Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation.

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.

Insecticidal activity of fluralaner (Exzolt®) administered to Gallus gallus domesticus against triatomines (Hemiptera, Reduviidae, Triatominae).

BACKGROUND: Triatoma infestans, Triatoma brasiliensis, Triatoma pseudomaculata and Rhodnius prolixus are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Chickens serve as an important blood food source for triatomines. This study aimed to assess the insecticidal activity of fluralaner (Exzolt®) administered to chickens against triatomines (R. prolixus, T. infestans, T. brasiliensis and T. pseudomaculata). METHODS: Twelve non-breed chickens (Gallus gallus domesticus) were randomized based on weight into three groups: negative control (n = 4); a single dose of 0.5 mg/kg fluralaner (Exzolt®) (n = 4); two doses of 0.5 mg/kg fluralaner (Exzolt®) (n = 4). Nymphs of 3rd, 4th and 5th instars of R. prolixus, T. infestans, T. brasiliensis and T. pseudomaculata (all n = 10) were allowed to feed on chickens before treatment, and at intervals of 1, 7, 14, 21, 28, 35 and 56 days after treatment, with insect mortality determined. RESULTS: Treatment with two doses of fluralaner showed higher insecticidal efficacy against R. prolixus, T. infestans and T. brasiliensis compared to the single-dose treatment. Similar insecticidal efficacy was observed for T. pseudomaculata for one and two doses of fluralaner. Insecticidal activity of fluralaner (Exzolt®) against triatomine bugs was noted up to 21 and 28 days after treatment with one and two doses of fluralaner, respectively. CONCLUSIONS: The results demonstrate that treatment of chickens with fluralaner (Exzolt®) induces insecticidal activity against triatomines for up to 28 days post-treatment, suggesting its potential use as a control strategy for Chagas disease in endemic areas.

Troponin T autoantibodies correlate with chronic cardiomyopathy in human Chagas disease.

OBJECTIVE: To evaluate the potential involvement of anti-Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease. METHODS: IgG total and isotypes were measured by ELISA, using epimastigote and trypomastigote forms of T. cruzi as antigens and human cardiac proteins (myosin and troponin T) in sera of patients with indeterminate (IND, n = 72), cardiac (CARD, n = 47) and digestive/cardiodigestive (DIG/CARD-DIG, n = 12) clinical forms of the disease. Samples from uninfected health individuals (CONT, n = 30) and patients with ischaemic cardiomyopathy (ISCH, n = 15) were used as controls. Autoantibody levels were correlated with parameters of cardiac function obtained by electrocardiographic, radiographic and echocardiographic examinations. RESULTS: Fifty five per cent of patients were classified as IND, 35.9% as CARD and 9.1% as DIG/CARD-DIG. Greater total IgG production was observed in IND, CARD and DIG/CARD-DIG chagasic patients than in CONT and ISCH, using trypomastigote, epimastigote and cardiac antigens. Moreover, patients with CARD and DIG/CARD-DIG presented greater total IgG production (trypomastigote and epimastigote antigen) than IND, and a negative correlation was determined between total IgG and left ventricular ejection fraction (LVEF). Patients with IND and CARD presented similar higher levels of total IgG specific to troponin T and myosin than CONT and ISCH individuals. Patients with chronic Chagas disease presented a negative correlation between left ventricular ejection fraction (LVEF) and the production of anti-myosin and troponin T autoantibodies. When grouped as low and high antibody producers and compared with LVEF, we observed that high anti-troponin T (P = 0.042) and myosin (P = 0.013) producers presented lower LVEF than low producers. Moreover, there was a positive correlation (r = 0.9508, P = 0.0001) between the production of troponin T and myosin autoantibodies. CONCLUSION: These findings indicate that increased production of anti-cardiac troponin T and myosin autoantibodies probably influences the left ventricular ejection fraction and could be related to chagasic cardiomyopathy.

Temporal assessment of entomological surveillance of Trypanosoma cruzi vectors in an endemic area of northeastern Brazil.

