A multicentric evaluation of dipstick test for serodiagnosis of visceral leishmaniasis in India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain.

Visceral leishmaniasis (VL) is one of the leading infectious diseases affecting developing countries. Colloidal gold-based diagnostic tests are rapid tools to detect blood/serum antibodies for VL diagnosis. Lack of uniformity in the performance of these tests in different endemic regions is a hurdle in early disease diagnosis. This study is designed to validate a serum-based dipstick test in eight centres of six countries, India, Nepal, Sri Lanka, Brazil, Ethiopia and Spain with archived and fresh sera from 1003 subjects. The dipstick detects antibodies against Leishmania donovani membrane antigens (LAg). The overall sensitivity and specificity of the test with 95% confidence intervals were found to be 97.10% and 93.44%, respectively. The test showed good sensitivity and specificity in the Indian subcontinent (>95%). In Brazil, Ethiopia, and Spain the sensitivity and specificity of the dipstick test (83.78-100% and 79.06-100%) were better as compared to the earlier reports of the performance of rK39 rapid test in these regions. Interestingly, less cross-reactivity was found with the cutaneous form of the disease in Spain, Brazil, and Sri Lanka demonstrating 91.58% specificity. This dipstick test can therefore be a useful tool for diagnosing VL from other symptomatically similar diseases and against cutaneous form of leishmaniasis.

Detection of anti-Trypanosoma cruzi antibodies by chimeric antigens in chronic Chagas disease-individuals from endemic South American countries.

BACKGROUND: Laboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries. METHODOLOGY/PRINCIPAL FINDINGS: IBMP-8.1 and IBMP-8.4 chimeric antigens were expressed as soluble proteins in E. coli and purified using chromatography methods. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house ELISA with sera from 122 non-infected and 215 T. cruzi-infected individuals from Argentina, Bolivia, and Paraguay. Cut-off values were based on ROC curves and performance parameters were determined using a dichotomous approach. Area under the curve values were > 99.7% for both IBMP-8.1 and IBMP-8.4 antigens. IgG levels in T. cruzi-positive and negative samples were higher for IBMP-8.4 than IBMP-8.1. Both IBMP-8.1 and -8.4 were 100% specific, while IBMP-8.4 were 100% sensitive compared to IBMP-8.1 (95.3%). Admitting RI values of 1.0 ± 0.10 as the inconclusive interval, 6.2% of the samples tested using IBMP-8.1 and 2.1% using IBMP-8.4 fell inside the grey zone. Based on accuracy and diagnostic odds ratio values, IBMP-8.4 presented the best performance. Differences in sensitivity and IgG levels among the samples from Argentina, Bolivia, and Paraguay were not significant. CONCLUSIONS/SIGNIFICANCE: Our findings showed a notable performance of IBMP-8.1 and -8.4 chimeric antigens in diagnosing chronic Chagas disease in individuals from endemic South American countries, confirming our hypothesis that these antigens could be used in geographical areas where distinct T. cruzi DTUs occur.

Maternal schistosomiasis: IL-2, IL-10 and regulatory T lymphocytes to unrelated antigen in adult offspring mice.

INTRODUCTION: We evaluated IL-10, IL-2 and regulatory T cells (Treg), in response to ovalbumin (OA), in offspring from schistosomotic mouse mothers. METHODS: We used animals born (BIM) or suckled (SIM) from infected mothers; and mice born/suckled from infected (BSIM) or non-infected mothers (CONTROL). After OA+adjuvant immunization, spleen cells were cultured, with or without OA, and doubly marked for cytometry. RESULTS: BIM showed fewer CD4+/IL-2+ and more B220+/IL-10+ cells, whereas the SIM group showed increased Treg frequency. BSIM had fewer B220+/IL-10+ and Treg cells. CONCLUSIONS: Separately, gestation or nursing induced immunosuppressive cells in infected mothers, but improved anti-OA immunity when combined.

Maternal schistosomiasis: IL-2, IL-10 and regulatory T lymphocytes to unrelated antigen in adult offspring mice

Abstract INTRODUCTION: We evaluated IL-10, IL-2 and regulatory T cells (Treg), in response to ovalbumin (OA), in offspring from schistosomotic mouse mothers. METHODS: We used animals born (BIM) or suckled (SIM) from infected mothers; and mice born/suckled from infected (BSIM) or non-infected mothers (CONTROL). After OA+adjuvant immunization, spleen cells were cultured, with or without OA, and doubly marked for cytometry. RESULTS: BIM showed fewer CD4+/IL-2+ and more B220+/IL-10+ cells, whereas the SIM group showed increased Treg frequency. BSIM had fewer B220+/IL-10+ and Treg cells. CONCLUSIONS: Separately, gestation or nursing induced immunosuppressive cells in infected mothers, but improved anti-OA immunity when combined.

