Abstract In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue ...patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14+ , CD32+ , CD86+ , CD11c+ ). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis.
Although heterotypic secondary infection with dengue virus (DENV) is associated with severe disease, the majority of secondary infections are mild or asymptomatic. The mechanisms of antibody-mediated ...protection are poorly understood. In 2010, 108 DENV3-positive cases were enrolled in a pediatric hospital-based study in Managua, Nicaragua, with 61 primary and 47 secondary infections. We analyzed DENV-specific neutralization titers (NT50), IgM and IgG avidity, and antibody titer in serum samples collected during acute and convalescent phases and 3, 6, and 18 months post-infection. NT50 titers peaked at convalescence and decreased thereafter. IgG avidity to DENV3 significantly increased between convalescent and 3-month time-points in primary DENV infections and between the acute and convalescent phase in secondary DENV infections. While avidity to DENV2, a likely previous infecting serotype, was initially higher than avidity to DENV3 in secondary DENV infections, the opposite relation was observed 3-18 months post-infection. We found significant correlations between IgM avidity and NT50 in acute primary cases and between IgG avidity and NT50 in secondary DENV infections. In summary, our findings indicate that IgM antibodies likely play a role in early control of DENV infections. IgG serum avidity to DENV, analyzed for the first time in longitudinal samples, switches from targeting mainly cross-reactive serotype(s) to the current infecting serotype over time. Finally, serum avidity correlates with neutralization capacity.
During August 2012-November 2014, we conducted a case ascertainment study to investigate household transmission of influenza virus in Managua, Nicaragua. We collected up to 5 respiratory swab samples ...from each of 536 household contacts of 133 influenza virus-infected persons and assessed for evidence of influenza virus transmission. The overall risk for influenza virus infection of household contacts was 15.7% (95% CI 12.7%-19.0%). Oseltamivir treatment of index patients did not appear to reduce household transmission. The mean serial interval for within-household transmission was 3.1 (95% CI 1.6-8.4) days. We found the transmissibility of influenza B virus to be higher than that of influenza A virus among children. Compared with households with <4 household contacts, those with >4 household contacts appeared to have a reduced risk for infection. Further research is needed to model household influenza virus transmission and design interventions for these settings.
Summary
Objective To evaluate the existing WHO dengue classification across all age groups and a wide geographical range and to develop a revised evidence‐based classification that would better ...reflect clinical severity.
Methods We followed suspected dengue cases daily in seven countries across South‐east Asia and Latin America and then categorised them into one of three intervention groups describing disease severity according to the overall level of medical and nursing support required. Using a pre‐defined analysis plan, we explored the clinical and laboratory profiles characteristic of these intervention categories and presented the most promising options for a revised classification scheme to an independent group of WHO dengue experts for consideration. Potential warning signs were also evaluated by comparing contemporaneous data of patients who progressed to severe disease with the data of those who did not.
Results A total of 2259 patients were recruited during 2006–2007 and 230 (13%) of the 1734 laboratory‐confirmed patients required major intervention. Applying the existing WHO system, 47/210 (22%) of patients with shock did not fulfil all the criteria for dengue haemorrhagic fever. However, no three‐tier revision adequately described the different severity groups either. Inclusion of readily discernible complications (shock/severe vascular leakage and/or severe bleeding and/or severe organ dysfunction) was necessary to devise a system that identified patients requiring major intervention with sufficient sensitivity and specificity to be practically useful. Only a small number of subjects (5%) progressed to severe disease while under observation; several warning signs were identified, but much larger studies are necessary to fully characterize features associated with disease progression.
Conclusions Based on these results, a revised classification system comprised of two entities, ‘Dengue’ and ‘Severe Dengue’, was proposed and has now been incorporated into the new WHO guidelines.
Objectif: Evaluer la classification actuelle de l’OMS pour la dengue dans tous les groupes d’âge et sur une vaste étendue géographique et élaborer une classification révisée, fondée sur des preuves permettant de mieux tenir compte de la sévérité clinique.
