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.

Chagas' disease and ageing: the coexistence of other chronic diseases with Chagas' disease in elderly patients / Doença de Chagas e envelhecimento: a associação de outras enfermidades crônicas em pacientes idosos chagásicos

This study aimed to identify the main comorbidities in elderly chagasic patients treated in a reference service and identify possible associations between the clinical form of Chagas' disease and chronic diseases. Ninety patients aged 60 years-old or over were interviewed and their clinical diagnoses recorded. The study population profile was: women (55.6 percent); median age (67 years); married (51.1 percent); retired (73.3 percent); up to four years' education (64.4 percent); and earning less than two minimum wages (67.8 percent). The predominant forms of Chagas' disease were the cardiac (46.7 percent) and mixed forms (30 percent). There was a greater proportion of mild cardiac dysfunction (84.1 percent), frequently in association with megaesophagus. The mean number of concurrent diseases was 2.856 ± 1.845, and 33 percent of the patients had four or more comorbidities. The most frequent were systemic arterial hypertension (56.7 percent), osteoporosis (23.3 percent), osteoarthritis (21.2 percent) and dyslipidemia (20 percent). Positive correlations were verified between sex and comorbidities and between age group and comorbidities.

Este trabalho objetivou avaliar o perfil sociodemográfico e identificar as principais co-morbidades de idosos chagásicos, buscando associação entre forma clínica da doença de Chagas e enfermidades crônicas. Foi realizada entrevista e levantamento dos diagnósticos clínicos de 90 chagásicos com idade > 60 anos. Encontrou-se: mulheres (55,6 por cento), mediana de 67 anos, casados (51,1 por cento) e renda mensal inferior a dois salários-mínimos (67,8 por cento). A forma clínica predominante foi a cardíaca (46,7 por cento), seguida da mista (30 por cento). Houve maior proporção de cardiopatia leve (84,1 por cento), sendo frequente a associação com megaesôfago. Trinta e três por cento apresentavam quatro ou mais co-morbidades, dentre elas: hipertensão arterial (56,7 por cento), osteoporose (23,3 por cento), osteoartrite (21,2 por cento) e dislipidemia (20 por cento). Obteve-se correlação positiva entre gênero e co-morbidades, faixa etária e co-morbidades.

Chagas' disease and ageing: the coexistence of other chronic diseases with Chagas' disease in elderly patients.

This study aimed to identify the main comorbidities in elderly chagasic patients treated in a reference service and identify possible associations between the clinical form of Chagas' disease and chronic diseases. Ninety patients aged 60 years-old or over were interviewed and their clinical diagnoses recorded. The study population profile was: women (55.6%); median age (67 years); married (51.1%); retired (73.3%); up to four years' education (64.4%); and earning less than two minimum wages (67.8%). The predominant forms of Chagas' disease were the cardiac (46.7%) and mixed forms (30%). There was a greater proportion of mild cardiac dysfunction (84.1%), frequently in association with megaesophagus. The mean number of concurrent diseases was 2.856 +/- 1.845, and 33% of the patients had four or more comorbidities. The most frequent were systemic arterial hypertension (56.7%), osteoporosis (23.3%), osteoarthritis (21.2%) and dyslipidemia (20%). Positive correlations were verified between sex and comorbidities and between age group and comorbidities.

Estudo do perfil clínico e evolutivo de indivíduos com a doença de Chagas crônica e neoplasias malignas / Clinic profile and evolution in chronic Chagasic patients with malign neoplasm

Objetivo: Verificar a apresentação clínica da doença de chagas em indivíduos com neoplasias malignas e a evolução em 12 meses. Métodos: Vinte chagásicos crônicos com neoplasias ma­lignas foram avaliados e comparados com outros 20 sem neoplasia em relação a alterações eletrocardiográficas, aumento de área cardíaca às radiográficas do tórax, xenodiagnóstico e forma clínica da doença de Chagas, em sua apresentação clíni­ca inicial e 12 meses após. A mesma avaliação foi feita em sub­grupos dos chagásicos com neoplasias com e sem tratamento quimioterápico. Resultados: Observou-se 55% de alterações ao eletrocar­diograma, 20% de aumento da área cardíaca às radiografias do torax, positividade de 20% dos xenodiagnósticos, sendo que 30% dos casos eram da forma indeterminada, 50% da cardíaca, 10% da digestiva e 10% de forma mista. Não houve diferença estatística com o grupo de chagásicos sem neoplasia. Após 12 meses essas variáveis não apresentaram alterações significati­vas. No subgrupo tratado com quimioterapia não foram obser­vadas alterações significantes nestas variáveis em relação ao subgrupo sem quimioterapia. Conclusão: Os dados sugerem que não houve mudança na evolução natural da doença de Chagas determinada pela neoplasia.

[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.

