Levels of angiopoietin-2 are predictive for mortality in yellow fever virus infected patients.

In 2018 there was a large yellow fever outbreak in São Paulo, Brazil, with a high fatality rate. Yellow fever virus can cause, among other symptoms, haemorrhage and disseminated intravascular coagulation, indicating a role for endothelial cells in the disease pathogenesis. Here, we conducted a case-control study and measured markers related to endothelial damage in plasma and its association with mortality. We found that angiopoietin-2 is strongly associated with a fatal outcome and could serve as a predictive marker for mortality. This could be used to monitor severe patients and provide care to improve disease outcome.

The tough process of unmasking the slow-growing mycobacterium: case report of Mycobacterium microti infection.

Mycobacterium microti belongs to the Mycobacterium tuberculosis complex (MTBC). It can cause pulmonary and extrapulmonary tuberculosis in humans. Compared to M. tuberculosis , which is the most prevalent subspecies of the MTBC, M. microti infection has a different etiology. Moreover, establishing the diagnosis with conventional bacteriology can be difficult. We will illustrate this with a case of an extrapulmonary tuberculosis of the hip caused by M .microti in an immunocompetent patient in The Netherlands.

Effect of Puumala hantavirus infection on human umbilical vein endothelial cell hemostatic function: platelet interactions, increased tissue factor expression and fibrinolysis regulator release.

Puumala virus (PUUV) infection causes over 5000 cases of hemorrhagic fever in Europe annually and can influence the hemostatic balance extensively. Infection might lead to hemorrhage, while a recent study showed an increased risk of myocardial infarction during or shortly after PUUV infection. The mechanism by which this hantavirus influences the coagulation system remains unknown. Therefore we aimed to elucidate mechanisms explaining alterations seen in primary and secondary hemostasis during PUUV infection. By using low passage PUUV isolates to infect primary human umbilical vein endothelial cells (HUVECs) we were able to show alterations in the regulation of primary- and secondary hemostasis and in the release of fibrinolysis regulators. Our main finding was an activation of secondary hemostasis due to increased tissue factor (TF) expression leading to increased thrombin generation in a functional assay. Furthermore, we showed that during infection platelets adhered to HUVEC and subsequently specifically to PUUV virus particles. Infection of HUVEC with PUUV did not result in increased von Willebrand factor while they produced more plasminogen activator inhibitor type-1 (PAI-1) compared to controls. The PAI-1 produced in this model formed complexes with vitronectin. This is the first report that reveals a potential mechanism behind the pro-coagulant changes in PUUV patients, which could be the result of increased thrombin generation due to an increased TF expression on endothelial cells during infection. Furthermore, we provide insight into the contribution of endothelial cell responses regarding hemostasis in PUUV pathogenesis.

Time since onset of disease and individual clinical markers associate with transcriptional changes in uncomplicated dengue.

BACKGROUND: Dengue virus (DENV) infection causes viral haemorrhagic fever that is characterized by extensive activation of the immune system. The aim of this study is to investigate the kinetics of the transcriptome signature changes during the course of disease and the association of genes in these signatures with clinical parameters. METHODOLOGY/PRINCIPLE FINDINGS: Sequential whole blood samples from DENV infected patients in Jakarta were profiled using affymetrix microarrays, which were analysed using principal component analysis, limma, gene set analysis, and weighted gene co-expression network analysis. We show that time since onset of disease, but not diagnosis, has a large impact on the blood transcriptome of patients with non-severe dengue. Clinical diagnosis (according to the WHO classification) does not associate with differential gene expression. Network analysis however, indicated that the clinical markers platelet count, fibrinogen, albumin, IV fluid distributed per day and liver enzymes SGOT and SGPT strongly correlate with gene modules that are enriched for genes involved in the immune response. Overall, we see a shift in the transcriptome from immunity and inflammation to repair and recovery during the course of a DENV infection. CONCLUSIONS/SIGNIFICANCE: Time since onset of disease associates with the shift in transcriptome signatures from immunity and inflammation to cell cycle and repair mechanisms in patients with non-severe dengue. The strong association of time with blood transcriptome changes hampers both the discovery as well as the potential application of biomarkers in dengue. However, we identified gene expression modules that associate with key clinical parameters of dengue that reflect the systemic activity of disease during the course of infection. The expression level of these gene modules may support earlier detection of disease progression as well as clinical management of dengue.

Hyperferritinaemia in dengue virus infected patients is associated with immune activation and coagulation disturbances.

