JP4852121B2 - Diagnosis of Gram-negative bacterial pneumonia using lipopolysaccharide in exhaled breath condensate - Google Patents

Abstract

A method for determining whether a subject has Gram negative bacterial pneumonia based on the presence of lipopolysaccharide in exhaled breath condensate collected from the subject. The collection devices utilized to collect exhaled breath condensate from both spontaneously breathing and mechanically ventilated subjects and the devices utilized to determine whether lipopolysaccharide is present in the collected exhaled breath condensate.

Description

  This application claims the benefit of US Provisional Application No. 60 / 576,641 filed Jun. 7, 2004 and claims priority, the entire contents of which are incorporated herein by reference.

  This application also claims the benefits of US Provisional Application No. 60/434916 filed on December 20, 2002 and US Provisional Application No. 60/44781 filed on February 14, 2003. It is also a continuation-in-part of US patent application Ser. No. 10 / 742,721. The entire contents of each of the above applications are incorporated herein by reference.

  Further, this application is a continuation-in-part of US patent application Ser. No. 10/778477 filed on Feb. 13, 2004, claiming the benefits of US Provisional Application No. 60/44781 filed on Feb. 14, 2003. . The entire contents of each of the above applications are similarly incorporated herein by reference.

  The present invention relates generally to methods and apparatus for diagnosing gram-negative bacterial pneumonia, and more particularly to diagnosing gram-negative bacterial pneumonia by detecting the presence of lipopolysaccharide in breath condensate.

  Pneumonia is representative of a common disease with significant morbidity and mortality. Pneumonia is the leading cause of death from infections in the United States and the sixth leading cause of death. The National Hospital Medical Care Survey (National Hospital Ambulatory Survey) revealed that in 2014, 1.48 million emergency department departments were involved in the diagnosis of pneumonia. The National Hospital Discharge Survey (National Hospital Discharge Survey) revealed that 1.32 million patients were discharged in 1998 after treating pneumonia.

  Pneumonia can be caused by pulmonary infections of many types of microorganisms, including viruses, chlamydia, mycoplasma, protozoa, fungi, and bacteria. For patients suspected of having pneumonia, identifying the infectious agent determines the choice of antibacterial treatment, so the clinician is obligated to determine the exact cause of the infection. The most common cause in Western society is bacterial pneumonia, and when bacterial pneumonia is suspected, clinicians generally attempt to classify the cause of bacterial pneumonia as gram positive, gram negative, or anaerobic.

  In particular, gram-negative lung infections are aggressive and lead to high complication rates and mortality, so clinicians are motivated to check for the presence of gram-negative bacterial infections. The gram-negative characteristics of bacteria relate to the color of the bacteria after the staining procedure, which is well understood by those skilled in the art. Gram-negative bacterial infections, including Gram-negative bacterial pneumonia, require specific antibacterial treatments that are different from the treatment of other types of bacterial infections and require high levels of financial reimbursement from third-party payers such as Medicare. to justify.

  In current clinical practice, pneumonia is diagnosed by combining clinical, laboratory, and radiographic information. In general, features such as patient complaints, patient vital signs, peripheral white blood cell count, and chest radiography results are used to determine the presence or absence of pneumonia. When these data sources are consistent with typical patterns, diagnosis can be made with reasonable clinical confidence and antimicrobial treatment can be initiated before bacterial culture results are available. Nonspecific clinical data is often used to initiate antibiotic therapy for Gram-negative infections, but in general practice, Gram-negative bacterial lung infections are the final diagnosis of Gram-negative bacterial pneumonia More specific evidence is needed.

  For this purpose, Gram staining can be performed immediately on sputum extracted from the lower respiratory tract, and microscopic analysis can reveal bacteria with morphology and color suggesting Gram negative infection. However, it is well known that it is difficult to obtain useful sputum samples from humans with pneumonia. If a blood sample culture from a patient with a clinical pattern consistent with pneumonia grows gram negative bacteria, this is a specific indicator of gram negative pneumonia. Unfortunately, in most cases, the blood of patients with Gram-negative bacterial pneumonia is sterile.

  In addition, the patient's blood can be tested for endotoxin concentration using chemical assays for endotoxin molecules. However, endotoxin concentration has been found to be an inaccurate predictor for either the cause or severity of the more common sepsis syndrome. Blood endotoxin levels can vary significantly over a short period of time. In addition, certain disease states, including liver disease, multiple trauma, hypertension, and hematological malignancies, are associated with chronic increases in endotoxin levels, even without clinically significant infections. Whether circulating endotoxin levels can predict gram-negative pathogenesis of pneumonia has not been studied.

  A sensitive and specific method of diagnosing Gram-negative pneumonia infection is to perform bronchoalveolar lavage and perform bacterial culture with lavage fluid. Another method is to chemically analyze the lipopolysaccharide components in the bronchoalveolar lavage fluid sample. Researchers using this method have found that high concentrations of lipopolysaccharide are associated with the co-growth of gram-negative bacteria in bronchoalveolar cultures. A complete description of this method can be found in Flanagan, P .; G. Jackson, S .; K. Findlay, G .; “Diagnosis of Gram negative, ventilator associated pneumonia by assigning endotoxin in bronchial lavage fluid”, J. Clin. Pathol. 2001, 54: 107-110, and Pugin, J. et al. Ackenthaler, R .; And Delaspre, O .; “Rapid Diagnosis of Gram-negative pneumonia by ass of endotoxin in bronchial lavage fluid”, Thorax 1992, 47: 547-549. Both methods have the disadvantage that special endoscopic devices and sub-specialties are required and they are relatively invasive and unpleasant procedures. In addition, known culture methods require at least 24 hours to obtain results. Thus, patients may wait at least 24 hours before receiving effective antibiotic treatment.

