Diagnosing Sepsis or Bacteremia by Detecting Peptidoglycan Associated Lipoprotein (PAL) in Urine

Abstract

A method, device and kit for detecting sepsis or bacteremia in a patient includes detecting peptidoglycan associated lipoprotein (Pal) from Gram-negative bacteria in the urine of the patient is disclosed.

Claims

1. A method for detecting septic or bacteremic levels of Gram-negative bacteria in a patient, comprising: obtaining the urine of a human patient; exposing the urine to a Gram-negative peptidoglycan associated lipoprotein specific binding agent; and detecting the Gram-negative peptidoglycan associated lipoprotein bound to the binding agent.

2. The method of claim 1, wherein the binding agent comprises a polyclonal antibody, monoclonal antibody, antibody fragment or molecule that binds specifically to a peptidoglycan associated lipoprotein from Gram-negative bacteria.

3. The method of claim 1, wherein the Gram-negative peptidoglycan associated lipoprotein comprises Enterobacteriaceae peptidoglycan associated lipoprotein.

4. The method of claim 1, wherein the Gram-negative peptidoglycan associated lipoprotein specific binding agent comprises Enterobacteriaceae peptidoglycan associated lipoprotein specific binding agent.

5. The method of claim 4, wherein the Enterobacteriaceae peptidoglycan associated lipoprotein specific binding agent comprises mouse monoclonal anti-Pal antibody (6D7).

6. The method of claim 1, wherein detecting peptidoglycan associated lipoprotein bound to the binding agent comprises a visual determination.

7. The method of claim 1, further comprising filtering out whole cell bacteria from the urine prior to exposing the urine to the binding agent.

8. A device comprising: a test window and optionally, a control window; an absorbent strip; an immunoassay strip, comprising a Gram-negative peptidoglycan associated lipoprotein-specific binding agent and optionally, a control-specific binding agent; a container housing the absorbent and immunoassay strips; and a cap covering the absorbent strip.

9. The device of claim 8, further comprising a sealed package enclosing the device.

10. The device of claim 8, wherein the control-specific binding agent comprises a creatinine-specific binding agent.

11. The device of claim 8, further comprising: a standard curve comprising samples of peptidoglycan associated lipoprotein protein at known concentrations; an output that correlates to protein concentration; a similar measurement performed on patient urine, as well as a control protein sample; and a calculation, which uses the standard curve and the urine sample measurements to estimate the peptidoglycan associated lipoprotein concentration in the urine sample.

12. The device of claim 11, wherein the output comprises an absorbance of light.

13. A kit comprising: a device comprising: a test window and optionally, a control window, an absorbent strip, an immunoassay strip, comprising a Gram-negative peptidoglycan associated lipoprotein-specific binding agent and optionally, a control-specific binding agent, a container housing the absorbent and immunoassay strips, and a cap covering the absorbent strip; a sterile wipe and clean catch urine collection cup; and a syringe and filter for optional removal of whole bacterial cells from the urine.

14. The kit of claim 13, wherein the device further comprises: a standard curve comprising samples of peptidoglycan associated lipoprotein protein at known concentrations; an output that correlates to protein concentration; a similar measurement performed on patient urine, as well as a control protein sample; and a calculation, which uses the standard curve and the urine sample measurements to estimate the peptidoglycan associated lipoprotein concentration in the urine sample.

15. The kit of claim 14, wherein the output comprises an absorbance of light.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows a Pal immunoblot of urine in accordance with the present disclosure; and

[0017] FIG. 2 shows an anti-Pal immunoblot of urine in accordance with the present disclosure.

DETAILED DESCRIPTION

[0018] The present disclosure relates to a method, device and kit for detecting sepsis or bacteremia in a patient. The method includes detecting peptidoglycan associated lipoprotein (Pal) from Gram-negative bacteria in the urine of the patient.

[0019] In an embodiment, a method for detecting septic or bacteremic levels of Gram-negative bacteria in a patient, includes: obtaining the urine of a human patient; optionally, filtering out whole cell bacteria from the urine; exposing the urine to a Gram-negative peptidoglycan associated lipoprotein (Pal) specific binding agent; and detecting the Gram-negative peptidoglycan associated lipoprotein (Pal) bound to the binding agent.