BACKGROUND: During a dengue outbreak on the Caribbean island Aruba, highly elevated levels of ferritin were detected in dengue virus infected patients. Ferritin is an acute-phase reactant and hyperferritinaemia is a hallmark of diseases caused by extensive immune activation, such as haemophagocytic lymphohistiocytosis. The aim of this study was to investigate whether hyperferritinaemia in dengue patients was associated with clinical markers of extensive immune activation and coagulation disturbances. METHODOLOGY/PRINCIPAL FINDINGS: Levels of ferritin, standard laboratory markers, sIL-2R, IL-18 and coagulation and fibrinolytic markers were determined in samples from patients with uncomplicated dengue in Aruba. Levels of ferritin were significantly increased in dengue patients compared to patients with other febrile illnesses. Moreover, levels of ferritin associated significantly with the occurrence of viraemia. Hyperferritinaemia was also significantly associated with thrombocytopenia, elevated liver enzymes and coagulation disturbances. The results were validated in a cohort of dengue virus infected patients in Brazil. In this cohort levels of ferritin and cytokine profiles were determined. Increased levels of ferritin in dengue virus infected patients in Brazil were associated with disease severity and a pro-inflammatory cytokine profile. CONCLUSIONS/SIGNIFICANCE: Altogether, we provide evidence that ferritin can be used as a clinical marker to discriminate between dengue and other febrile illnesses. The occurrence of hyperferritinaemia in dengue virus infected patients is indicative for highly active disease resulting in immune activation and coagulation disturbances. Therefore, we recommend that patients with hyperferritinaemia are monitored carefully.

Serum angiopoietin-2 and soluble VEGF receptor 2 are surrogate markers for plasma leakage in patients with acute dengue virus infection.

BACKGROUND: Endothelial cell dysfunction is believed to play an important role in the pathogenesis of plasma leakage in patients with acute dengue virus (DENV) infection. Several factors, produced by activated endothelial cells, have been associated with plasma leakage or severe disease in patients with infectious diseases. OBJECTIVES: The aim of this study was to investigate which of these markers could serve as a surrogate marker for the occurrence of plasma leakage in patients with acute DENV infection. STUDY DESIGN: A case-control study was performed in patients with acute DENV infection in Santos, Brazil. Plasma leakage was detected with X-ray and/or ultrasound examination at admission. Serum levels of soluble endoglin, endothelin-1, angiopoietin-2, VEGF, soluble VEGFR-2, MMP-2, MMP-9, TIMP-1 and TIMP-2 were determined using commercially available ELISAs. RESULTS: Increased levels of angiopoietin-2, endothelin-1 and MMP-2 and decreased levels of soluble VEGFR-2 were significantly associated with the occurrence of plasma leakage. An unsupervised cluster analysis confirmed that angiopoietin-2 and soluble VEGFR-2 were strongly associated with clinical apparent vascular leakage. CONCLUSION: Angiopoietin-2 and soluble VEGFR-2 can serve as surrogate markers for the occurrence of plasma leakage in patients with acute DENV infection.

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.

Evaluation of the 2009 WHO dengue case classification in an Indonesian pediatric cohort.

The classification of dengue virus-infected patients continues to be a challenge to researchers and clinicians in the field. The accuracy of the 1997 World Health Organization (WHO) dengue case definition has been debated for a decade, because the definition was very stringent, for instance, several researchers showed that apparently severe cases were misclassified as not severe. Therefore the WHO issued revised guidelines in 2009. Here, we retrospectively compared the performance of the WHO case definition of 2009 with the WHO case definition of 1997 in a detailed documented pediatric cohort from Indonesia. Intensive treatment intervention was used as an indicator of severity of disease. In line with our expectations, the 2009 WHO case classification proved to be significantly more specific, albeit less sensitive than the WHO case classification of 1997. We conclude that the revised classification is promising both from research and clinical perspectives, but validation of the classification criteria still needs to be addressed.

Lipopolysaccharide levels are elevated in dengue virus infected patients and correlate with disease severity.

BACKGROUND: Although in the majority of cases dengue virus (DENV) infection results in a self-limiting febrile disease, it can cause severe plasma leakage in a minority of patients. The appearance of plasma leakage indicates an increased permeability of the vascular wall. In this study we investigated if DENV infection can lead to leakage of lipopolysaccharide (LPS) from the intestine into the blood of the patient, indicative of an increased permeability of the intestinal mucosal barrier. OBJECTIVES: The aim of this study was to investigate if LPS levels were elevated in DENV infected patients and if these levels correlated with disease severity. STUDY DESIGN: LPS levels in the blood of DENV infected children were determined using the Limulus Amebocyte Lysate assay. To determine disease severity we used the 1997-WHO criteria, the expert physician's judgement and a score that focused on plasma leakage in particular. Furthermore, the modulatory factors LPS binding protein (LBP) and sCD14, as well as the immune activation marker neopterin were determined. RESULTS: We showed significantly elevated LPS levels in plasma of DENV infected children compared to healthy controls. The plasma leakage severity score had the strongest correlation with levels of LPS. LBP, sCD14 and neopterin were elevated compared to healthy controls. CONCLUSION: In this study we show evidence of elevated LPS levels during DENV infection. Moreover, a correlation between LPS levels and disease severity was found, especially when disease severity was determined in terms of plasma leakage.