Entomological surveillance is essential for the control of triatomines and the prevention of Trypanosoma cruzi infection in humans and domestic animals. Thus, the objective of this study was to evaluate entomological indicators and triatomine control during the period from 2005 to 2015 in an endemic area in the state of Rio Grande do Norte, Brazil. This observational and retrospective study was developed based on data analysis related to active entomological surveillance activities and chemical control of infested housing units (HU) in the Agreste mesoregion of the state of Rio Grande do Norte, Brazil, in the period between 2005 to 2015. The quantitative analysis of housing units surveyed for entomological indicators was performed by linear regression of random effects (p < 0.05). The effect of the number of HU surveyed on the entomological indicators was analyzed by fitting a linear random effects regression model and an increasing intradomiciliary colonization rate was significant. In the period evaluated 92,156 housing units were investigated and the presence of triatomines was reported in 4,639 (5.0%). A total of 4,653 specimens of triatomines were captured and the species recorded were Triatoma pseudomaculata (n = 1,775), Triatoma brasiliensis (n = 1,569), Rhodnius nasutus (n = 741) and Panstrongylus lutzi (n = 568), with an index of natural infection by T. cruzi of 2.2%. Only 53.1% of the infested HU were subjected to chemical control. Moreover, there was a decrease in the total number of HU surveyed over time associated with an increase in the index of intradomiciliary colonization (p = 0.004). These data demonstrated that entomological surveillance and control of vectors in the Agreste mesoregion of the state has been discontinued, emphasizing the need for more effective public policies to effectively control the vectors, in order to avoid the exposure of humans and domestic animals to the risk of T. cruzi infection.

Accessing the Variability of Multicopy Genes in Complex Genomes using Unassembled Next-Generation Sequencing Reads: The Case of Trypanosoma cruzi Multigene Families.

Repetitive elements cause assembly fragmentation in complex eukaryotic genomes, limiting the study of their variability. The genome of Trypanosoma cruzi, the parasite that causes Chagas disease, has a high repetitive content, including multigene families. Although many T. cruzi multigene families encode surface proteins that play pivotal roles in host-parasite interactions, their variability is currently underestimated, as their high repetitive content results in collapsed gene variants. To estimate sequence variability and copy number variation of multigene families, we developed a read-based approach that is independent of gene-specific read mapping and de novo assembly. This methodology was used to estimate the copy number and variability of MASP, TcMUC, and Trans-Sialidase (TS), the three largest T. cruzi multigene families, in 36 strains, including members of all six parasite discrete typing units (DTUs). We found that these three families present a specific pattern of variability and copy number among the distinct parasite DTUs. Inter-DTU hybrid strains presented a higher variability of these families, suggesting that maintaining a larger content of their members could be advantageous. In addition, in a chronic murine model and chronic Chagasic human patients, the immune response was focused on TS antigens, suggesting that targeting TS conserved sequences could be a potential avenue to improve diagnosis and vaccine design against Chagas disease. Finally, the proposed approach can be applied to study multicopy genes in any organism, opening new avenues to access sequence variability in complex genomes. IMPORTANCE Sequences that have several copies in a genome, such as multicopy-gene families, mobile elements, and microsatellites, are among the most challenging genomic segments to study. They are frequently underestimated in genome assemblies, hampering the correct assessment of these important players in genome evolution and adaptation. Here, we developed a new methodology to estimate variability and copy numbers of repetitive genomic regions and employed it to characterize the T. cruzi multigene families MASP, TcMUC, and transsialidase (TS), which are important virulence factors in this parasite. We showed that multigene families vary in sequence and content among the parasite's lineages, whereas hybrid strains have a higher sequence variability that could be advantageous to the parasite's survivability. By identifying conserved sequences within multigene families, we showed that the mammalian host immune response toward these multigene families is usually focused on the TS multigene family. These TS conserved and immunogenic peptides can be explored in future works as diagnostic targets or vaccine candidates for Chagas disease. Finally, this methodology can be easily applied to any organism of interest, which will aid in our understanding of complex genomic regions.

Virulence of Trypanosoma cruzi Strains Is Related to the Differential Expression of Innate Immune Receptors in the Heart.