[0020] Gram-negative bacteria containing Pal include the following: Escherichia coli and all other Enterobacteriaceae; Haemophilus influenzae; Chlamydia pneumoniae; Helicobacter; Pseudomonas; Moraxella catarrhalis; Leptospira interrogans; Cupriavidus; Thermococcus kodakarensis; Corynebacterium glutamicum; Listeria inoocua; Legionella; Fluoribacter; Tatlockia; Gammaproteobacteria; Halomonas; Chromohalobacter; Plasticicumulans; Methylobacter; Cobetia; Halotalea; Neptuniibacter; Oceanospirillaceae; Solimonas; Halovibrio; Salinisphaera; Kushneria; Ketobacter; Alcanivoracaceae; Xanthomonadales; candidatus; Salinisphaera; Aquicella; Wohlfahrtiimonas; Coxiellaceae; Xenorhabdus; Thiolapillus; Paracoccus; Ewingella americana; Serratia. These Gram-negative bacteria all contain a known and identified peptidoglycan associated lipoprotein (Pal) that is similar in sequence and/or structure to other peptidoglycan associated lipoproteins, including peptidoglycan associated lipoprotein (Pal) from E. coli.

[0021] E. coli peptidoglycan associated lipoprotein (Pal) has been shown to be released from the bacterium under certain conditions, such as in the presence of human serum. Peptidoglycan associated lipoprotein (Pal) released by E. coli can be found in the urine of patients with E. coli sepsis. Therefore, it is reasonable to expect that peptidoglycan associated lipoprotein (Pal) from other Gram-negative bacteria behave in a similar manner when exposed to human serum. That is, when a person is infected with a Gram-negative bacterium that contains peptidoglycan associated lipoprotein (Pal), that peptidoglycan associated lipoprotein (Pal) is likely to be released by the bacterium and filtered into that person's urine for excretion, thus allowing for detection of peptidoglycan associated lipoprotein (Pal) in that person's urine in accordance with the present methods.

[0022] A binding agent that is specific for Pal from one or more Gram-negative bacteria(um) can be prepared according to the following. Such a binding agent can be obtained by understanding the primary sequence of the Pal protein and/or the tertiary structure of the Pal protein and/or producing the Pal protein using known recombinant protein expression methods or native purification methods. Once a purified Gram-negative Pal is obtained, animals could be with immunized the purified protein to obtain a monoclonal or polyclonal antibody specific for Pal. As an example, a monoclonal antibody (6D7) was produced in mice. That monoclonal antibody binds specifically to Pal from E. coli, and cross-reacts with Pal from any Enterobacteriaceae. After immunizing mice with the purified E. coli Pal protein, the spleens were harvested from those mice to obtain B cells. Those B cells were fused with immortal B cells to produce hybridoma cells, which produced the 6D7 monoclonal antibody, which can be used as a binding agent.

[0023] In cases where patients are catheterized, one can obtain urine from the drainage bag; in cases where patients are not catheterized, urine will be obtained using normal clean catch methods collected in a sterile cup. Optionally, urine may be filtered to remove whole cell bacteria using a syringe and 0.45 μm attached filter. Total volume required will vary depending on the specific detection test, but 5-10 mL would be a suitable amount.

[0024] In accordance with the procedure, the urine is exposed to a Gram-negative peptidoglycan associated lipoprotein (Pal)-specific binding agent, such as a polyclonal antibody, monoclonal antibody, antibody fragment or molecule that binds specifically to the Gram-negative Pal. For example, the urine can be exposed to an Enterobacteriaceae peptidoglycan associated lipoprotein (Pal)-specific binding agent, such as a polyclonal or monoclonal antibody, antibody fragment or molecule that binds specifically to Pal from Enterobacteriaceae. For example, the urine can be exposed to mouse monoclonal anti-Pal antibody (6D7), which binds specifically to Pal from E. coli, and also cross-reacts (binds) with Pal from any Enterobacteriaceae.

