Evaluation of Molecular Methods to Identify Chagas Disease and Leishmaniasis in Blood Donation Candidates in Two Brazilian Centers.

In Brazil, blood donation is regulated by the Brazilian Ministry of Health, and all States follow the same protocol for clinical and laboratory screening. Brazil is an endemic country for Chagas disease (CD), caused by Trypanosoma cruzi, and for leishmaniasis, caused by a species of Leishmania spp. Screening for leishmaniosis is not routinely performed by blood banks. Given the antigenic similarity between T. cruzi and Leishmania spp., cross-reactions in serological tests can occur, and inconclusive results for CD have been found. The objective of this study was to apply molecular techniques, e.g., nPCR, PCR, and qPCR, to clarify cases of blood donation candidates with non-negative serology for CD and to analyze the difference between the melting temperature during real-time PCR using SYBR Green. Thirty-seven cases that showed non-negative results for CD using chemiluminescent microparticle immunoassay (CMIA) tests from blood banks in Campo Grande, MS, and Campinas, SP, were analyzed. In the serum samples, 35 samples were evaluated by ELISA, and 24.3% (9/35) showed positive results for CD. nPCR was able to detect 12 positive results in 35 samples (34.28%). qPCR for T. cruzi was quantifiable in the samples that showed a value ≥0.002 par eq/mL (parasite equivalents per milliliter), and in 35 samples, 11 (31.42%) were positive. Of all evaluated samples using the described tests (CMIA, ELISA, nPCR, and qPCR), 18 (48.6%) were positive for CD. For MCA by qPCR, the melting temperature was 82.06 °C ± 0.46 for T. cruzi and 81.9 °C ± 0.24 for Leishmania infantum. The Mann-Whitney test showed a significant value of p < 0.0001. However, the differentiation between T. cruzi and L. infantum could not be considered due to temperature overlap. For leishmaniasis, of the 35 samples with non-negative serology for CD tested by the indirect fluorescent antibody test (IFAT), only one sample (2.85%) was positive (1:80). The PCR for Leishmania spp. was performed on 36 blood samples from donation candidates, and all were negative. qPCR for L. infantum showed 37 negative results for the 37 analyzed samples. The data presented here show the importance of performing two different tests in CD screening at blood banks. Molecular tests should be used for confirmation, thereby improving the blood donation system.

Parasite load evaluation by qPCR and blood culture in Chagas disease and HIV co-infected patients under antiretroviral therapy.

Chagas disease also known as American trypanosomiasis, is caused by Trypanosoma cruzi and transmitted by triatominae-contaminated feces. It is considered a neglected tropical disease that affects 6 to 7 million people worldwide. The reactivation of Chagas disease occurs when the chronically infected hosts are not able to control T. cruzi infection, generating recurrence of the acute phase. HIV is the main immunosuppressive infection that can lead to the reactivation of chronic Chagas disease in AIDS conditions. In co-infected patients, the reactivation of Chagas disease is related to their high parasite load, high HIV viral load, and CD4 T-cell counting less than 200/mm3, which may evolve to meningoencephalitis and myocarditis. Eight T. cruzi/HIV co-infected patients under antiretroviral therapy (ART) and ten Chagas disease patients without HIV infection that attended at Study Group of Chagas Disease, Hospital de Clínicas, University of Campinas (GEdoCh/HC/UNICAMP-SP) and Pontifical Catholic University of Campinas SP (PUCC/SP) were evaluated. Tests for Chagas disease were performed, such as qPCR and T. cruzi blood culture. The patient's medical records were analyzed to verify clinical and epidemiological data, viral load, and CD4 T-cell counting since the outset of ART. For both groups, we found no statically significant differences between parasite load via blood culture and qPCR. In T. cruzi/HIV co-infected subjects, we observed a significant increase of CD4 T-cells counting and viral load decrease, which became undetectable over the years after ART. Parasites isolated from the patient's blood culture were genotyped, being the majority of them infected with TcII and one case of mixed infection (TcII and TcV/TcVI). These results were expected according to the region of origin of the patients. We suggest that the parasite load be monitored through qPCR in T.cruzi/HIV co-infected patients. We conclude that ART in people living with HIV improves infection and immunosuppression control, enabling the natural evolution of the American trypanosomiasis.

Serological and molecular inquiry of Chagas disease in an Afro-descendant settlement in Mato Grosso do Sul State, Brazil.