  The cell wall of gram-negative bacteria contains endotoxins. Endotoxins are toxic substances released from bacteria upon cell lysis. Endotoxin was first recognized for its ability to induce heat, but is now known to have a wide range of biological activities. Lysis releases endotoxins composed of lipopolysaccharides and aggregates of proteins and lipids from the bacteria to the surrounding medium. Endotoxins are composed primarily of lipopolysaccharide (“LPS”) and varying amounts of proteins and lipids. The terms “endotoxin” and “lipopolysaccharide” are used interchangeably as almost all of the biological activity normally attributed to bacterial endotoxins can be induced by isolated chemically pure lipopolysaccharides. .

  From the viewpoint of pathogenicity, the presence of lipopolysaccharide is one of the most important effects of gram-negative infection. Therefore, the detection of lipopolysaccharide in a patient's body fluid is an indicator of Gram-negative bacterial infection. More specifically, the presence of lipopolysaccharide in a patient's body fluid is an important potential indicator of Gram-negative pneumonia.

  The present invention overcomes the clinical disadvantages noted above in the diagnosis of Gram-negative bacterial pneumonia by performing lipopolysaccharide analysis on fluids derived from breath condensate. Using the novel devices and methods described herein, exhaled breath condensate can be obtained from a spontaneously breathing subject through a mouthpiece, face mask, or other similar means, or with mechanical ventilator assistance. Can be obtained from a subject breathing on the basis of a connection to the exhalation piping of a mechanical ventilator. An additional advantage of the present invention is that the time to diagnose a Gram-negative bacterial infection such as pneumonia is shorter than the time required for other diagnostic methods.

  Alternative devices for collecting exhaled breath condensate are also known. These devices are disclosed in U.S. Pat. Nos. 6,033,368 and 6,419,634 to Gaston et al., U.S. Pat. No. 6,585,661 to Hunt et al., U.S. Patent Application No. 10/257912 to Badour, and European Patent 0759169 to Winsel et al. The entire contents of which are hereby incorporated by reference. In addition, Kline's US Patent Applications Nos. 10/742721 and 10/778477, two non-provisional patent applications assigned to the same applicant, disclose devices for collecting exhaled breath, The contents are incorporated herein by reference.

  Exhaled breath condensate is known to contain many molecules that can serve as markers for many lung diseases, as outlined by Kharitinov et al. In 2001 (Biomarkers 7 (1): 1-32, 2002). ). However, the concept of measuring lipopolysaccharide in breath condensate for the purpose of diagnosing Gram-negative bacterial pneumonia or other Gram-negative bacterial infections has never been disclosed.

  The present invention includes a method for determining whether a subject has gram-negative bacterial pneumonia based on the presence or absence of lipopolysaccharide in breath condensate collected from the subject. The present invention further provides a collection device utilized to collect exhaled breath condensate from both spontaneously breathing subjects and artificially ventilated subjects, and whether lipopolysaccharide is present in the collected exhaled breath condensate. Includes devices used to determine.

  In a broad sense, the present invention, in one aspect, collects exhaled breath condensate from an air-breathing vertebrate subject, measures the concentration of lipopolysaccharide in the collected exhaled breath condensate, and Diagnosing pulmonary gram-negative bacterial infection in an air-breathing vertebrate subject, including determining whether the subject has an pulmonary gram-negative bacterial infection based on the measured concentration of lipopolysaccharide It is a method for monitoring.

  A feature of this aspect is that the intrapulmonary gram-negative bacterial infection is pneumonia, the intrapulmonary gram-negative bacterial infection is bronchitis, and the method determines that the subject has an intrapulmonary gram-negative bacterial infection. Selecting an antibiotic treatment in response, wherein collecting the breath condensate from the air-breathing vertebrate subject comprises collecting the breath condensate from the mammalian subject, the method against antibiotic treatment Monitoring the response of a mammalian subject, the method further comprising identifying a particular strain of pulmonary Gram-negative bacteria, wherein the identifying comprises determining the O-monosaccharide portion of the lipopolysaccharide molecule. Identifying a specific strain of gram-negative bacteria in the lung based, wherein measuring the concentration of lipopolysaccharide comprises measuring the concentration of lipopolysaccharide using Limulus modified cell lysate analysis, and at least about A measured concentration of lipopolysaccharide of 20 endotoxin units / mL (EU / mL) indicates the presence of a Gram-negative bacterial infection, and collection of exhaled breath condensate from an air-breathing vertebrate subject is from a spontaneously breathing subject Collecting breath condensate from a breathing vertebrate subject including collecting breath condensate from an artificially breathing subject and from an air breathing vertebrate subject Collection of exhaled breath condensate involves utilizing an exhaled breath condensate collecting device with a chamber having an inner wall that can be cooled to a temperature of about 32 ° F. or less to facilitate condensation.

  In another aspect, the present invention provides a reaction chamber in which a breath condensate is collected from a vertebrate subject breathing in air, a reaction reagent is disposed therein, and at least a part of the collected breath condensate is provided in the reaction chamber. To determine whether the subject has an intrapulmonary gram-negative bacterial infection based on the physical changes that occur when at least a portion of the collected breath condensate is pumped into the reaction chamber. A method for diagnosing and monitoring an intrapulmonary gram-negative bacterial infection in an air-breathing vertebrate subject, wherein the physical change is caused by the presence of lipopolysaccharide in the breath condensate.

  In a feature of this aspect, the reaction reagent includes a Limulus deformed cell lysate, a chromogenic substrate, and / or a fluorescent substrate, the reaction reagent includes a chemical that releases heat through an exothermic reaction by feeding at least a portion of the breath condensate, The physical change is the formation of a gel, and the method further comprises visually matching the color change in the reaction chamber to a standard, the standard comprising a print strip of color patches of increasing hue intensity, Each color patch corresponds to an increasing concentration of lipopolysaccharide, and a hue intensity corresponding to a concentration of 0.2 endotoxin units per milliliter indicates the presence of an intrapulmonary gram-negative bacterial infection from an air-breathing vertebrate subject. Collection of exhaled breath condensate includes collecting exhaled breath condensate from a subject breathing spontaneously, and exhaled breath condensate from a vertebrate subject breathing air Collecting includes collecting exhaled breath condensate from a mechanically ventilated subject, and collecting exhaled condensate from an air breathing vertebrate subject to a temperature below about 32 ° F. to facilitate condensation Using a breath condensate collection device with a chamber having an inner wall that can be cooled to at least a portion of the breath condensate is fed into the reaction chamber via a hypodermic needle. Including injection.

