Fernando Rosas

Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients.

Chagas disease caused by Trypanosoma cruzi is a complex disease that is endemic and an important problem in public health in Latin America. The T. cruzi parasite is classified into six discrete taxonomic units (DTUs) based on the recently proposed nomenclature (TcI, TcII, TcIII, TcIV, TcV and TcVI). The discovery of genetic variability within TcI showed the presence of five genotypes (Ia, Ib, Ic, Id and Ie) related to the transmission cycle of Chagas disease. In Colombia, TcI is more prevalent but TcII has also been reported, as has mixed infection by both TcI and TcII in the same Chagasic patient. The objectives of this study were to determine the T. cruzi DTUs that are circulating in Colombian chronic Chagasic patients and to obtain more information about the molecular epidemiology of Chagas disease in Colombia. We also assessed the presence of electrocardiographic, radiologic and echocardiographic abnormalities with the purpose of correlating T. cruzi genetic variability and cardiac disease. Molecular characterization was performed in Colombian adult chronic Chagasic patients based on the intergenic region of the mini-exon gene, the 24Sα and 18S regions of rDNA and the variable region of satellite DNA, whereby the presence of T.cruzi I, II, III and IV was detected. In our population, mixed infections also occurred, with TcI-TcII, TcI-TcIII and TcI-TcIV, as well as the existence of the TcI genotypes showing the presence of genotypes Ia and Id. Patients infected with TcI demonstrated a higher prevalence of cardiac alterations than those infected with TcII. These results corroborate the predominance of TcI in Colombia and show the first report of TcIII and TcIV in Colombian Chagasic patients. Findings also indicate that Chagas cardiomyopathy manifestations are more correlated with TcI than with TcII in Colombia.

Concomitant mitral valve or atrial septal defect surgery and the modified Cox-maze procedure

Atrial fibrillation (AF) is generally associated with rheumatic valve disease and atrial septal defects (ASD) in young adults. Surgical correction of both disorders fails to convert to sinus rhythm or prevent further episodes of paroxysmal or chronic AF in most patients. The role and efficacy of combining mitral valve surgery or ASD correction with AF surgery in this setting has not been widely addressed and remains to be established. The present study prospectively assessed the recovery of sinus rhythm, functional status, and atrial function in 21 patients (mean age 42 +/- 9.2 years) who underwent a modified Cox-maze procedure concomitant with mitral valve or ASD surgery at our institution between March 1993 and February 1995. Seventeen (81%) had chronic AF, and 4 (19%) had paroxysmal AF, with a mean AF duration of 3.5 +/- 3.6 years (range 0.6 to 15.3). Concomitant surgery was performed in 9 patients (42.9%) with mitral stenosis, 5 (23.8%) with mitral regurgitation, 1 (4.8%) with mitral and aortic regurgitation, and 3 (14.3%) with ASD. Eighteen patients (86%) were in New York Heart Association class II to IV before operation. Doppler echocardiography was performed in all patients before surgery, and 1 week, and 3 and 6 months after surgery in patients maintaining sinus rhythm. One patient with severe mitral stenosis and depressed ventricular function died in the immediate postoperative period. Sinus rhythm was restored in the immediate postoperative period in 7 patients (35%), and in another 10 patients (50%) before discharge (mean 5.8 +/- 2 days). Overall, sinus rhythm was restored before discharge in 17 patients (85%); 3 (15%) patients required antiarrhythmic therapy. Doppler echocardiography performed 3 months after surgery documented atrial contractility (A and E waves) in 12 patients (71%). After a mean follow-up period of 8 months (range 3 to 23), 18 (90%) remained in sinus rhythm. Sinus rhythm was successfully restored and maintained in most patients with drug refractory AF undergoing a concomitant Cox-maze procedure with mitral valve or ASD surgery improving atrial function and New York Heart Association class.

Time- and Frequency-Domain Analyses of the Signal-Averaged ECG in Patients With Arrhythmogenic Right Ventricular Dysplasia

BACKGROUND Arrhythmogenic right ventricular dysplasia (ARVD) is characterized by recurrent ventricular tachycardia of right ventricular origin and a cardiomyopathy with hypokinetic areas involving the free wall of the right ventricle. Subjects have a risk of sudden cardiac death, particularly during sports and strenuous exercise. Routine clinical examinations may be normal, but fragmented or delayed electrograms are usually recorded in the right ventricle of these patients. However, the frequency with which late potentials are detected by conventional time-domain analysis of the signal-averaged ECG (SAECG) is not high. This study evaluated the usefulness of the frequency-domain analysis of the SAECG in addition to the conventional time-domain analysis for a screening test to detect patients with ARVD. METHODS AND RESULTS SAECG was recorded by using a bipolar X, Y, and Z lead system in 28 patients with ARVD (mean age, 38 +/- 13 years) and 35 age-matched normal subjects (mean age, 35 +/- 11 years). The conventional time-domain analysis of the SAECG was performed at two different high-pass filter settings, 25 and 40 Hz, and the low-pass cutoff frequency was fixed at 250 Hz. The fast-Fourier transform analysis of SAECG was performed using a Blackman-Harris window. Area ratio 1 (area of 20 to 50 Hz)/(area of 0 to 20 Hz) and area ratio 2 (area of 40 to 100 Hz)/(area of 0 to 40 Hz) were calculated. In the conventional time-domain analysis, 20 (71%) and 18 (64%) patients had positive criteria at filter settings of 25 and 40 Hz, respectively. In the frequency-domain analysis, 18 (64%) and 20 (71%) patients had abnormal values in area ratios 1 and 2, respectively. Combining the time- and frequency-domain analyses, all patients were judged positive, with a sensitivity of 100% and a specificity of 94%. CONCLUSIONS Each result of the time- and frequency-domain analyses revealed that both methods had equivalent value. Combining the two domain analyses improved the sensitivity without reducing the specificity. These findings suggest that combining the time- and frequency-domain analyses of the SAECG may be useful as a screening test to detect patients with ARVD.

