RESUMEN
Chagas cardiomyopathy is the most harmful complication of Chagas disease. The electrocardiogram is a well-studied exam and has been considered an important tool for detection and evaluation of Chagas cardiomyopathy since the first years of its description. Many of its abnormalities have been described as associated with a worse prognosis. Serum BNP levels were described as inversely related to the left ventricular ejection fraction and as an independent predictor of death. It was not reported how electrocardiographic alterations correlate to NT-proBNP and its analog. The present study aims to describe the baseline electrocardiograms of a large cohort of patients with Chagas disease from endemic area and to establish an association between the number of electrocardiogram alterations and high levels of NT-ProBNP in Chagas disease patients. This study selected 1959 Chagas disease patients in 21 municipalities within a limited region in the northern part of the State of Minas Gerais (Brazil), 1084 of them had Chagas cardiomyopathy. NT-proBNP levels were suggestive of heart failure in 11.7% of this population. One or more electrocardiographic alterations have an Odds Ratio of 9.12 (CI 95% 5.62-14.80) to have NT-proBNP elevation. Considering the association between the number of 1, 2, and 3 or more alterations in electrocardiogram and NT-proBNP elevation, the ORs were 7.11 (CI 95% 4.33-11.67); 16.04 (CI 95% 9.27-27.77) and 47.82 (CI 95% 17.98-127.20), respectively. The presence and the number of typical electrocardiographic alterations of Chagas disease are independently associated with the severity of the cardiomyopathy.
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Cardiomiopatía Chagásica/fisiopatología , Electrocardiografía , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Anciano , Biomarcadores/sangre , Brasil , Cardiomiopatía Chagásica/sangre , Enfermedad de Chagas/epidemiología , Estudios de Cohortes , Estudios Transversales , Enfermedades Endémicas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores SexualesRESUMEN
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects 7 million people in Latin American areas of endemicity. About 30% of infected patients will develop chronic Chagas cardiomyopathy (CCC), an inflammatory cardiomyopathy characterized by hypertrophy, fibrosis, and myocarditis. Further studies are necessary to understand the molecular mechanisms of disease progression. Transcriptome analysis has been increasingly used to identify molecular changes associated with disease outcomes. We thus assessed the whole-blood transcriptome of patients with Chagas disease. Microarray analysis was performed on blood samples from 150 subjects, of whom 30 were uninfected control patients and 120 had Chagas disease (1 group had asymptomatic disease, and 2 groups had CCC with either a preserved or reduced left ventricular ejection fraction [LVEF]). Each Chagas disease group displayed distinct gene expression and functional pathway profiles. The most different expression patterns were between CCC groups with a preserved or reduced LVEF. A more stringent analysis indicated that 27 differentially expressed genes, particularly those related to natural killer (NK)/CD8+ T-cell cytotoxicity, separated the 2 groups. NK/CD8+ T-cell cytotoxicity could play a role in determining Chagas disease progression. Understanding genes associated with disease may lead to improved insight into CCC pathogenesis and the identification of prognostic factors for CCC progression.
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Cardiomiopatía Chagásica/genética , Disfunción Ventricular/genética , Linfocitos T CD8-positivos/inmunología , Cardiomiopatía Chagásica/sangre , Cardiomiopatía Chagásica/fisiopatología , Citotoxicidad Inmunológica/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Células Asesinas Naturales/inmunología , Análisis por Micromatrices , Persona de Mediana Edad , Miocardio/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Disfunción Ventricular/sangre , Disfunción Ventricular/parasitologíaRESUMEN
Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens.