[0025] Binding of Gram-negative peptidoglycan associated lipoprotein (Pal) to the binding agent can be detected with a known output or measurement. Detection methods include fluorescence, a change in color, a change in light scattering, or an enzyme assay that is sensitive to the binding of Pal to its binding agent. For example, a strip will change colors or another visual output will appear when Pal is present in the urine sample. A test may be designed to detect a certain level of Gram-negative Pal in the urine above a specific threshold concentration.

[0026] Alternatively, the specific Pal levels (protein concentration) may be measured using a more complex test. In that case, Pal levels may be normalized to a standard urine component, such as creatinine. Such a normalization factor would be preferred since each person's urine is different and may be more or less diluted with water. By quantifying the creatinine levels in the urine, a specific Pal concentration can be determined and normalized to that creatinine concentration. The quantitative Pal levels may be measured to determine the severity of the patient's sepsis/bacteremia or to track the patient's disease and recovery after the initial diagnosis.

[0027] An embodiment of the disclosure includes a point-of-care (POC) assay, similar to a pregnancy test, which detects the presence of Pal in the urine of the patient. The assay can be performed with a Pal antibody or Pal-specific binding agent coated or bound to a strip. When Pal is present in the urine, the Pal would bind to the strip, resulting in a color change or some sort of visual change in the strip, notifying the clinician of the presence of Pal in the patient's urine.

[0028] An embodiment of the disclosure includes a device that can be used as a point of care for sepsis diagnosis, which can be similar to a dipstick pregnancy test. Components of the device may include a test window and optionally, a control window; an absorbent strip; an immunoassay strip, which contains the Pal-specific binding agent and optionally, a second binding agent to detect creatinine or another control; a container that houses the strips; and a cap to cover the absorbent strip. The device can be stored in a sealed package.

[0029] A more complex method/device could be used to quantify Pal levels in a patient's urine. The device would include the creation of a standard curve using samples of Pal protein at known concentrations; an output (such as absorbance of light) that correlates to protein concentration; a similar measurement performed on patient urine, as well as a control protein sample; and a calculation, which uses the standard curve and the urine sample measurements to estimate the actual Pal concentration in the urine sample.

[0030] The components of a kit that can be used as a point of care for sepsis diagnosis would be the device as described above, with the addition of a sterile wipe and cup for clean catch urine collection and a syringe and filter for optional removal of whole bacterial cells from the urine.

[0031] Methods in accordance with the present disclosure to detect Gram-negative sepsis in human patients preferably would be able to detect Pal from one or more Gram-negative bacteria(um) in the urine of those patients at an early stage of sepsis. Pal release is known to be enhanced by certain antibiotics, but a background level of Pal is released in the presence of human sera without antibiotics; therefore the Pal levels detected in urine should, in general, correlate with the amount of bacteria in the blood.

[0032] A more complex test (e.g., an enzyme-linked immunosorbent assay-ELISA) could be used to quantify the Pal in urine and therefore help determine the severity of sepsis and/or “track” the progression of the disease and/or recovery of the patient.

[0033] The present concept uses peptidoglycan associated lipoprotein (Pal) from Gram-negative bacteria as a urine biomarker for sepsis or bacteremia. Pal is commonly found in Gram-negative bacteria and is localized to the outer membrane via its lipid anchor (which embeds itself in the outer membrane of the bacterium). Much is known about Pal from E. coli, which is the most commonly studied Enterobacteriaceae. E. coli Pal is known to be shed from E. coli under certain conditions, such as in the presence of human blood or sera or when the bacteria are exposed to antibiotics. During an E. coli infection, Pal is released from the bacterium. When Pal is released by E. coli in the blood of human patients, Pal may also be filtered into urine. Since urine contains far fewer proteins than human serum, low levels of Pal in urine are detectable.

[0034] Anti-Pal or another molecule that binds specifically to Pal is used to detect Pal that is shed into the urine of patients with E. coli sepsis. Because E. coli Pal is highly similar in structure to Pal from other Enterobacteriaceae, an antibody/molecule used to detect E. coli Pal would likely cross-react with Pal from any Enterobacteriaceae. It is important to note that E. coli is a commensal organism found in the intestines of healthy humans. This E. coli, as part of the healthy flora, does not shed Pal that is detectable in the urine of healthy humans.