Increased CD4+/CD8+ Double-Positive T Cells in Chronic Chagasic Patients

Background CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out. Methodology/Principal Findings Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor. Conclusions Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can migrate to the heart tissue.

Evaluation of IFN-gamma production by CD8 T lymphocytes in response to the K1 peptide from KMP-11 protein in patients infected with Trypanosoma cruzi.

The cellular response mediated by MHC class I restricted CD8 + T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP‐11 protein has a high binding affinity to the HLA‐A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+T lymphocytes to produce IFN‐γ. Therefore, CD8+T lymphocytes from 22 HLA‐A*0201+individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN‐γ‐secreting CD8+T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+T cells. In contrast, none of HLA‐A*0201+uninfected controls responded to K1 peptide. Responses to HLA‐A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1‐specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+T lymphocytes during the natural course of Chagas disease.

Frequency of specific CD8+ T cells for a promiscuous epitope derived from Trypanosoma cruzi KMP‐11 protein in chagasic patients

The K1 peptide is a CD8 + T cell HLA‐A*0201‐restricted epitope derived from the Trypanosoma cruzi KMP‐11 protein. We have previously shown that this peptide induces IFN‐γ secretion by CD8+T cells. The aim of this study was to characterize the frequency of K1‐specific CD8+T cells in chagasic patients. Nineteen HLA‐A2+individuals were selected from 50 T. cruzi infected patients using flow cytometry and SSP‐PCR assays. Twelve HLA‐A*0201+noninfected donors were included as controls. Peripheral blood mononuclear cells were stained with HLA‐A2‐K1 tetramer, showing that 15 of 19 infected patients have K1‐specific CD8+T cells (0·09–0·34% frequency) without differences in disease stages or severity. Of note, five of these responders were A*0205, A*0222, A*0226, A*0259 and A*0287 after molecular typing. Thus, a phenotypic and functional comparison of K1‐specific CD8+T cells from non‐HLA‐A*0201 and HLA‐A*0201+infected patients was performed. The results showed that both non‐HLA‐A*0201 and HLA‐A*0201+individuals have a predominant effector memory CD8+T cell phenotype (CCR7−, CD62L−). Moreover, CD8+T cells from non‐HLA‐A*0201 and HLA‐A*0201+individuals expressed IL‐2, IFN‐γ and perforin without any differences. These findings support that K1 peptide is a promiscuous epitope presented by HLA‐A2 supertype molecules and is highly recognized by chagasic patients.

Cytokine Profiling in Chagas Disease: Towards Understanding the Association with Infecting Trypanosoma cruzi Discrete Typing Units (A BENEFIT TRIAL Sub-Study).

Background Chagas disease caused by the protozoan Trypanosoma cruzi is an important public health problem in Latin America. The immunological mechanisms involved in Chagas disease pathogenesis remain incompletely elucidated. The aim of this study was to explore cytokine profiles and their possible association to the infecting DTU and the pathogenesis of Chagas disease. Methods 109 sero-positive T. cruzi patients and 21 negative controls from Bolivia and Colombia, were included. Flow cytometry assays for 13 cytokines were conducted on human sera. Patients were divided into two groups: in one we compared the quantification of cytokines between patients with and without chronic cardiomyopathy; in second group we compared the levels of cytokines and the genetic variability of T. cruzi. Results Significant difference in anti-inflammatory and pro-inflammatory cytokines profiles was observed between the two groups cardiac and non-cardiac. Moreover, serum levels of IFN-γ, IL-12, IL-22 and IL-10 presented an association with the genetic variability of T.cruzi, with significant differences in TcI and mixed infections TcI/TcII. Conclusion Expression of anti-inflammatory and pro-inflammatory cytokines may play a relevant role in determining the clinical presentation of chronic patients with Chagas disease and suggests the occurrence of specific immune responses, probably associated to different T. cruzi DTUs.

