In the present study the sensitivity of detection of

In the present study, the sensitivity of detection of the clinical isolates in vitro by the FcMBL ELLecSA was enhanced upon release of PAMPs by either mechanical disruption or treatment with bacteriocidal antibiotics, and this correlated with the detection of PAMPs in blood samples in both our studies with animals and human patients when blood cultures were negative but infections remained present at distant sites. These observations demonstrate that PAMPs released as a result of immune cell killing reactions or antibiotic therapy can be detected within blood samples using the FcMBL ELLecSA assay. We could detect PAMPs in blood samples of animals that were injected intravenously with live pathogen, in animals that were injected locally in the peritoneum with pathogens, as well as in blood culture-negative animals that were later shown to have local infection sites within internal purchase PLX4720 (e.g., liver, spleen and lung). More importantly, we showed that we could detect infections in blood culture-negative humans who demonstrated clinical symptoms of sepsis. Thus, the FcMBL-based PAMP blood assay clearly can be used to detect the presence of infections where living pathogens are localized to specific organ sites as well as blood-borne infections. While the assay cannot currently discriminate between these types of infection, this could be explored in future studies (e.g., based on PAMP levels or analysis of their molecular composition). Also, although we did not detect a significant increase in PAMP signal in paired infection samples of patients between time zero (when patients purchase PLX4720 first received broad spectrum antibiotics) and 24h post-admittance, the overall mean PAMP level appeared to increase between these time points, but a more targeted trial is needed to determine significance. This raises the possibility that the FcMBL ELLecSA could be used to track the efficacy of antibiotic treatments in infected patients in vivo, either alone or in combination with other biomarkers, such as PCT, which are currently being explored as a means to monitor antibiotic therapy for sepsis in the ICU (Carr, 2015; Schuetz et al., 2013; Jensen et al., 2011).
Molecular diagnostics that purport to detect and identify specific pathogens directly in blood are currently in development or entering the clinic. For example, the molecular diagnostic T2Candida Panel (T2 Biosytems) has demonstrated improved specificity, sensitivity and speed of detection (~4h) for identifying invasive candidiasis in blood culture negative patients (Mylonakis et al., 2015). But due to the low frequency of fungal infections in septic patients, the assay faces challenges for adaptation into the clinic as a general screening test. While the multiplexed PCR methods, SeptiFast (Roche, Diagnostics GmbH), and hybcell® Pathogens (Anagnostics Bioanalysis GmbH), and the PCR-ESI-MS method, IRIDICA (Abbott Molecular), can detect and identify bacteria and fungi in blood within 4–6h with >90% sensitivity and specificity in patients identified as positive by either blood culture or PCR (Knabl et al., 2016; Bacconi et al., 2014), the sensitivity of these assays drops dramatically to 10–40% for all patients with suspected bloodstream infections (Knabl et al., 2016; Bacconi et al., 2014) (Supplemental Fig. 2). In contrast, the FcMBL ELLecSA along with immunoassays such as PCT, offer an even more rapid (<1h) and generic test for identification of infection by a broad range of different types of pathogens. The FcMBL ELLecSA does not identify the specific type of pathogen; however, one can envision combining molecular diagnostic tools, such as mass spectrometry, to distinguish specific pathogen types based on the molecular composition of the captured PAMPs. This assay is also unique in that quantification of PAMP levels over time also might be used to track antibiotic efficacy in vivo without the need for pathogen identification (Carr, 2015; Schuetz et al., 2013; Jensen et al., 2011), and thus, rapidly assist physicians in their selection of an optimal therapy.