Use of lipoteichoic acid (LTA) and / or lipopolysaccharide (LPS) positive extracellular vesicles in plasma

Abstract

The present application belongs to the field of medicine and health, and relates to the use of lipoteichoic acid (LTA) and / or lipopolysaccharide (LPS) positive extracellular vesicles in plasma in the detection of bacterial inflammation and / or bacteremia. Specifically, the present application provides an in vitro method for detecting bacterial inflammation and / or bacteremia, comprising the following steps: providing a sample to be tested from a subject; isolating extracellular vesicles from the sample to be tested; detecting the level of LPS positive extracellular vesicles (LPS + EVs) and / or the level of LTA positive extracellular vesicles (LTA + EVs); if the levels of LPS + EVs and / or LTA + EVs increase, it indicates that the subject has bacterial inflammation and / or bacteremia.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to the use of extracellular vesicles positive for lipoteichoic acid (LTA) and / or lipopolysaccharide (LPS) in plasma. Background Technology

[0002] Accurately identifying bacterial inflammation and differentiating it from viral inflammation is crucial for effective treatment and preventing antibiotic overuse. Specific biomarker testing provides physicians with objective diagnostic evidence, ensuring antibiotics are used only for patients with bacterial infections. This significantly reduces unnecessary drug use, slows the development of antibiotic resistance, and protects patients from potential drug side effects. This not only optimizes treatment strategies but also safeguards public health.

[0003] Further differentiation between Gram-negative and Gram-positive bacterial infections can significantly improve the precision and efficiency of treatment. Due to the different cell wall structures of these two types of bacteria, the pathogenic mechanisms of inflammation they cause and the appropriate therapeutic drugs also differ significantly. Rapid identification of bacterial strains can guide physicians in selecting more targeted antibiotics (such as vancomycin for Gram-positive bacteria or specific cephalosporins for Gram-negative bacteria), enabling a shift from empirical broad-spectrum therapy to targeted narrow-spectrum therapy. This leads to faster infection control, improved patient prognosis, and further reduction of the risk of drug resistance.

[0004] Early differentiation and diagnosis of bacterial infections are of paramount clinical importance. The initial stage of infection is the golden window for controlling disease progression. Early and precise medication can effectively prevent the proliferation of pathogens and the release of toxins, preventing mild cases from developing into life-threatening complications such as sepsis or septic shock. Therefore, the process of early detection, early identification, and early intervention directly affects the success rate of treatment, not only shortening the course of the disease and alleviating patient suffering, but also demonstrating immeasurable value in reducing medical costs and saving lives.

[0005] Therefore, there is an urgent need to provide a method for detecting bacterial inflammation in the early stages of bacterial infection and for effectively distinguishing between bacterial inflammation caused by Gram-negative and Gram-positive bacteria. Summary of the Invention

[0006] This invention provides a method for detecting LPS-positive extracellular vesicles (LPS). + The levels of EVs and / or LTA-positive extracellular vesicles (LTA) + Methods for detecting bacterial inflammation and / or bacteremia at the level of EVs.

[0007] In a first aspect of the present invention, an in vitro detection method for bacterial inflammation and / or bacteremia is provided, comprising the following steps:

[0008] (S1) Provide test samples from the test subjects;

[0009] (S2) Isolate extracellular vesicles from the sample to be tested;

[0010] (S3) Detection of LPS-positive extracellular vesicles (LPS) + The levels of EVs and / or LTA-positive extracellular vesicles (LTA) + The level of EVs; if LPS + EVs and / or LTA + An increase in EVs levels indicates the presence of bacterial inflammation and / or bacteremia in the subject.

[0011] In another preferred embodiment, the bacterial inflammation and / or bacteremia refers to bacterial inflammation and / or bacteremia in the early stages of a bacterial infection.

[0012] In another preferred embodiment, the LPS + EVs and / or LTA + Increased EVs levels include: LPS + The proportion of EVs is ≥8%, preferably ≥10%, and most preferably ≥12%; and / or

[0013] LTA + The proportion of EVs is ≥5%, preferably ≥8%, even better ≥10%, and optimally ≥12%.

[0014] In another preferred embodiment, the in vitro detection method further includes: verifying the presence of bacterial inflammation and / or bacteremia in the sample to be tested by blood culture.

[0015] In another preferred embodiment, step (S3) includes: comparing the LPS of the test samples. + Levels of EVs and LPS in normal control + The level of EVs, if the LPS of the sample to be tested + The level of EVs was significantly higher than that of LPS in the normal control group. + If the level of EVs is high, then the sample indicates bacterial inflammation and / or bacteremia due to Gram-negative bacterial infection.

[0016] The normal control is a reference sample that is not infected with Gram-negative bacteria.

[0017] In another preferred embodiment, the significant increase refers to the LPS of the test sample. + EVs level Y1 and normal control LPS + Compared to the level Y0 of EVs, Y1 / Y0≥2, better is ≥3, even better is ≥4, and best is ≥5.

[0018] In another preferred embodiment, the Gram-negative bacteria include: Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Salmonella spp., Neisseria gonorrhoeae, Neisseria meningitidis, or combinations thereof.

[0019] In another preferred embodiment, the Gram-negative bacteria include: Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, or a combination thereof.

[0020] In another preferred embodiment, step (S3) includes: comparing the LTA of the test sample. + Levels of EVs and LTA in normal controls + The level of EVs, if the LTA of the test sample + The level of EVs was significantly higher than that of LTA in the normal control group. + If the level of EVs is high, then the sample is infected with Gram-positive bacteria;

[0021] The normal control is a reference sample that is not infected with Gram-positive bacteria.

[0022] In another preferred embodiment, the significant increase refers to the LTA of the test sample. + EVs level X1 and normal control LTA + Compared to the level X0 of EVs, Y1 / Y0 ≥ 2, preferably ≥ 3, and optimally ≥ 5.

[0023] In another preferred embodiment, the Gram-positive bacteria include: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Clostridium difficile, Bacillus anthracis, or combinations thereof.

[0024] In another preferred embodiment, the Gram-positive bacteria include: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, or a combination thereof.

[0025] In another preferred embodiment, the sample comprises: plasma, blood, saliva, urine, cerebrospinal fluid, feces, ascites, sputum, semen, breast milk, or sweat.

[0026] In another preferred embodiment, the subject does not suffer from gastrointestinal inflammation.

[0027] In another preferred embodiment, the gastrointestinal inflammation includes: upper gastrointestinal inflammation and / or lower gastrointestinal inflammation.

[0028] In another preferred embodiment, the lower gastrointestinal inflammation includes: enteritis, colitis, appendicitis, diverticulitis, or a combination thereof.

[0029] In another preferred embodiment, the upper gastrointestinal inflammation includes esophagitis, gastritis, duodenitis, or a combination thereof.

[0030] In another preferred embodiment, the subject does not suffer from enteritis, colitis, or a combination thereof.

[0031] In another preferred embodiment, the subject has an intact intestinal barrier.

[0032] In another preferred embodiment, the bacteremia is not bacteremia caused by the intestines.

[0033] In another preferred embodiment, the bacteremia is not caused by the entry of intestinal flora into the bloodstream.

[0034] In another preferred embodiment, the bacterial inflammation includes: bacterial pneumonia, bacterial meningitis, or a combination thereof.

[0035] In another preferred embodiment, in step (S2), the separation comprises separation based on molecular size and / or molecular density.

[0036] In another preferred embodiment, in step (S2), the separation comprises separation using one or more methods selected from the group consisting of: differential centrifugation, immunoaffinity capture, density gradient ultracentrifugation, size exclusion chromatography.

[0037] In another preferred embodiment, in step (S3), the detection refers to detection using nanoflow cytometry.

[0038] In another preferred embodiment, the subject does not suffer from autoimmune inflammation.

[0039] In a second aspect of the invention, detection of LPS is provided. + EVs and / or LTA + The reagents for EVs are used to prepare products for the detection of bacterial inflammation and / or bacteremia.

[0040] In another preferred embodiment, the detection of LPS + EVs and / or LTA + The reagents for EVs include:

[0041] (i) Reagents for separating EVs; and

[0042] (ii) Reagents for detecting LPS and / or LTA on the surface of EVs.

[0043] In another preferred embodiment, the bacterial inflammation and / or bacteremia refers to bacterial inflammation and / or bacteremia in the early stages of a bacterial infection.

[0044] In another preferred embodiment, the bacterial inflammation and / or bacteremia includes bacterial inflammation and / or bacteremia caused by Gram-positive bacteria, bacterial inflammation and / or bacteremia caused by Gram-negative bacteria, or a combination thereof.

[0045] In another preferred embodiment, the Gram-negative bacteria include: Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Salmonella spp., Neisseria gonorrhoeae, Neisseria meningitidis, or combinations thereof.

[0046] In another preferred embodiment, the Gram-positive bacteria include: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Clostridium difficile, Bacillus anthracis, or combinations thereof.

[0047] In another preferred embodiment, the bacterial inflammation and / or bacteremia does not include gastrointestinal inflammation.

