Microbial Translocation Is Associated with Extensive Immune Activation in Dengue Virus Infected Patients with Severe Disease

Background:Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection.Methods:Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of São Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles.Results:Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease.Conclusions:The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis

Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

Objective: The soluble guanylate cyclase stimulator riociguat is approved for the treatment of adult patients with pulmonary arterial hypertension (PAH) and inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) following Phase

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results Triagem primária de doadores de sangue por teste de ácidos nucléicos: desenvolvimento de um método não-comercial e resultados

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.<br>Desenvolveu-se uma metodologia própria ("in-house") baseada em RT-PCR, que permite detectar simultaneamente o RNA do vírus HCV e de um RNA artificial empregado como controle externo. As amostras são analisadas em pools de 6-12 doações, cada doação sendo incluída em dois pools diferentes, um horizontal e um vertical, permitindo a identificação imediata de uma doação reativa, sem a necessidade de desmembrar-se um pool reativo. O processo todo consumiu de 6-8 horas diárias e os resultados foram emitidos em paralelo à sorologia. O método detectou os seis genótipos de HCV, com um limite de sensibilidade de 500 UI/mL (95% hit rate). Até julho de 2005 haviam sido testadas 139.678 doações com a detecção de 315 (0,23%) doações reativas para HCV-RNA. Exceto cinco falso-positivas, todas estas doações também apresentavam o respectivo anticorpo, portanto não se detectou nenhuma doação em janela imunológica. A especificidade foi de 99,83%. A detecção de amostra em janela imunológica, nesta população de doadores, provavelmente demandará a análise de um número maior de doações, espelhando-se na experiência internacional que tem mostrado a detecção de amostras HCV-RNA isoladas em 1:200.000 - 1:500.000 doações

Use of selective medium for Burkholderia cepacia isolation in respiratory samples from cystic fibrosis patients Uso de meio seletivo para identificação de cepas de Burkholderia cepacia em amostras de trato respiratório de pacientes portadores de fibrose cística

Burkholderia cepacia colonizes cystic fibrosis (CF) patients. We evaluated the impact of the use of a selective medium in the rate of B. cepacia recovery from respiratory samples of CF patients. During a 6-month period, respiratory samples were collected from 106 CF patients and cultivated on selective media including a B. cepacia selective medium. Confirmation of the identity of B. cepacia isolates was carried out by species specific PCR and determination of genomovar status performed by a sequential PCR approach. Results of B. cepacia isolation during this period were compared to the preceding two years, when the sample processing was identical except for the lack of the B. cepacia selective medium. B. cepacia was isolated in 11/257 (4.2%) of the samples using the selective medium, in contrast with the preceding two years, when it was isolated in 6/1029 samples (0.58%), p < 0.0001. Identity of all 11 isolates was confirmed by PCR and genomovar determination was accomplished in all but one isolate. These results suggest that the use of a selective medium increases recovery rate of B. cepacia from respiratory samples.<br>A Burkholderia cepacia coloniza os pacientes portadores de fibrose cística (FC). Avaliamos o impacto do uso de um meio seletivo no isolamento de B. cepacia em amostras de secreção respiratória de pacientes portadores de FC. Durante um período de 6 meses, amostras de trato respiratório foram colhidas de 106 pacientes com FC e cultivadas em meios seletivos incluindo um meio para isolamento de B. cepacia. A identidade das cepas de B. cepacia foi confirmada através de PCR espécie específica e a determinação do genomovar ou subespécie realizada através de reações seqüenciais de PCR. Os resultados de isolamento de B. cepacia durante este período foram comparados com os dois anos precedentes, quando o processamento das amostras era idêntico, exceto pela utilização do meio seletivo para B. cepacia. B. cepacia foi isolada em 11/257 (4,2%) amostras usando o meio seletivo, e em apenas 6/1029 (0,58%) nos dois anos precedentes (p < 0,0001). A identidade destas 11 cepas foi confirmada e a determinação do genomovar obtida em 10/11. Estes resultados sugerem que o uso do meio seletivo aumenta a freqüência de isolamento de B. cepacia em amostras de trato respiratório de pacientes portadores de FC

Detection of GBV-C/HGV RNA in cervico-vaginal smears from healthy individuals

The purpose of the present study was to evaluate the sexual transmission of GBV-C/HGV, through RNA detection in cervicovaginal smears. Therefore the GBV-C/HGV RNA in cervicovaginal smears from apparently healthy women was investigated using routine proceedings for prophylactic screening to cervical cancer. GBV-C/HGV RNA was detected by reverse transcriptase and polymerase chain reaction (RT-PCR). Only one woman presented co-infection with human papilloma virus (HPV). The GBV-C/HGV RNA was detected in 13/73 (17.57%) healthy women and it's prevalence in participating women between 28-43 years old was 53.85%. No association was found with GBV-C/HGV for the age of first sexual intercourse and number of pregnancies. In GBV-C/HGV RNA positive women, 69.23% were married. In conclusion, the present findings show that cervical and vaginal specimens could contain the GBV-C/HGV RNA.<br>O objetivo do presente estudo foi avaliar a transmissão sexual de GBV-C/HBV, através da detecção do RNA viral em raspados cérvico-vaginais. Portanto, a presença do RNA GBV-C/HGV foi investigada em raspados cérvico-vaginais em mulheres aparentemente saudáveis que realizaram exames preventivos para câncer cervical. GBV-C/HGV RNA foi detectado por reação de transcriptase reversa e reação em cadeia da polimerase (RT-PCR). Apenas uma mulher apresentou a co-infecção com o papiloma vírus humano (HPV). O RNA GBV-C/HGV foi detectado em 13/73 (17,57%) mulheres saudáveis e sua prevalência entre participantes da idade de 28-43 anos foi de 53,85%. Não foi observada relação entre a presença do RNA GBV-C/HGV com a idade de primeira relação sexual, nem com o número de gestações. Entre as mulheres que apresentavam o RNA viral, 69,23% eram casadas. O presente estudo demonstrou que secreções cérvico-vaginais podem conter o RNA viral GBV-C/HBV