Resistance or susceptibility to T. cruzi infection is dependent on the host immunological profile. Innate immune receptors, such as Toll-like receptors (TLRs/TLR2, TLR4, TLR7, and TLR9) and Nod-like receptors (NLRs/NOD1 and NLRP3 inflammasome) are involved with the resistance against acute experimental T. cruzi infection. Here, we evaluated the impact of T. cruzi virulence on the expression of innate immune receptors and its products in mice. For that, we used six T. cruzi strains/isolates that showed low (AM64/TcIV and 3253/Tc-V), medium (PL1.10.14/TcIII and CL/TcVI), or high (Colombian/Tc-I and Y/TcII) virulence and pathogenicity to the vertebrate host and belonging to the six discrete typing units (DTUs)-TcI to TcVI. Parasitemia, mortality, and myocarditis were evaluated and correlated to the expression of TLRs, NLRs, adapter molecules, cytokines, and iNOS in myocardium by real time PCR. Cytokines (IL-1ß, IL-12, TNF-α, and IFN-γ) were quantified in sera 15 days after infection. Our data indicate that high virulent strains of T. cruzi, which generate high parasitemia, severe myocarditis, and 100% mortality in infected mice, inhibit the expression of TLR2, TLR4, TLR9, TRIF, and Myd88 transcripts, leading to a low IL-12 production, when compared to medium and low virulent T. cruzi strains. On the other hand, the high virulent T. cruzi strains induce the upregulation of NLRP3, caspase-1, IL-1ß, TNF-α, and iNOS mRNA in heart muscle, compared to low and medium virulent strains, which may contribute to myocarditis and death. Moreover, high virulent strains induce higher levels of IL-1ß and TNF-α in sera compared to less virulent parasites. Altogether the data indicate that differential TLR and NLR expression in heart muscle is correlated with virulence and pathogenicity of T cruzi strains. A better knowledge of the immunological mechanisms involved in resistance to T. cruzi infection is important to understand the natural history of Chagas disease, can lead to identification of immunological markers and/or to serve as a basis for alternative therapies.

Triatomine and Trypanosoma cruzi discrete typing units distribution in a semi-arid area of northeastern Brazil.

The occurrence of triatomine species, their bloodmeal sources and the discrete typing units (DTUs) of Trypanosoma cruzi isolated from them were determined in different municipalities of the state of Rio Grande do Norte, Brazil. Triatomine captures were carried out in the rural areas of 23 municipalities. The genotyping of T. cruzi isolates was performed using the mitochondrial cytochrome c oxidase subunit 2 (coii) gene, the D7 region of the 24Sα rDNA, and the spliced leader intergenic region (SL-IR). Five triatomine species were captured, and the most frequent was Triatoma brasiliensis (84.3%; 916/1086), which was found in 16 of the 23 municipalities surveyed, and infested all types of environment investigated. The TcI DTU was found in all mesoregions surveyed in 51.5% (17/33) of the culture-positive samples. In contrast, TcII (9.1%; 3/33) was detected in the Central mesoregion, while TcIII (27.3%; 9/33) was found in all mesoregions. The geographic distribution and spatial overlap of different DTUs was inferred using the superposition of the radius of occurrence of isolates and using ecological niche distribution modelling. Triatoma brasiliensis was found infected in all mesoregions and with all three T. cruzi DTUs, including mixed infections. With regard to bloodmeal sources, the DNA of rodents was found in triatomines infected with either TcI or TcIII, while that of domestic animals and humans was associated with both single and mixed infections. Our findings demonstrate that different DTUs of T. cruzi are widely dispersed among triatomines in our study area. The association of T. brasiliensis with several different mammalian hosts, as well as overlapping areas with different DTUs, suggests that this triatomine species may have an important role as a vector in both anthropic and sylvatic environments.

Insecticidal efficacy of fluralaner (Bravecto®) against Triatoma brasiliensis, a major vector of Trypanosoma cruzi in Brazil.