[0048] In another preferred embodiment, the bacterial inflammation and / or bacteremia does not include enteritis, colitis, or a combination thereof.

[0049] In another preferred embodiment, the patient with bacterial inflammation and / or bacteremia has an intact intestinal barrier.

[0050] In another preferred embodiment, the bacteremia is not bacteremia caused by the intestines.

[0051] In another preferred embodiment, the bacteremia is not caused by the entry of intestinal flora into the bloodstream.

[0052] In another preferred embodiment, the bacterial inflammation includes: bacterial pneumonia, bacterial meningitis, or a combination thereof.

[0053] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0054] Figure 1 Characterization analysis of Staphylococcus aureus cell membrane vesicles (S. aureus-CMVs) and Escherichia coli outer membrane vesicles (E. coli-OMVs) is shown.

[0055] Figure 1 Image a shows a transmission electron microscope (TEM) image of a Staphylococcus aureus cell membrane vesicle (S. aureus-CMVs).

[0056] Figure 1 b shows the particle size distribution of Staphylococcus aureus cell membrane vesicles (S. aureus-CMVs) as determined by nano-flow cytometry (nano-FCM).

[0057] Figure 1c shows a transmission electron microscope (TEM) image of E. coli outer membrane vesicles (E. coli-OMVs).

[0058] Figure 1 Figure d shows the particle size distribution of E. coli outer membrane vesicles (OMVs) as determined by nano-flow cytometry (nano-FCM).

[0059] Figure 1 The image shows the presence of lipoteichoic acid (LTA) on Staphylococcus aureus cell membrane vesicles (S. aureus-CMVs) confirmed by immunoelectron microscopy (IEM). Cell membrane vesicles were detected using an anti-LTA antibody; arrows indicate LTA-positive cell membrane vesicles (LTA...). + CMVs).

[0060] Figure 1 f shows the immunoelectron microscopy isotype control of lipoteichoic acid (LTA) (Staphylococcus aureus cell membrane vesicles + isotype IgG).

[0061] Figure 1 g shows the effect of nano-flow cytometry (nano-FCM) on the detection of LTA-positive cell membrane vesicles (LTA) derived from Gram-positive bacteria. + Quantitative analysis was performed using CMVs. Data are expressed as mean ± standard deviation, n = 6.

[0062] Figure 1 h shows the binding of lipopolysaccharide (LPS) antibody to E. coli outer membrane vesicles (E. coli-OMVs) as observed by immunoelectron microscopy (IEM). The outer membrane vesicles were detected using anti-LPS antibody; the arrows indicate LPS-positive outer membrane vesicles (LPS...). + OMVs).

[0063] Figure 1 i shows the immunoelectron microscopy isotype control of lipopolysaccharide (LPS) (E. coli outer membrane vesicles + isotype IgG).

[0064] Figure 1 j shows the cytometry (nano-FCM) used to detect LPS-positive outer membrane vesicles (LPS) derived from Gram-negative bacteria. + Quantitative analysis was performed using OMVs. Data are expressed as mean ± standard deviation, n = 6.

[0065] Figure 2 The results showed LTA after co-incubation with the isotype control (antibody). + and LPS + vesicle ratio

[0066] Figure 2 a shows the LPS after co-incubation with the control antibody.+ The proportion of extracellular vesicles (EVs). Data are expressed as mean ± standard deviation (n = 6).

[0067] Figure 2 b shows the LTA after co-incubation with the control antibody. + The proportion of extracellular vesicles (EVs). Data are expressed as mean ± standard deviation (n = 6).

[0068] Figure 3 The study showed changes in cytokine release and mRNA expression in mouse macrophages after treatment with lipopolysaccharide-positive extracellular vesicles (LPS+EVs) and lipoteichoic acid-positive extracellular vesicles (LTA+EVs).

[0069] Figure 3 Figure a shows the effects of LPS+EVs on the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mouse macrophage culture supernatants. The left figure investigates the dynamic changes in TNF-α levels in mouse macrophage culture supernatants after LPS+EVs treatment. The right figure observes the effects of LPS+EVs on IL-6 levels in mouse macrophage culture supernatants.

[0070] Figure 3 Figure b shows the effect of LTA+EVs on TNF-α and IL-6 levels in mouse macrophage culture supernatant. The left panel analyzes the trend of TNF-α levels in mouse macrophage culture supernatant after LTA+EVs treatment. The right panel assesses the effect of LTA+EVs on IL-6 levels in mouse macrophage culture supernatant.

[0071] Figure 3 c shows the effect of LPS+EVs on the relative mRNA levels of TNF-α and IL-6 in mouse macrophages. Left panel: Relative mRNA level of TNF-α in mouse macrophages after LPS+EVs treatment. Right panel: Relative mRNA level of IL-6 in mouse macrophages after LPS+EVs treatment.

[0072] Figure 3 Figure d shows the effect of LTA+EVs on the relative mRNA levels of TNF-α and IL-6 in mouse macrophages. Left panel: Relative mRNA level of TNF-α in mouse macrophages after LTA+EVs treatment. Right panel: Relative mRNA level of IL-6 in mouse macrophages after LTA+EVs treatment. Data are expressed as mean ± standard deviation. Statistical significance was assessed using unpaired t-tests. Sample size (n) = 3. P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001.

[0073] Figure 4The results of blood bacterial cultures from mouse models infected with different pathogens are shown.

[0074] Figure 4 a shows the results of blood bacterial cultures in mice with Staphylococcus aureus-induced pneumonia;

[0075] Figure 4 b shows the results of blood bacterial culture in mice with Staphylococcus aureus-induced meningitis;

[0076] Figure 4 c shows the results of blood bacterial cultures in mice with E. coli-induced pneumonia;

[0077] Figure 4 d shows the results of blood bacterial cultures in mice with E. coli-induced meningitis;

[0078] Figure 4 e shows the results of blood bacterial culture in mice with Staphylococcus aureus (S. aureus)-induced bloodstream infection;

[0079] Figure 4 f shows the results of blood bacterial culture in mice with E. coli-induced bloodstream infection. Note: The black shading visible on the back of the blood agar plate is a watermark indicating the plate's production date and main ingredients.

[0080] Figure 5 The results of hematoxylin-eosin (H&E) staining of tissues from mouse models infected with different pathogens are shown.

[0081] Figure 5 a shows the H&E staining results of mouse lung tissue induced by Staphylococcus aureus (S. aureus).

[0082] Figure 5 b shows the H&E staining results of mouse lung tissue induced by E. coli pneumonia.

[0083] Figure 5 c shows the H&E staining results of mouse brain tissue induced by Staphylococcus aureus (S. aureus) meningitis.

[0084] Figure 5 Image d shows the H&E staining results of mouse brain tissue induced by Escherichia coli (E. coli) meningitis.

[0085] Figure 6 The study showed that LTA levels in the blood of mice infected with Staphylococcus aureus / Escherichia coli were... + / LPS +Analysis of extracellular vesicles (EVs)

[0086] Figure 6 a shows the detection of LPS in the blood of mice with different E. coli infection models using nano-flow cytometry (Nano-FCM). + The proportion of extracellular vesicles.

[0087] Figure 6 b shows the detection of LTA in the blood of mice with different Staphylococcus aureus infection models using nano-flow cytometry (Nano-FCM). + The proportion of extracellular vesicles.

[0088] Figure 6 c shows the LPS levels in the blood of mice infected with E. coli. + Immunoelectron microscopy (IEM) images of extracellular vesicles.

[0089] Figure 6 d shows the LTA in the blood of mice infected with Staphylococcus aureus. + Immunoelectron microscopy (IEM) images of extracellular vesicles.

[0090] Figure 6 e shows the analysis of LTA in the blood of mice co-infected with Staphylococcus aureus and Escherichia coli using nano-flow cytometry (Nano-FCM). + / LPS + The proportion of extracellular vesicles.

[0091] Figure 6 f shows the LTA levels in the blood of mice co-infected with Staphylococcus aureus and Escherichia coli. + Immunoelectron microscopy (IEM) images of extracellular vesicles.

[0092] Figure 6 g showed the LPS levels in the blood of mice co-infected with Staphylococcus aureus and Escherichia coli. + Immunoelectron microscopy (IEM) images of extracellular vesicles.

[0093] Data are expressed as mean ± standard deviation, and the sample size (n) is 6.

[0094] Figure 7 The results show the LTA before and after Triton X-100 treatment. + Extracellular vesicles (EVs) / LPS + Tracking analysis of extracellular vesicles (EVs).

[0095] Figure 7 a shows raw data from nano-flow cytometry (Nano-FCM), demonstrating LTA in extracellular vesicles of plasma before 1% Triton X-100 treatment. +The number of particles.

[0096] Figure 7 b shows raw data from nano-flow cytometry (Nano-FCM), demonstrating LTA levels in extracellular vesicles of plasma after treatment with 1% Triton X-100. + The number of particles.

[0097] Figure 7 c shows Figure 7 a and Figure 7 The statistical graph for the three independent experiments is shown in Figure b. Statistical analysis was performed using an independent samples t-test.