Méthodes: Nous avons suivi quotidiennement des cas suspects de dengue dans 7 pays d’Asie du sud‐est et d’Amérique latine, puis les avons classé en trois groupes d’intervention décrivant la sévérité de la maladie en fonction du niveau général du soutien médical et infirmier nécessaire. En utilisant un plan d’analyse prédéfini, nous avons exploré les profils cliniques et de laboratoire, caractéristiques de ces catégories d’intervention et avons soumis pour avis, les options les plus prometteuses pour un système révisé de classification, à un groupe d’experts indépendants de l’OMS pour la dengue. Les signes avant‐coureurs potentiels ont également étéévalués en comparant les données contemporaines de patients qui ont évolué vers une maladie sévère avec les données de ceux qui n’ont pas évolué de cette façon.
Résultats: 2259 patients ont été recrutés en 2006–2007 et 230 (13%) des 1734 patients avec une confirmation de laboratoire ont nécessité une intervention majeure. En appliquant le système actuel de l’OMS, 47/210 (22%) patients atteints de choc ne remplissaient pas tous les critères de dengue hémorragique. Toutefois, aucune révision tertiaire non plus n’a pu décrire adéquatement les différents groupes de sévérité. L’inclusion de complications facilement reconnaissables (choc/pertes vasculaires sévères et/ou saignements sévères et/ou dysfonctionnement sévère d’un organe) a été nécessaire pour concevoir un système permettant d’identifier les patients nécessitant une intervention majeure, avec une sensibilité et une spécificité suffisantes pour être utiles dans la pratique. Seul un petit nombre de sujets (5%) a progressé vers une maladie sévère alors qu’ils étaient sous observation; plusieurs signes d’alerte ont été identifiés, mais beaucoup plus d’études sont nécessaires pour caractériser complètement les caractéristiques associées à la progression de la maladie.
Conclusions: Sur base de ces résultats, un système de classification révisé, composé de deux entités, “Dengue” et “ dengue sévère “, a été proposé et a été intégré dans les nouvelles directives de l’OMS.
The four serotypes of dengue virus (DENV) cause dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Severe disease has been associated with heterotypic secondary DENV ...infection, mediated by cross-reactive antibodies (Abs) and/or cross-reactive T cells. The role of cross-reactive immunity in mediating enhanced disease versus cross-protection against secondary heterotypic DENV infection is not well defined. A better understanding of the cross-reactive immune response in natural infections is critical for development of safe and effective tetravalent vaccines. We studied the B cell phenotype of circulating B cells in the blood of pediatric patients suspected of dengue during the 2010-2011 dengue season in Managua, Nicaragua (n = 216), which was dominated by the DENV-3 serotype. We found a markedly larger percentage of plasmablast/plasma cells (PB/PCs) circulating in DENV-positive patients as compared to patients with Other Febrile Illnesses (OFIs). The percentage of DENV-specific PB/PCs against DENV-3 represented 10% of the circulating antibody-producing cells (ASCs) in secondary DENV-3 infections. Importantly, the cross-reactive DENV-specific B cell response was higher against a heterotypic serotype, with 46% of circulating PB/PCs specific to DENV-2 and 10% specific to DENV-3 during acute infection. We also observed a higher cross-reactive DENV-specific IgG serum avidity directed against DENV-2 as compared to DENV-3 during acute infection. The neutralization capacity of the serum was broadly cross-reactive against the four DENV serotypes both during the acute phase and at 3 months post-onset of symptoms. Overall, the cross-reactive B cell immune response dominates during secondary DENV infections in humans. These results reflect our recent findings in a mouse model of DENV cross-protection. In addition, this study enabled the development of increased technical and research capacity of Nicaraguan scientists and the implementation of several new immunological assays in the field.
The abundance and diversity of antibiotic resistance genes (ARGs) in the human respiratory microbiome remain poorly characterized. In the context of influenza virus infection, interactions between ...the virus, the host, and resident bacteria with pathogenic potential are known to complicate and worsen disease, resulting in coinfection and increased morbidity and mortality of infected individuals. When pathogenic bacteria acquire antibiotic resistance, they are more difficult to treat and of global health concern. Characterization of ARG expression in the upper respiratory tract could help better understand the role antibiotic resistance plays in the pathogenesis of influenza-associated bacterial secondary infection.