BACKGROUND: Triatomines are responsible for the vector transmission of the protozoan parasite Trypanosoma cruzi, which causes Chagas disease. Triatoma brasiliensis is the main vector of the parasite in Brazil, and dogs are an important reservoir of the parasite. The aim of this study was to evaluate the insecticidal effect of fluralaner (Bravecto®) on T. brasiliensis after a blood meal in treated dogs. METHODS: Healthy mongrel dogs (n = 8) were recruited from the Zoonoses Control Center (ZCC) in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups, a fluralaner (Bravecto®)-treated group (n = 4) and a control group (n = 4). Colony-reared third-, fourth- and fifth-instar nymphs of T. brasiliensis nymphs (n = 10) were allowed to feed on dogs from both groups for 30-40 min, once monthly, for up to 12 months. Bug mortality was observed up to 5 days after each blood meal. RESULTS: Mortality in triatomines which had a blood meal on fluralaner (Bravecto®)-treated dogs was 100% for up to 7 months after treatment, with mortality decreasing to 66.4% after 8 months, 57% after 9 months, 35% after 10 months, 10% after 11 months and 0% after 12 months. The mortality of triatomines that fed on non-treated control dogs was always ≤ 2.5%. CONCLUSIONS: Our results suggest that fluralaner (Bravecto®) treatment of dogs induces long-term mortality of T. brasiliensis after the blood meal. This is a potential approach to be used to control vector transmission of T. cruzi, the etiological agent of Chagas disease, especially in endemic areas.

Comparison of phenol-chloroform and a commercial deoxyribonucleic acid extraction kit for identification of bloodmeal sources from triatomines (Hemiptera: Reduviidae).

INTRODUCTION: Knowledge of triatomine bloodmeal sources is essential for understanding vector-host interactions in Trypanosoma cruzi transmission cycles. Expensive commercial deoxyribonucleic acid (DNA) extraction kits are widely used for bloodmeal identification. This study assessed the performance of an inexpensive phenol-chloroform DNA extraction protocol for identification of triatomine bloodmeal sources, comparing it with a commercially available kit. METHODS: Both methods were used to obtain DNA from the intestinal contents of Triatoma brasiliensis blood-fed on either Columba sp., Mus musculus, or Gallus gallus. Subsequently, the mitochondrial 12S ribosomal ribonucleic acid (rRNA) gene was amplified by polymerase chain reaction, sequenced, and compared with GenBank data. RESULTS: Twelve (80%) samples extracted with the commercial kit and four (26.7%) with phenol-chloroform were pure (according to the A260/A280 ratio). Samples extracted with phenol-chloroform, except for Columba sp. samples, had higher DNA concentration than those extracted with the commercial kit. Samples extracted using phenol-chloroform and blood-fed on G. gallus had significantly higher DNA concentration than those blood-fed on Columba sp. (p-value <0.001) and M. musculus (p-value <0.001). The 215-base-pair 12S rRNA fragment was amplified from all samples and produced reliable sequences, enabling the identification of the bloodmeal source, most of which showed identity and coverage above 95%. The phenol-chloroform method was much less expensive than the commercial kit but took considerably more time to perform. CONCLUSIONS: Our data showed that both DNA extraction methods produced reliable sequences enabling identification of triatomine bloodmeal sources but differed greatly in cost and time required.

Epidemiological profile of acute Chagas disease in individuals infected by oral transmission in northern Brazil.

INTRODUCTION: Oral infection by Trypanosoma cruzi is currently the most important route of transmission of acute Chagas disease (ACD) in the North region of Brazil, and the reported outbreaks are usually related to ingestion of contaminated food, especially unprocessed açaí pulp. METHODS: A retrospective cohort study was performed to analyze the epidemiological profile of individuals with suspected cases of ACD in the municipality of Breves, located in the state of Pará, Brazil. Therefore, notifications of suspected cases of ACD were collected from the Municipal Health Department of Breves from January 2007 to December 2017. RESULTS: A total of 265 individuals were registered, and the majority were male (54.7%; 145/265). Age ranged from nine months to 79 years, with a greater number of notifications for individuals aged between 1 and 39 years (71.3%; 189/265). Most of them had a low level of education (74.3%, 197/265), were living in rural and urban areas (58.9%; 156/265 and 37.7%; 100/265, respectively). Infection occurred mainly in the domestic environment (96.2%; 255/265) through oral transmission (98.1%; 260/265). There were a greater number of notifications in November, December and January. CONCLUSIONS: These data showed that oral transmission of T. cruzi has become increasingly high in the study region, and health education programs need to be implemented as strategies to ensure good manufacturing practices of unprocessed food.