[0098] Figure 7 d shows raw data from nano-flow cytometry (Nano-FCM), showing LPS in extracellular vesicles of plasma before 1% Triton X-100 treatment. + The number of particles.

[0099] Figure 7 e shows raw data from nano-flow cytometry (Nano-FCM), demonstrating LPS levels in extracellular vesicles of plasma after treatment with 1% Triton X-100. + The number of particles.

[0100] Figure 7 f shows Figure 7 d and Figure 7 Figure e shows the statistical graphs of the three independent experiments. Statistical analysis was performed using an independent samples t-test. In all graphs, ns indicates p > 0.05; ns indicates p < 0.05; ns indicates p < 0.01; ns indicates p < 0.001; **** indicates p < 0.0001.

[0101] Figure 8 The study showed that LTA levels in the plasma of mice co-infected with Staphylococcus aureus and Escherichia coli were elevated. + Detection of extracellular vesicles (EVs).

[0102] Figure 8 Image a shows an electron micrograph of extracellular vesicles in the plasma of co-infected mice co-incubated with LTA isotype antibody.

[0103] Figure 8 b shows an electron micrograph of extracellular vesicles in the plasma of co-infected mice co-incubated with LPS isotype antibody.

[0104] Figure 9The results of blood cultures in mice infected with a mixed bloodstream of Staphylococcus aureus and Escherichia coli are shown. The left panel shows the blood culture results of mice at the initial time of infection (0 hours); the right panel shows the blood culture results of mice 24 hours after infection. Note: The black shading visible on the back of the blood agar plates is a watermark indicating the production date and main components of the plates.

[0105] Figure 10 The study showed that LPS levels in the plasma of mice co-infected with Staphylococcus aureus and Escherichia coli were elevated. + Detection of extracellular vesicles (EVs).

[0106] Figure 10 Image a shows an electron micrograph of extracellular vesicles in the plasma of co-infected mice co-incubated with LTA isotype antibody.

[0107] Figure 10 b shows an electron micrograph of extracellular vesicles in the plasma of co-infected mice co-incubated with LPS isotype antibody.

[0108] Figure 11 The skin-bloodstream infection model showed LTA + / LPS + Dynamic changes in extracellular vesicles (EVs) and inflammatory markers

[0109] Figure 11 Figure 1 shows a schematic diagram of a mouse skin-bloodstream infection model. This figure illustrates the process of constructing the mouse skin-bloodstream infection model.

[0110] Figure 11 b shows the blood culture results at different time points after Staphylococcus aureus skin infection in mice.

[0111] Figure 11 c shows the blood culture results at different time points after Escherichia coli infection in mice.

[0112] Figure 11 d shows the LTA levels in the blood of mice after Staphylococcus aureus infection. + Dynamic changes in the proportion of extracellular vesicles.

[0113] Figure 11 e shows the dynamic changes in C-reactive protein (CRP) levels in the blood of mice after Staphylococcus aureus infection.

[0114] Figure 11 f shows the dynamic changes in the level of interleukin-6 (IL-6) in the blood of mice after Staphylococcus aureus infection.

[0115] Figure 11g shows the LPS levels in the blood of mice at different time points after E. coli infection. + Dynamic changes in the proportion of extracellular vesicles.

[0116] Figure 11 h shows the dynamic changes in C-reactive protein (CRP) levels in the blood of mice after Escherichia coli infection (similar to (11e)).

[0117] Figure 11 i shows the dynamic changes in the level of interleukin-6 (IL-6) in the blood of mice after Escherichia coli infection (similar to (11f)).

[0118] Data are expressed as mean ± standard deviation, and the sample size (n) is 3.

[0119] Note: The dark shadow visible behind the blood agar plate is a watermark, indicating the plate's production date and main ingredients.

[0120] Figure 12 LTA was displayed + / LPS + The diagnostic value of extracellular vesicles in clinical bacterial infections of Staphylococcus aureus and Escherichia coli.

[0121] Figure 12 a shows the workflow for processing human blood samples.

[0122] Figure 12 b shows the LTA levels in plasma samples from different patients. + Nanoflow cytometry analysis of EV proportions.

[0123] Figure 12 c shows LPS in plasma samples from different patients. + Nanoflow cytometry analysis of EV proportions.

[0124] Figure 12 d shows the C-reactive protein (CRP) levels in plasma samples from different patients.

[0125] Figure 12 e shows the neutrophil levels in plasma samples from different patients.

[0126] in, Figure 12 b、 Figure 12 c. Figure 12 d、 Figure 12 The statistical analysis in e uses one-way ANOVA.

[0127] Figure 12 b、 Figure 12 c. Figure 12 d、 Figure 12Sample size for e: Staphylococcus aureus bloodstream infection (n=17), Escherichia coli bloodstream infection (n=16), viral infection (n=15), Crohn's disease (n=15), healthy control group (n=15).

[0128] All data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0129] Figure 12 f shows 11 days of continuous monitoring in patients with Staphylococcus aureus bloodstream infection, including LTA. + The percentage of EVs, CRP levels, white blood cell (WBC) count, and body temperature. In the chart, body temperature data is plotted on the right vertical axis, while all other parameters are plotted on the left vertical axis. LTA + The proportion of EVs is expressed as a percentage (%), CRP levels as milligrams per milliliter (mg / ml), and WBC counts as billions per liter (10^9 / L). All data are expressed as mean ± standard deviation. Sample size (n) = 5.

[0130] Figure 12 g shows 11 days of continuous monitoring in patients with E. coli bloodstream infection, including LPS + EVs, CRP levels, WBC count, and body temperature. This chart is constructed in a manner consistent with ( Figure 12 f) Same, body temperature data is on the right vertical axis, and other parameters are on the left. LPS + The units of EVs ratio, CRP level, and WBC count are related to ( Figure 12 Consistent with f). All data are expressed as mean ± standard deviation. Sample size (n) = 5.

[0131] Figure 12 h shows the receiver operating characteristic (ROC) curve used to evaluate LTA. + EVs and LPS + Diagnostic performance of EVs in bloodstream infections. Detailed Implementation

[0132] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that LTA plays a crucial role in the early stages of bacterial inflammation. + / LPS + Extracellular vesicles can serve as biomarkers to effectively differentiate between viral and bacterial infections, and further, between Gram-positive and Gram-negative bacterial infections. More importantly, LTA... + / LPS +The detection of extracellular vesicles is of great significance for the diagnosis of meningitis, pneumonia, and bacteremia, and holds promise as an auxiliary tool for the diagnosis of these diseases. This invention was developed based on this understanding.

[0133] the term

[0134] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0135] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0136] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0137] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value 20, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered by this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0138] As used in this article, “bloodstream infection,” “bacteremia,” and “sepsis” can be used interchangeably, all referring to infections caused by pathogens in the blood and their associated systemic inflammatory responses.

[0139] As used herein, the term "LTA" + / LPS + "Extracellular vesicles" refers to LTA + Extracellular vesicles and / or LPS + Extracellular vesicles. In a specific embodiment, the "LTA" + / LPS + Extracellular vesicles contain LTA + Extracellular vesicles, LPS + Extracellular vesicles, or LTA + and LPS + Extracellular vesicles.

[0140] Bacterial infection

[0141] Bacterial infections remain a leading cause of high morbidity and mortality rates worldwide, with sepsis being the most severe clinical manifestation of bacterial invasion. Despite significant advancements in sepsis diagnostic techniques, a rapid, sensitive, and specific gold standard diagnostic method is still lacking.

[0142] Bacterial culture, as a traditional diagnostic method, takes too long to produce results. Studies have shown that as many as 70.1% of bloodstream infection cases cannot be identified in a timely manner.

[0143] A retrospective analysis of 282 blood culture specimens from 196 patients with bloodstream infections in the intensive care unit (ICU) showed that the median time to positive detection was day 4 after admission.

[0144] The emergence of molecular biology techniques, especially polymerase chain reaction (PCR) and metagenomic sequencing, has revolutionized pathogen identification through rapid and accurate nucleic acid detection, thereby improving diagnostic sensitivity and expanding the range of detectable pathogens. However, molecular biology techniques face numerous challenges and limitations in practical applications. The complex sample collection and processing procedures are a primary concern, requiring efficient extraction and purification of bacterial nucleic acids from patient samples. Furthermore, in specific situations such as early infection or after antibiotic treatment, the sensitivity and specificity of detection may be affected due to low pathogen nucleic acid loads in the blood. Moreover, current technologies struggle to fully represent the host's inflammatory response, which is crucial for developing personalized treatment plans.

[0145] Observational studies have shown that for every hour the administration of antibiotics is delayed in ICU patients, the mortality rate increases by 1.5%. Notably, existing diagnostic techniques based on mononuclear cell volume have failed to shorten the diagnostic window for sepsis.

