Direct detection of bacteremia by exploiting host-pathogen interactions of lipoteichoic acid and lipopolysaccharide

Abstract

Bacteremia is a leading cause of death in sub-Saharan Africa where childhood mortality rates are the highest in the world. The early diagnosis of bacteremia and initiation of treatment saves lives, especially in high-disease burden areas. However, diagnosing bacteremia is challenging for clinicians, especially in children presenting with co-infections such as malaria and HIV. There is an urgent need for a rapid method for detecting bacteremia in pediatric patients with co-morbidities to inform treatment. In this manuscript, we have developed and clinically validated a novel method for the direct detection of amphiphilic pathogen biomarkers indicative of bacteremia, directly in aqueous blood, by mimicking innate immune recognition. Specifically, we have exploited the interaction of amphiphilic pathogen biomarkers such as lipopolysaccharides (LPS) from Gram-negative bacteria and lipoteichoic acids (LTA) from Gram-positive bacteria with host lipoprotein carriers in blood, in order to develop two tailored assays – lipoprotein capture and membrane insertion – for their direct detection. Our assays demonstrate a sensitivity of detection of 4 ng/mL for LPS and 2 ng/mL for LTA using a waveguide-based optical biosensor platform that was developed at LANL. In this manuscript, we also demonstrate the application of these methods for the detection of LPS in serum from pediatric patients with invasive Salmonella Typhimurium bacteremia (n = 7) and those with Staphylococcal bacteremia (n = 7) with 100% correlation with confirmatory culture. Taken together, these results demonstrate the significance of biochemistry in both our understanding of host-pathogen biology, and development of assay methodology, as well as demonstrate a potential new approach for the rapid, sensitive and accurate diagnosis of bacteremia at the point of need.

Pediatric Tuberculosis: The Impact of “Omics” on Diagnostics Development

“…Previous work from our group has demonstrated that LAM is associated with high-density lipoproteins (HDL) in host blood [ 45 ]. This association should be considered when developing diagnostics, as with membrane insertion and lipoprotein capture methodologies [ 43 , 137 , 141 , 142 , 143 , 144 ], because traditional strategies for measuring the monomeric antigen are likely to be unsuccessful in this conformation. To date there are only a few studies showing detection of LAM in blood from adults [ 46 , 47 , 133 ] and none in a pediatric population.…”

A centrifugal microfluidic cross-flow filtration platform to separate serum from whole blood for the detection of amphiphilic biomarkers

“…Because of this sequestration, it is necessary to liberate the amphiphilic biomarkers from their lipoprotein carriers in order to enable sensitive measurement. Detection can then occur via enzyme-linked immunosorbent assays (ELISA’s), waveguide-based biosensors 5 , 6 , 8 , 10 , or other methods 9 .…”

Interaction of Amphiphilic Lipoarabinomannan with Host Carrier Lipoproteins in Tuberculosis Patients: Implications for Blood-based Diagnostics

“…This article is a US Government work. It is not subject to copyright under 17 USC The copyright holder for this preprint this version posted November 20, 2020. ; https://doi.org/10.1101/2020.11.20.391037 doi: bioRxiv preprint 7 directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane which forms the functional surface of a biosensor (22,24,25). Although both of these assays are platform ambivalent, we used enzyme linked immunosorbent assays (ELISA) and fluorescence measurements from a waveguide-based biosensor platform developed at the Los Alamos National Laboratory for this study (19,26).…”

“…To evaluate the impact of serum sequestration of LAM in host lipoprotein complexes, we measured serum LAM using two methods tailored for the detection of amphiphilic biomarkers in aqueous matrices (Figure 1) – lipoprotein capture and membrane insertion. The lipoprotein capture assay (Figure 1) relies on capture of host lipoproteins, exploiting their biological association with the pathogen amphiphile to “concentrate” LAM (19,24). In contrast, the membrane insertion assay (Figure 1) is independent of that host lipoprotein/LAM association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane which forms the functional surface of a biosensor (22,24,25).…”

“…The lipoprotein capture assay (Figure 1) relies on capture of host lipoproteins, exploiting their biological association with the pathogen amphiphile to “concentrate” LAM (19,24). In contrast, the membrane insertion assay (Figure 1) is independent of that host lipoprotein/LAM association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane which forms the functional surface of a biosensor (22,24,25). Although both of these assays are platform ambivalent, we used enzyme linked immunosorbent assays (ELISA) and fluorescence measurements from a waveguide-based biosensor platform developed at the Los Alamos National Laboratory for this study (19,26).…”