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    • To measure the presence of a wide range of pathogens

    2018-11-05

    To measure the presence of a wide range of pathogens directly from blood samples we leveraged a recently developed engineered version of the natural human blood opsonin, Mannose Binding Lectin, as a broad-spectrum agent that can capture a wide range of pathogens and released toxins. This engineered protein contains the carbohydrate recognition domain of Mannose Binding Lectin linked to the Fc portion of human IgG1, and hence, it is known as FcMBL (Kang et al., 2014). FcMBL binds many Pathogen-Associated Molecular Patterns (PAMPs),(Parlato and Cavaillon, 2015) which are carbohydrate cell wall materials including lipopolysaccharide endotoxin (LPS), lipoteichoic imidazoline receptors and outer membrane vesicles, that are present on, or released by, various types of live and dead pathogens, including more than 90 different Gram negative and positive bacteria, fungi, viruses, and parasites (Kang et al., 2014; Schultz et al., 2008; Dommett et al., 2006). PAMP release can occur spontaneously (e.g., with Escherichia coli), and it increases significantly when pathogens are killed by antibiotics (Didar et al., 2015) and likely by immune cells as well. We previously showed that FcMBL can be used in dialysis-like devices to efficiently remove living Staphylococcus aureus and Escherichia coli bacteria as well as dead pathogens and cell wall-derived LPS from blood in rats based on its carbohydrate binding activity (Kang et al., 2014; Didar et al., 2015). Given its broad spectrum binding capabilities, we set out here to develop a simple and rapid infection diagnostic based on the use of FcMBL to detect the presence of PAMPs in whole blood of infected animals and human patients that might be used in the future to triage patients, accelerate antibiotic administration, and serve as a companion diagnostic to identify patients with high levels of PAMPs for emerging dialysis-like sepsis therapies (Basu et al., 2014; Tullis et al., 2010; Mitaka and Tomita, 2011; McCrea et al., 2014).
    Methods
    Procedures
    Results
    Discussion The need for rapid and accurate diagnosis of infection is great as delayed administration of antimicrobial therapy increases mortality by the hour as the infection rapidly evolves from local infection to sepsis, and eventually, to severe sepsis and septic shock (Martin et al., 2003; Kumar et al., 2006; Ferrer et al., 2014; Liu et al., 2014). A positive blood culture remains the current gold standard for laboratory diagnosis of systemic infection; unfortunately, most (>70%) septic patients have negative blood cultures (Tsalik et al., 2012; Gille-Johnson et al., 2013) and the sepsis and infection biomarkers currently in clinical use that measure the patient\'s inflammatory status, such as CRP, are of limited value in patient populations with inflammation induced by non-infectious illnesses or trauma. Thus, to address the critical need for an infection-specific biomarker that can assist physicians to decide on whether hospitalization is critical, antibiotics should be administered, or if recently developed dialysis-like sepsis therapies (Basu et al., 2014; Tullis et al., 2010; Mitaka and Tomita, 2011; McCrea et al., 2014) are appropriate, we leveraged the wide pathogen- and toxin-binding capabilities of the engineered FcMBL opsonin (Kang et al., 2014) to develop a broad-spectrum rapid diagnostic that directly detects the presence of pathogen-released PAMPs in whole blood. The FcMBL ELLecSA detects the cell wall materials of a broad range of living and dead pathogens, including negative and positive bacteria, fungi, viruses and parasites. Importantly, in our studies with human subjects, the FcMBL-based ELLecSA proved to be a sensitive and specific systemic biomarker for infections, detecting PAMPs in >80% of blood samples from emergency department patients with infection-related disease aetiologies, even though <20% of these patients had positive blood cultures. The FcMBL ELLecSA\'s ability to detect PAMPs directly also can be leveraged to distinguish patients with infection from those with non-infection related inflammatory conditions. Our ability to use this PAMP assay to discriminate between patients with infection and those with trauma is in line with the new Sepsis 3 guidelines in which SIRS criteria have been questioned because of issues related to determining the source of infection (Singer et al., 2016). The PAMP infection diagnostic also can be implemented in settings outside of central hospitals where qSOFA/SOFA scores are more challenging to determine, and it potentially can enable clinicians to identify patients with infection even when they have low qSOFA or SOFA scores in the hospital setting (Singer et al., 2016). Thus, the FcMBL-based PAMP assay may serve as a new rapid diagnostic for infection in patients who are suspected to have a blood-borne infection or early-stage sepsis, and thereby help to reduce unnecessary use of antibiotics.