Antigen-Independent Restriction of Pneumococcal Density by Mucosal Adjuvant Cholera Toxin Subunit B.

For many bacterial respiratory infections, development of (severe) disease is preceded by asymptomatic colonization of the upper airways. For Streptococcus pneumoniae, the transition to severe lower respiratory tract infection is associated with an increase in nasopharyngeal colonization density. Insight into how the mucosal immune system restricts colonization may provide new strategies to prevent clinical symptoms. Several studies have provided indirect evidence that the mucosal adjuvant cholera toxin subunit B (CTB) may confer nonspecific protection against respiratory infections. Here, we show that CTB reduces the pneumococcal load in the nasopharynx, which required activation of the caspase-1/11 inflammasome, mucosal T cells, and macrophages. Our findings suggest that CTB-dependent activation of the local innate response synergizes with noncognate T cells to restrict bacterial load. Our study not only provides insight into the immunological components required for containment and clearance of pneumococcal carriage, but also highlights an important yet often understudied aspect of adjuvants.

High pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants. A small percentage of the infected infants develops a severe infection, while most of these severely ill patients were previously healthy. It remains unclear why these children develop severe RSV infections. In this study, we investigate whether pneumococcal nasopharyngeal carriage patterns correlate with mucosal inflammation and severity of disease. METHODS: In total, 105 infants hospitalized with RSV infection were included and recovery samples were taken from 42 patients. The presence and density of Streptococcus pneumoniae was determined by RT qPCR to study its relation to viral load, inflammation (MMP-9 and IL-6) and severity of RSV disease. RESULTS: We show that pneumococcal presence or absence in the nasopharynx does not correlate with viral load, inflammation or severity of disease. However, when pneumococcus is present in patients, a higher nasopharyngeal pneumococcal density was correlated with a higher RSV load, higher MMP-9 levels and a less severe course of disease. CONCLUSIONS: Our results show correlations between S. pneumoniae density and viral load, inflammation and disease severity, suggesting that pneumococcal density may be an indicator for severity in paediatric RSV disease.

Nasopharyngeal gene expression, a novel approach to study the course of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) causes mild infections in the vast majority of children. However, in some cases, it causes severe disease, such as bronchiolitis and pneumonia. Development of severe RSV infection is determined by the host response. Therefore, the main aim of this study was to identify biomarkers associated with severe RSV infection. To identify biomarkers, nasopharyngeal gene expression was profiled by microarray studies, resulting in the selection of five genes: ubiquitin D, tetraspanin 8, mucin 13, ß-microseminoprotein and chemokine ligand 7. These genes were validated by real-time quantitative PCR in an independent validation cohort, which confirmed significant differences in gene expression between mildly and severely infected and between recovery and acute patients. Nasopharyngeal aspirate samples are regularly taken when a viral respiratory tract infection is suspected. In this article, we describe a method to discriminate between mild and severe RSV infection based on differential host gene expression. The combination of pathogen detection and host gene expression analysis in nasopharyngeal aspirates will significantly improve the diagnosis and prognosis of respiratory tract infections.

Aptasensors for viral diagnostics.

Novel viral diagnostic tools need to be affordable, fast, accurate and easy to use with sensitivity and specificity equivalent or superior to current standards. At present, viral diagnostics are based on direct detection of viral components or indirect detection by measuring antibodies generated in response to viral infection. While sensitivity of detection and quantification are still important challenges, we expect major advances from new assay formats and synthetic binding molecules, such as aptamers. Compared to traditional antibody-based detection, aptamers could provide faster adaptation to continuously evolving virus strains and higher discriminating capacity between specific virus serotypes. Aptamers are very stable and easily modifiable, so are ideal molecules for detection and chemical sensing applications. Here, we review the use of aptasensors for detection of viral pathogens and consider the feasibility of aptasensors to become standard devices for point-of-care diagnostics of viruses.

Non-invasive diagnostics of pathogenic bacteria using a breath sampler in children with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease causing thick, viscous secretions leading to pulmonary infections with pathogenic bacteria. As part of routine patient care, colonization and infection with these bacteria is monitored with cough swab or sputum cultures and sometimes bronchoalveolar lavage. In this cross-sectional proof-of-concept study in a cohort of CF patients we collected swabs or sputa and exhaled breath samples with the modular breath sampler (MBS), a newly developed two-way non-rebreathing sampling device. Pathogen specific polymerase chain reactions (PCRs) were performed on the MBS samples and compared with the results obtained with conventional diagnostics (i.e. culturing of swabs and sputa). A control group of stable asthma patients was used as negative control for the MBS measurements. The pathogens detected using MBS and conventional culturing differed:S. aureuswas found more often in swab or sputum samples whereasPseudomonas aeruginosaandS. pneumoniaewere found more often in MBS samples. We hypothesize that this is due to sampling of different compartments, MBS samples are derived from the lower respiratory tract while cultures from cough swabs and sputa are dominated by pathogens residing in the upper respiratory tract. Another important difference is the readout, i.e. culture versus PCR. The majority of CF patients in whomP. aeruginosawas found did not have recent positive cultures suggesting higher sensitivity of MBS-based than conventional diagnostics. The majority of parents/patients found the MBS easy to use and less of a burden than respiratory sampling.

SARS-CoV-2 RNA in exhaled air of hospitalized COVID-19 patients.

Knowledge about contagiousness is key to accurate management of hospitalized COVID-19 patients. Epidemiological studies suggest that in addition to transmission through droplets, aerogenic SARS-CoV-2 transmission contributes to the spread of infection. However, the presence of virus in exhaled air has not yet been sufficiently demonstrated. In pandemic situations low tech disposable and user-friendly bedside devices are required, while commercially available samplers are unsuitable for application in patients with respiratory distress. We included 49 hospitalized COVID-19 patients and used a disposable modular breath sampler to measure SARS-CoV-2 RNA load in exhaled air samples and compared these to SARS-CoV-2 RNA load of combined nasopharyngeal throat swabs and saliva. Exhaled air sampling using the modular breath sampler has proven feasible in a clinical COVID-19 setting and demonstrated viral detection in 25% of the patients.

Isolation and characterization of Alicycliphilus denitrificans strain BC, which grows on benzene with chlorate as the electron acceptor.

A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 microm wide and 1 to 2 microm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.

Short-term repeated HRV-16 exposure results in an attenuated immune response in vivo in humans.

INTRODUCTION: Naturally, development of adaptive immunity following HRV infection affects the immune response. However, it is currently unclear whether or not HRV re-exposure within a short time frame leads to an altered innate immune response. The "experimental cold model" is used to investigate the pathogenesis of HRV infection and allows us to investigate the effects of repeated exposure on both local and systemic innate immunity. METHODS: 40 healthy male and female (1:1) subjects were nasally inoculated with HRV-16 or placebo. One week later, all subjects received HRV-16. Baseline seronegative subjects (n = 18) were included for further analysis. RESULTS: Infection rate was 82%. Primary HRV infection induced a marked increase in viral load and IP-10 levels in nasal wash, while a similar trend was observed for IL-6 and IL-10. Apart from an increase in IP-10 plasma levels, HRV infection did not induce systemic immune effects nor lower respiratory tract inflammation. With similar viral load present during the second HRV challenge, IP-10 and IL-6 in nasal wash showed no increase, but gradually declined, with a similar trend for IL-10. CONCLUSION: Upon a second HRV challenge one week after the first, a less pronounced response for several innate immune parameters is observed. This could be the result of immunological tolerance and possibly increases vulnerability towards secondary infections.