[0146] Accurate differentiation between bacterial and viral infections is the cornerstone of the rational use of antimicrobial drugs in clinical practice. Its core significance lies in guiding initial treatment decisions, avoiding antibiotic overuse, and thus curbing the global spread of antibiotic resistance. Bacterial infections typically require targeted antibiotic treatment, while the vast majority of viral infections are self-limiting diseases, ineffective with antibiotics, and only require symptomatic support or specific antiviral drugs. Misdiagnosing a viral infection as a bacterial infection and administering antibiotics not only fails to provide clinical benefit but also causes adverse drug reactions, disrupts the patient's gut microbiota balance, increases medical costs, and, most importantly, exerts selective pressure, promoting the generation and spread of drug-resistant strains. Furthermore, differentiating between the two helps in determining disease prognosis (viral infections are usually self-limiting, while bacterial infections may progress) and implementing appropriate infection control measures (such as different isolation methods). To aid in differentiation, clinicians often rely on biomarkers such as procalcitonin (PCT), which significantly increases in bacterial infections but does not increase or only slightly increases in viral infections, effectively guiding the initiation or discontinuation of antibiotics. However, a single differentiation method cannot be cross-validated, easily leading to misdiagnosis or missed diagnosis. Secondly, bacterial infections cause highly heterogeneous diseases, and multi-dimensional information helps in more accurate assessment and prognosis. Therefore, providing a way to assist in differentiating between viral and bacterial infections could effectively reduce or mitigate treatment delays, erroneous medical decisions, waste of medical resources, and worsening patient outcomes.

[0147] After confirming a bacterial infection, further differentiation is needed to target Gram-positive bacteria (G bacteria). + The presence of Gram-negative bacteria (G-) in infections is of vital immediate importance in guiding the selection of empirical antibiotic therapy, understanding the pathogenesis, and predicting clinical prognosis.

[0148] G + Gram-negative bacteria and Gram-negative bacteria differ fundamentally in their cell wall structure, leading to differences in their susceptibility to antibiotics. For example, vancomycin is a Gram-negative antibiotic used to treat methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-negative bacteria. + This is a key drug for bacteria, but ineffective against most Gram-negative bacteria; while third-generation cephalosporins are effective against many Gram-negative bacteria, but not against some Gram-negative bacteria. + Bacteria (such as enterococci) have limited effectiveness. Before obtaining pathogen culture and drug sensitivity results (usually requiring 48-72 hours), physicians must immediately select antibiotics that cover the most probable pathogen, based on the site of infection, patient risk factors, and local epidemiological data. Incorrect selection can delay treatment, leading to infection worsening or even death. Furthermore, rapid differentiation of Gram-negative bacteria is crucial. + Gram-negative bacteria and Gram-negative bacteria are crucial.

[0149] Bacterial vesicles

[0150] In recent years, bacterial extracellular vesicles (EVs) have been found to contain pathogen-specific components such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), proteins, nucleic acids, and metabolites, which remain stable in the bloodstream even under antibiotic stress.

[0151] Extracellular vesicles of microorganisms have been detected in a variety of clinical samples, including blood, urine, and cerebrospinal fluid. Techniques such as enzyme-linked immunosorbent assay (ELISA), nanoflow cytometry, mass spectrometry, and nucleic acid detection have been applied to characterize extracellular vesicles of microorganisms in infectious diseases.

[0152] Today, nanoflow cytometry enables single-particle resolution quantitative analysis of microbial extracellular vesicles in clinical samples, facilitating rapid, phenotype-related pathogen detection. These advances highlight the potential of microbial extracellular vesicle-based detection methods to shorten diagnostic time windows and improve early decision-making.

[0153] With the continuous advancement and innovation of nanoflow cytometry technology, we are now able to precisely characterize the molecular features of extracellular vesicles at the single-particle level. However, despite the presence of LPS in plasma... + / LTA + The detection of dynamic changes in EV numbers holds great promise, but the level of LPS in plasma... + / LTA + The specific correlation between dynamic changes in EV numbers and the disease infection process and outcome remains largely unexplored.

[0154] The main advantages of this invention include:

[0155] (a) This invention provides LTA + EVs and LPS + The application of EVs in detecting bacterial infections and effectively distinguishing between Gram-positive and Gram-negative bacterial infections provides an auxiliary diagnostic method for the diagnosis, prognosis, and disease surveillance of bacterial infections.

[0156] (b)LTA + / LPS + Extracellular vesicles appear earlier than bacteria enter the bloodstream, therefore LTA + / LPS + The proportion of extracellular vesicles can serve as an early marker of infection; the method of this invention can provide diagnostic results in about 3 hours, making up for the long time required for blood culture. In addition, it has the advantages of low cost and ease of operation.

[0157] (c) The method of the present invention is of great significance for the diagnosis of pneumonia, meningitis and bloodstream infections. It has a low false positive rate, high detection rate and high detection sensitivity. It can also diagnose pneumonia and meningitis when bacteria cannot be detected in blood culture.

[0158] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0159] Example 1: Materials and Methods.

[0160] 1) Bacterial culture

[0161] The bacterial strains used in this invention include Gram-positive and Gram-negative bacteria.

[0162] Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae.

[0163] Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii.

[0164] When culturing Gram-positive bacteria, tryptic soy broth (TSB) was used as the culture medium. The bacteria were cultured at 37°C with shaking at 200 rpm to ensure uniform growth under optimal temperature and aeration conditions.

[0165] When culturing Gram-negative bacteria, Luria-Bertani (LB) medium is used. The bacteria are also cultured at 37°C with shaking at 200 rpm to promote rapid bacterial proliferation and growth.

[0166] 2) Density gradient centrifugation was used to separate Staphylococcus aureus cell membrane vesicles (S. aureus-CMVs) and Escherichia coli outer membrane vesicles (E. coli-OMVs).

[0167] Staphylococcus aureus and Escherichia coli were cultured overnight in TSB / LB medium at 37°C and 200 rpm with shaking. The overnight culture was diluted 1:100 into fresh TSB / LB medium and cultured for another 8 hours under the same conditions to reach the logarithmic growth phase and promote the secretion of Staphylococcus aureus cell membrane vesicles and Escherichia coli outer membrane vesicles. The bacterial culture was centrifuged at 4°C and 4000×g for 30 minutes (Beckman Coulter Allegra X-15R centrifuge) to precipitate the bacterial cells. The resulting supernatant was filtered through a 0.22 μm pore size filter membrane (Millibert & Co., #SLGPR33RB) to remove residual bacteria. The clarified supernatant was concentrated using an Amicon Ultra centrifuge filter (molecular weight cutoff 100 kDa; Millibert & Co., UFC9100) to reduce volume and remove small molecules and free proteins. First, the concentrated sample was ultracentrifuged for 4 hours at 174,900 × g using an SW 32Ti rotor (Optima XE-90 centrifuge, Beckman Coulter, USA) at 4°C to precipitate total vesicles. The precipitate was resuspended in 50% OptiPrep (iodixanol) solution (Sigma-Aldrich, Germany, #D1556), and then successively covered with 40% and 10% OptiPrep solution to establish a discontinuous density gradient. The sample was then ultracentrifuged for 16 hours at 200,000 × g using an SW 41Ti rotor (Beckman Coulter, USA). Visible bands at the interface between the 10% and 40% OptiPrep layers were carefully aspirated and transferred to clean centrifuge tubes, stored at -80°C for subsequent experiments.

[0168] 3) Differential centrifugation method for separating plasma extracellular vesicles (EVs)

[0169] Peripheral venous blood was collected from patients (2 mL) and mice (500 μL), respectively, and placed in tubes coated with EDTA for processing over 2 hours. Plasma was obtained by centrifugation at 1000 × g for 10 minutes at 4 °C, followed by two centrifugations at 2500 × g for 15 minutes each at 4 °C to remove residual cells, platelets, and debris. The supernatant was centrifuged at 10,000 × g for 20 minutes at 4 °C to remove apoptotic bodies and large vesicles, then filtered through a 0.45 μm filter and ultracentrifuged at 100,000 × g for 2 hours at 4 °C. The precipitate was washed once with phosphate-buffered saline under the same conditions, resuspended in 100 μL PBS, aliquoted, and stored at -80 °C.

[0170] 4) Nanoflow cytometry detection of extracellular vesicles carrying lipoteichoic acid (LTA) and lipopolysaccharide (LPS)

[0171] Vesicles carrying LTA or LPS are called "LTA". + "EVs" and "LPS" + "EVs", not "LTA" + CMVs or LPS + "OMVs" are extracellular vesicles derived from plasma, and without direct observation, they cannot be definitively classified into a specific bacterial subtype.