Thirty-seven individuals participating in the Household Influenza Transmission Study (HITS) in Managua, Nicaragua, were selected for this study. We performed metatranscriptomics and 16S rRNA gene sequencing analyses on nasal and throat swab samples, and host transcriptome profiling on blood samples. Individuals clustered into two groups based on their microbial gene expression profiles, with several microbial pathways enriched with genes differentially expressed between groups. We also analyzed antibiotic resistance gene expression and determined that approximately 25% of the sequence reads that corresponded to antibiotic resistance genes mapped to Streptococcus pneumoniae and Staphylococcus aureus. Following construction of an integrated network of ARG expression with host gene co-expression, we identified several host key regulators involved in the host response to influenza virus and bacterial infections, and host gene pathways associated with specific antibiotic resistance genes.
This study indicates the host response to influenza infection could indirectly affect antibiotic resistance gene expression in the respiratory tract by impacting the microbial community structure and overall microbial gene expression. Interactions between the host systemic responses to influenza infection and antibiotic resistance gene expression highlight the importance of viral-bacterial co-infection in acute respiratory infections like influenza. Video abstract.
Highlights • DENV and CHIKV co-circulate and result in overlapping clinical presentations. • A single-reaction pan-DENV–CHIKV rRT-PCR was designed. • The pan-DENV–CHIKV rRT-PCR was evaluated using ...serum samples from Nicaragua. • The pan-DENV–CHIKV rRT-PCR proved as sensitive as individual reference tests. • This DENV–CHIKV multiplex test may reduce costs and improve molecular workflow.
Disruption of the microbial community in the respiratory tract due to infections, like influenza, could impact transmission of bacterial pathogens. Using samples from a household study, we determined ...whether metagenomic-type analyses of the microbiome provide the resolution necessary to track transmission of airway bacteria. Microbiome studies have shown that the microbial community across various body sites tends to be more similar between individuals who cohabit in the same household than between individuals from different households. We tested whether there was increased sharing of bacteria from the airways within households with influenza infections as compared to control households with no influenza.
We obtained 221 respiratory samples that were collected from 54 individuals at 4 to 5 time points across 10 households, with and without influenza infection, in Managua, Nicaragua. From these samples, we generated metagenomic (whole genome shotgun sequencing) datasets to profile microbial taxonomy. Overall, specific bacteria and phages were differentially abundant between influenza positive households and control (no influenza infection) households, with bacteria like Rothia, and phages like Staphylococcus P68virus that were significantly enriched in the influenza-positive households. We identified CRISPR spacers detected in the metagenomic sequence reads and used these to track bacteria transmission within and across households. We observed a clear sharing of bacterial commensals and pathobionts, such as Rothia, Neisseria, and Prevotella, within and between households. However, due to the relatively small number of households in our study, we could not determine if there was a correlation between increased bacterial transmission and influenza infection.
We observed that airway microbial composition differences across households were associated with what appeared to be different susceptibility to influenza infection. We also demonstrate that CRISPR spacers from the whole microbial community can be used as markers to study bacterial transmission between individuals. Although additional evidence is needed to study transmission of specific bacterial strains, we observed sharing of respiratory commensals and pathobionts within and across households. Video Abstract.
Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection ...against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF.
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•DENV-specific IL-10+IFN-γ+ DP CD4 T cells are prominent during acute disease•Most DP cell DE genes are non-cytotoxic/Tr1 and include IL21, IL22, CD109, and CCR1•DP cells have similar gene expression in DF and DHF, despite higher frequency in DHF•Disease severity is not associated with altered DP cell phenotype or functionality
Tian et al. identify and characterize antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells in acute dengue patients. DP cells display similar transcriptomic profiles in mild DF and severe DHF, despite their increased frequency in DHF, suggesting that DHF is not associated with the altered phenotype or functionality of DP cells.
Serologic testing remains crucial for Zika virus diagnosis. We found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus ...infection with previous dengue (sensitivity 87.5%, specificity 93.8%). This test will aid serodiagnosis, serosurveillance, and monitoring of Zika complications in dengue-endemic regions.
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