[0035] The present methods are the first known to be able to detect Pal in the urine of sepsis patients. Most studies on Pal/sepsis have focused on Pal's role in sepsis and its potential role as a therapeutic. The inventors had access to sepsis patient urine and Pal monoclonal antibody, and therefore were able to confirm Pal's presence in the urine of sepsis patients.

[0036] Successful diagnosis of sepsis can be a difficult task, as no single method currently provides a definitive diagnosis. As described above, in many cases, patient outcomes are greatly dependent on efficient and early diagnosis of the disease. The method provides a quick and reliable alternative for sepsis diagnosis. The Pal detection test could also be employed in combination with the current sepsis diagnostic tests in order to create a more accurate and comprehensive diagnosis protocol.

[0037] The present technology allows for point of care, low-cost, quick, reliable diagnosis of sepsis. This process also removes the need for a blood draw, which can be challenging in elderly or very young patients. Many sepsis patients already have catheters, making the collection of urine even more efficient. Since the test requires urine and not blood, this test greatly reduces the risk of contracting blood borne diseases for healthcare professionals.

[0038] The disclosure will be further illustrated with reference to the following specific examples. It is understood that these examples are given by way of illustration and are not meant to limit the disclosure or the claims to follow.

Example

[0039] This example detected recombinant Pal (genetically modified to remove the N-terminal lipid attachment) spiked into healthy urine at levels as low as 0.2 ng/μl, without any purification step. This procedure also detected (with monoclonal anti-Pal) a putative Pal band in the urine of patients with diagnosed E. coli sepsis (FIG. 1). FIG. 1 shows a Pal immunoblot of healthy urine and urine from a patient with E. coli sepsis. The first two samples were syringe filtered to remove any potential whole bacterial cells, and the last two samples were gently centrifuged (5000×g) to remove any whole bacterial cells. Bands were detected at the same MW of native Pal. The same protein band was not detected in urine from healthy donors or elderly patients with acute inflammation, but no sepsis or urinary tract infections (UTI) (not shown). A similar 18-kDa protein was detected in additional urine samples from E. coli sepsis patients using monoclonal Pal antibody (FIG. 2). In FIG. 2 an anti-Pal immunoblot detects proteins in urine samples from three E. coli sepsis patients. All urine samples were filtered (with a 0.2 μm filter) to remove intact cells. The ˜16 kDa bands were detected in the urine of Patients #1 and #2 using monoclonal anti-Pal. Also considered was the possibility that this procedure was detecting Pal from a UTI. However, as seen in FIG. 2, Patient #3 was diagnosed with a UTI, but Pal was not observed in that sample. Putative Pal bands were observed in Patients #1 and #2, only one of whom was diagnosed with a UTI, suggesting that the putative Pal bands were not associated with UTI.

[0040] All urine samples were obtained from Rochester General Hospital; patients were confirmed GNS patients unless otherwise noted. The urine was kept at 4° C. until prepared, as described below. The urine was either filtered (0.2 μm filter) or gently centrifuged (5000×g) to remove intact cells. The samples were then combined at a 1:1 ratio with 2× Sample Buffer (recipe: 0.12 M Tris/HCl pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue) and boiled for 10 minutes. The urine samples were then separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10% gel). Proteins were transferred to a Nitrocellulose membrane (Pierce) and blocked with 5% milk in Tris buffered saline (TBS). The membrane was incubated with monoclonal anti-Pal at a 1:4000 dilution in 1% milk and TBS and then horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Bethyl Laboratories) at a 1:12,000 dilution in 1% milk and TBST (TBS with 0.05% Tween-20). The membrane was washed with TBS or TBST between antibody incubations. The blot was visualized using the Lumiglo Reserve HRP chemiluminscent substrate kit (KPL) according to the manufacturer's instructions.

[0041] In summary, the present experimental data suggest that Gram-negative Pal can act as a biomarker for Gram-negative bacterial sepsis in the urine of human patients.

[0042] Although various embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the disclosure and these are therefore considered to be within the scope of the disclosure as defined in the claims which follow.