Ex vivo T-lymphocyte chemokine receptor phenotypes in patients with chronic Chagas disease.

INTRODUCTION: Elucidating the molecules involved in the inflammatory process of chronic Chagas disease may allow identification of treatment targets. METHODS: The ex vivo phenotypic expression of chemokine receptors CCR1, CCR3, CCR4, CCR5, CXCR2, CXCR3, CXCR4, and CXCR5 on the CD4+ and CD8+ T-cells of patients with chronic Chagas cardiomyopathy of varying severity was evaluated using flow cytometry. RESULTS: Differential expression of CD4+CCR3+ and CD8+CCR4+ T-cells was observed in patients with mild cardiac involvement compared, respectively, with patients with severe cardiac and asymptomatic forms of Chagas disease. CONCLUSIONS: These receptors are possibly involved in the pathogenesis of chronic Chagas cardiomyopathy.

Ex vivo T-lymphocyte chemokine receptor phenotypes in patients with chronic Chagas disease

Abstract INTRODUCTION: Elucidating the molecules involved in the inflammatory process of chronic Chagas disease may allow identification of treatment targets. METHODS: The ex vivo phenotypic expression of chemokine receptors CCR1, CCR3, CCR4, CCR5, CXCR2, CXCR3, CXCR4, and CXCR5 on the CD4+ and CD8+ T-cells of patients with chronic Chagas cardiomyopathy of varying severity was evaluated using flow cytometry. RESULTS: Differential expression of CD4+CCR3+ and CD8+CCR4+ T-cells was observed in patients with mild cardiac involvement compared, respectively, with patients with severe cardiac and asymptomatic forms of Chagas disease. CONCLUSIONS: These receptors are possibly involved in the pathogenesis of chronic Chagas cardiomyopathy.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

Accuracy of chimeric proteins in the serological diagnosis of chronic chagas disease - a Phase II study.

BACKGROUND: The performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results. METHODOLOGY/PRINCIPAL FINDINGS: Here, four chimeric proteins (IBMP-8.1 to -8.4) comprising immunodominant regions of different Trypanosoma cruzi antigens were tested by enzyme-linked immunosorbent assay. The proteins were used to detect specific anti-T. cruzi antibodies in the sera of 857 chagasic and 689 non-chagasic individuals to evaluate their accuracy for chronic CD diagnosis. The antigens were recombinantly expressed in Escherichia coli and purified by chromatographic methods. The sensitivity and specificity values ranged from 94.3% to 99.3% and 99.4% to 100%, respectively. The diagnostic odds ratio (DOR) values were 6,462 for IBMP-8.1, 3,807 for IBMP-8.2, 32,095 for IBMP-8.3, and 283,714 for IBMP-8.4. These chimeric antigens presented DORs that were higher than the commercial test Pathozyme Chagas. The antigens IBMP-8.3 and -8.4 also showed DORs higher than the Gold ELISA Chagas test. Mixtures with equimolar concentrations were tested in order to improve the diagnosis accuracy of negative samples with high signal and positive samples with low signal. However, no gain in accuracy was observed relative to the individual antigens. A total of 1,079 additional sera were used to test cross-reactivity to unrelated diseases. The cross-reactivity rates ranged from 0.37% to 0.74% even for Leishmania spp., a pathogen showing relatively high genome sequence identity to T. cruzi. Imprecision analyses showed that IBMP chimeras are very stable and the results are highly reproducible. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the IBMP-8.4 antigen can be safely used in serological tests for T. cruzi screening in blood banks and for chronic CD laboratory diagnosis.

Expression of co-stimulatory molecules CD80 and CD86 is altered in CD14 + HLA-DR + monocytes from patients with Chagas disease following induction by Trypanosoma cruzi recombinant antigens.