RESUMEN
BACKGROUND: Chagas disease remains a major cause of cardiovascular death in endemic areas. Focused echocardiography (FoCUS) is a point-of-care means of assessing cardiac function which can be useful for the diagnosis of cardiac involvement. OBJECTIVE: This study aims evaluating the characteristics of validity and reliability of FoCUS applied on Chagas disease patients. METHODS: Patients with Chagas disease coming from an endemic area were selected from a large cohort (SaMi-Trop). A simplified echocardiogram with only three images was extracted from the conventional echocardiogram performed in this cohort. The images were evaluated by an observer who was blinded to the clinical and echocardiographic data, to determine the accuracy and reliability of FoCUS for cardiac assessment. The analysis constituted of 5 prespecified variables, dichotomized in absence or presence: left ventricular (LV) size and systolic function, right ventricular (RV) size and systolic function, and LV aneurysm. RESULTS: We included 725 patients with a mean age of 63.4 ± 12.3 years, 483 (67%) female. Abnormal electrocardiogram was observed in 81.5% of the patients. Left and right ventricular dysfunctions were found in 103 (14%) and 49 (7%) of the patients, respectively. Sensitivity, specificity, positive predictive value and negative predictive value were 84%, 94%, 70% and 97% for LV enlargement and 81%, 93%, 68% and 97% for LV systolic dysfunction, respectively, and 46%, 99%, 60% and 98% for RV dilatation, and 37%, 100%, 100% and 96% for RV dysfunction, respectively. Inter and intraobserver agreement were 61% and 87% for LV enlargement and 63% and 92% for LV dysfunction, respectively, and 50% and 49% for RV size and 46% and 79% for RV dysfunction, respectively. LV apical aneurysm was found in 45 patients (6.2%) with the lowest sensitivity of FoCUS study (11%; 95% CI 2-28%). CONCLUSIONS: FoCUS showed satisfactory values of validity and reliability for assessment of cardiac chambers in patients with Chagas disease, except for apical aneurysm. This tool can identify heart disease with potential impact on patient management in the limited-resource setting.
Asunto(s)
Enfermedad de Chagas/diagnóstico , Ecocardiografía , Corazón/diagnóstico por imagen , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Derecha/diagnóstico , Anciano , Cardiomiopatía Chagásica/diagnóstico , Cardiomiopatía Chagásica/diagnóstico por imagen , Cardiomiopatía Chagásica/fisiopatología , Enfermedad de Chagas/diagnóstico por imagen , Enfermedad de Chagas/fisiopatología , Femenino , Corazón/fisiopatología , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Sístole/fisiología , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/fisiopatología , Disfunción Ventricular Derecha/diagnóstico por imagen , Disfunción Ventricular Derecha/fisiopatologíaRESUMEN
Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi. Cardiomyopathy and damage to gastrointestinal tissue are the main disease manifestations. There are data suggesting that the immune response to T. cruzi depends on the intestinal microbiota. We hypothesized that Chagas disease is associated with an altered gut microbiome and that these changes are related to the disease phenotype. The stool microbiome from 104 individuals, 73 with Chagas disease (30 with the cardiac, 11 with the digestive, and 32 with the indeterminate form), and 31 healthy controls was characterized using 16S rRNA amplification and sequencing. The QIIME (Quantitative Insights Into Microbial Ecology) platform was used to analyze the data. Alpha and beta diversity indexes did not indicate differences between the groups. However, the relative abundance of Verrucomicrobia, represented primarily by the genus Akkermansia, was significantly lower in the Chagas disease groups, especially the cardiac group, compared to the controls. Furthermore, differences in the relative abundances of Alistipes, Bilophila, and Dialister were observed between the groups. We conclude that T. cruzi infection results in changes in the gut microbiome that may play a role in the myocardial and intestinal inflammation seen in Chagas disease.
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Enfermedad de Chagas , Microbioma Gastrointestinal , Trypanosoma cruzi , Disbiosis , Heces , Humanos , ARN Ribosómico 16S/genéticaRESUMEN
Chagas is a neglected disease endemic in Latin America. Vector transmission control had been aggressively performed. Recent entomological surveillance in Brazil has revealed natural infection rates ranging from 0.40% to 0.52%. Although serological surveys are complex to develop, they are important for disease control. In this study, we validated the use of saliva in ELISA commercial kits with a cohort of 100 patients with Chagas disease followed at Hospital das Clinicas in São Paulo, Brazil, and 50 healthy controls. Five ELISA kits for detecting antibodies against Trypanosoma cruzi were tested. The best discrimination between Chagas patients and controls was observed with the Wiener kit, which yielded a sensitivity of 97% and a specificity of 100%. Our findings reveal that the use of saliva may be an alternative to large-scale screening surveys in detecting T. cruzi antibodies; it is a noninvasive sample collection method potentially key to large-scale screening in children.