  The present invention, in another aspect, collects exhaled breath condensate from an air-breathing vertebrate subject and provides a reaction chamber having an internal surface that binds to a lipopolysaccharide disposed therein. The substance has a substance, and at least a part of the collected breath condensate is sent to the reaction chamber, the reaction chamber is washed with a buffer solution, the reaction reagent is sent to the reaction chamber, and at least a part of the collected breath condensate is sent to the reaction chamber. Determining whether the subject is pulmonary Gram-negative bacterial infection based on physical changes that occur when delivered, said physical changes being due to the presence of lipopolysaccharide in the breath condensate A method for diagnosing and monitoring induced pulmonary Gram-negative bacterial infections in an air-breathing vertebrate subject.

  In a feature of this embodiment, the reaction reagent includes a chromogenic substrate and / or a fluorescent substrate, the binding agent is an antibody that binds to a specific type of lipopolysaccharide, the antibody is disposed within the surface of the reaction chamber, and the binding agent is It is an insoluble polymer. The binder is embedded in a substrate surface disposed within the reaction chamber, and the method further includes visually matching the color change in the reaction chamber to a standard, the standard being a print band of color patches of increasing hue intensity. Each color patch corresponds to an increasing concentration of lipopolysaccharide, and a hue intensity corresponding to a concentration of 0.2 endotoxin units per milliliter indicates the presence of intrapulmonary gram-negative bacterial infection and breathes air Collecting exhaled breath condensate from a vertebrate subject includes collecting exhaled breath condensate from a spontaneously breathing subject, and collecting exhaled breath condensate from an air breathing vertebrate subject exhaled from a subject breathing artificially Collecting condensate, including collecting breath condensate from an air-breathing vertebrate subject because the temperature is cooled below about 32 ° F. to facilitate condensation Using at least a portion of the breath condensate with a chamber having an inner wall, wherein the feeding of at least a portion of the breath condensate comprises injecting at least a portion of the breath condensate into the reaction chamber via a hypodermic needle. Including.

  In another aspect, the present invention provides a fiber plug impregnated with an enzyme that collects exhaled breath condensate from a vertebrate subject breathing with air with a collection device having a capillary tube and causes gelation when exposed to lipopolysaccharide. Insert into the tubule and introduce at least a portion of the collected breath condensate sample into the tubule to wet the fiber plug. For diagnosing and monitoring an intrapulmonary gram-negative bacterial infection in an air-breathing vertebrate subject, wherein said gelation is caused by the presence of lipopolysaccharide in exhaled breath condensate Is the method.

  In a feature of this aspect, the fiber plug is impregnated with an enzyme derived from Limulus deformed cell lysate, and the method further comprises providing a syringe disposed in the collection device, wherein the syringe is for measuring hydraulic pressure. It has a manometer located on one side and the capillary is a hypodermic needle placed at the end of the syringe.

  In another aspect, the present invention provides a mechanical ventilator circuit for facilitating breathing of a subject, the ventilator circuit including an expiratory flow tube that serves as a conduit for removing the subject's exhalation and having a central chamber having an interior. An expiratory input assembly disposed at one end of the central chamber that can be cooled to a temperature of about 32 ° F. or less to facilitate condensation and is in fluid communication with the interior of the central chamber and the expiratory flow tube. An expiratory input assembly that connects the expiratory flow tube with the central chamber, an outlet assembly disposed at the other end of the central chamber so as to be in fluid communication with the interior of the central chamber, and expiratory condensate is collected from the central chamber A device for collecting exhaled breath condensate from a subject breathing with the help of a mechanical ventilation circuit.

  The present invention, in another aspect, comprises a central chamber having an interior and first and second opposing ends, an exhalation input assembly in fluid communication with the interior of the central chamber, and an outlet assembly in fluid communication with the interior of the central chamber. The outlet assembly includes a capillary tube, the capillary tube having a fiber plug disposed therein, impregnated in the plug with a reaction reagent that causes gelation when exposed to lipopolysaccharide. Exhalation condensate collection device.

  In a feature of this embodiment, the plug is a fiber substrate impregnated with an enzyme derived from Limulus deformed cell lysate, the apparatus further comprising a plunger assembly having a piston and a handle, the piston sliding into the interior of the central chamber. Arranged freely, the handle extends from the first end of the central chamber so that the piston can be moved in the central chamber, so that the breath condensate collected can come into contact with the fiber plug disposed in the capillary tube. it can.

  In another aspect, the present invention provides a reaction chamber, a feed port for feeding the breath condensate into the reaction chamber, a reaction reagent that reacts with the lipopolysaccharide present in the breath condensate, and a physical change in the reaction chamber. A reaction chamber assembly for measuring the concentration of lipopolysaccharide in exhaled breath condensate, including a viewing portion that allows visual detection of

  In a feature of this aspect, the delivery port is a resealable lid that can puncture a hypodermic needle for delivery of exhaled breath condensate. The viewing part is a transparent wall. The reaction chamber further includes an internal surface, and the lipopolysaccharide immobilizing agent is bound to at least a portion of the internal surface of the reaction chamber, the lipopolysaccharide immobilizing agent being a monoclonal antibody, a polyclonal antibody, a polymyxin antibiotic, a lipopolysaccharide binding protein, and The reaction chamber further comprises a chemical substance selected from the group consisting of horseshoe crab anti-lipopolysaccharide factors, the chemical substance releasing heat by a hydration reaction, thereby hydrating the chemical substance To achieve a temperature in the range of 34-43 ° C. for 15-30 minutes, the chemical is a salt in a semipermeable substrate, the chemical is a salt in crystalline form, and the chemical is sodium thiosulfate pentahydrate It is a Japanese product.