[0172] Under standardized conditions, extracellular vesicles were immunostained and analyzed using a nano-flow cytometry system (Nano-Flow Cytometry, Nano-FCM, model N30E). All samples were diluted to 5 × 10⁻⁶ with phosphate-buffered saline (PBS) before antibody incubation. 8 Particles / mL (reaction volume is 100 μL, approximately 5 × 10⁻⁶ particles / mL, each assay is approximately 5 × 10⁻⁶). 7 To ensure the same antigen-to-antibody ratio in each experiment, the experimental samples were aliquoted and incubated at 37°C for 60 minutes with either rabbit polyclonal anti-LTA primary antibody (LifeSpan Biosciences, LS-C760114) or rabbit polyclonal anti-LPS primary antibody (LifeSpan Biosciences, LS-C343196). Excess primary antibody was removed by two consecutive rounds of ultracentrifugation at 120,000×g for 60 minutes at 4°C using an SW55Ti rotor. The precipitate was gently resuspended in 100 μL of PBS and then incubated at 37°C for 1 hour with Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (Abcam, ab150113). Two more rounds of ultracentrifugation were performed under the same conditions to remove unbound secondary antibody, and finally the precipitate was resuspended in 20 μL of PBS. Simultaneously, negative controls were set up and treated in parallel, including: (1) extracellular vesicles incubated with secondary antibody only; (2) extracellular vesicles incubated with primary antibody only; and (3) extracellular vesicles incubated with isotype-matched IgG (Life Span Biosciences, LS-C292295) and then incubated with secondary antibody.

[0173] Each sample was precisely analyzed using nanoflow cytometry, detecting 10,000 single extracellular vesicle particles. The sheath fluid flow rate was set to 40 pL / min, the sample flow rate to 2 nL / min, and the detection volume to 25 fL. Absolute particle counts were converted to a percentage of positive events using calibration curves generated from 100 nm uncoated fluorescent polystyrene microspheres. All samples were analyzed under the same instrument settings to ensure comparability between samples.

[0174] 5) Observation using transmission electron microscopy (TEM)

[0175] 10 μL of extracellular vesicle suspension was dropped onto a 200-mesh carbon-coated copper grid and allowed to adsorb for 1 minute at room temperature. Excess liquid was blotted off with filter paper, and the vesicle was fixed with 0.4 μL of 25% glutaraldehyde for 60 seconds. It was then negatively stained with 2% uranium acetate for 90 seconds, air-dried, and observed using a Tecnai G2 Spirit BioTWIN transmission electron microscope. Images were acquired using a Megaview G2 CCD camera and processed using Adobe Photoshop.

[0176] 6) Immunoelectron microscopy (IEM) observation

[0177] Extracellular vesicles were mixed with 4% paraformaldehyde at a 1:1 (v / v) ratio, and 10-20 μL of the mixture was dropped onto a Parafilm membrane. A carbon-coated copper mesh (membrane side down) was placed on the droplet for 20 minutes to capture the extracellular vesicles, and then the following steps were performed: (1) Washing: Wash twice with 100 μl PBS for 3 minutes each time; (2) Aldehyde quenching: Wash three times with 50 mM glycine for 3 minutes each time; (3) Blocking: Block overnight at 4°C with 5% bovine serum albumin (BSA); (4) Primary antibody incubation: Incubate at room temperature with blocking buffer diluted 1:20 with mouse anti-LPS (Novus, catalog number #NB100-62450) or anti-LTA ( (5) Washing: Wash six times with PBS, 3 minutes each time; (6) Secondary antibody incubation: Incubate with colloidal gold-labeled secondary antibody (Abcam, #ab29619; 1:200) for 1 hour at room temperature; (7) Washing: Wash six times with 0.5% BSA, 3 minutes each time, then wash six times with PBS, 2 minutes each time; (8) Post-fixation: Fix with 1% glutaraldehyde for 2 minutes; (9) Final rinse: Rinse six times with double-distilled water, 2 minutes each time. Counterstain copper mesh with 2% uranium acetate for 90 seconds, air dry, and image according to the description of transmission electron microscopy observation.

[0178] 7) Detection experiment of extracellular vesicle-induced cytokine release

[0179] RAW264.7 macrophages (2 × 10⁶ cells per well) 5 Cells were cultured overnight in serum-free medium (approximately 1 × 10⁶ cells / well) in a humidified environment at 37°C with 5% carbon dioxide, and then exposed to 10 μg / mL of purified extracellular vesicles (approximately 1 × 10⁶ cells / well). 9Cells were cultured in phosphate-buffered saline (PBS, negative control) or an equal volume of phosphate-buffered saline (PBS) for 12 hours. After treatment, the culture supernatant was collected by centrifugation at 300×g for 5 minutes at 4°C to remove cell debris. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the culture supernatant were quantified using a commercially available mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems; IL-6 assay kit: M600B, TNF-α assay kit: MTA00B) according to the manufacturer's instructions.

[0180] 8) Gene expression analysis using quantitative polymerase chain reaction (qPCR).

[0181] Total RNA was isolated from cells using the TRIzol-chloroform method, followed by precipitation with isopropanol, washing with 75% ethanol, and resuspending in diethyl pyrocarbonate (DEPC)-treated water. RNA concentration and purity (A) were assessed using a NanoDrop spectrophotometer. 260 / A 280 (Ratio between 1.9 and 2.1), using only A. 260 / A 280 Samples with a ratio between 1.9 and 2.1 should be analyzed further. RNA samples can be reverse transcribed immediately or stored at -80°C until use.

[0182] During cDNA synthesis, 1 μg of total RNA was reverse transcribed using the HiScript II Reverse Transcription Ultramix Kit (Vazyme, R222-01; Vazyme Biotechnology Co., Ltd., Nanjing, China) according to the manufacturer's instructions. Quantitative PCR was performed in a 20 μL reaction system using reagents containing SYBR Green Master Mix (Vazyme, Q711-02) on a Roche LightCycler 96 system under the following cycling conditions: 95°C pre-denaturation for 30 seconds; 40 cycles of 95°C denaturation for 5 seconds, followed by 60°C annealing and extension for 30 seconds; subsequently, melting curve analysis was performed from 65°C to 95°C. The primer sequences are as follows: Interleukin-6 (IL-6): forward primer 5'-GAGGATACCACTCCCAACAGACC-3' (SEQ ID NO:1), reverse primer 5'-AAGTGCATCATCGTTGTTCATACA-3' (SEQ ID NO:2); Tumor necrosis factor-α (TNF-α): forward primer 5'-TCCTTCTCATTCCTGCTTGTGG-3' (SEQ ID NO:3), reverse primer 5'-GGTCTGGGCCATAGAACTGA-3' (SEQ ID NO:4); β-actin: forward primer 5'-GGAGGGGGTTGAAGGTCTTTA-3' (SEQ ID NO:5), reverse primer 5'-AGGTGTAAAACGCAGCTCAGTA-3' (SEQ ID NO:6). Relative expression levels were standardized using β-actin as an internal reference and calculated using the 2^-ΔΔCt method; all qPCR reactions were performed in triplicate.

[0183] 9) BALB / c mouse pneumonia model

[0184] Prepare bacterial suspensions of Staphylococcus aureus or Escherichia coli (4 × 10⁻⁶) using sterile phosphate-buffered saline (PBS). 8 (CFU / mL). Gently restrain the mouse and use a pipette to drop 20 μL of the suspension into the mouse's left nostril. After the drop, keep the mouse upright for about 1 minute to promote the distribution of the suspension.

[0185] Mice were then divided into nine post-infection time groups: 0 hours (healthy control group), 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, and 72 hours (n=6 per group). At each time point, 1 mL of blood was collected from the posterior ocular venous plexus into a test tube coated with ethylenediaminetetraacetic acid (EDTA). The mice were then euthanized by carbon dioxide inhalation.

[0186] Half (500 μL) of each blood sample was inoculated onto blood agar plates and incubated at 37°C for 24 hours to assess bacterial growth. The remaining 500 μL of blood was used to isolate extracellular vesicles (EVs) from the plasma using differential centrifugation. The isolated EVs were stored at -80°C until further analysis.

[0187] 10) BALB / c mouse meningitis model

[0188] Anesthetize the mouse until its reflexes are completely lost, then place the mouse on a stereotactic surgical platform. Make a precise skin incision 1-2 mm posterior to the anterior fontanelle and approximately 1 mm lateral to the midline. Using ophthalmic scissors, drill a small hole in the skull, taking care to avoid damaging brain tissue. With the aid of a needle holder, gently insert the needle into the hole and accurately advance it to the lateral ventricle of the brain.

[0189] Mice were randomly assigned to experimental groups (n=6 per group). 2 μL of solution containing 1×10⁻⁶ mice was administered. 9 A bacterial suspension of CFU (Cytocinonized Fusarium oxysporum) or Escherichia coli was slowly injected into the lateral ventricle of the brain. Control animals were injected with an equal volume of sterile PBS. After injection, the needle was carefully withdrawn to minimize cerebrospinal fluid leakage and prevent air bubbles from forming within the cranial cavity. The cranial foramen and skin incision were carefully sutured to close the surgical site. Antibiotic ointment was applied to the wound to prevent postoperative infection. Twenty-four hours after injection, 1 ml of blood was collected via retroocular vein puncture, and the mice were then euthanized to collect brain tissue. The blood sample was divided into two aliquots: 500 μL was used for culture to confirm asepticity, and the remaining 500 μL was used for differential centrifugation to separate extracellular vesicles (EVs). The purified EVs were stored at -80°C for subsequent analysis. The brain tissue was fixed and stained with hematoxylin and eosin (H&E) to assess bacterial infection and histopathological status.