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

Com o objetivo de avaliar a apresentação clínica da doença de Chagas em idosos foi realizado estudo retrospectivo utilizando-se os prontuários de doentes atendidos em ambulatório de referência. A casuística foi dividida em idosos (> 60 anos) e não idosos. Avaliou-se: sexo, co-morbidades, forma clínica, eletrocardiograma e títulos das sorologias. Idosos (61 casos): média de idade de 66,0 ± 5 anos, 67,2 por cento do sexo feminino; comorbidades em 59 por cento, mais freqüente a hipertensão arterial sistêmica (HAS)= 39,3 por cento; forma indeterminada= 1,6 por cento, forma cardíaca= 88,5 por cento, forma digestiva= 36,1 por cento; alterações freqüentes no eletrocardiograma: bloqueio divisional ântero-superior esquerdo (BDASE)= 41 por cento, bloqueio completo de ramo direito (BCRD)= 32,8 por cento, extra-sístole ventricular (EV)=22,9 por cento. Não idosos (61 casos): média de idade: 39,30±8,36 anos, 54,1 por cento do sexo feminino; comorbidades em 50,8 por cento, mais freqüente a HAS (26,2 por cento); forma indeterminada= 18 por cento (p<0,05), forma cardíaca= 78,7 por cento, forma digestiva= 32,8 por cento; alterações freqüentes no eletrocardiograma: BDASE= 24,6 por cento, BCRD= 21,3 por cento, EV =18 por cento. Concluindo, não houve diferenças clínicas entre indivíduos idosos e não idosos e a forma indeterminada predominou nos indivíduos abaixo dos 60 anos.

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 (> 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 percent were female; 59 percent presented comorbidities (most frequently systemic arterial hypertension, in 39.3 percent); 1.6 percent had the indeterminate clinical form, 88.5 percent the cardiac form and 36 percent the digestive form; and abnormalities were frequently found on electrocardiograms: 41 percent presented anterosuperior left bundle branch block (AS-LBBB), 32.8 percent presented right bundle branch block (RBBB) and 22.9 percent presented ventricular ectopic beats (VEB). In the non-elderly group (61 cases), the mean age was 39.30 ± 8.36 years; 54.1 percent were female; 50.8 percent presented comorbidities (most frequently systemic arterial hypertension, in 26.2 percent); 18 percent had the indeterminate clinical form (p<0.05), 78.7 percent the cardiac form and 32.8 percent the digestive form; and abnormalities were frequently found on electrocardiogram: 24.6 percent presented AS-LBBB, 21.3 percent RBBB and 18 percent 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.

Implantação de um Sistema de Avaliação Estruturada de Performance em Escola Médica: Resultados do Projeto-Piloto / Implementation of a Structured Evaluation System in a Medical School: Pilot Project Results

Resumo: A avaliação de um processo de ensino-aprendizagem é fundamental para rever objetivos, metodologia e conteúdo. Avaliações de competência clínica através de exames clínicos objetivos estruturados têm-se firmado como instrumentos válidos, fidedignos e efetivos de avaliação de habilidades, além de permitir apreciar as atitudes dos alunos de Medicina. A Faculdade de Ciências Médicas da Unicamp vem implementando extensa reforma curricular nos últimos anos, baseada em módulo de aprendizagem que incluem o ensino de técnicas de entrevista aos aluno do 2° ano de graduação. Descrevemos a implantação de um método baseado em pacientes-padrão, treinados para avaliar conhecimento adquirido, habilidades e as atitudes dos alunos. Os resultados do projeto-piloto aplicado a 10 voluntários no módulo de habilidades de entrevista mostram que o método é factível, relativamente simples e barato, permite um avaliação objetiva das habilidades adquiridas além de identificar precocemente indivíduos com possíveis problemas na relação médico-paciente. O método permitiu ainda identificar pontos que necessitam de reforço por parte do corpo docente assim como rever os objetivos propostos pelo módulo em questão.

Abstract: Evaluation of the teaching and learning process is essential in order to review objetives, methods and contentes. The use of objective, structured clinical examination methods is well established as a valid, reliable and effective process for the assessment of clinical skills, besides allowing the perception ofmedical students' atitudes. The Faculty of Medical Science of Unicamp has implementd na extensive curriculum restructuring process based on learning units that include the teaching of interview techniques to second grade students. We describe the implementation of na assessment method based on standart-pacients trained to evaluate students' acquantance, skills and atitudes. The results of the pilot Project applied to 10 voluntees demosntrate that the method is feasible, relatively simple and inexpansive and allows na objective assessment of the students' skills besides providing precocious indications of possible patient-doctor relationship problems. The method allowed us to identify points that need to be reinforced by the teachers during the course as well as to review our aims in the module evaluated.

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.

Aspectos médicos trabalhalistas de pacientes chagásicos com vínculo empregatício

Resumo: Foram avaliados 250 indivíduos portadores da infecçäo pelo T. cruzzi com pelo menos duas sorologias positivas, com vínculo empregatício regulamentado. Divididos em dois grupos após entrevista, exame clínico e complementares: grupo F1 com 143 indivíduos (forma indeterminada) e FC com 99 indivíduos (com algum grau de cardiopatia), sendo retirados os casos com megavísceras isoladas. Avaliados quanto ao sexo, raça grau de escolaridade, tipo de serviço, vínculo previdenciário, salários, exame médico admissional, recusa ao exame admissional. O estudo contatou a existência de inadequaçäo quanto a capacidade de trabalho dos portadores de T. cruzi e a funçäo desempenhada. Alguns elementos sugestivos de discriminaçäo com trabalhadores soropositivos. Seria necessário adotar-se estratégias de esclarecimento aos profissionais da área de saúde quanto a capacidade de trabalhodos portadores de infecçäo pelo T.cruzi, bem como os peritos da Previdência Social.

Metástase óssea de carcinoma cutâneo: relato de um caso / Bone mestastasis of cutaneous carcinoma: a case report

Apresenta-se o caso de um paciente de carcinoma basocelular com acometimento neoplásico tardio da segunda vértebra lombar, cujo diagnóstico foi de carcinoma baso-epidermóide