  In another aspect, the present invention provides a housing, and a test module disposed in the housing, wherein the test module detects the presence of lipopolysaccharide in the breath condensate using a user-initiated chemical reaction; Comparison with a test module, which is a positive control module located in the enclosure adjacent to the test module and which has a lipopolysaccharide placed therein to show a positive positive result for the presence of the lipopolysaccharide A test kit cartridge for detecting the presence of lipopolysaccharide in breath condensate, comprising the positive control module for

  In a feature of this embodiment, the chemical placed in the enclosure causes a controlled exothermic reaction, whereby a temperature between 34 ° C. and 43 ° C. is achieved for a period of 15-30 minutes, the chemical being sodium thiosulfate Each module of the test kit includes a resealable injection port, a reaction well, a fluid connection between the reaction well and the injection port, and a first one disposed in the fluid connection. Including a directional valve, an outlet tube providing an outlet of the reaction well, and a second one-way valve disposed in the outlet tube, each reaction well adapted to allow visual determination of physical changes Each reaction well contains an insoluble polymeric lipopolysaccharide binding substrate, the reaction well of the positive control module contains lipopolysaccharide derived from the appropriate species of Gram-negative bacteria, and the test kit cartridge reacts Room Further comprising a reconstituted chromogenic substrate, the test kit cartridge further comprises a color strip having color patches of increasing hue intensity, each color patch corresponding to a predetermined range of endotoxin units, and the color of the reaction well Allows visual comparison with changes, the enclosure is formed from a polymer and the polymer is plastic.

Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Further features, embodiments and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the drawings.

1 is a schematic diagram of the basic structure of a lipopolysaccharide. It is side surface sectional drawing of the expiration condensate collection apparatus which concerns on the 1st preferable embodiment of this invention. 3A is a partial cross-sectional schematic side view of the exhaled breath condensate collection device of FIG. 2 with the plunger assembly in a partially depressed position. 3B is a partial side cross-sectional schematic view of the exhaled breath condensate collection device of FIG. 2 with the plunger assembly in a fully depressed position. It is a partial side sectional view of a breath condensate collection device according to a second preferred embodiment of the present invention. It is side surface sectional drawing of the breath condensate collection apparatus which concerns on the 3rd preferable embodiment of this invention. 1 is a schematic diagram of an alternative ventilation system of a preferred embodiment of the present invention. 6 is a side cross-sectional schematic view of a reaction chamber apparatus suitable for use with the collection apparatus of FIGS. It is a top view of the reaction chamber apparatus of FIG. FIG. 3 is a side perspective view of a first alternative reaction chamber apparatus. FIG. 10 is a partial side cross-sectional view of the first alternative reaction chamber apparatus of FIG. 9. FIG. 10 is a partial side cross-sectional view of the first alternative reaction chamber apparatus of FIG. 9. It is a perspective view of the test | inspection kit incorporating the 2nd alternative reaction chamber apparatus. 12 is a side cross-sectional view taken along line 12-12 of the inspection module of FIG. FIG. 13 is a side cross-sectional view taken along line 13-13 of the positive control module of FIG. It is a partial side sectional view of a breath condensate collection device according to a fourth preferred embodiment of the present invention. FIG. 15 is an enlarged partial side sectional view of the needle of FIG. 14. FIG. 6 is a scatter plot showing the measured endotoxin concentration of patients in each of the three test groups.

  The method of the present invention utilizes breath condensate to determine whether LPS is present in the breath condensate, thus determining whether the subject has an intrapulmonary gram-negative bacterial infection. The present application is directed to diagnosing Gram-negative bacterial pneumonia. However, one of ordinary skill in the art will appreciate that the present invention can be used to detect any Gram negative bacterial infection that is effective. Patients diagnosed with a gram-negative bacterial infection are treated with the same antimicrobial therapy regardless of whether the diagnosis is gram-negative bacterial pneumonia or another gram-negative bacterial infection. The methodology for detecting LPS described herein is a Limulus modified cell lysate assay, but in the present invention to detect the presence of LPS, including but not limited to an ELISA assay and a rabbit pyrogen test. Those skilled in the art will appreciate that any of these methodologies can be utilized.

  Intrapulmonary LPS content can vary based on the lung health of the subject providing the breath condensate sample. Generally, a concentration of LPS in the breath condensate of at least about 0.2 EU / mL corresponds to a laboratory test positive threshold and indicates the presence of a gram-negative bacterial infection in the lung. EU is an endotoxin unit. Unlike typical mass concentrations that cannot be standardized due to variable potency of lipopolysaccharides that vary between bacterial types, the EU can standardize, so USDA reports endotoxin concentrations in endotoxin units. It is recommended to do. Endotoxin units refer to the amount of LPS reactivity contained in an unknown sample. This amount of reactivity is determined by interpolation from a standard curve derived from an FDA approved strain of E. coli having a known amount of LPS activity.

  The device used to collect exhaled breath condensate can include a “background” level of LPS with naturally occurring LPS. To account for this background concentration, a “simulated” standard can be implemented on the device used for breath collection by injecting water into the device without LPS. The revealed LPS concentration value in the water sample is then divided from the revealed LPS concentration value in the breath condensate sample to remove the background “noise” of the system. The background concentration of LPS varies depending on the equipment used for collection.

  An exhaled breath condensate collection device, that is, as described in more detail herein below, in some embodiments thereof, from a patient exhibiting symptoms synonymous with Gram-negative bacterial pneumonia, an LPS content of an amount indicative of infection. For detection, it can be used to collect exhaled breath condensate.