NOD2 receptor is crucial for protecting against the digestive form of Chagas disease.

Digestive and cardiodigestive forms of Chagas' disease are observed in 2% to 27% of the patients, depending on their geographic location, Trypanosoma cruzi strain and immunopathological responses. The aim of this work was to evaluate the role of NOD2 innate immune receptor in the pathogenesis of the digestive system in Chagas' disease. Patients with digestive form of the disease showed lower mRNA expression of NOD2, higher expression of RIP2 and α-defensin 6, compared to indeterminate form, detected by Real-time PCR in peripheral blood mononuclear cells. In addition, there was a negative correlation between the expression of NOD2 and the degree of dilation of the esophagus, sigmoid and rectum in those patients. The infection of NOD2-/- mice with T. cruzi strain isolated from the digestive patient induced a decrease in intestinal motility. Histopathological analysis of the colon and jejunum of NOD2-/- and wild type C57BL/6 animals revealed discrete inflammatory foci during the acute phase of infection. Interestingly, during the chronic phase of the infection there was inflammation and hypertrophy of the longitudinal and circular muscular layer more pronounced in the colon and jejunum from NOD2-/- animals, when compared to wild type C57BL/6 mice. Together, our results suggest that NOD2 plays a protective role against the development of digestive form of Chagas' disease.

Synanthropic triatomines (Hemiptera: Reduviidae): infestation, colonization, and natural infection by trypanosomatids in the State of Rio Grande do Norte, Brazil.

INTRODUCTION: The ecoepidemiological situation in the State of Rio Grande do Norte, Brazil is characterized by frequent invasion and colonization of domiciliary units (DUs) by several triatomine species, with high rates of natural infection by Trypanosoma cruzi. METHODS: We evaluated the possibility of vector transmission of T. cruzi based on records of the occurrence of domiciled triatomines collected by the Secretariat of State for Public Health from 2005 to 2015. During this period, 67.7% (113/167) of municipalities conducted at least one active search and 110 recorded the presence of insects in DUs. These activities were more frequent in municipalities considered to have a high and medium-level risk of T. cruzi transmission. RESULTS: Of 51,569 captured triatomines, the most common species were Triatoma brasiliensis (47.2%) and T. pseudomaculata (40.2%). Colonies of T. brasiliensis, T. pseudomaculata, T. petrocchiae, Panstrongylus lutzi, and Rhodnius nasutus were also recorded in the intradomicile and peridomicile. Natural infection by trypanosomatids was detected in 1,153 specimens; the highest rate was found in R. nasutus (3.5%), followed by T. brasiliensis (2.5%) and T. pseudomaculata (2.4%). There have been high levels of colonization over the years; however, not all infested DUs have been sprayed. CONCLUSIONS: This is the first report of intradomicile and peridomicile colonization by P. lutzi. These results demonstrate the risk of new cases of infection by T. cruzi and reinforce the need for continuous entomological surveillance in the State of Rio Grande do Norte.

Innate immune receptors over expression correlate with chronic chagasic cardiomyopathy and digestive damage in patients.