T Lymphocytes from Chagasic Patients Are Activated but Lack Proliferative Capacity and Down-Regulate CD28 and CD3ζ

Background Chronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3ζ chain on T lymphocytes between chronic chagasic patients and uninfected controls. Methodology/Principal Findings Forty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3ζ. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3ζ chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1%±6.1) and CD8+/HLA-DR+/CD38+ (19.8±8.9) T cells in comparison with healthy (1.6±1.0; 10.6±8.0) and non-chagasic cardiomyopathy donors (2.9±2.9; 5.8±6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7±0.3) was lower when compared with healthy (2.3±0.3) and non-chagasic cardiomyopathy individuals (3.1±1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3ζbright/CD3ζdim% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3ζbright/CD3ζdim% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3ζ. Conclusions CD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3ζ down-regulation and diminished CD28 expression on CD4 T cells.

Follow-up of an asymptomatic Chagas disease population of children after treatment with nifurtimox (Lampit) in a sylvatic endemic transmission area of Colombia.

Background Chagas disease is an anthropozoonosis caused by Trypanosoma cruzi. Two drugs are currently used for the etiological treatment of the disease: Nifurtimox (Lampit) and Benznidazole. This study presents a quasi-experimental trial (non-control group) of sixty-two patients who were treated for Chagas disease with Nifurtimox (Lampit), and were then followed for 30 months post-treatment. The safety of Nifurtimox (Lampit) for Chagas disease in this group of children primarily between 4 and 19 years old was also evaluated. Materials and methods The 62 patients included in the study were selected when resulted seropositive for two out of three fundamentally different serological tests. All children were treated during two months according to protocols established by WHO. Monitoring was performed every twenty days to evaluate treatment safety. In 43 patients, two different serological tests: ELISA and IFAT; and two parasitological tests: blood culture, and real time PCR, (qPCR) were performed to assess therapeutic response, defined as post-treatment serological negativization. Principal findings All patients completed the treatment successfully, and six patients abandoned the post-treatment follow-up. Adverse effects occurred in 74% of patients, but only 4.8% of cases required temporary suspension to achieve 100% adherence to the 60-day treatment, and all symptoms reverted after treatment completion. Both parasite load (measured through qPCR) and antibodies (ELISA absorbance) evidenced a significant median reduction 6 months after treatment from 6.2 to 0.2 parasite equivalents/mL, and from 0.6 to 0.2 absorbance units respectively (p<0.001). Serological negativization by ELISA was evident since 6 months post-treatment, whereas by IFAT only after 18 months. Serological negativization by the two tests (ELISA and IFAT) was 41.9% (95%CI: 26.5–57.3) after 30 months post-treatment. qPCR was positive in 88.3% of patients pre-treatment and only in 12.1% of patients after 30 months. Survival analysis indicated that only 26.3% (95%CI: 15.5–44.8) persisted with negative qPCR during the whole follow-up period. Conclusions Nifurtimox was very well tolerated and successfully reduced parasite load and antibody titers. Re-infection, lysed parasites or a lack of anti-parasitic activity could explain these persistently positive qPCR cases.

Trypanosoma cruzi paraflagellar rod proteins 2 and 3 contain immunodominant CD8+ T-cell epitopes that are recognized by cytotoxic T cells from Chagas disease patients

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease. To date, no vaccine is available for protection against T. cruzi infection. The CD8(+) T cells immune response against specific antigens has shown to efficiently control the spread of the parasite in murine experimental infection. However, data concerning CD8(+) response in Chagas patients are still restricted to a few epitopes. We have studied the existence of immunodominant CD8(+) T cell epitopes in the paraflagellar rod proteins 2 and 3 (PFR2 and PFR3) from T. cruzi in a mouse model, and analyzed their recognition by cytotoxic T lymphocytes from Chagas disease patients. Immunization of C57BL/6-A2/K(b) transgenic mice with plasmids coding for the fusion proteins PFR2-HSP70 and PFR3-HSP70 induced a specific CTL response against two PFRs epitopes (PFR2(449-457) and PFR3(481-489)), and showed specific lysis percentages of 24 and 12, respectively. Moreover, the PFR2(19-28), PFR2(156-163), PFR2(449-457), PFR3(428-436), PFR3(475-482) and PFR3(481-489) peptides were observed to have a high binding affinity to the HLA-A*02:01 molecule. Remarkably, these HLA-A*02:01-binding peptides are successfully processed and presented during natural infection by T. cruzi in the context of MHC class I as evidenced by using peptide-pulsed K562-A2 cells as antigen presenting cells. The T cells from Chagas disease chronic patients specific for PFR2/PFR3 selected CD8(+) epitopes showed a pro-inflammatory cytokine secretion profile (IFN-γ, TNF-α and IL-6). A positive Granzime B secretion was observed in three out of 16 patients in response to PFR2(156-163) and PFR2(449-457) peptides, two out of 11 patients in response to PFR2(19-28) peptide and one out of 14 and 11 patients in response to PFR3(428-436) and PFR3(481-489) peptides, respectively. The PFRs-specific cytotoxic activity in purified PBMC was only detected in patients in the indeterminate phase of the disease.