  Preferred embodiments of the present invention will now be described with reference to the drawings, wherein like numerals represent like elements throughout the several views. The following description of preferred embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

  FIG. 1 is a schematic diagram of the basic structure of lipopolysaccharide. This structure is found in the cell wall of all Gram negative bacteria. Lipopolysaccharide (also referred to as endotoxin) is a complex glycolipid weighing about 10 Kd. The basic structure of lipopolysaccharide contains three relatively well defined regions and is similar in all gram-negative bacteria. These regions are the O-antigen part, the core polysaccharide, and lipid A. The O-antigen part is composed of repeating polysaccharide units each having 2 to 6 monosaccharides. The O-antigen portion varies considerably among gram-negative species and can therefore serve as a marker for individual bacterial species based on the binding of specific monoclonal antibodies. The core is a branched polysaccharide that exists between the O-antigen part and lipid A and has typical carbohydrates such as glucose, N-acetylglucosamine, and galactose. Unlike the O-antigen region, there are only minor changes throughout the core region, and the structure of the inner core region adjacent to lipid A is highly conserved. The most highly conserved part of the LPS molecule is lipid A, a diphosphorylated disaccharide with long chain fatty acids attached. The lipid A portion of the molecule is toxic in virtually all eukaryotic tissues. In humans, this includes cytokine induction, fever, leukocytosis, leukocyte recruitment and activation, vascular damage, vasodilation, intravascular coagulation, and tissue damage.

  One accepted method of detecting and quantifying LPS takes advantage of the toxic effects of LPS on an aqueous extract of deformed cells from horseshoe crab (Limulus polyphemus). As used herein, the term “deformed cell” refers to a blood cell. This method, commonly known as the Limulus Modified Cell Lysate (“LAL”) assay, is described in US Pat. Nos. 4,322,217, 5,310,657, and 5,702,882. Other recognized methods include the in vitro rabbit pyrogen assay and the human pyrogen assay, which are well known to those skilled in the art and will not be described in detail here.

Briefly summarized, LAL forms a coagulated gel when cultured in LPS, which allows the detection of small amounts of LPS. More specifically, in the LAL assay, LPS activates an enzyme commonly known as factor C contained in deformed cells. Activated factor C then activates factor B by hydrolyzing specific sites of factor B. Activated factor B activates the proclotting enzyme and converts it to a clotting enzyme. The coagulation enzyme then cleaves coagulogen (coagulation protein, molecular weight: 19723) at specific sites (ie Arg ⁸ -Thr ¹⁹ and Arg ⁴⁶ -Gly ⁴⁷ ), causing the mixture to gel. In addition, cofactors such as calcium, magnesium, and phosphate, or hydroxymethyl to maintain a pH between 6.5 and 7.4 that allows the clotting enzyme to function Other organic compounds must be present, such as ("TRIS") aminomethane buffer.

  Various techniques have been utilized to detect LPS based on the formation of a coagulated gel in the LAL assay. Some techniques include endpoint assays that simply wait for gel formation to determine the presence of LPS. Others are more complex and use turbidimetric time analysis to measure turbidity that increases as clotting occurs in the LAL assay.

  To overcome the problems associated with accurately determining gel formation, LPS has been detected using alternative analytical methodologies using chromogenic or fluorescent substrates. This alternative methodology relies on the clotting enzyme formed in the cascade reaction described above, which hydrolyzes the amide bond of the synthetic substrate. Synthetic substrates can be covalently linked to marker molecules or compounds that are released when hydrolyzed by clotting enzymes. The liberated marker can then be detected colorimetrically or spectrophotometrically, thus indicating the presence of LPS. Examples of synthetic substrates include t-butoxycarbonyl-leucyl-glycyl-arginine-paranitroanilide (Nt-Boc-Leu-Gly-Arg-pNA), Nt-Boc-Val-Leu-Gly-Arg- pNA (SEQ ID NO: 1), benzyloxycarbonyl-leucyl-glycyl-arginine-paranitroanilide (Z-Leu-Gly-Arg-pNA), Boc-Ile-Glu-Gly-Arg-pNA (SEQ ID NO: 2) , Boc-Val-Ser-Gly-Arg-pNA (SEQ ID NO: 3), or Boc-Ser-Gly-Arg-pNA, but is not limited thereto. These all liberate paranitroaniline, which makes the reaction mixture yellow. Specific examples of chromogenic assays are described in Ling US Pat. No. 6,645,724 and Tamura et al. US Pat. No. 5,702,882.

  In addition, instead of obtaining enzymes from horseshoe crabs, it is also possible to synthesize enzymes necessary for the in vitro coagulation cascade by recombinant biotechnology. Such synthases can be used to detect and quantify LPS in unknown solutions using chromogenic methods.

  Other alternative methods for capturing and detecting LPS are also known. An LPS binding agent, such as a polymyxin antibiotic or resin in the form of an insoluble matrix polymer, supports the LPS so that it can be immobilized and subsequently detected by any of the turbidimetric, chromogenic, or fluorogenic methods described above. Can be combined. In addition, LPS can be immobilized by any number of antibodies with high affinity for LPS. The antibody has a Y-shaped structure and contains two basic units. The first unit is a fragment-antigen binding site (“Fab”), which binds to an antigen, in this context LPS. The second unit is a fragment crystallization moiety (“Fc”). The Fc unit can function as a handle that binds the antibody to the immobilization surface. Potentially useful antibodies include polyclonal or monoclonal antibodies such as lipid A reactive monoclonal antibody (“HA-IA”) and mouse anti-endotoxin immunoglobulin M (“E5”), lipopolysaccharide binding protein (“LBP”). ) And related proteins, bactericidal / permeability increasing protein (“BPI”), and Limulus anti-LPS factor (“LALF”).

  Methods utilizing enzyme-linked immunoassay techniques are also known, and a variety of selected matrix-bound antibodies can be used to capture and detect specific endotoxins and specific bacterial types. Examples of endotoxins from bacteria that can be detected include Escherichia, Bordetella, Blanchamera, Salmonella, Haemophilus, Klebsiella, Proteus, Enterobacter, Pseudomonas, Pasteurella, Acinetobacter, Chlamydia, and Neisseria, and generally their LPS Any bacterium that is capable of binding to a specific antibody includes, but is not limited to.

  Furthermore, it is recognized that the presence of β-1,3 glucans (β-D-glucans) can also activate the coagulation cascade described above. One skilled in the art will appreciate that certain measures can be taken to limit this false positive effect.

