 10 patients and massive liver enlargement in 1 patient. Epicardial access was achieved in all patients. Complications included 1 severe cardiogenic shock and 1 phrenic nerve paralysis. No intra-abdominal organ injury occurred; only 1 death, which was caused by progressive heart failure, was reported more than 1 month after the procedure. Conclusions Laparoscopic-guided epicardial access in the setting of ventricular tachycardia ablation and enlarged intra-abdominal organ is a simple alternative to more complex surgical access and can be performed with low complication rates.

A Leishmania infantum hypothetical protein evaluated as a recombinant protein and specific B-cell epitope for the serodiagnosis and prognosis of visceral leishmaniasis.

The serodiagnosis of visceral leishmaniasis (VL) presents problems related to the sensitivity and/or specificity of the tests. In this context, more refined antigens should be identified and applied for the improvement of disease diagnosis. In the present study, DNA with an encoding of a Leishmania infantum hypothetical protein, LiHyC, was cloned, and the recombinant protein was expressed, purified, and evaluated for the serodiagnosis of canine and human VL. In addition, a specific B-cell epitope present in the LiHyC sequence was predicted; the peptide was both synthetized and evaluated in the ELISA experiments. For comparison, commercial diagnostic kits were used against positive (VL hosts) and negative (healthy hosts) samples. Results showed that the recombinant protein (rLiHyC) and synthetic peptide (PeptC) were highly sensitive and specific to diagnose canine and human VL, with 100% sensitivity and specificity, while no false-positive or false-negative result was detected. When the DPP® CVL kit was used to identify canine samples, 44 and 52 of the 60 L. infantum-infected animals, without or with clinical signals of disease, respectively, were identified, while eight and four samples were considered as false-negatives, respectively. For human VL, an IT LEISH® kit was used, and 33 of the 40 VL patients were identified, while seven samples were considered to be false-negatives. Post-therapeutic serological follow-up testing sera samples from treated and untreated VL patients showed a significant drop in the anti-PeptC and anti-rLiHyC antibody levels, thus suggesting the feasibility to use the recombinant protein and/or synthetic peptide in future studies as diagnostic and/or prognostic markers for VL.

Ischemic cerebrovascular events in patients with Chagas cardiomyopathy: a prospective follow-up study.

BACKGROUND: Chagas disease cardiomyopathy is a common form of dilated cardiomyopathy worldwide, and an important cause of stroke in Latin America. The long-term cumulative risk of ischemic cerebrovascular event (ICE) and its relation to left ventricular (LV) dysfunction have not been determined. The aims of this study were to describe the incidence and to evaluate the effect of LV ejection fraction on the risk for ICE in patients with Chagas cardiomyopathy. METHODS: A total of 213 consecutive patients with Chagas disease and LV systolic dysfunction, 131 males, mean age 48+/-12 years, were prospectively enrolled. The use of anticoagulation was based on clinical indications. The end point was ICE, which included fatal or nonfatal stroke and transient ischemic attack. Risk factors for events were assessed by Cox proportional-hazards analysis. RESULTS: Mean follow-up was 36 months; 69 patients died and seven underwent cardiac transplantation. The overall incidence of ICE was 2.67 events per 100 patient/years. Independent risk factors for ICE included LV ejection fraction (HR 0.95, 95% CI 0.91 to 0.99, p=0.009) and left atrial volume corrected for body surface area (HR 1.04, 95% CI 1.01 to 1.07, p=0.007), which persisted after adjustment for anticoagulation use. Patients with ejection fractions

Correlation between BNP levels and Doppler echocardiographic parameters of left ventricle filling pressure in patients with Chagasic cardiomyopathy.

UNLABELLED: Cardiomyopathy is the most important manifestation of Chagas' disease. Brain natriuretic peptide (BNP) level and Doppler echocardiographic parameters for diastolic dysfunction have shown correlation with left ventricle (LV) filling pressures. OBJECTIVES: The purpose of this study is to compare BNP levels with Doppler echocardiographic parameters in patients with chagasic cardiomyopathy. METHODS: Forty-three patients (69.8% men; mean age 41.0 +/- 10.4 years) were submitted to an echocardiographic study and 39 had their BNP levels measured. RESULTS: BNP levels increased with the deterioration of the diastolic function (P=0.025). Pulmonary venous flow parameters were correlated with BNP levels, but E/E'ratio (E'measured at the inferior mitral annulus) was the only diastolic parameter that remained an independent predictor of elevated BNP levels in the multivariate analysis. The area under the receiver-operating curve for BNP to detect E/E' >15 was 0.875. A BNP value of 280.4 pg/ml had a sensitivity of 96% and a specificity of 75% for predicting E/E' >15. CONCLUSIONS: In a group of patients with chagasic cardiomyopathy, BNP levels correlated with diastolic function patterns regardless of systolic function. The E/E'ratio (inferior wall) was the only isolated parameter of diastolic function that was independently associated with BNP levels.

MMP-2 and MMP-9 plasma levels are potential biomarkers for indeterminate and cardiac clinical forms progression in chronic Chagas disease.