Furnas do Dionísio is a Brazilian Afro-descendant settlement in the city of Jaraguari, 21.4 miles from Campo Grande, Mato Grosso do Sul, Brazil. Approximately 96 families live in this quilombola (Maroon) settlement, also known in Brazil as a remnant community of descendants of African slaves. Recent studies found 20% of households were infested by triatomines, 18% of insects captured in the community were infected by Trypanosoma cruzi, and 22.7% of dogs presented T. cruzi antibodies. The low prevalence of Chagas disease observed in humans in Mato Grosso do Sul State is attributed to its arrival via colonist migration and subsequent transplacental transmission. In order to gain a better understanding of the T. cruzi cycle in residents of the study community, serological and molecular tests were carried out to diagnose Chagas disease. In the present study, 175 residents between 2 and 80 years old were included. A total of 175 participants were interviewed and 170 provided blood samples, which were tested for T. cruzi antibodies with serological tests. Molecular diagnosis was performed in 167 participants by PCR (KDNA) and NPCR (satellite DNA) tests. One of the 170 samples tested positive for all serological tests performed. The overall frequency of Chagas disease in the community was low (0.6%). Interview responses revealed that 66.3% knew of triatomine insects and 65.7% reported having had no contact with them. Physical improvements to residences, together with vector surveillance and control by the State and municipal governments and local ecological conservation contribute to the low frequency of the Chagas disease in this quilombola community.

The detection of Trypanosoma cruzi by nested-PCR in elderly patients: relationship to the clinical and epidemiological profile.

Chagas disease, which is caused by Trypanosoma cruzi, is transmitted primarily by triatomine bugs, although the incidence of new cases has decreased as a result of vector control. In Brazil, most of those affected have the chronic form of the disease and are generally elderly individuals who require appropriate clinical follow-up. In this work, we undertook a descriptive study in which 85 patients were interviewed and blood samples were collected for molecular analyses based on the amplification of parasite satellite DNA. The cardiac form of the disease was the most prevalent among the patients and hypertension was the most frequent comorbidity; polypharmacy was detected in 34% of the cases. Serological tests were positive in 95% of cases while 36% were positive in nested-polymerase chain reaction. These findings indicate an increased use of medications and a larger number of age-related diseases in elderly patients with Chagas disease, even in patients with low parasitemia. We conclude that elderly patients with Chagas disease require special attention and that further studies should be done with elderly individuals who carry this disease.

Intake of heat-expanded amaranth grain reverses endothelial dysfunction in hypercholesterolemic rabbits.

This study reports the new functional property of amaranth grain against diet-induced endothelial dysfunction in rabbits. Twenty-seven New Zealand rabbits were fed either a standard diet (SD/G1) or a hypercholesterolemic diet (Hichol) for 28 days. On day 29, the Hichol group was subdivided into four groups and begun receiving the following diets for 21 days: G2: SD + amaranth, G3: Hichol + amaranth, G4: SD alone, and G5: Hichol alone, while G1 continued to receive SD for 21 days. Amaranth intake restored endothelial function (G2, G3) to nearly normal during the 21-day recovery besides substantially lowering total and LDL blood cholesterol levels. This effect was not seen by simply correcting the diet (G4). Upon continuance of Hichol, however, amaranth supplementation did show some contribution to the cholesterol-lowering effect (G4 vs. G3). On day 49, feeding Hichol without the help of amaranth, harm was further magnified by lowering HDL-cholesterol (G5). Fecal cholesterol was found increased in groups that ingested amaranth (G2, G3), but no significant impact from either supplementation or diet reversal was found in fecal bile acids. Amaranth supplementation granted some protection against tissue cholesterol (G5) and tissue peroxidation (G3). It is concluded that even in concurrence with a hypercholesterolemic diet, intake of heat-expanded amaranth can revert an associated endothelial dysfunction besides incrementing fecal cholesterol excretion and lowering blood and tissue cholesterol oxidation in dyslipidemic rabbits. These results supported the notion of a lipid peroxidation process occurring with high cholesterol intakes.

Evolution and involution of atherosclerosis and its relationship with vascular reactivity in hypercholesterolemic rabbits.

