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Summary Objectives Animal studies suggest that RSV increases nasopharyngeal (NP) bacterial colonization facilitating bacterial infections. We investigated the influence of antibiotic treatment and colonization with potentially pathogenic bacteria on inflammatory markers and disease severity in RSV-infected in infants. Methods Healthy young infants hospitalized with RSV bronchiolitis (n = 136) and age-matched healthy controls (n = 23) were enrolled and NP samples cultured for potentially pathogenic bacteria including: Gram-positive bacteria (GPB): Staphylococcus aureus , Streptococcus pneumoniae , β-hemolytic Streptococcus ; and Gram-negative bacteria (GNB): Moraxella catarrhalis and Haemophilus influenzae . Clinical parameters and plasma IL-8, IL-6 and TNF-α concentrations were compared according to the bacterial class and antibiotic treatment. Results Antibiotic treatment decreased by 10-fold NP bacterial recovery. Eighty-one percent of RSV infants who did not receive antibiotics before sample collection were colonized with pathogenic bacteria. Overall, GNB were identified in 21% of patients versus 4% of controls who were mostly colonized with GPB. Additionally, in RSV patients NP white blood cell counts (p = 0.026), and blood neutrophils (p = 0.02) were higher in those colonized with potentially pathogenic bacteria versus respiratory flora. RSV patients colonized with GNB had higher plasma IL-8 (p = 0.01) and IL-6 (p Conclusions Infants with RSV bronchiolitis colonized with potentially pathogenic bacteria had increased numbers of mucosal and systemic inflammatory cells. Specifically, colonization with GNB was associated with higher concentrations of proinflammatory cytokines and a trend towards increased disease severity.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection (LRTI) in young children worldwide and an increasingly recognized cause of death in developing countries [1, 2]. Despite that selected groups of infants have increased risk for severe disease and mortality, the majority of children hospitalized with RSV LRTI are previously healthy with no known risk factors for severe disease [3, 4]. Of those hospitalized infants, 10%–20% will develop a disease severe enough to require admission to the pediatric intensive care unit (PICU) [5, 6]. It has been proposed that the combination of both viral factors and the host immune response probably contribute to the severity of RSV disease [7–11]. The importance of the innate immune response in the pathogenesis of severe RSV disease is being increasingly recognized. To date, however, studies addressing the role of innate immunity cytokines on RSV disease severity have been focused on determining concentrations of blood and respiratory cytokines, with inconsistent results [10–13]. Studies in critically ill patients with bacterial sepsis suggest that innate immune hyporesponsiveness, defined by decreased whole blood tumor necrosis factor α (TNF-α) production after ex vivo lipopolysaccharide (LPS) stimulation, is associated with worse clinical outcomes [14, 15]. Whether the functional capacity of the innate immune response is impaired in children with severe RSV disease, and how it correlates with plasma concentrations of innate immunity cytokines has not been characterized. The overall aims of this study were to determine whether patients with severe RSV bronchiolitis admitted to the PICU had decreased whole blood functional innate immune responses and to evaluate the relationships between innate immune dysfunction and disease outcomes.

In this study, Mejias and colleagues found that specific blood RNA profiles of infants with RSV LRTI allowed for specific diagnosis, better understanding of disease pathogenesis, and better assessment of disease severity. Please see later in the article for the Editors' Summary, Background Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity. Methods and Findings This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%–98%]) and specificity (98% [95% CI 88%–99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (, Editors' Summary Background Lower respiratory tract infections (LRTIs)—bacterial and viral infections of the lungs and airways (the tubes that take oxygen-rich air to the lungs)—are major causes of illness and death in children worldwide. Pneumonia (infection of the lungs) alone is responsible for 14% of all child deaths. The leading cause of viral LTRIs in children is respiratory syncytial virus (RSV), which is readily transmitted from person to person by direct contact with nasal fluids or airborne droplets. Almost all children have an RSV infection before their second birthday, but most have only minor symptoms similar to those of a common cold and are cared for at home. Unfortunately, some children develop more serious conditions when they become infected with RSV, such as pneumonia or bronchiolitis (swelling and mucus build-up in the bronchioles, the smallest air passages in the lungs). These children have to be admitted to the hospital for supportive care—there is no specific treatment for RSV infection—such as the provision of supplemental oxygen. Why Was This Study Done? The lack of a treatment (and of a vaccine) for RSV is largely due to our incomplete understanding of the cellular events and reactions, including the host immune response, that occur during the development of an RSV infection (disease pathogenesis). Moreover, based on physical examination and available diagnostic tools, it is impossible to predict which children infected with RSV will develop a serious condition that requires hospitalization and which ones can be safely nursed at home. Here, the researchers use microarrays to analyze the global host response to acute RSV LTRI in infants, to define gene expression patterns that are specific to RSV infection rather than infection with other common respiratory viruses, and to identify biomarkers that indicate the severity of RSV infection. “Microarray” analysis allows researchers to examine gene expression patterns (“RNA transcriptional profiles”) in, for example, whole blood; a biomarker is a molecule whose level in bodily fluids or tissues indicates how a disease might develop and helps with patient classification. What Did the Researchers Do and Find? The researchers compared the RNA transcriptional profile in whole blood taken from children less than two years old hospitalized with RSV, human rhinovirus, or influenza virus infection (rhinovirus and influenza are two additional viral causes of LRTI), and from healthy infants. Using “statistical group comparisons,” they identified more than 2,000 transcripts that were differentially expressed in blood from 45 infants with RSV infection and from 14 healthy matched controls. Genes related to interferon function (interferons are released by host cells in response to the presence of disease-causing organisms) and neutrophil function (neutrophils are immune system cells that, like interferons, are involved in the innate immune response, the body's first line of defense against infection) were among the most overexpressed genes in infants infected with RSV. Genes regulating T and B cells (components of the adaptive immune response, the body's second-line of defense against infection) were among the most underexpressed genes. This specific transcriptional profile, which was validated in three additional groups of infants, accurately distinguished between infants infected with RSV and those infected with human rhinovirus or influenza virus. Finally, a “molecular distance to health” score (a numerical score that quantifies the transcriptional perturbation associated with an illness) was correlated with the clinical disease severity score of the study participants, with how long they needed supplemental oxygen, and with their duration of hospitalization. What Do These Findings Mean? These findings suggest that it might be possible to use whole blood RNA transcriptional profiles to distinguish between infants infected with RSV and those with other viruses that commonly cause LRTI. Moreover, if these findings can be replicated in more patients (including non-hospitalized children), gene expression profiling might provide a strategy for triaging patients with RSV infections when they first present to an emergency department and for monitoring clinical changes during the course of the infection, particularly given the development of molecular tools that might soon enable the “real time” acquisition of transcriptional profiles in the clinical setting. Finally, although certain aspects of the study design limit the accuracy and generalizability of the study's findings, these data provide new insights into the pathogenesis of RSV infection and open new avenues for the discovery of biomarkers for RSV infection and for the identification of therapeutic and preventative targets. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001549. This study is further discussed in a PLOS Medicine Perspective by Peter Openshaw The US Centers for Disease Control and Prevention provides information about RSV infection The US National Heart, Lung, and Blood Institute provides information about the respiratory system and about RSV infections The UK National Health Service Choices website provides information about bronchiolitis The British Lung Foundation also provides information on RSV and on bronchiolitis MedlinePlus provides links to other resources about RSV infections and about pneumonia (in English and Spanish); the MedlinePlus encyclopedia has a page on bronchiolitis (in English and Spanish) PATH is an international non-profit organization investigating new RSV vaccines