[0190] 11) BALB / c mouse bacteremia model

[0191] A sepsis model was established in BALB / c mice via posterior ocular vein injection. Under anesthesia, bacteria (Staphylococcus aureus ATCC25923 or Escherichia coli ATCC25923) were injected into the posterior ocular venous sinus of the mice at a concentration of 10 at a 45° angle. 8 CFU / ml. Each experimental group consisted of six mice, with the same number of healthy mice serving as a control group.

[0192] Twenty-four hours after injection, 1 ml of blood was collected from each mouse via posterior ocular vein puncture and immediately transferred to EDTA-coated tubes. The mice were then euthanized. Of the collected blood, 500 μL was used for bacterial culture to assess the success of the sepsis model, and the remaining 500 μL was used to isolate extracellular vesicles (EVs) from mouse plasma. The isolated EVs were stored at -80°C for subsequent analysis.

[0193] 12) BALB / c mouse model of invasive bloodstream infection of the skin

[0194] A skin abscess model was established in BALB / c nude mice via subcutaneous injection. After anesthetizing the mice, the skin at the injection site was gently lifted, and a sterile needle was inserted into the subcutaneous tissue with the needle facing upwards. Injection was rapid to prevent leakage of the bacterial suspension. A standardized dose of 200 μL suspension containing 2 × 10⁻⁶ bacteria was used in this experiment. 8 CFU / ml of Staphylococcus aureus or Escherichia coli. Control mice were injected with an equal volume of PBS.

[0195] Mice were then randomly divided into 13 groups (n=6 per group). At specified 2-hour intervals, 1 mL of blood was collected from each group of mice via retroocular vein puncture, and the mice were then euthanized. Of the collected blood, 500 μL was used for bacterial culture to monitor infection status, and the remaining 500 μL was used to measure and record the levels of inflammatory factors in the plasma. Extracellular vesicles (EVs) were isolated from mouse plasma using differential centrifugation. The isolated EVs were aliquoted and stored at -80°C for subsequent analysis.

[0196] Example 2: Expression characteristics of lipopolysaccharide (LPS) on outer membrane vesicles (OMVs) and lipoteichoic acid (LTA) on cytoplasmic membrane vesicles (CMVs).

[0197] outer membrane vesicles (OMVs) were isolated from the Gram-negative bacterium *Escherichia coli* (E. coli), and cytoplasmic membrane vesicles (CMVs) were isolated from the Gram-positive bacterium *Staphylococcus aureus* (S. aureus) using density gradient centrifugation.

[0198] Figure 1 This study showcases the morphology and structure of Staphylococcus aureus cell membrane vesicles (S. aureus-CMVs). Nanoflow cytometry (nano-FCM) further reveals that the vesicle size is primarily concentrated in the 50-80 nanometer range. Figure 1 b).

[0199] The morphology and structure of E. coli outer membrane vesicles (E. coli-OMVs) are as follows: Figure 1 As shown in c, nanoflow cytometry results indicate that the vesicle size is mainly 75-120 nanometers. Figure 1 d).

[0200] Immunoelectron microscopy (IEM) further confirmed the specific expression of surface markers on the cytoplasmic membrane vesicles of Staphylococcus aureus and the outer membrane vesicles of Escherichia coli. Specifically, such as Figure 1 h、 Figure 1 As shown in i, lipopolysaccharide (LPS) antigen is significantly expressed on the surface of Escherichia coli outer membrane vesicles; Figure 1 e Figure 1 f indicates that Staphylococcus aureus cell membrane vesicles significantly express lipoteichoic acid (LTA) antigen.

[0201] In addition, a comprehensive analysis was performed using nanoflow cytometry to quantify LTA (long-term active ingredient) on the plasma membrane vesicles of Gram-positive bacteria. Figure 1 g) and LPS on the outer membrane vesicles of Gram-negative bacteria ( Figure 1 The expression level of LPS was analyzed. The scope of analysis was expanded to include outer membrane vesicles of Gram-negative bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa), cytoplasmic membrane vesicles of Gram-positive bacteria (Staphylococcus epidermidis and Streptococcus pneumoniae), and extracellular vesicles (EVs) of human A549 cells and mouse NIH-3T3 cells. Nanoflow cytometry results showed that among the outer membrane vesicles secreted by different Gram-negative bacteria, LPS-positive (LPS) expression levels were significantly higher. + The proportion of extracellular vesicles ranged from 24.8% to 40.2%. Figure 1 g and Figure 2 a). This proportion is significantly higher than that detected in mammalian cells (1.3% to 1.7%) and Gram-positive bacterial strains (1.6% to 2.0%). Conversely, for cytoplasmic membrane vesicles secreted by Gram-positive bacteria, LTA-positive (LTA) + The proportion of extracellular vesicles ranged from 30.6% to 48.3%. Figure 1 j and Figure 2 b). LTA is almost undetectable in the outer membrane vesicles of Gram-negative bacteria and the extracellular vesicles of mammalian cells, with detection rates of only 1.6% to 2.2% and 0.6% to 1.5%, respectively.

[0202] These findings suggest that LPS and LTA can serve as potential biomarkers for outer membrane vesicles of Gram-negative bacteria and cytoplasmic membrane vesicles of Gram-positive bacteria, respectively, and have broad prospects for future applications.

[0203] Example 3: LPS + / LTA + Interactions between extracellular vesicles (EVs) and host cells.

[0204] To explore LPS in depth + / LTA + To investigate whether extracellular vesicles can induce inflammatory responses or regulate cell function, this study used the mouse mononuclear / macrophage cell line RAW264.7 (ATCC TIB-71) as an experimental model.

[0205] The first step of the experiment was to extract cell membrane vesicles (CMVs) from Staphylococcus aureus and outer membrane vesicles (OMVs) from Escherichia coli, obtaining LTA from each. + Extracellular vesicles and LPS +Extracellular vesicles. After extraction, these LTAs... + / LPS + Extracellular vesicles and mouse macrophages were co-incubated for 12 hours under standard macrophage culture conditions (37°C, 5% CO2) to fully observe their interactions.

[0206] To quantitatively assess the impact of these vesicles on macrophage inflammatory responses, enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of key pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant of macrophage cultures treated with LTA / LPS. LTA / LPS-treated macrophages release large amounts of IL-6 and TNF-α; by detecting and measuring the concentrations of these cytokines, the impact of vesicles on macrophage inflammatory responses can be quantitatively assessed.

[0207] like Figure 3 a and Figure 3 As shown in b, compared with the control group, LPS + / LTA + The levels of IL-6 and TNF-α were significantly increased in the culture supernatant of macrophages treated with extracellular vesicles, which strongly suggests that LPS + / LTA + Extracellular vesicles can activate macrophages and trigger inflammatory responses.

[0208] In addition, monitoring was also conducted via LPS. + / LTA + Changes in the expression of inflammation-related genes in macrophages after treatment with extracellular vesicles. Specifically, the expression of macrophages treated with LPS was examined. + Extracellular vesicles and LTA + Expression of TNF-α and IL-6 genes in macrophages after treatment with extracellular vesicles.

[0209] like Figure 3 c and Figure 3 As shown in d, the experimental data clearly demonstrate that the expression of these genes is upregulated after vesicle treatment, further confirming that these vesicles have immunostimulatory properties.

[0210] The results of this embodiment show that LPS + / LTA + Extracellular vesicles significantly influence the host immune response by activating immune cells, providing strong evidence for their potential role as inflammatory biomarkers.

[0211] Example 4: LPS in the plasma of infected animals + / LTA + Dynamic changes of extracellular vesicles.

[0212] To further investigate the level of LTA in plasma during bacterial infection + / LPS + The dynamic changes in the proportion of extracellular vesicles were investigated using two common clinical pathogens: Staphylococcus aureus (Gram-positive cocci) and Escherichia coli (Gram-negative bacilli). Mouse models of pneumonia, meningitis, and bloodstream infection were established. Twelve hours after infection, mouse blood samples were collected and divided into two parts: one part was used for blood culture to confirm the infection status, and the other part was used to monitor LPS levels in the plasma using nano-flow cytometry (nano-FCM). + Extracellular vesicles and LTA + The level of extracellular vesicles.

[0213] Bacterial culture results showed that pneumonia ( Figure 4 a and Figure 4 c) and meningitis Figure 4 b and Figure 4 d) No bacteria were detected in the blood of mice, but bacteria were cultured from the blood of mice in a bloodstream infection model. Figure 4 e and Figure 4 f).

[0214] To further confirm the successful establishment of the pneumonia and meningitis models, lung and brain tissues were collected from the corresponding mice and stained with hematoxylin and eosin (H&E). The staining results showed that the lungs ( Figure 5 a and Figure 5 b) and brain tissue ( Figure 5 c and Figure 5 d) A significant inflammatory response was observed at the bacterial inoculation site.