One of the major challenges in chronic Chagas disease is to understand the mechanisms that predict the clinical evolution from asymptomatic to severe cardiac clinical forms. Our cohort consisted of twenty-eight Chagas disease patients followed for twenty years. Plasma levels of MMP-2 and MMP-9 gelatinases and TIMPs were evaluated by multiplexed immunoassay at two points in time with an average interval of six years. MMP-2 plasma levels, but not MMP-9, increased in cardiac patients over time. TIMP-1 levels diminished in cardiac patients, while TIMP-3 dropped in asymptomatic patients in the course of the evaluated interval. An inversion of time lines was observed relative to the clinical asymptomatic and cardiac forms for MMP-2. Receiver Operating Characteristic (ROC) curve analysis identified MMP-2 as a biomarker to distinguish asymptomatic from cardiac clinical forms, while MMP-9 is a biomarker that segregates infected from non-infected patients. We have pointed out that MMP-2 and MMP-9 together can predict clinical evolution in Chagas disease. MMP-2 was suggested as a biomarker for fibrosis replacement in early remodeling and a sensitive predictor for initial changes in asymptomatic patients that may evolve into the cardiac clinical form. MMP-9 seems to be a biomarker for late fibrosis and severe cardiac remodeling in cardiac patients.

PD1 and PDL1 molecules control suppressor activity of regulatory T cells in chronic Chagas cardiomyopathy patients.

INTRODUCTION: Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), is the fourth most important tropical disease, which affects approximately 7 million people worldwide. The mechanisms involved in the development of this disease are not completely well understood. An important protective role of regulatory T cells (Treg) in Chagas disease has been observed; however, the specific mechanisms remain unclear. We evaluated apoptosis as a possible mechanism mediated by Treg cells (CD4+CD25HighFOXP3+) to orchestrate the immune response in chronic Chagas disease. METHODS AND RESULTS: Patients with Chagas disease were grouped as the indeterminate (IND; asymptomatic patients with Chagas disease; n = 10) and dilated cardiomyopathy (CARD; n = 10). Healthy T. cruzi-negative individuals (NI; n = 10) were included as a control group. In order to evaluate the apoptotic cell profile, the expression of PD1, PD1L, CD39, CD95, CD95L molecules were investigated. We also evaluated the proportion of CD14+ cells expressing caspase 3. The IND group presented a substantially higher expression of CD39 by Treg cells as compared to the CARD group. On the other hand, the CARD group showed higher expression of PD-1 by Treg cells than both NI and IND groups. Significant positive correlations were observed between Treg CD95L+ cells and CD14 cells expressing caspase 3 as well as between Treg CD39 cells and CD14+ Caspase3+ cells in the IND group. CONCLUSION: Our data indicate that the expressions of different molecules that induce apoptosis are associated with suppressive mechanisms mediated by Treg cells and suggest a possible role for PD1 and PDL1 molecules in the morbidity of chronic Chagas disease.

Increased frequencies of circulating CCR5+ memory T cells are correlated to chronic chagasic cardiomyopathy progression.

The infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease, a neglected tropical disease in Latin America and an imported emerging disease worldwide. Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, culminating in heart failure and high rates of sudden death. CCC pathogenesis is influenced by both host and parasite factors and is proposed to be mostly immune-driven. Chemokines are crucial players in orchestrating immune cell recruitment to infected tissues and inflammation. Herein, we investigated inflammatory chemokine receptor expression on circulating T cells in patients stratified by CCC severity. Compared to asymptomatic individuals, we found increased percentages of effector CD4+ T cells and central memory CD4+ and CD8+ T cells expressing CCR5 in patients with structural cardiopathy, but normal global ventricular function and no symptoms of chronic heart failure. Even naïve T cells expressed CCR5 in these patients. In contrast, reduced frequencies of CD4+ and CD8+ effector T cells expressing CXCR3 were observed in patients presenting with severe heart disease. Patients with increased left ventricular diameter, heart enlargement, and insufficiency had higher frequencies of CCR5+ effector and effector memory CD8+ T cells. Moreover, the percentage of effector CCR5+ CD8+ T cells was increased in patients with a reduced ejection fraction. Our results show that high expression CCR5 and low expression of CXCR3 on circulating T cells are associated with worse prognosis, possibly reflecting immune-mediated cardiac remodeling of CCC.

A comparison of ML Flow serology and slit skin smears to assess the bacterial load in newly diagnosed leprosy patients in Brazil.

INTRODUCTION: The ML Flow test is an immunochromatographic assay that detects IgM antibodies against M. leprae-specific anti-phenolic glycolipid I (PGL-I). In addition to slit skin smears stained by the Ziehl-Neelsen technique, it can be helpful in the operational classification of leprosy patients for treatment purposes. OBJECTIVE: This work studied the relationship between antibody levels as detected by semi-quantitative ML Flow serologic test and bacterial load as quantified by slit skin smear. PATIENTS AND METHODS: 135 patients with newly detected leprosy at the reference service in Sanitary Dermatology in Brazil had slit skin smears (registered as bacillary index - BI) and an ML Flow test (registered qualitatively and semi-quantitatively) performed at admission. A logistic regression and agreement measures (kappa index) were calculated. RESULTS: Slit skin smears were positive in 35.9% of patients and 57% of patients were seropositive for PGL-1 antibodies. Among the seropositive patients, 416% had five or fewer skin lesions, and 65.8% had more than one peripheral nerve involved. Slit skin smears were positive in only three seronegative patients (5.6%), and negative in 41.9% of seropositive patients. Patients with a BI of 4 + had an OR of 33 for being seropositive in comparison to those with a low BI. CONCLUSIONS: There is a correlation between serologic test and slit skin smear results. Therefore, an ML Flow test may become a useful tool in the clinical classification of leprosy, besides slit skin smears, which require a proper laboratory infrastructure and experienced personnel.