A Flowthrough Assay for Rapid Bedside Stratification of Bloodstream Bacterial Infection in Critically Ill Patients: a Pilot Study

Abstract

Bacterial infections affect more than 2 million people annually. Of these, systemic infections caused by bacteria in critically ill patients may lead to life-threatening conditions such as sepsis. We have developed a point-of-care (POC) device called Septiflo that can detect and stratify the Gram status of bloodstream bacterial infections in less than 10 min from a drop of human plasma. It works on the principle of identifying pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) and lipoteichoic acid (LTA) that are released into the bloodstream at the onset of Gram-negative and Gram-positive bacterial infections, respectively. The biomarkers are captured on a membrane without a receptor, and the Gram status specificity is conferred by the ligands attached to gold nanoparticles (AuNPs) used as signal amplification probes. The ultrasensitive colorimetric results are read by eye down to a 100-fg/ml detection limit without an instrument. No cross-interference between the PAMPs is seen during Gram stratification. Septiflo results also display better performance than commercial enzyme-linked immunosorbent assays (ELISAs). Tests performed on 60 clinical samples from patients showed a correlation accuracy of 70% against procalcitonin (PCT), an accepted surrogate biomarker for sepsis. A direct comparison with eubacterial PCR yielded up to 94% accuracy in 31 patients at a chosen cutoff level for LPS and LTA and area under the curve (AUC) values of 0.927 and 0.885, respectively, though blood culture was negative for most samples. The high sensitivity, low cost, and simple bedside utility of the assay may aid in better sepsis management apparently at the presymptomatic stage, lowering empirical therapy, medical costs, antimicrobial resistance, and mortality.

Sodium Butyrate More Effectively Mitigates the Negative Effects of High-Concentrate Diet in Dairy Cows than Sodium β-Hydroxybutyrate via Reducing Free Bacterial Cell Wall Components in Rumen Fluid and Plasma

“…The PGN concentrations in rumen fluid and plasma were measured by the GPB Peptidoglycan Chromogenic Assay Kit (Autobio Diagnostics Co., Ltd., Zhengzhou, China), a patent product from Autobio (patent number: CN201610230254.1). The LTA concentrations in rumen fluid and plasma were measured using the LTA ELISA Kit (J&L Biological Industrial Co., Ltd., Shanghai, China), as described by previous studies [ 58 , 59 ]. The rumen fluid samples were centrifuged and diluted to the detective ranges using pyrogen-free water and depyrogenated glass tubes.…”

The Future of Biomarkers

“…Importantly, markers of sepsis should be distinguished from tests to identify the presence of microorganisms. There is no doubt that rapidly identifying the presence of microorganisms will be possible in the near future, [11][12][13] without having to rely on time-consuming microbiological cultures. As these tests are refined and they are more widely used, the time needed to obtain a result will decrease from several hours to minutes, and the price, which remains a strong limitation to their use today, will go down.…”

A Single Step in vitro Bioassay Mimicking TLR4-LPS Pathway and the Role of MD2 and CD14 Coreceptors

“…Four ligands were chosen for this purpose. Alexidine and sushi peptide were selected as positive controls due to their specific affinity toward LPS as shown in our recent studies (3,4). Vancomycin and bacitracin were used as negative controls for their lack of reported interaction with LPS (41,42).…”

“…Among the TLRs, TLR4 is a type I integral membrane glycoprotein that is specifically produced against Gram-negative bacterial endotoxins lipopolysaccharides (LPS). These LPS molecules exist in million copies per cell and are released into the bloodstream, in the form of random clusters or outer membrane vesicles (OMVs), as the cells multiply or die in response to antibiotic treatment (3,4). It is now well-established that activation of TLR4 is one of the two innate immune pathways triggered during Gram-negative bacterial sepsis in immunocompromised patients (the other one is caspase 11 activation in macrophages) (5,6).…”