  The present invention relates to LPS concentrations following rapid (eg, less than 30 minutes) non-invasive collection of exhaled breath condensate (“EBC”) from a subject who breathes spontaneously or who is undergoing mechanical ventilation procedures. It includes several collection devices designed to allow one-step or semi-quantitative analysis of the condensate. In spontaneously breathing subjects, exhaled breath condensate is typically collected via a mouthpiece held by the lips. However, in patients with severe dyspnea, the sample is fitted to the patient with a tightly fitting face mask that allows oxygen to be delivered while diverting exhaled gas and aerosol into the condensation chamber as described below. Can be collected.

  In general, each breath condensate collection device comprises a collection chamber having an inner wall free of sterile LPS that can be cooled to a temperature of less than 32 ° F. to allow condensation of the breath. These breath condensate collection devices are preferably disposable and lightweight. Each device includes a coaxial chamber and a region through which a coolant that can be cooled from the outside or through an internal endothermic reaction is interposed. Such breath condensate collection devices are outlined in the above-mentioned US patent application Ser. Nos. 10 / 742,721 and 10/778477, which the inventor and assignee have in common with the present application. However, the devices described and illustrated herein have additional new and useful features not included in any prior art device.

  FIG. 2 is a schematic side sectional view of the breath condensate collection device 10 according to a preferred embodiment of the present invention. This configuration is particularly suitable for use with cooperative humans. The breath condensate collection device 10 includes a double walled syringe 20 and a breath input assembly 50. The inner wall 22 of the syringe 20 defines a cylindrical central chamber 24 in which a plunger assembly 25 comprising a piston 26, a rubber gasket 28, and a handle 30 extending from one end of the syringe 20 is mounted. The outer wall 32 is disposed around the inner wall 22 so that a narrow space is formed between the inner wall 22 and the outer wall 32. During manufacturing, the space between the inner wall 22 and the outer wall 32 can be filled with a jacket of coolant 34, and the outer wall 32 can then be sealed to the inner wall 22 to prevent leakage. In a preferred embodiment, water can be used as the coolant 34, but it is clear that other materials such as polyethylene glycol ("PEG") can be used as well.

  The syringe 20 further includes an inlet 36, an outlet 38, and a pair of one-way valves 40, 42. To facilitate the passage of exhaled air through the central chamber 24 in only a single direction, the first valve 40 is a suction valve that can be positioned adjacent to the inlet 36 and the second valve 42 is adjacent to the outlet 38. It is an outlet valve that can be arranged. The outlet 38 is preferably located at the opposite end of the plunger handle 30 so that material collected in the central chamber 24 can be pushed out of the outlet 38 by the piston 26. The outlet 38 can be arranged at the end of a nipple nozzle 39. The nozzle 39 can also be equipped with a fitting such as a luer lock female part at its distal end. Such a fixture allows a protective cover or other accessory to be attached to the nozzle 39. The valves 40, 42 are only schematically shown in the various figures, but they can include, for example, two or three self-sealing leaves formed from plastic or another deformable polymer. Such valve designs will be apparent to those skilled in the art.

  Piston 26 includes a tip or projection 27 of a size and shape suitable for a snug fit within nozzle 39 when plunger assembly 25 is fully depressed. The tip 27 includes a plurality of radial conduits 31 disposed around the base of the tip 27 and connected to a hollow central shaft or conduit 33 in which a needle 35, such as a hypodermic needle, is disposed. The radial conduit 31 and the central conduit 33 are preferably between about 1 mm and 2 mm in diameter, and the outer diameter of the needle 35 is also between about 1 mm and 2 mm to fit snugly within the central conduit 33. A diameter is preferred.

  Further, since the piston 26 fills one end of the syringe 20 and an outlet 38 is disposed at the opposite end, the inlet 36 is configured to penetrate both the inner wall and the outer walls 22 and 32 of the side portion of the syringe 20. It is preferable. The inlet 36 is preferably located as close as possible to the piston 26 to cause maximum interaction between the exhaled air passing through the central chamber 24 and the inner surface 44 of the inner wall 22, but the other components of these components It will be apparent that arrangements are equally possible without departing from the scope of the invention.

  The exhalation input assembly 50 includes a mouthpiece 52, a filter 54, and any tubing 56 required to direct exhalation from the mouthpiece 52 to the inlet 36 of the syringe 20. The mouthpiece 52 is appropriately sized and shaped so that it can comfortably contact the area of the patient's mouth. In order to prevent saliva and other liquids or minimally sized solids from passing through and into the syringe 20, a filter 54 comprising a polymeric material with perforations and continuous penetration is provided with a mouthpiece 52. And a pipe 56 between the syringe inlet 36. By arranging the exhalation input assembly 50 below the syringe 20 so that the air passing through the exhalation input assembly 50 moves upward, saliva can be further prevented from reaching the central chamber 24. With this arrangement, the action of gravity on saliva and other liquids or solids tends to prevent such substances from being fed into the central chamber 24 because they tend to collect instead in the tubing 56. Useful.

  The tubing 56 is preferably configured to avoid interference between the mouthpiece 52 or other portions of the tubing 56 and operation of the plunger assembly 25, such as that described below. More preferably, the mouthpiece 52 is oriented generally parallel to the syringe 20 and the plunger assembly 25 therein, in other words, the mouthpiece 52 is oriented parallel to the major axis defined by the syringe 20. In this orientation, exhaled air can be received from the patient without causing interference with the operation of the plunger assembly 25, and the condensate formed inside the syringe 20 as the patient uses the device 10 is directed toward the outlet 38. Easily discharged downward.