INTRODUCTION: The relationships between monocytes and lymphocytes through MHC class II molecules and costimulatory, are of utmost importance for the production of an efficient immune response. In this work, we assessed the expression of surface molecules CD80 and CD86 on CD14+HLA-DR+ monocytes from patients with Chagas disease. METHODS:: The study population consisted of 31 patients with chronic clinical forms of Chagas disease. Patient blood samples were cultured in the presence of recombinant cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA). RESULTS:: We found considerable differences in the expression profile of surface molecules involved in antigen presentation. CONCLUSIONS:: CRA and FRA may contribute to host immune response evasion by Trypanozoma cruzi.

Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

Gestation and breastfeeding in schistosomotic mothers differently modulate the immune response of adult offspring to postnatal Schistosoma mansoni infection.

Schistosoma mansoni antigens in the early life alter homologous and heterologous immunity during postnatal infections. We evaluate the immunity to parasite antigens and ovalbumin (OA) in adult mice born/suckled by schistosomotic mothers. Newborns were divided into: born (BIM), suckled (SIM) or born/suckled (BSIM) in schistosomotic mothers, and animals from noninfected mothers (control). When adults, the mice were infected and compared the hepatic granuloma size and cellularity. Some animals were OA + adjuvant immunised. We evaluated hypersensitivity reactions (HR), antibodies levels (IgG1/IgG2a) anti-soluble egg antigen and anti-soluble worm antigen preparation, and anti-OA, cytokine production, and CD4+FoxP3+T-cells by splenocytes. Compared to control group, BIM mice showed a greater quantity of granulomas and collagen deposition, whereas SIM and BSIM presented smaller granulomas. BSIM group exhibited the lowest levels of anti-parasite antibodies. For anti-OA immunity, immediate HR was suppressed in all groups, with greater intensity in SIM mice accompanied of the remarkable level of basal CD4+FoxP3+T-cells. BIM and SIM groups produced less interleukin (IL)-4 and interferon (IFN)-g. In BSIM, there was higher production of IL-10 and IFN-g, but lower levels of IL-4 and CD4+FoxP3+T-cells. Thus, pregnancy in schistosomotic mothers intensified hepatic fibrosis, whereas breastfeeding diminished granulomas in descendants. Separately, pregnancy and breastfeeding could suppress heterologous immunity; however, when combined, the responses could be partially restored in infected descendants.

Gestation and breastfeeding in schistosomotic mothers differently modulate the immune response of adult offspring to postnatal Schistosoma mansoni infection

Schistosoma mansoni antigens in the early life alter homologous and heterologous immunity during postnatal infections. We evaluate the immunity to parasite antigens and ovalbumin (OA) in adult mice born/suckled by schistosomotic mothers. Newborns were divided into: born (BIM), suckled (SIM) or born/suckled (BSIM) in schistosomotic mothers, and animals from noninfected mothers (control). When adults, the mice were infected and compared the hepatic granuloma size and cellularity. Some animals were OA + adjuvant immunised. We evaluated hypersensitivity reactions (HR), antibodies levels (IgG1/IgG2a) anti-soluble egg antigen and anti-soluble worm antigen preparation, and anti-OA, cytokine production, and CD4+FoxP3+T-cells by splenocytes. Compared to control group, BIM mice showed a greater quantity of granulomas and collagen deposition, whereas SIM and BSIM presented smaller granulomas. BSIM group exhibited the lowest levels of anti-parasite antibodies. For anti-OA immunity, immediate HR was suppressed in all groups, with greater intensity in SIM mice accompanied of the remarkable level of basal CD4+FoxP3+T-cells. BIM and SIM groups produced less interleukin (IL)-4 and interferon (IFN)-g. In BSIM, there was higher production of IL-10 and IFN-g, but lower levels of IL-4 and CD4+FoxP3+T-cells. Thus, pregnancy in schistosomotic mothers intensified hepatic fibrosis, whereas breastfeeding diminished granulomas in descendants. Separately, pregnancy and breastfeeding could suppress heterologous immunity; however, when combined, the responses could be partially restored in infected descendants.

Spatiotemporal analysis of reported cases of acute Chagas disease in the State of Pernambuco, Brazil, from 2002 to 2013.