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Anticuerpos Antiprotozoarios/análisis , Enfermedad de Chagas/diagnóstico , Enfermedades Endémicas , Ensayo de Inmunoadsorción Enzimática/métodos , Saliva/inmunología , Trypanosoma cruzi/inmunología , Brasil/epidemiología , Estudios de Casos y Controles , Enfermedad de Chagas/epidemiología , Estudios de Cohortes , Humanos , Sensibilidad y Especificidad , Estudios SeroepidemiológicosRESUMEN
Background Risk stratification of Chagas disease patients in the limited-resource setting would be helpful in crafting management strategies. We developed a score to predict 2-year mortality in patients with Chagas cardiomyopathy from remote endemic areas. Methods and Results This study enrolled 1551 patients with Chagas cardiomyopathy from Minas Gerais State, Brazil, from the SaMi-Trop cohort (The São Paulo-Minas Gerais Tropical Medicine Research Center). Clinical evaluation, ECG, and NT-proBNP (N-terminal pro-B-type natriuretic peptide) were performed. A Cox proportional hazards model was used to develop a prediction model based on the key predictors. The end point was all-cause mortality. The patients were classified into 3 risk categories at baseline (low, <2%; intermediate, ≥2% to 10%; high, ≥10%). External validation was performed by applying the score to an independent population with Chagas disease. After 2 years of follow-up, 110 patients died, with an overall mortality rate of 3.505 deaths per 100 person-years. Based on the nomogram, the independent predictors of mortality were assigned points: age (10 points per decade), New York Heart Association functional class higher than I (15 points), heart rate ≥80 beats/min (20 points), QRS duration ≥150 ms (15 points), and abnormal NT-proBNP adjusted by age (55 points). The observed mortality rates in the low-, intermediate-, and high-risk groups were 0%, 3.6%, and 32.7%, respectively, in the derivation cohort and 3.2%, 8.7%, and 19.1%, respectively, in the validation cohort. The discrimination of the score was good in the development cohort (C statistic: 0.82), and validation cohort (C statistic: 0.71). Conclusions In a large population of patients with Chagas cardiomyopathy, a combination of risk factors accurately predicted early mortality. This helpful simple score could be used in remote areas with limited technological resources.
Asunto(s)
Cardiomiopatía Chagásica/mortalidad , Técnicas de Apoyo para la Decisión , Enfermedades Endémicas , Indicadores de Salud , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Brasil/epidemiología , Cardiomiopatía Chagásica/diagnóstico , Cardiomiopatía Chagásica/terapia , Toma de Decisiones Clínicas , Electrocardiografía , Femenino , Estado de Salud , Humanos , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Riesgo , Adulto JovenRESUMEN
OBJECTIVES: The gut microbiota is associated with obesity and weight loss after bariatric surgery and has been related to its changing pattern. Exactly how the bacterial population affects weight loss and the results of surgery remain controversial. This study aimed to evaluate the intestinal microbiota of superobese patients before and after gastric bypass surgery (RYGB). METHOD: DNA fragments for the microbiota obtained from stool samples collected from nine superobese patients before and after bariatric surgery were sequenced using Ion Torrent. RESULTS: We observed that with a mean follow-up of 15 months, patients achieved 55.9% excess weight loss (EWL). A significant population reduction in the Proteobacteria phylum (11 to 2%, p=0.0025) was observed after surgery, while no difference was seen in Firmicutes and Bacteroidetes. Further analyses performed with two specific individuals with divergent clinical outcomes showed a change in the pattern between them, with a significant increase in Firmicutes and a decrease in Bacteroidetes in the patient with less weight loss (%EWL 50.79 vs. 61.85). CONCLUSIONS: RYGB affects the microbiota of superobese patients, with a significant reduction in Proteobacteria in patients with different weight loss, showing that different bacteria may contribute to the process.