  Using a device 10 that is easily held by the patient or attendant and does not require the patient to change its breathing pattern and that is sufficiently small and light, a sufficient amount in a relatively short time. The dimensions of the device 10 are selected so that condensate can be collected. The walls 22, 32 and other structures of the device 10 are preferably constructed from a material that does not tend to bind to proteins, such as platinum-cured silicon. Other suitable materials include, but are not limited to glass, plastic, polyethylene, polycarbonate, or polyvinyl polymers or other synthetic polymers. Plunger assembly 25 is similarly preferably constructed from a non-protein binder, but can be made from any suitable inert material, including but not limited to plastic, vinyl, polyethylene, rubber, platinum cured silicon, or fluorine-containing polymers. Can be configured. In addition, it is a fluorine-containing polymer and is manufactured by the company E.I. I. TEFLON (registered trademark), a registered trademark owned by Du Pont De Nemours and Company, can be used. In a preferred embodiment, the syringe 20 is between 10 cm and 20 cm in length and between 2 cm and 5 cm in diameter. The thickness of the coolant jacket 34 can be between 1 mm and 10 mm, and the sample volume pumped from a single use is preferably between 100 μL and 1000 μL, but useful results can be obtained from as little as 25 μL of sample. It may be possible to obtain.

  The plunger assembly 25 is an improvement over the plunger assembly disclosed in the prior application by including the hypodermic needle 35 or similar structure described above. This improvement facilitates aseptic delivery of the condensate into the specialized reaction chamber apparatus described below in some embodiments. The needle 35 is centered in the central conduit 33 of the piston tip 27 and the interior of the needle 35 is configured in fluid communication with the radial conduit 31 of the tip 27 for purposes that will become apparent below.

  In operation, such as a conventional home or commercial freezer, which can cool the jacket of the coolant 34 contained between the inner and outer walls 22, 32 of the syringe 20 by reducing the temperature to about 0 ° F. One or more syringes 20 are initially stored in the refrigeration apparatus. When examining a patient, a single syringe 20 is first withdrawn from the freezer. The exhalation input assembly 50 or mouthpiece 52 is stored separately from the rest of the device 10, and the device 10 is then assembled for use by combining the various components together. Next, the patient places the mouthpiece 52 so as to seal the mouth area of the patient, and exhales into the mouthpiece 52. Exhaled air is guided into the central chamber 24 through the pipe 56 and through the inlet 36. Suction valve 40 is opened by positive pressure, but without such pressure, it prevents air in central chamber 24 from escaping through inlet 36. The exhaled air then drains through the needle 35 from the outlet 38 at the end of the central chamber 24 opposite the intake end. The outlet valve 42 allows air to pass out of the central chamber 24 only when positive pressure is present on the cylinder side of the valve 42, and when there is no such pressure, the valve 42 allows the ambient air to pass through the outlet 38. Through the central chamber 24 is prevented.

  As the patient exhales through the device 10, moisture in the exhalation begins to condense on the interior surface 44 of the central chamber 24. Due to the reduced temperature of the coolant 34 and the syringe 24, the condensate can be immediately frozen on the inner surface 44. The diameters of nozzle 39 and needle 35 are small enough to create resistance to patient exhalation. The diameter can preferably be selected to reduce the rate of exhalation until each exhalation takes about 5 seconds to complete, or until a water pressure resistance of up to about 5 cm is provided. . Those skilled in the art will appreciate that the nozzle 39 may alternatively be equipped with an end expiratory positive pressure valve (“PEEP” valve) having a dial that varies resistance to exhalation. PEEP valves are commercially available from Life Assist Inc, Rancho Cordova, California. This improvement increases the amount of time for expiration to equilibrate with the inner surface 44 of the central chamber 24.

  As the patient continues to exhale through the device 10, the frozen coolant 34 disposed in the space between the inner wall 22 and the outer wall 32 of the device 10 begins to melt. The composition, volume, and thickness of the coolant jacket 34 surrounding the central chamber 24 is preferably calibrated such that the coolant 34 begins to dissolve after approximately 10-15 exhalations by the patient. As the coolant 34 dissolves or thaws after the desired number of exhalations, the condensate can begin to dissolve as well. When the condensate is dissolved, the plunger assembly 25 can be pushed in and the collected condensate sample can be pressed out from the outlet 38.

  3A and 3B are partial side cross-sectional schematic views of the breath condensate collection device 10 of FIG. 2 with the plunger assembly 25 in a partially depressed position and a fully depressed position, respectively. When the plunger assembly 25 is depressed, the needle 35 is pushed out of the outlet 38. On the other hand, as the volume of the space between the piston 26 and the outlet 38 contracts, the condensate is pushed through the radial conduit 31 into the central conduit 33 and from there into the hypodermic needle 35. The condensate is then ready to be discharged into a suitable reaction chamber apparatus. Some examples of reaction chamber devices are described below in connection with FIGS. Ejection can be facilitated by depressing the plunger assembly 25 completely. A clip assembly may be provided at the opposite end of the syringe 20 to capture the handle 30 of the plunger assembly 25 and thereby indicate to the user that the plunger assembly 25 has been fully depressed. Finally, once the EBC is collected and ejected through the needle 35 according to the inspection procedure described herein below, the entire device 10 can be disposed of according to conventional waste disposal procedures.

  FIG. 4 is a partial side cross-sectional view of the breath condensate collection device 60 according to the second preferred embodiment of the present invention. Similar to the first embodiment, this breath condensate collection device 60 includes a central chamber 24, a plunger assembly 65, a rubber gasket 28, a double wall syringe 62 comprising a handle 30, and a breath input assembly (FIG. Not shown). The body of the syringe 62, including the central chamber 24 and its various components, is similar to that of the first syringe 20 with some important exceptions. Initially, the outlet 38 of the first embodiment is replaced with a bleed port 68 having a diameter of about 1 mm that penetrates the inner wall 22 and outer wall 32 of the syringe 62 near its distal end. When the plunger assembly is pushed down but holding the EBC, the bleed port 68 allows air to escape from the central chamber 24. Also, the plunger assembly 65 does not include a nipple-shaped tip or a needle extending therefrom on the front surface of the piston 66. However, the front face of the piston 66 has a slight conical shape and the end of the central chamber 24 has a corresponding shape. Together, this configuration can guide the collected condensate to the outlet 38 more efficiently. However, the nipple tip 27 of the previous device 10 and the conical shape disclosed in the device of FIG. 4 are not mutually exclusive, ie, the conical shape of FIG. 4 is coupled to the nipple tip 27 of FIG. It will be apparent that the conical shape can be easily incorporated into the device of FIG.

