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Background Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes. Methods We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients. Results We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative. Conclusion Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

Respiratory syncytial virus (RSV) infection results in millions of hospitalizations and thousands of deaths each year. Variations in the adaptive and innate immune response appear to be associated with RSV severity. To investigate the host response to RSV infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. We implemented a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation, which enabled us to identify cellular pathways associated with RSV severity. In both airway and immune cells, we found an association between RSV severity and activation of pathways controlling Th17 and acute phase response signaling, as well as inhibition of B cell receptor signaling. Dysregulation of both the humoral and mucosal response to RSV may play a critical role in determining illness severity., Author summary This paper presents a novel approach to understanding the localized molecular responses to respiratory syncytial virus (RSV) and the system-level correlates of clinical outcomes. To do this, we developed a novel statistical method able to integrate high dimensional molecular data characterizing the host airway microbota and immune and nasal gene expression. We show that this integrative approach facilitates superior performance in estimating clinical outcome as opposed to any single data type. Using this approach, we identified both cell type-specific and shared biomarkers and regulatory pathways associated with RSV severity. Specifically, we identified an association between RSV severity, activation of pathways controlling Th17, and inhibition of B cell receptor signaling, which were present in both the site of infection airway and in peripheral immune cells. These results can guide future efforts to identify biomarkers for identifying or predicting illness severity following infant RSV infection. They may also be useful as biomarkers to inform the efficacy of future interventions (e.g., therapies) or preventative measures to suppress the rate of severe disease (e.g., vaccines).

Rationale Lower respiratory tract illness (LRTI) frequently causes adult hospitalization and antibiotic overuse. Procalcitonin (PCT) treatment algorithms have been used successfully in Europe to safely reduce antibiotic use for LRTI but have not been adopted in the United States. We recently performed a feasibility study for a randomized clinical trial (RCT) of PCT and viral testing to guide therapy for non-pneumonic LRTI. Objective The primary objective of the current study was to understand factors influencing PCT algorithm adherence during the RCT and evaluate factors influencing provider antibiotic prescribing practices for LRTI. Study Design From October 2013-April 2014, 300 patients hospitalized at a community teaching hospital with non-pneumonic LRTI were randomized to standard or PCT-guided care with viral PCR testing. Algorithm adherence data was collected and multivariate stepwise logistic regression of clinical variables used to model algorithm compliance. 134 providers were surveyed anonymously before and after the trial to assess knowledge of biomarkers and viral testing and antibiotic prescribing practices. Results Diagnosis of pneumonia on admission was the only variable significantly associated with non-adherence [7% (adherence) vs. 26% (nonadherence), p = 0.01]. Surveys confirmed possible infiltrate on chest radiograph as important for provider decisions, as were severity of illness, positive sputum culture, abnormal CBC and fever. However, age, patient expectations and medical-legal concerns were also at least somewhat important to prescribing practices. Physician agreement with the importance of viral and PCT testing increased from 42% to 64% (p = 0.007) and 49% to 74% (p = 0.001), respectively, after the study. Conclusions Optimal algorithm adherence will be important for definitive PCT intervention trials in the US to determine if PCT guided algorithms result in better outcomes than reliance on traditional clinical variables. Factors influencing treatment decisions such as patient age, presence of fever, patient expectations and medical legal concerns may be amenable to education to improve PCT algorithm compliance for LRTI.

Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization., Author Summary Respiratory syncytial virus (RSV) is the second most common infectious cause of infant death worldwide. Despite this great clinical impact, no effective antivirals or vaccines against RSV are available. Here we find that the RSV attachment (G) glycoprotein uses CX3CR1 as a receptor on primary human airway epithelial (HAE) cultures, an excellent model of RSV infection of the human lung. The G protein contains a CX3C motif and we find that this region is critical for its role in infection of HAE cultures, but not of immortalized cells. Furthermore, we find that antibodies against the G protein neutralize RSV infection of HAE cultures differently from immortalized cells. These insights suggest that HAE cultures should be used to quantify neutralizing antibodies, including during vaccine development, that the CX3CR1 interaction with the RSV G protein could be a target for antiviral drug development, and that the G protein should be considered for inclusion in vaccines.