[0215] Another portion of the blood samples was used to extract plasma extracellular vesicles, which were then analyzed using nanoflow cytometry. The results showed that LPS was detected in the plasma samples from infected mice. + / LTA + Extracellular vesicles, and the infection group LTA + / LPS + The level of extracellular vesicles was significantly higher in the bloodstream infection group than in the uninfected group. LTA + / LPS + The level of extracellular vesicles was higher in the local infection group than in the local infection group (see...) Figure 6 a and Figure 6 b). Specifically, as shown in Tables 1 and 2.

[0216] Table 1. LTA levels in the blood of mice infected with E. coli + / LPS + The proportion of extracellular vesicles (EVs)

[0217] pneumonia meningitis bacteremia E. coli 14.5％ 10.2％ 26.0％ Staphylococcus aureus 1.01％ 3.80％ 0.87％ Normal 1.57％ 1.60％ 1.20％ E. coli / Normal 9.2 6.3 21.7

[0218] Table 2. LTA levels in the blood of mice infected with Staphylococcus aureus + / LPS + The proportion of extracellular vesicles (EVs)

[0219] pneumonia meningitis bacteremia E. coli 0.98％ 4.10％ 0.72％ Staphylococcus aureus 13.8％ 10.4％ 19.1％ Normal 1.45％ 1.50％ 1.43％ Staphylococcus aureus / Normal 9.5 6.9 13.4

[0220] To confirm that the LTA / LPS fluorescence signal detected by nanoflow cytometry originated from vesicles, the extracted vesicles were treated with 1% Triton X-100 (a nonionic surfactant). Triton X-100 has a structure consisting of a hydrophilic head and a hydrophobic tail, which can embed into the lipid bilayer and disrupt the membrane structure. + The results of the particles are as follows Figure 7 a and Figure 7 As shown in b, LPS + The results of the particles are as follows Figure 7 d and Figure 7 As shown in e. After processing, LTA + / LPS + The proportion of particles was reduced to approximately 2% of the pre-treatment level (see...). Figure 7 c and Figure 7 f). This finding indicates that most LTAs + or LPS + The particles do indeed originate from vesicles, not lipoproteins.

[0221] Immunoelectron microscopy (IEM) further revealed the presence of LPS in the plasma of infected mice. + or LTA + Extracellular vesicles (see) Figure 6 c and Figure 6 d). The isotype antibody control obtained by immunoelectron microscopy is shown in [reference needed]. Figure 8 a and Figure 8 b.

[0222] LPS was detected in a mixed bloodstream infection model of Gram-negative and Gram-positive bacteria. + Extracellular vesicles and LTA + The proportion of extracellular vesicles was increased (see...) Figure 6 (e and Table 3). Bacterial culture results are shown in [reference]. Figure 9 Immunoelectron microscopy showed that LPS was present in the plasma of mixed-infected mice. + Extracellular vesicles (see) Figure 6 f) and LTA + Extracellular vesicles (see) Figure 6 g) Coexistence. See the isotype antibody controls for these experiments. Figure 10 a and Figure 10 b.

[0223] Table 3. LTA in the mixed bloodstream infection model+ / LPS + The proportion of extracellular vesicles (EVs)

[0224] <![CDATA[LTA + EVs]]> <![CDATA[LPS + EVs]]> E. coli + Staphylococcus aureus 19.2％ 23.8％ E. coli 1.30％ 25.0％ Staphylococcus aureus 16.1％ 1.15％ Normal 0.92％ 1.60％

[0225] These results indicate that during local infection, bacteria at the site of infection can secrete vesicles into the bloodstream, increasing plasma LTA levels. + / LPS + The proportion of extracellular vesicles can serve as an indicator of bacterial infection. Compared to localized infections, patients with bloodstream infections have higher levels of LTA in their plasma. + / LPS + The proportion of extracellular vesicles was significantly increased, and therefore this proportion could serve as a potential biomarker for assessing the severity of bacterial infections.

[0226] Further investigation was conducted in nude mice using a skin-invasive bloodstream infection model, and Staphylococcus aureus and Escherichia coli were used to explore the effects of LTA during sepsis. + / LPS + Dynamic changes of extracellular vesicles. Experimental procedure is as follows: Figure 11 a. Within 24 hours of infection, blood samples were collected from mice every two hours for blood culture and plasma vesicle extraction, and the mice were then sacrificed for tissue collection.

[0227] Blood culture results showed that Staphylococcus aureus-infected nude mice developed bloodstream infection 12 hours after skin infection, while Escherichia coli-infected mice developed bloodstream infection 14 hours after infection (see...). Figure 11 b and Figure 11 c).

[0228] Monitoring of LTA in plasma vesicles over 24 hours using nanoflow cytometry + / LPS + The proportion of extracellular vesicles (results as follows) Figure 11 d and Figure 11 (as shown in g), while simultaneously measuring the inflammatory marker interleukin-6 (IL-6) (results are shown in g). Figure 11 f and Figure 11 The levels of C-reactive protein (CRP) (as shown in i) and C-reactive protein (CRP) (results are shown in...) Figure 11 e and Figure 11 (as shown in h). The results indicated that with prolonged infection time, plasma LTA levels decreased. + / LPS + The proportion of extracellular vesicles increased, consistent with the temporal patterns of early inflammatory markers IL-6 and CRP. Specifically, 2–4 hours post-infection, LTA… + / LPS + The proportion of extracellular vesicles begins to rise, while inflammatory markers only increase after 8-10 hours. After the infection progresses to bacteremia, plasma LTA levels...+ / LPS + The proportion of extracellular vesicles was significantly higher than in the early stages of infection.

[0229] LTA + / LPS + Extracellular vesicles appear before bacteria enter the bloodstream. LTA + / LPS + The proportion of extracellular vesicles can serve as an early marker of infection and reflect the intensity of inflammation. This finding provides new insights and methods for the early diagnosis and monitoring of sepsis.

[0230] Example 5: LTA in patients with bacterial infections + / LPS + Clinical analysis of extracellular vesicles.

[0231] To further explore LTA + / LPS + To explore the potential clinical value of extracellular vesicles in the diagnosis of bacterial infections, this embodiment collected 78 clinical blood samples for in-depth analysis.

[0232] These samples covered a variety of disease states, including 17 patients with Staphylococcus aureus bloodstream infection, 16 patients with Escherichia coli bloodstream infection, 15 patients with viral infection, 15 patients with Crohn's disease, and 15 healthy controls. Their clinical characteristics are shown in Table 4.

[0233] Table 4. Clinical characteristics of enrolled patients (n=78)

[0234]

[0235]

[0236] To ensure the homogeneity and comparability of the study, all samples were balanced in terms of gender and age. The processing procedures for each group of clinical samples are detailed below. Figure 12 a.

[0237] Nano-flow cytometry (Nano-FCM) analysis showed that in the Staphylococcus aureus bloodstream infection group, LTA + The number of extracellular vesicles was significantly higher in the E. coli bloodstream infection group, Crohn's disease group, virus infection group, and healthy control group. Figure 12 b). Conversely, in the E. coli bloodstream infection group, LPS + The number of extracellular vesicles was significantly higher than in other groups. Figure 12 c). These results highlight the specific differences in vesicle-positive markers under different types of bacterial infections.

[0238] Furthermore, it was observed that in the Staphylococcus aureus and Escherichia coli bloodstream infection groups, C-reactive protein (CRP) levels were higher. Figure 12 d) and neutrophil count ( Figure 12 e) were significantly higher than those in the virus-infected group and the healthy control group.

[0239] LTA + / LPS + The changing trends of extracellular vesicles are highly consistent with the changing trends of these commonly used clinical inflammatory markers, which further validates the reliability of vesicles as inflammatory biomarkers.

[0240] In addition, we clinically followed up 5 patients in the Staphylococcus aureus and Escherichia coli bloodstream infection group. The results showed that plasma LTA levels... + / LPS + The proportion of extracellular vesicles is correlated with changes in clinical indicators such as body temperature, white blood cell count, and inflammatory factors (see [link to relevant documentation]). Figure 12 f and Figure 12 g).

[0241] This discovery further highlights the importance of LTA. + / LPS + Extracellular vesicles have the potential value as an indicator of disease activity. Following effective clinical antibiotic intervention on day 5, all relevant indicators showed significant improvement. Plasma LTA... + / LPS + The proportion of extracellular vesicles changes dynamically with improvement in infection (see...). Figure 12 f and Figure 12 g).

[0242] To further explore LTA + Extracellular vesicles and LPS + The potential diagnostic applications of extracellular vesicles in bloodstream infections were further investigated by plotting receiver operating characteristic (ROC) curves. The results showed that LTA (Large-Terminal Acid) plays a significant role in the diagnosis of bloodstream infections. + The area under the curve (AUC) for extracellular vesicles reached 0.8013, while LPS... + Extracellular vesicles had a higher AUC, at 0.9350 ( Figure 12 h).