Chronic chagasic cardiomyopathy (CCC) is observed in 30% to 50% of the individuals infected by Trypanosoma cruzi and heart failure is the important cause of death among patients in the chronic phase of Chagas disease. Although some studies have elucidated the role of adaptive immune responses involving T and B lymphocytes in cardiac pathogenesis, the role of innate immunity receptors such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in CCC pathophysiology has not yet been determined. In this study, we evaluated the association among innate immune receptors (TLR1-9 and nucleotide-binding domain-like receptor protein 3/NLRP3), its adapter molecules (Myd88, TRIF, ASC and caspase-1) and cytokines (IL-1ß, IL-6, IL-12, IL-18, IL-23, TNF-α, and IFN-ß) with clinical manifestation, digestive and cardiac function in patients with different clinical forms of chronic Chagas disease. The TLR8 mRNA expression levels were enhanced in the peripheral blood mononuclear cells (PBMC) from digestive and cardiodigestive patients compared to indeterminate and cardiac patients. Furthermore, mRNA expression of IFN-ß (cytokine produced after TLR8 activation) was higher in digestive and cardiodigestive patients when compared to indeterminate. Moreover, there was a positive correlation between TLR8 and IFN-ß mRNA expression with sigmoid and rectum size. Cardiac and cardiodigestive patients presented higher TLR2, IL-12 and TNF-α mRNA expression than indeterminate and digestive patients. Moreover, cardiac patients also expressed higher levels of NLRP3, ASC and IL-1ß mRNAs than indeterminate patients. In addition, we showed a negative correlation among TLR2, IL-1ß, IL-12 and TNF-α levels with left ventricular ejection fraction, and positive correlation between NLRP3 with cardiothoracic index, and TLR2, IL-1ß and IL-12 with left ventricular mass index. Together, our data suggest that high expression of innate immune receptors in cardiac and digestive patients may induce an enhancement of cytokine expression and participate of cardiac and digestive dysfunction.

Combined parasitological and molecular-based diagnostic tools improve the detection of Trypanosoma cruzi in single peripheral blood samples from patients with Chagas disease.

INTRODUCTION: In order to detect Trypanosoma cruzi and determine the genetic profiles of the parasite during the chronic phase of Chagas disease (ChD), parasitological and molecular diagnostic methods were used to assess the blood of 91 patients without specific prior treatment. METHODS: Blood samples were collected from 68 patients with cardiac ChD and 23 patients with an indeterminate form of ChD, followed by evaluation using blood culture and polymerase chain reaction. T . cruzi isolates were genotyped using three different genetic markers. RESULTS:: Blood culture was positive in 54.9% of all patients, among which 60.3% had the cardiac form of ChD, and 39.1% the indeterminate form of ChD. There were no significant differences in blood culture positivity among patients with cardiac and indeterminate forms. Additionally, patient age and clinical forms did not influence blood culture results. Polymerase chain reaction (PCR) was positive in 98.9% of patients, although comparisons between blood culture and PCR results showed that the two techniques did not agree. Forty-two T . cruzi stocks were isolated, and TcII was detected in 95.2% of isolates. Additionally, one isolate corresponded to TcIII or TcIV, and another corresponded to TcV or TcVI. CONCLUSIONS: Blood culture and PCR were both effective for identifying T. cruzi using a single blood sample, and their association did not improve parasite detection. However, we were not able to establish an association between the clinical form of ChD and the genetic profile of the parasite.

Myocarditis in different experimental models infected by Trypanosoma cruzi is correlated with the production of IgG1 isotype.

This study was designed to verify the relationship between IgG antibodies isotypes and myocarditis in Trypanosoma cruzi infection using mice and dogs infected with different T. cruzi strains. The animals were infected with benznidazole-susceptible Berenice-78 and benznidazole-resistant AAS and VL-10 strains. The IgG subtypes were measured in serum samples from dogs (IgG, IgG1, and IgG2) and mice (IgG, IgG1, IgG2a, and IgG2b). The infection of dogs with VL-10 strain induced the highest levels of heart inflammation while intermediate and lower levels were detected with Berenice-78 and AAS strains, respectively. Similar results were found in mice infected with VL-10, but not in those infected with AAS or Berenice-78 strains. The AAS strain induced higher levels of heart inflammation in mice, while Berenice-78 strain was not able to induce it. Correlation analysis between myocarditis and antibody reactivity index revealed very interesting results, mainly for IgG and IgG1, the latter being the most exciting. High IgG1 showed a significant correlation with myocarditis in both experimental models, being more significant in dogs (r=0.94, p<0.0001) than in mice (r=0.58, p=0.047). Overall, our data suggest that IgG1 could be a good marker to demonstrate myocarditis intensity in Chagas disease.