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Cirugía Bariátrica , Microbioma Gastrointestinal , Obesidad Mórbida/microbiología , Obesidad Mórbida/cirugía , Pérdida de Peso , Adolescente , Adulto , Heces/microbiología , Femenino , Estudios de Seguimiento , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , ARN Ribosómico 16S/análisis , Adulto JovenRESUMEN
BACKGROUND: Trypanosoma cruzi parasite, the causative agent of Chagas disease, infects about six million individuals in more than 20 countries. Monitoring parasite persistence in infected individuals is of utmost importance to develop and evaluate treatments to control the disease. Routine screening for infected human individuals is achieved by serological assays; PCR testing to monitor spontaneous or therapy-induced parasitological cure has limitations due to the low and fluctuating parasitic load in circulating blood. The aim of the present study is to evaluate a newly developed antibody profiling assay as an indirect method to assess parasite persistence based on waning of antibodies following spontaneous or therapy-induced clearance of the infection. METHODOLOGY/PRINCIPAL FINDINGS: We designed a multiplex serology assay, an array of fifteen optimized T. cruzi antigens, to evaluate antibody diversity in 1654 serum samples from chronic Chagas patients. One specific antibody response (antibody 3, Ab3) showed a strong correlation with T. cruzi parasite persistence as determined by T. cruzi PCR positive results. High and sustained Ab3 signal was strongly associated with PCR positivity in untreated patients, whereas significant decline in Ab3 signals was observed in BZN-treated patients who cleared parasitemia based on blood PCR results. CONCLUSION/SIGNIFICANCE: Ab3 is a new surrogate biomarker that strongly correlates with parasite persistence in chronic and benznidazole-treated Chagas patients. We hypothesize that Ab3 is induced and maintained by incessant stimulation of the immune system by tissue-based and shed parasites that are not consistently detectable by blood based PCR techniques. Hence, a simple immunoassay measurement of Ab3 could be beneficial for monitoring the infectious status of seropositive patients.
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Anticuerpos Antiprotozoarios/sangre , Biomarcadores/sangre , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/inmunología , Pruebas Serológicas/métodos , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/aislamiento & purificación , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Brasil , Enfermedad de Chagas/sangre , Enfermedad de Chagas/terapia , ADN Protozoario , Humanos , Inmunoensayo/métodos , Nitroimidazoles/uso terapéutico , Carga de Parásitos , Parasitemia/parasitología , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidadRESUMEN
Chagas disease (CD) is a neglected tropical disease that affects individuals in almost every country in Latin America. There are two available drugs with antiparasitic profiles; however, only benznidazole (BZN) has been approved for commercialization in Brazil. The usefulness of prescribing BZN for patients with chronic Chagas cardiomyopathy (CCC) is controversial. There are no studies in the literature describing the extent of BZN use at this stage or the profile of patients using this drug. The present study aimed to determine the prevalence and factors associated with previous BZN use among individuals with CCC. This cross-sectional study was conducted with 1,812 individuals with CCC from 21 Brazilian cities endemic for CD. The dependent variable was "prior use of BZN" (no vs. yes). The independent variables were grouped into socioeconomic, lifestyle and medical history aspects. Binary logistic regression (α ≥ 0.05) was used. Among the evaluated individuals, 27.2% reported previous use of BZN. The likelihood of prior use of BZN was higher among younger individuals (OR = 2.7), individuals with a higher education (OR = 2.7), individuals with a lower monthly per capita income (OR = 1.3), individuals who practiced physical exercise (OR = 1.5), individuals who had prior knowledge of the CD diagnosis (OR = 2.5), individuals without hypertension (OR = 1.3) and individuals with a longer time to the CD diagnosis (OR = 6.1). The present study revealed a small proportion of therapeutic BZN use among Brazilian CCC patients. This finding suggests a late diagnosis and undertreatment of the disease. BZN use was higher among individuals with better clinical and demographic conditions but with a lower income and a longer time to the CD diagnosis. Knowledge of the BZN usage profile may help reduce the current state of neglect of this disease and pave the way for future studies.