INTRODUCTION: Control strategies to eliminate the transmission of Chagas disease by insect vectors have significantly decreased the number of reported acute cases in Brazil. However, data regarding the incidence and distribution of acute Chagas disease cases in the State of Pernambuco are unavailable in the literature. METHODS: A geographical information system was used to delineate the spatiotemporal distribution profile of the cases from 2002 to 2013 in 185 municipalities of Pernambuco based on the municipality where notification occurred. The results were presented in digital maps generated by the TerraView software (INPE). RESULTS: A total of 302 cases of acute disease were recorded in 37.8% of the municipalities, for a total of 0.13 cases per 1,000,000 inhabitants per year. Out of the 302 cases, 99.3% were reported between 2002 and 2006. The most affected municipalities were Carnaubeira da Penha, Mirandiba and Terra Nova. The risk maps showed a significant decrease in the number of notifications and a concentration of cases in the Midwest region. CONCLUSIONS: This study highlights a significant decrease in new cases of acute Chagas disease in Pernambuco starting in 2006 when Brazil received an international certification for the interruption of vectorial transmission by Triatoma infestans. However, control strategies should still be encouraged because other triatomine species can also transmit the parasite; moreover, other transmission modes must not be neglected.

Spatiotemporal analysis of reported cases of acute Chagas disease in the State of Pernambuco, Brazil, from 2002 to 2013

INTRODUCTION: Control strategies to eliminate the transmission of Chagas disease by insect vectors have significantly decreased the number of reported acute cases in Brazil. However, data regarding the incidence and distribution of acute Chagas disease cases in the State of Pernambuco are unavailable in the literature. METHODS: A geographical information system was used to delineate the spatiotemporal distribution profile of the cases from 2002 to 2013 in 185 municipalities of Pernambuco based on the municipality where notification occurred. The results were presented in digital maps generated by the TerraView software (INPE). RESULTS: A total of 302 cases of acute disease were recorded in 37.8% of the municipalities, for a total of 0.13 cases per 1,000,000 inhabitants per year. Out of the 302 cases, 99.3% were reported between 2002 and 2006. The most affected municipalities were Carnaubeira da Penha, Mirandiba and Terra Nova. The risk maps showed a significant decrease in the number of notifications and a concentration of cases in the Midwest region. CONCLUSIONS: This study highlights a significant decrease in new cases of acute Chagas disease in Pernambuco starting in 2006 when Brazil received an international certification for the interruption of vectorial transmission by Triatoma infestans. However, control strategies should still be encouraged because other triatomine species can also transmit the parasite; moreover, other transmission modes must not be neglected. .

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease. .

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens.

INTRODUCTION: CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. METHODS: Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. RESULTS: It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. CONCLUSIONS: Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Chagas disease in the State of Pernambuco, Brazil: analysis of admissions and mortality time series.

INTRODUCTION: A time series study of admissions, deaths and acute cases was conducted in order to evaluate the context of Chagas disease in Pernambuco. METHODS: Data reported to the Information Technology Department of the Brazilian National Health Service between 1980 and 2008 was collected for regions and Federal Units of Brazil; and microregions and municipalities of Pernambuco. Rates (per 100,000 inhabitants) of hospitalization, mortality and acute cases were calculated using a national hospital database (SIH), a national mortality database (SIM) and the national Information System for Notifiable Diseases (SINAN), respectively. RESULTS: The national average for Chagas disease admissions was 0.99 from 1995 to 2008. Pernambuco obtained a mean of 0.39 in the same period, with the highest rates being concentrated in the interior of the state. The state obtained a mean mortality rate of 1.56 between 1980 and 2007, which was lower than the national average (3.66). The mortality rate has tended to decline nationally, while it has remained relatively unchanged in Pernambuco. Interpolating national rates of admissions and deaths, mortality rates were higher than hospitalization rates between 1995 and 2007. The same occurred in Pernambuco, except for 2003. Between 2001 and 2006, rates for acute cases were 0.56 and 0.21 for Brazil and Pernambuco, respectively. CONCLUSIONS: Although a decrease in Chagas mortality has occurred in Brazil, the disease remains a serious public health problem, especially in the Northeast region. It is thus essential that medical care, prevention and control regarding Chagas disease be maintained and improved.

Chagas disease in the State of Pernambuco, Brazil: analysis of admissions and mortality time series / Doença de Chagas no Estado de Pernambuco, Brasil: análise de séries históricas das internações e da mortalidade