BACKGROUND AND OBJECTIVES: The aim of this study is to verify the evolution and involution of experimental atherosclerosis in rabbits through the study of endothelial function, lipids and tissue lipid peroxidation, macro and microscopic quantification of aortic atherosclerosis. METHODS: Thirty male New Zealand white rabbits were divided into six groups (n=5): G1 normal diet; G2: hypercholesterolemic receiving 0.5% of cholesterol diet for 4 months; G3: hypercholesterolemic diet for 4 months after normal diet for more 4 months; G4: hypercholesterolemic diet for 4 months plus normal diet and rosuvastatin for 1 month, G5: hypercholesterolemic diet for 4 months plus normal diet and rosuvastatin for 2 months, G6: hypercholesterolemic diet for 4 months plus normal diet and rosuvastatin for 4 months. Rosuvastatin was administered at a dosage of 5mg dissolved in 150 ml of water daily. At the end of the experiment were measured: total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C), tissue cholesterol (CAO), lipid peroxidation tissue (MDA). Endothelial function (RMAX) was studied in a segment of thoracic aorta, through curve-effect of acetylcholine and sodium nitroprusside. The amount of atherosclerosis was determined by measurement of the arterial lesion, through software, after staining with Sudan IV and histological staining. RESULTS: In relation the water the rabbits drank 60-70 ml all day. It was seen significantly increase in all parameters at G2 both biochemical and tissue. In the group G3 it was seen significantly decrease in plasma lipids levels and tissue cholesterol. Treated groups G4, G5 and G6 all showed a decreased plasma lipid levels, only at G6 group it was noted a tissue cholesterol, tissue peroxidation and quantification of atherosclerosis, which showed a significant decrease. In relation the endothelial function only G6 improve significantly. INTERPRETATION AND CONCLUSIONS: Our findings indicated that the treatment with rosuvastatin for 4 months is more efficient because improve the endothelial function significantly.

Recommendations for management of Chagas disease in organ and hematopoietic tissue transplantation programs in nonendemic areas.

The substantial immigration into Spain from endemic areas of Chagas disease such as Latin America has increased the number of potential donors of organs and tissues. In addition, an increasing number of patients with advanced Chagas heart disease may eventually be eligible to receive a heart transplant, a universally accepted therapeutic strategy for the advanced stages of this disease. Therefore, it is necessary to establish protocols for disease management. This document is intended to establish the guidelines to be followed when a potential donor or a tissue or organ recipient is potentially affected by Chagas disease and summarizes the action criteria against the possibility of Chagas disease transmission through the donation of organs, tissues, or hematopoietic stem cells and aims to help professionals working in this field. A single registry of transplants in Trypanosoma cruzi infected donors and/or recipients will provide and disseminate experience in this area, which has shown a low recorded incidence to date.

Quality of life and depressive symptoms in Chagas disease patients.

BACKGROUND: Besides the typical organic aspects of Chagas disease, the patients need to face physical, psychological, social and economic difficulties, which can compromise their quality of life. Consequently they may develop feelings of sadness, grief and loss, and depressive symptoms. METHODS: This study assessed the quality of life and depressive symptoms in 110 Chagas disease patients using WHOQOL-BREF for measuring the quality of life; also it was used Beck Depression Inventory (BDI) for depressive symptom evaluation. RESULTS: Among all patients, 51% were women; 42.5% considered their quality of life as positive and 40.9% presented depressive symptoms. Chagas disease clinical form distribution was: cardiac 49.09%, indeterminate 26.36%, digestive 12.73%, and mixed 11.82%. There was no significant difference comparing the depressive symptom intensity on gender, age and marital status, although there was a significant difference comparing depressive symptoms in indeterminate clinical form patients with others. All domains of WHOQOL-BREF showed significant correlation coefficients (Pearson). CONCLUSIONS: At digestive form it was reported minor scores in the quality of life domains and greater ones on depressive symptom levels. There was a negative correlation between BDI and WHOQOL-BREF, suggesting that, among Chagas disease patients, the higher is the quality of life the lower is the intensity of depressive symptoms.

Tc-99m sestamibi thyroid imaging in patients on chronic amiodarone treatment: a comparison with Tc-99m pertechnetate imaging.

