Use of PET/CT to detect myocardial inflammation and the risk of malignant arrhythmia in chronic Chagas disease.

BACKGROUND: Chagas heart disease (CHD) is characterized by progressive myocardial inflammation associated with myocardial fibrosis and segmental abnormalities that may lead to malignant ventricular arrhythmia and sudden cardiac death. This arrhythmia might be related to the persistence of parasitemia or inflammation in the myocardium in late-stage CHD. Positron emission tomography/computed tomography (PET/CT) has been used to detect myocardial inflammation in non-ischemic cardiomyopathies, such as sarcoidosis, and might be useful for risk prediction in patients with CHD. METHODS AND RESULTS: Twenty-four outpatients with chronic CHD were enrolled in this prospective cross-sectional study between May 2019 and March 2022. The patients were divided into two groups: those with sustained ventricular tachycardia and/or aborted sudden cardiac death who required implantable cardioverter-defibrillators, and those with the same stages of CHD and no complex ventricular arrhythmia. Patients underwent 18F-fluorodeoxyglucose (18F-FDG) and 68Ga-DOTATOC PET/CT, and blood samples were collected for qualitative parasite assessment by polymerase chain reaction. Although similar proportions of patients with and without complex ventricular arrhythmia showed 18F-FDG and 68Ga-DOTATOC uptake, 68Ga-DOTATOC corrected SUVmax was higher in patients with complex arrhythmia (3.4 vs 1.7; P = .046), suggesting that inflammation could be associated with the presence of malignant arrhythmia in the late stages of CHD. We also detected Trypanosoma cruzi in both groups, with a nonsignificant trend of increased parasitemia in the group with malignant arrhythmia (66.7% vs 33.3%). CONCLUSION: 18F-FDG and 68Ga-DOTATOC uptake on PET/CT may be useful for the detection of myocardial inflammation in patients with Chagas cardiomyopathy, and 68Ga-DOTATOC uptake may be associated with the presence of malignant arrhythmia, with potential therapeutic implications.

Overexpression of TcNTPDase-1 Gene Increases Infectivity in Mice Infected with Trypanosoma cruzi.

Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

Physical Exercise Promotes a Reduction in Cardiac Fibrosis in the Chronic Indeterminate Form of Experimental Chagas Disease.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a neglected tropical disease and a health problem in Latin America. Etiological treatment has limited effectiveness in chronic CD; thus, new therapeutic strategies are required. The practice of physical exercises has been widely advocated to improve the quality of life of CD patients. The most frequent clinical CD manifestation is the chronic indeterminate form (CIF), and the effect of physical exercises on disease progression remains unknown. Here, in a CIF model, we aimed to evaluate the effect of physical exercises on cardiac histological, parasitological, mitochondrial, and oxidative metabolism, electro and echocardiographic profiles, and immunological features. To establish a CIF model, BALB/c and C57BL/6 mice were infected with 100 and 500 trypomastigotes of the Y T. cruzi strain. At 120 days postinfection (dpi), all mouse groups showed normal PR and corrected QT intervals and QRS complexes. Compared to BALB/c mice, C57BL/6 mice showed a lower parasitemia peak, mortality rate, and less intense myocarditis. Thus, C57BL/6 mice infected with 500 parasites were used for subsequent analyses. At 120 dpi, a decrease in cardiac mitochondrial oxygen consumption and an increase in reactive oxygen species (ROS) were detected. When we increased the number of analyzed mice, a reduced heart rate and slightly prolonged corrected QT intervals were detected, at 120 and 150 dpi, which were then normalized at 180 dpi, thus characterizing the CIF. Y-infected mice were subjected to an exercise program on a treadmill for 4 weeks (from 150 to 180 dpi), five times per week in a 30-60-min daily training session. At 180 dpi, no alterations were detected in cardiac mitochondrial and oxidative metabolism, which were not affected by physical exercises, although ROS production increased. At 120 and 180 dpi, comparing infected and non-infected mice, no differences were observed in the levels of plasma cytokines, indicating that a crucial biomarker of the systemic inflammatory profile was absent and not affected by exercise. Compared with sedentary mice, trained Y-infected mice showed similar parasite loads and inflammatory cells but reduced cardiac fibrosis. Therefore, our data show that physical exercises promote beneficial changes that may prevent CD progression.

Toward the Establishment of a Single Standard Curve for Quantification of Trypanosoma cruzi Natural Populations Using a Synthetic Satellite Unit DNA Sequence.

Accurate diagnostic tools and surrogate markers of parasitologic response to treatment are needed for managing Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (TcI) and CL-Brener (TcVI) stocks similar 95% limit of detection values [0.903 (0.745 to 1.497) and 0.667 (CI, 0.113 to 3.927) copy numbers/µL, respectively] when synthetic DNA was the standard for quantification, allowing direct comparison of loads in samples infected with different discrete typing units. This standard curve was evaluated in 205 samples (38 acute oral and 19 chronic Chagas disease patients) from different geographical areas infected with various genotypes, including samples obtained during treatment follow-up; high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase 3 clinical trials, to follow up patients under treatment or at risk of reactivation, and in experimental models using different parasite strains.

Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi.

Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.

TGF-ß inhibitor therapy decreases fibrosis and stimulates cardiac improvement in a pre-clinical study of chronic Chagas' heart disease.

TGF-ß involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-ß signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TßR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-ß signaling pathway reduced TGF-ß/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß inhibitors.

Nucleoside triphosphate diphosphohydrolase1 (TcNTPDase-1) gene expression is increased due to heat shock and in infective forms of Trypanosoma cruzi.

BACKGROUND: Ecto-Nucleoside Triphosphate Diphosphohydrolases (Ecto-NTPDases) are enzymes that hydrolyze tri- and/or di-phosphate nucleotides. Evidences point to their participation in Trypanosoma cruzi virulence and infectivity. In this work, we evaluate TcNTPDase-1 gene expression in comparison with ecto-NTPDase activity, in order to study the role of TcNTPDase-1 in parasite virulence, infectivity and adaptation to heat shock. FINDINGS: Comparison between distinct T. cruzi isolates (Y, 3663 and 4167 strains, and Dm28c, LL014 and CL-14 clones) showed that TcNTPDase-1 expression was 7.2 ± 1.5 times higher in the Dm28c than the CL-14 avirulent clone. A remarkable expression increase was also observed in the trypomastigote and amastigote forms (22.5 ± 5.6 and 16.3 ± 3.8 times higher than epimastigotes, respectively), indicating that TcNTPDase-1 is overexpressed in T. cruzi infective forms. Moreover, heat shock and long-term cultivation also induced a significant increment on TcNTPDase-1 expression. CONCLUSIONS: Our results suggest that TcNTPDase-1 plays an important role on T. cruzi infectivity and adaptation to stress conditions, such as long-term cultivation and heat shock.