Characteristics of Triatomine infestation and natural Trypanosoma cruzi infection in the State of Rio Grande do Norte, Brazil.

INTRODUCTION Natural and artificial ecotope infestation by the kissing bug triatomines and their colonization and infection by Trypanosoma cruzi , the Chagas disease agent, were evaluated in nine municipalities of the State of Rio Grande do Norte, Brazil. METHODS Following identification, triatomine intestinal contents were analyzed by direct microscopic examination, xenoculture, and polymerase chain reaction (PCR) for parasite detection. Trypanosoma cruzi isolates were genotyped using three different markers. RESULTS Of 842 triatomines captured, 65% were Triatoma brasiliensis , 17.8% Triatoma pseudomaculata , 12.5% Panstrongylus lutzi , and 4.7% Rhodnius nasutus . Triatoma brasiliensis and P. lutzi adults were found in the intradomicile. T. brasiliensis, T. pseudomaculata , and R. nasutus nymphs and adults were found in the peridomicile and wild environment. Intradomiciliary and peridomiciliary infestation indexes were 5.6% and 33.7%, respectively. In the peridomicile, chicken coops were the most infested ecotope. The T. cruzi triatomine infection rate was 30.2%, of which PCR detected 29%. P . lutzi (78.1%), T . brasiliensis (24.5%), and T . pseudomaculata (22.7%) were the most infected species. TcII and III genotypes were detected in T. brasiliensis and TcIII in P. lutzi . CONCLUSIONS T. brasiliensis was found in all environments and most ecotopes with high T. cruzi infection rates. High infection rates were also detected in T . pseudomaculata and P. lutzi , suggesting their role in the interchange between the wild and peridomestic transmission cycles. The combination of PCR, microscopic examination, and xenoculture contributed to improving T. cruzi infection evaluation in triatomine bugs. The TcII and TcIII genotypes were predominant in the study area.

Inflammation Enhances the Risks of Stroke and Death in Chronic Chagas Disease Patients.

Ischemic strokes have been implicated as a cause of death in Chagas disease patients. Inflammation has been recognized as a key component in all ischemic processes, including the intravascular events triggered by vessel interruption, brain damage and repair. In this study, we evaluated the association between inflammatory markers and the death risk (DR) and stroke risk (SR) of patients with different clinical forms of chronic Chagas disease. The mRNA expression levels of cytokines, transcription factors expressed in the adaptive immune response (Th1, Th2, Th9, Th17, Th22 and regulatory T cell), and iNOS were analyzed by real-time PCR in peripheral blood mononuclear cells of chagasic patients who exhibited the indeterminate, cardiac, digestive and cardiodigestive clinical forms of the disease, and the levels of these transcripts were correlated with the DR and SR. Cardiac patients exhibited lower mRNA expression levels of GATA-3, FoxP3, AHR, IL-4, IL-9, IL-10 and IL-22 but exhibited higher expression of IFN-γ and TNF-α compared with indeterminate patients. Digestive patients showed similar levels of GATA-3, IL-4 and IL-10 than indeterminate patients. Cardiodigestive patients exhibited higher levels of TNF-α compared with indeterminate and digestive patients. Furthermore, we demonstrated that patients with high DR and SR exhibited lower GATA-3, FoxP3, and IL-10 expression and higher IFN-γ, TNF-α and iNOS mRNA expression than patients with low DR and SR. A negative correlation was observed between Foxp3 and IL-10 mRNA expression and the DR and SR. Moreover, TNF-α and iNOS expression was positively correlated with DR and SR. Our data suggest that an inflammatory imbalance in chronic Chagas disease patients is associated with a high DR and SR. This study provides a better understanding of the stroke pathobiology in the general population and might aid the development of therapeutic strategies for controlling the morbidity and mortality of Chagas disease.