INTRODUCTION: A time series study of admissions, deaths and acute cases was conducted in order to evaluate the context of Chagas disease in Pernambuco. METHODS: Data reported to the Information Technology Department of the Brazilian National Health Service between 1980 and 2008 was collected for regions and Federal Units of Brazil; and microregions and municipalities of Pernambuco. Rates (per 100,000 inhabitants) of hospitalization, mortality and acute cases were calculated using a national hospital database (SIH), a national mortality database (SIM) and the national Information System for Notifiable Diseases (SINAN), respectively. RESULTS: The national average for Chagas disease admissions was 0.99 from 1995 to 2008. Pernambuco obtained a mean of 0.39 in the same period, with the highest rates being concentrated in the interior of the state. The state obtained a mean mortality rate of 1.56 between 1980 and 2007, which was lower than the national average (3.66). The mortality rate has tended to decline nationally, while it has remained relatively unchanged in Pernambuco. Interpolating national rates of admissions and deaths, mortality rates were higher than hospitalization rates between 1995 and 2007. The same occurred in Pernambuco, except for 2003. Between 2001 and 2006, rates for acute cases were 0.56 and 0.21 for Brazil and Pernambuco, respectively. CONCLUSIONS: Although a decrease in Chagas mortality has occurred in Brazil, the disease remains a serious public health problem, especially in the Northeast region. It is thus essential that medical care, prevention and control regarding Chagas disease be maintained and improved.

INTRODUÇÃO: Foi realizado estudo de séries históricas de internações, óbitos e casos agudos por doenças de Chagas objetivando avaliar o contexto desta enfermidade em Pernambuco. MÉTODOS: Foram coletados dados notificados de 1980 a 2008 ao Departamento de Informática do Sistema Único de Saúde (DATASUS/MS) para regiões e unidades federativas do Brasil, microrregiões e municípios pernambucanos. As taxas (por 100.000 habitantes) de internações, mortalidade e casos agudos foram obtidas por consulta ao Sistema de Informações Hospitalares (SIH), Sistema de Informação sobre Mortalidade (SIM) e Sistema de Informações de Agravos de Notificação (SINAN), respectivamente. RESULTADOS: A média de internações nacional por doença de Chagas ficou em 0,99 no período de 1995 a 2008. Pernambuco, neste intervalo, apresentou média de 0,39, com as maiores taxas concentradas no interior do estado. Este estado obteve média de óbitos 1,56 entre 1980 e 2007, valor inferior a brasileira (3,66). O país demonstrou declínio de óbitos na análise de tendência, com Pernambuco encontrando-se em estado estacionário para esta taxa. Interpolando os dados referentes a internações e óbitos, evidenciou-se mortalidade em valores superiores as taxas de internações nacionais, entre 1995 e 2007. O mesmo fato ocorreu em Pernambuco, exceto em 2003. Entre 2001 e 2006, a taxa de casos agudos foi de 0,56 e 0,21 respectivamente para Brasil e para Pernambuco. CONCLUSÕES: Mesmo o Brasil demonstrando redução na mortalidade, a doença permanece como grave problema de saúde pública, principalmente no nordeste. Desta forma, é fundamental a manutenção e melhoria das ações de atenção médica, controle e prevenção já existentes.

A comparative analysis of the suitability of different peripheral blood samples for reverse transcriptase polymerase chain reaction / Análise comparativa da adequação de diferentes amostras de sangue periférico em ensaios de reação em cadeia da polimerase via transcriptase reversa

Venipuncture is one of the easiest clinical procedures to obtain viable blood samples to evaluate gene expression using mRNA analysis. However, the use of this sample type in reverse transcriptase polymerase chain reaction tests (RT-PCR) without prior treatment is controversial. We therefore propose to compare the suitability of different peripheral blood samples (whole blood without treatment, whole blood with hemolysis, peripheral blood mononuclear cells and frozen whole blood) for RT-PCR analysis. The results showed that, despite the blood sample being peripheral, it is possible to extract a fair amount of RNA and perform target gene amplification. Thus, peripheral blood without prior treatment could be used to investigate the gene expression using Real Time PCR.

A punção venosa representa um dos procedimentos clínicos mais simples na obtenção de amostras de sangue periférico e avaliação da expressão gênica através da análise do RNA mensageiro. Contudo, a utilização desta amostra, sem um tratamento prévio, em ensaios de Transcrição Reversa (RT-PCR) é controverso. Desta forma, propomos comparar a adequação de diferentes amostras de sangue periférico (sangue total sem tratamento, sangue total após hemólise, células mononucleares do sangue periférico e sangue total congelado) em ensaios de Transcrição Reversa Os resultados mostraram que independente da amostra de sangue periférico é possível extrair RNA em quantidade adequada e realizar a amplificação do gene alvo. Desta forma, o sangue periférico sem tratamento prévio pode ser utilizado em abordagens que envolvam a avaliação da expressão gênica por reação em cadeia da polimerase (PCR) em tempo real.