PURPOSE OF THE REPORT: To compare thyroid imaging using Tc-99m sestamibi with the standard Tc-99m pertechnetate scintigraphy in patients on chronic use of amiodarone. MATERIALS AND METHODS: A total of 23 patients on oral amiodarone for at least 4 months had thyroid scintigraphy and uptake measurement using Tc-99m pertechnetate and Tc-99m sestamibi. Thyroid function was evaluated by measuring serum concentrations of thyrotropin, free thyroxine, and free triiodothyronine, and antithyroglobulin and antithyroperoxidase antibodies. RESULTS: Ten of the 23 patients were euthyroid, 9 hypothyroid, and 4 hyperthyroid, with normal, increased, and decreased serum thyrotropin, respectively. All euthyroid patients had markedly decreased thyroid Tc-99m pertechnetate uptake and normal or slightly increased Tc-99m sestamibi uptake, except for one patient who had increased uptake of both radiotracers. One of the 4 hyperthyroid patients had Graves' disease and markedly increased thyroid uptake of both tracers. The other 3 hyperthyroid patients had normal or decreased Tc-99m pertechnetate uptake and increased Tc-99m sestamibi uptake. Differently than expected, all 9 hypothyroid patients had normal or increased uptake of both radiopharmaceuticals. CONCLUSIONS: This study suggests that Tc-99m sestamibi may be an alternative tracer for thyroid scintigraphy and uptake measurement of patients on chronic use of amiodarone. Tc-99m sestamibi seems to be better than Tc-99m pertechnetate for the scintigraphic evaluation of the thyroid of euthyroid and hyperthyroid patients.

Neuropathy of gastrointestinal Chagas' disease: immune response to myelin antigens.

Most reports of autoimmune response during infection with the parasite Trypanosoma cruzi have dealt with the cardiomyopathic form of Chagas' disease, but little is known about the mechanisms of tissue damage involved in the gastrointestinal form, which was studied here. Chronically infected patients with a severe gastrointestinal form of Chagas' disease present increased antibody production and proliferative responses to peripheral myelin components, such as myelin basic protein (MBP), which is homologous to the P1 protein fraction of peripheral myelin. T lymphocytes preferentially recognize a region on the MBP molecule (1-30), which suggests that the MBP is a potential target on the peripheral nerve for autoimmune reactions in patients with gastrointestinal lesions resulting from Chagas' disease.

[Clinical presentation of chronic Chagas disease in elderly individuals]. / Apresentação clínica da doença de Chagas crônica em indivíduos idosos.

This study had the aim of evaluating the clinical presentation of chronic Chagas disease among the elderly. It was a retrospective analysis of clinical records at an outpatient referral service. The sample was divided into two groups: elderly (>or= 60 years old) and non-elderly. Sex, comorbidities, clinical form, electrocardiogram and serological titers were evaluated. In the elderly group (61 cases), the mean age was 66.03+/-5 years; 67.2% were female; 59% presented comorbidities (most frequently systemic arterial hypertension, in 39.3%); 1.6% had the indeterminate clinical form, 88.5% the cardiac form and 36% the digestive form; and abnormalities were frequently found on electrocardiograms: 41% presented anterosuperior left bundle branch block (AS-LBBB), 32.8% presented right bundle branch block (RBBB) and 22.9% presented ventricular ectopic beats (VEB). In the non-elderly group (61 cases), the mean age was 39.30+/-8.36 years; 54.1% were female; 50.8% presented comorbidities (most frequently systemic arterial hypertension, in 26.2%); 18% had the indeterminate clinical form (p<0.05), 78.7% the cardiac form and 32.8% the digestive form; and abnormalities were frequently found on electrocardiogram: 24.6% presented AS-LBBB, 21.3% RBBB and 18% VEB. It was concluded that there were no clinical differences between elderly and non-elderly Chagas patients. The indeterminate clinical form predominated in patients less than 60 years old.

Clinical evaluation and hepatic laboratory assessment in individuals with congestive heart failure.

OBJECTIVES: To verify the clinical alterations and, in particular, hepatic laboratory alterations in patients in each of the heart failure (HF) functional classes. METHODS: The clinical and laboratory data--alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma-glutamyl transpeptidase (GGT), bilirubin and coagulogram--of 50 patients admitted in 2002 to a tertiary hospital with the diagnosis of heart failure were researched by means of a cross sectional study. The patients were separated in accordance with their HF class and their data were compared statistically. Patients with hepatopathy of any etiology were excluded. RESULTS: Analysis of the mean transaminase values revealed a significant increase only for the Class IV patients. On the other hand, alkaline phosphatase and GGT presented a progressive increase in accordance with the HF class. CONCLUSION: HF is characterized by a progressive cholestatic profile of laboratory alterations, while transaminase values are only elevated in advanced HF. It is of utmost importance to understand these alterations in order to avoid unnecessary hepatic investigations in individuals with heart failure.

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies.

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.