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Cardiomiopatía Chagásica/tratamiento farmacológico , Cardiomiopatía Chagásica/epidemiología , Nitroimidazoles/uso terapéutico , Anciano , Brasil/epidemiología , Cardiomiopatía Chagásica/diagnóstico , Enfermedad Crónica , Comorbilidad , Estudios Transversales , Femenino , Estudios de Seguimiento , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Factores Socioeconómicos , Tripanocidas/uso terapéuticoRESUMEN
PURPOSE: We have established a prospective cohort of 1959 patients with chronic Chagas cardiomyopathy to evaluate if a clinical prediction rule based on ECG, brain natriuretic peptide (BNP) levels, and other biomarkers can be useful in clinical practice. This paper outlines the study and baseline characteristics of the participants. PARTICIPANTS: The study is being conducted in 21 municipalities of the northern part of Minas Gerais State in Brazil, and includes a follow-up of 2â years. The baseline evaluation included collection of sociodemographic information, social determinants of health, health-related behaviours, comorbidities, medicines in use, history of previous treatment for Chagas disease, functional class, quality of life, blood sample collection, and ECG. Patients were mostly female, aged 50-74â years, with low family income and educational level, with known Chagas disease for >10â years; 46% presented with functional class >II. Previous use of benznidazole was reported by 25.2% and permanent use of pacemaker by 6.2%. Almost half of the patients presented with high blood cholesterol and hypertension, and one-third of them had diabetes mellitus. N-terminal of the prohormone BNP (NT-ProBNP) level was >300â pg/mL in 30% of the sample. FINDINGS TO DATE: Clinical and laboratory markers predictive of severe and progressive Chagas disease were identified as high NT-ProBNP levels, as well as symptoms of advanced heart failure. These results confirm the important residual morbidity of Chagas disease in the remote areas, thus supporting political decisions that should prioritise in addition to epidemiological surveillance the medical treatment of chronic Chagas cardiomyopathy in the coming years. The São Paulo-Minas Gerais Tropical Medicine Research Center (SaMi-Trop) represents a major challenge for focused research in neglected diseases, with knowledge that can be applied in primary healthcare. FUTURE PLANS: We will continue following this patients' cohort to provide relevant information about the development and progression of Chagas disease in remotes areas, with social and economic inequalities. TRIAL REGISTRATION NUMBER: NCT02646943; Pre-results.
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Cardiomiopatía Chagásica/epidemiología , Enfermedad Crónica/epidemiología , Insuficiencia Cardíaca/epidemiología , Inmunosupresores/uso terapéutico , Péptido Natriurético Encefálico/sangre , Nitroimidazoles/uso terapéutico , Fragmentos de Péptidos/sangre , Anciano , Biomarcadores/sangre , Brasil/epidemiología , Cardiomiopatía Chagásica/sangre , Cardiomiopatía Chagásica/tratamiento farmacológico , Cardiomiopatía Chagásica/fisiopatología , Enfermedad Crónica/tratamiento farmacológico , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/prevención & control , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Vigilancia de la Población , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Calidad de Vida , Factores SocioeconómicosRESUMEN
Pediatric autoimmune hepatitis (AIH) patients present hypergammaglobulinemia, periportal CD8(+) cytotoxic T cell infiltration, and cirrhosis. Autoantibody profile defines AIH types 1 and 2 in addition to strong association with HLA-DRB1. We previously detected increased IgE serum levels and sought to compare clinical and histological features according to IgE levels in AIH (n = 74, ages 1-14 years) patients. Additionally, we typed 117 patients and 227 controls for functional polymorphisms of IL4, IL13, IL5, and IL4RA genes involved in IgE switching and eosinophil maturation that might contribute to overall genetic susceptibility to AIH. Serum IgE levels were high in 55% of AIH-1, but only in 12% of AIH-2 (P = 0.003) patients. Liver IgE was present in 91.3% of AIH-1 patients. The A alleles at both IL13 rs20541 and IL4RA rs1805011 were associated with AIH-1 (P = 0.024, OR = 1.55 and P < 0.0001, OR = 2.15, resp.). Furthermore, individuals presenting homozygosis for the A allele at IL4RA rs1805011 and HLA-DRB1(∗)03 and/or (∗)13 allele had sixfold greater risk to develop the disease (OR = 14.00, P < 0.001). The novel association suggests an additional role for IgE-linked immune response genes in the pathogenesis of AIH.