Insecticidal efficacy of fluralaner (Bravecto®) against Triatoma brasiliensis, a major vector of Trypanosoma cruzi in Brazil

Background: Triatomines are responsible for the vector transmission of the protozoan parasite Trypanosoma cruzi, which causes Chagas disease. Triatoma brasiliensis is the main vector of the parasite in Brazil, and dogs are an important reservoir of the parasite. The aim of this study was to evaluate the insecticidal effect of fluralaner (Bravecto®) on T. brasiliensis after a blood meal in treated dogs. Methods: Healthy mongrel dogs (n = 8) were recruited from the Zoonoses Control Center (ZCC) in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups, a fluralaner (Bravecto®)-treated group (n = 4) and a control group (n = 4). Colony-reared third-, fourth- and fifth-instar nymphs of T. brasiliensis nymphs (n = 10) were allowed to feed on dogs from both groups for 30-40 min, once monthly, for up to 12 months. Bug mortality was observed up to 5 days after each blood meal. Results: Mortality in triatomines which had a blood meal on fluralaner (Bravecto®)-treated dogs was 100% for up to 7 months after treatment, with mortality decreasing to 66.4% after 8 months, 57% after 9 months, 35% after 10 months, 10% after 11 months and 0% after 12 months. The mortality of triatomines that fed on non-treated control dogs was always ≤ 2.5%. Conclusions: Our results suggest that fluralaner (Bravecto®) treatment of dogs induces long-term mortality of T. brasiliensis after the blood meal. This is a potential approach to be used to control vector transmission of T. cruzi, the etiological agent of Chagas disease, especially in endemic areas.

Insecticidal efficacy of fluralaner (Bravecto®) against Triatoma brasiliensis, a major vector of Trypanosoma cruzi in Brazil

Background: Triatomines are responsible for the vector transmission of the protozoan parasite Trypanosoma cruzi, which causes Chagas disease. Triatoma brasiliensis is the main vector of the parasite in Brazil, and dogs are an important reservoir of the parasite. The aim of this study was to evaluate the insecticidal efect of furalaner (Bravecto®) on T. brasiliensis after a blood meal in treated dogs. Methods: Healthy mongrel dogs (n=8) were recruited from the Zoonoses Control Center (ZCC) in the city of Natal, Rio Grande do Norte, Brazil, and randomized into two groups, a furalaner (Bravecto®)-treated group (n=4) and a control group (n=4). Colony-reared third-, fourth- and ffth-instar nymphs of T. brasiliensis nymphs (n=10) were allowed to feed on dogs from both groups for 30­40 min, once monthly, for up to 12 months. Bug mortality was observed up to 5 days after each blood meal. Results: Mortality in triatomines which had a blood meal on furalaner (Bravecto®)-treated dogs was 100% for up to 7 months after treatment, with mortality decreasing to 66.4% after 8 months, 57% after 9 months, 35% after 10 months, 10% after 11 months and 0% after 12 months. The mortality of triatomines that fed on non-treated control dogs was always ≤ 2.5%. Conclusions: Our results suggest that furalaner (Bravecto®) treatment of dogs induces long-term mortality of T. brasiliensis after the blood meal. This is a potential approach to be used to control vector transmission of T. cruzi, the etiological agent of Chagas disease, especially in endemic areas.

[Brazilian Consensus on Chagas Disease, 2015]. / II Consenso Brasileiro em Doença de Chagas, 2015.

Chagas disease is a neglected chronic condition that presents high morbidity and mortality burden, with considerable psychological, social, and economic impact. The disease represents a significant public health issue in Brazil, with different regional patterns. This document presents the evidence that resulted in the Brazilian Consensus on Chagas Disease. The objective was to review and standardize strategies for diagnosis, treatment, prevention, and control of Chagas disease in the country, based on the available scientific evidence. The consensus is based on collaboration and contribution of renowned Brazilian experts with vast knowledge and experience on various aspects of the disease. It is the result of close collaboration between the Brazilian Society of Tropical Medicine and the Ministry of Health. This document shall strengthen the development of integrated control measures against Chagas disease in the country, focusing on epidemiology, management, comprehensive care (including families and communities), communication, information, education, and research.