Despite viral etiologies driving most cases of acute

Despite viral etiologies driving most cases of acute respiratory infection, definitive diagnostic tools for these syndromes are lacking. Even highly sensitive pathogen-specific tests such as PCR are dependent upon proper sampling technique and inclusion of virus-type-specific reagents and processing methods. Moreover, detection of a specific microbe in a clinical sample does not necessarily indicate the cause of the acute clinical syndrome. For example, it has been reported that HRV has been detected in up to 44% of asymptomatic individuals (Byington et al., 2015; Johnston et al., 1993). Therefore, better tools that help providers define the etiology of a suspected infectious syndrome in a safe, rapid, accurate, and cost-effective manner are of paramount importance for both individual and public health as recently noted by the Presidential Advisory Council on Combating Antibiotic-Resistant Bacteria (House, 2014), and others (O\’Neill, 2015; Organization, 2015). A complementary diagnostic strategy to pathogen detection could focus on utilizing the varied (but pathogen-class specific) host-response to infection (Ramilo and Mejias, 2009; Zaas et al., 2014). This approach discriminates between infection and colonization. It is pathogen-agnostic and therefore circumvents another limitation of pathogen detection assays, which due to technical limitations are only capable of detecting a Cyt 387 manufacturer limited subset of microorganisms. Furthermore, categorizing infection based on host response provides additional insights into the mechanisms of infection and disease response, and may offer new targets, pathways, or strategies for therapeutic intervention.
We recently identified gene Cyt 387 manufacturer patterns in peripheral whole blood capable of differentiating (Zaas et al., 2009; Woods et al., 2013; McClain et al., 2016; Tsalik et al., 2016; Huang et al., 2011) individuals with symptomatic infection due to influenza H3N2, HRV, or RSV from uninfected individuals with >90% accuracy. Moreover, this ARV signature was validated in an independent population of patients with influenza A infection, demonstrating an ability to distinguish from bacterial respiratory infections (93% accuracy) and healthy controls (100% accuracy) (Zaas et al., 2009). Thus, host derived biomarkers are capable of making these types of distinction. However, considering the technical challenges inherent in developing peripheral blood host gene expression classifiers as a diagnostic tool – including semi-invasive venipuncture, RNA instability, processing complexity, relatively high cost of RNA profiling, and time to result – we sought to extend this host response paradigm for ARV diagnosis to an alternative and potentially more suitable sample matrix and analyte class.
Upon contact with the respiratory epithelium, respiratory viruses incite activation of type I interferons (IFNs) and pro-inflammatory cytokines, orchestrate proliferation of inflammatory cells and the innate immune response, and regulate induction of adaptive immunity (Yoneyama and Fujita, 2010; Koyama et al., 2008; Bhoj et al., 2008). Based on the prominent role of the nasopharyngeal epithelium in mediating ARV infections, we hypothesized that nasopharyngeal lavage (NPL) would reflect the in situ host response and serve as a potential target for diagnostic development. Furthermore, the NPL protein fraction represents an accessible sample matrix, providing a highly tractable diagnostic analyte class. Multiple reaction monitoring (MRM), a quantitative mass spectrometry (MS) platform for facile development of multiplexed, quantitative assays for measuring specific protein levels in biologic fluids and is routinely used for biomarker verification in clinical cohorts (Kiyonami et al., 2011; Gerszten et al., 2010; Boja and Rodriguez, 2011). In addition to being customizable for nearly any target protein, MRM assays provide a more specific quantitation of individual proteins and protein isoforms by targeting multiple unique peptides per protein target. Combined with internal stable-isotope labeled (SIL) peptide standards, these assays match or exceed the quantitative precision of ELISA assays with low femtomole limits of quantitation and analytical precision coefficient of variation<10% across clinically sized cohorts (Addona et al., 2009; Aebersold et al., 2013).