[0243] In summary, by employing nanoflow cytometry, this invention enables the study of LPS during infection at single-particle resolution. + / LTA + The dynamic changes of EVs should be studied to elucidate their dynamic behavior throughout the infection process as much as possible.

[0244] Specifically, this invention explores the diagnostic potential of plasma extracellular vesicles carrying bacterial lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Immunoelectron microscopy (IEM) and nano-flow cytometry (nano-FCM) confirmed the presence of LTA on cytoplasmic membrane vesicles (CMVs) of Gram-positive bacteria and the presence of LPS on outer membrane vesicles (OMVs) of Gram-negative bacteria. The levels of LTA-positive or LPS-positive LTA in plasma were also quantitatively measured. + / LPS + Extracellular vesicles.

[0245] Bacterial infection models showed that, compared with the control group, the infection group had lower LTA levels. + / LPS + Extracellular vesicle levels were higher and associated with elevated levels of inflammatory markers such as interleukin-6 (IL-6) and C-reactive protein (CRP). Clinical samples from patients with bloodstream infections also showed LTA. + / LPS + Extracellular vesicle levels were elevated. However, inflammatory markers such as interleukin-6 (IL-6) and C-reactive protein (CRP) were not effective in distinguishing between Gram-positive and Gram-negative bacteria, while LTA... + / LPS + Extracellular vesicle level can enable precision treatment.

[0246] These extracellular vesicles can distinguish between bacterial and non-bacterial infections, differentiate between Gram-positive and Gram-negative bacterial infections, and reflect disease progression and treatment response.

[0247] The research results of this invention show that LPS + / LTA + Extracellular vesicles (LTA) hold great potential as novel diagnostic and prognostic biomarkers, providing new methods for the early detection and management of bacterial infections. Through in-depth analysis of clinical samples, this study further validates the efficacy of LTA. + / LPS + The clinical value of extracellular vesicles in the diagnosis of sepsis. These findings deepen our understanding of the dynamic changes of extracellular vesicles during bacterial infection and provide new insights and methods for the early diagnosis, disease surveillance, and prognostic assessment of bacterial infections.

[0248] discuss

[0249] This invention systematically investigated the expression characteristics of lipoteichoic acid (LTA) in cell membrane vesicles (CMVs) of Gram-positive bacteria and lipopolysaccharide (LPS) in outer membrane vesicles (OMVs) of Gram-negative bacteria, and evaluated their potential in the diagnosis and prognostic assessment of bacterial infections. Using nanoflow cytometry (nano-FCM), we performed high-resolution, multi-parameter analysis on individual vesicles, sensitively detecting LTA even in samples with low bacterial loads. + and LPS + Extracellular vesicles (EVs).

[0250] Using "LTA" + "EVs" and "LPS" + The term "EVs" is used to describe plasma-derived extracellular vesicles carrying these bacterial components. Although immunoelectron microscopy (IEM) confirms that LTA is primarily associated with CMVs of Gram-positive bacteria and LPS is primarily associated with OMVs of Gram-negative bacteria, plasma-derived EVs can originate from diverse sources, including bacterial secretion, bacterial lysis, adsorption of bacterial components onto host EVs, or processing by immune cells. Given this heterogeneity and the inability to directly trace their origin without visualization, biomarker-based nomenclature more accurately reflects our detection methods and avoids over-interpreting their exact biogenetic processes.

[0251] This study contributes in the following ways: (i) directly comparing LTA in Gram-positive and Gram-negative bacterial infections. + EVs and LPS + (ii) Analyzing the dynamic changes of EVs in bloodstream infections, pulmonary infections, and meningitis; (iii) Performing rapid (within 3 hours) and highly sensitive single vesicle analysis using nano-FCM. This method, combining bacterial specificity, speed, and multi-parameter analysis, expands upon previous research.

[0252] Extracellular vesicles (UC-EVs) isolated by ultracentrifugation may contain heterogeneous mixtures of vesicles from host cells, bacteria, and even fungi. This complexity reflects the diversity of EV sources in biofluids. LTA and LPS are absent in mammalian cells, supporting their application as specific diagnostic targets; patients with nonbacterial gastrointestinal diseases exhibit only extremely low or undetectable LTA. + and LPS + EVs levels. Triton X-100 disruption assays further supported the specificity of these biomarkers, with >95% LTA remaining after membrane disruption. + / LPS +The disappearance of the particle signal confirmed their vesicle origin. Although potential cross-reactivity from non-target microbial EVs or structurally similar host components cannot be completely ruled out, plasma LTA... + / LPS + There is no doubt that particle signal detection can be used for rapid auxiliary diagnosis of meningitis, pneumonia, and bacteremia.

[0253] Even LTA + and LPS + The sensitivity of EV detection can be affected by a variety of factors, but the nano-FCM platform can still detect LTA in diluted clinical samples. + and LPS + EVs demonstrate strong analytical sensitivity. Notably, LPS + EVs have high diagnostic accuracy for Gram-negative bacteremia (AUC = 0.9350; sensitivity = 90–95%; specificity = 85–90%), while LTA... + EVs exhibit moderate but complementary performance against Gram-positive bacterial infections (AUC = 0.8013; sensitivity = 70–80%; specificity = 70–85%). Compared to traditional blood culture methods (high specificity but limited sensitivity, approximately 60–80%, and long turnaround time, >24 hours) and molecular detection methods such as PCR^52 (sensitivity up to 80–95%, but requiring prior identification of the target pathogen), the nano-FCM method enables rapid, broad-spectrum detection of microbial EVs without prior pathogen identification. Additional purification strategies, such as immunoaffinity separation or density gradient centrifugation, may further improve specificity.

[0254] Isolating pure microbial EVs remains a technical challenge. While ultracentrifugation is widely used, it can co-isolate heterogeneous EV populations. Additional enrichment methods, such as immunoaffinity capture or density gradient ultracentrifugation, may help improve purity and specificity, particularly for LTA. + or LPS + During vesicle formation. Future strategies may combine multiple methods, for example, first using size exclusion chromatography to remove large amounts of impurities, and then capturing LTA using antibody-based methods. + or LPS + EVs are used to achieve high purity and specificity. Combined with nano-FCM, direct, rapid, and multi-parameter analysis of enriched microbial EV populations can be achieved.

[0255] This invention emphasizes LTA + and LPS +EVs hold promise as rapid, specific, and sensitive biomarkers for bacterial infections, particularly in differentiating between Gram-negative and Gram-positive bacteremia. By addressing the complexities of UC-EVs, assessing their sensitivity and specificity, and proposing purification strategies, this study provides insights for practical diagnosis and a roadmap for future research aimed at clinical translation.

[0256] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An in vitro detection method for bacterial inflammation and / or bacteremia, characterized in that, Includes the following steps: (S1) Provide test samples from the test subjects; (S2) Isolate extracellular vesicles from the sample to be tested; (S3) Detection of LPS-positive extracellular vesicles (LPS) + The levels of EVs and / or LTA-positive extracellular vesicles (LTA) + The level of EVs; if LPS + EVs and / or LTA + An increase in EVs levels indicates that the subject has bacterial inflammation and / or bacteremia.

2. The in vitro detection method as described in claim 1, characterized in that, Step (S3) includes: comparing the LPS of the test samples. + Levels of EVs and LPS in normal control + The level of EVs, if the LPS of the sample to be tested + The level of EVs was significantly higher than that of LPS in the normal control group. + If the level of EVs is high, then the sample indicates bacterial inflammation and / or bacteremia due to Gram-negative bacterial infection. The normal control is a reference sample that is not infected with Gram-negative bacteria.

3. The in vitro detection method as described in claim 1, characterized in that, Step (S3) includes: comparing the LTA of the test samples. + Levels of EVs and LTA in normal controls + The level of EVs, if the LTA of the test sample + The level of EVs was significantly higher than that of LTA in the normal control group. + If the level of EVs is high, then the sample is infected with Gram-positive bacteria; The normal control is a reference sample that is not infected with Gram-positive bacteria.

4. The in vitro detection method as described in claim 1, characterized in that, The samples include: plasma, blood, saliva, urine, cerebrospinal fluid, feces, ascites, sputum, semen, breast milk, or sweat.

5. The in vitro detection method as described in claim 1, characterized in that, The subjects did not suffer from enteritis, colitis, or a combination thereof.

6. The in vitro detection method as described in claim 1, characterized in that, The subjects had an intact intestinal barrier.

7. The in vitro detection method as described in claim 1, characterized in that, The bacteremia described is not caused by the entry of intestinal flora into the bloodstream.

8. The in vitro detection method as described in claim 1, characterized in that, The bacterial inflammation includes: bacterial pneumonia, bacterial meningitis, or a combination thereof.

9. Detect LPS + EVs and / or LTA + The uses of EVs as reagents are characterized by, Used to prepare products for the detection of bacterial inflammation and / or bacteremia.

10. The use as described in claim 9, characterized in that, The detection of LPS + EVs and / or LTA + The reagents for EVs include: (i) Reagents for separating EVs; and (ii) Reagents for detecting LPS and / or LTA on the surface of EVs.

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