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Eosinófilos/fisiología , Cadenas HLA-DRB1/genética , Hepatitis Autoinmune/inmunología , Inmunoglobulina E/metabolismo , Interleucina-13/genética , Adolescente , Autoanticuerpos/sangre , Brasil , Niño , Preescolar , Predisposición Genética a la Enfermedad , Hepatitis Autoinmune/diagnóstico , Hepatitis Autoinmune/genética , Humanos , Lactante , Polimorfismo de Nucleótido Simple , RiesgoRESUMEN
OBJECTIVES: The gut microbiota is associated with obesity and weight loss after bariatric surgery and has been related to its changing pattern. Exactly how the bacterial population affects weight loss and the results of surgery remain controversial. This study aimed to evaluate the intestinal microbiota of superobese patients before and after gastric bypass surgery (RYGB). METHOD: DNA fragments for the microbiota obtained from stool samples collected from nine superobese patients before and after bariatric surgery were sequenced using Ion Torrent. RESULTS: We observed that with a mean follow-up of 15 months, patients achieved 55.9% excess weight loss (EWL). A significant population reduction in the Proteobacteria phylum (11 to 2%, p=0.0025) was observed after surgery, while no difference was seen in Firmicutes and Bacteroidetes. Further analyses performed with two specific individuals with divergent clinical outcomes showed a change in the pattern between them, with a significant increase in Firmicutes and a decrease in Bacteroidetes in the patient with less weight loss (%EWL 50.79 vs. 61.85). CONCLUSIONS: RYGB affects the microbiota of superobese patients, with a significant reduction in Proteobacteria in patients with different weight loss, showing that different bacteria may contribute to the process.
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Humanos , Masculino , Adolescente , Adulto , Persona de Mediana Edad , Adulto Joven , Obesidad Mórbida/cirugía , Obesidad Mórbida/microbiología , Pérdida de Peso , Cirugía Bariátrica , Microbioma Gastrointestinal , ARN Ribosómico 16S/análisis , Estudios de Seguimiento , Estudios Longitudinales , Heces/microbiologíaRESUMEN
Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.
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Bartonella henselae/genética , Enfermedad por Rasguño de Gato/microbiología , ADN Bacteriano/análisis , ARN Ribosómico 16S/análisis , Adolescente , Adulto , Anciano de 80 o más Años , Bartonella henselae/aislamiento & purificación , Enfermedad por Rasguño de Gato/diagnóstico , Chaperonina 60/análisis , Niño , Preescolar , ADN Espaciador Ribosómico/análisis , Femenino , Humanos , Inmunocompetencia , Huésped Inmunocomprometido , Ganglios Linfáticos/microbiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: The aim of this cross-sectional study was to evaluate whether interleukin 10 (IL10) and transforming growth factor β1 (TGFβ1) gene polymorphisms were associated with persistent IgE-mediated cow's milk allergy in 50 Brazilian children. The diagnostic criteria were anaphylaxis triggered by cow's milk or a positive double-blind, placebo-controlled food challenge. Tolerance was defined as the absence of a clinical response to a double-blind, placebo-controlled food challenge or cow's milk exposure. METHOD: The genomic DNA of the 50 patients and 224 healthy controls (HCs) was used to investigate five IL10 gene polymorphisms (-3575A/T, -2849A/G, -2763A/C, -1082G/A, -592C/A) and one TGFβ1 polymorphism (-509C/T). RESULTS: Among the five IL10 polymorphisms analyzed, homozygosis for the G allele at the -1082 position was significantly higher in the patients compared with the healthy controls (p = 0.027) and in the persistent cow's milk allergy group compared with the healthy controls (p = 0.001). CONCLUSIONS: Homozygosis for the G allele at the IL10 -1082G/A polymorphism is associated with the persistent form of cow's milk allergy. .