  Another difference between the first device 10 and the second device 60 is that the second device comprises a threaded nozzle 61 to which the hypodermic needle 35 can be attached. Needle 35 is similar to needle 35 of first device 10, but is not attached to plunger assembly 65 but is fixed to nozzle 61. The inside of the needle 35 is connected in communication with the inside of the central chamber 24. The needle 35 can be protected by a removable plastic cover 69 having a threaded fitting that facilitates easy connection and removal of the cover 69 from the threaded nozzle 61. When attached to the syringe 62, the cover 69 preferably provides a relatively hermetic seal for the purposes described below.

  The use of this second device 60 is somewhat similar to that of the first device 10, but the cover 69 must be removed before the subject can breathe through the needle 35. However, with the improvements described above, this device 60 can be better adapted to deliver a calibrated amount of condensate than the first device 10. After the subject has breathed through the device 60 a sufficient number of times to ensure that a minimal amount of condensate has been collected, the cover 69 can be returned to the end of the syringe 62 and the plunger assembly 65 Can be pushed down carefully. While the cover 69 effectively prevents gas or liquid from passing through the needle 35, the conventionally designed bleed port 68 allows air and excess condensate to drain until the front of the piston 66 passes the calibration line 78. Enable. To prevent the EBC from escaping to the environment, a cotton ball or other absorbent material, if desired, and an appropriate support structure, if desired, can be placed over the bleed port 68. By holding the device 60 with the bleed port 68 oriented upwards, most or all of the air can be removed from the central chamber 24 and a calibration amount of condensate therein, as measured by the calibration line 78. Remain in. As shown in FIG. 4, the syringe 62 can use a single wall design in the area of the calibration line 78 to make it easier to see the liquid contained therein. Preferably, the syringe 62 is calibrated to take an amount between 250-500 μL. The cover 69 can then be removed again and this calibration amount can be fed by needle 35 to the appropriate reaction chamber apparatus.

  FIG. 5 is a schematic side sectional view of a breath condensate collection device 80 according to a third preferred embodiment of the present invention. It may be advantageous to partition the exhaled breath into airway and alveolar components and generate condensation only from the part of the exhaled air that originates from the alveoli. This segmentation process may help differentiate lower respiratory pulmonary infection from bronchitis. As described in Kline US patent application Ser. No. 10 / 778,477, described above, an apparatus for collection of alveolar breath condensate incorporates a gating mechanism that operates, for example, by raising the partial pressure of exhaled carbon dioxide. be able to. The breath condensate collection device 80 shown in FIG. 5 is an example of a device suitable for use in a preferred embodiment of the device or a comparable device. The device 80 of FIG. 5 is capable of inserting the device 80 into a housing as included in the device disclosed in US patent application Ser. No. 10 / 778,477, with the exhalation input assembly 50 of the first device 10 removed. Is the same as the apparatus 10 of FIG. As will be apparent to those skilled in the art, to facilitate fluid communication from the gating mechanism, or for other purposes, the direction of air flow can also be reversed during the condensation process, as shown in FIG. it can. In this case, the respective positions of the outlet 36 and inlet 38 and their respective one-way valves 81, 83 are now reversed relative to the device 10 of FIG.

  Although not illustrated herein, airflow reversal can also be applied to the apparatus 60 shown in FIG. However, even if the air flow direction is reversed, the condensate can still be pumped into the reaction chamber device via the hypodermic needle 35 as described with respect to either FIGS. 3A and 3B or FIG.

  In the preceding embodiment, each device 10, 60, 80 is designed to collect EBC from a subject that breathes spontaneously, and the subject can pass through a mouthpiece or face mask (not shown), as shown in FIG. Exhale into a pipe that directs flow to an appropriate inlet, such as that shown schematically in FIG. It may also be advantageous to adapt the present invention to allow collection from patients undergoing mechanical ventilation. In most hospitals, humans are often ventilated against pressurized air mixed with oxygen and humidified with excess water vapor. This excess water vapor accumulates condensation in the outflow tubing that directs exhaled breath away from the patient's lungs during the breathing cycle. It is standard practice for a respiratory therapist to attach a small cylinder of about 20-100 mL to the bottom of most of the exhalation circuit and collect this condensate. Using the present invention, a certain amount of collected condensate can be analyzed for LPS content to diagnose Gram-negative bacterial lung infections in patients undergoing artificial respiration. In the simplest embodiment, a commercially available syringe of the type used for drug delivery (eg, an insulin syringe) is used to set a condensate volume (eg, in the condensate tank of the exhalation line of the ventilator circuit (eg, 100-200 microliters) can be withdrawn and this amount can be injected into the reaction chamber apparatus of the examples described below.

  FIG. 6 is a schematic diagram of an alternative ventilation system that implements a preferred embodiment of the present invention. Using the device 80 of FIG. 5 as an example, collection from a patient undergoing artificial respiration adds a vacuum system 92 to the outlet 36 of the device 80 and connects the Y or T joint 94 to the aforementioned luer at the end of the nozzle 39. Can be facilitated by connecting to a lock fitting. A Y or T joint 94 is connected to the conduit along the outflow path from the patient 96 to the conventional ventilator 98. The outflow path includes two polyethylene tubes 102, 104, the first tube being connected from the conventional endotracheal tube 100 or other patient interface to the inlet of the Y or T fitting 94 and the second tube being Y or T. Connected between the outlet of the coupling 94 and a port such as is normally provided on the outflow track of the ventilator 110. The third polyethylene tube 106 is connected to the endotracheal tube 100 from the inflow track of the artificial respirator 110. Each polyethylene tube 102, 104, 106 has an inner diameter of about 10-30 mm and a length of 1 meter or more. Overall, such a device allows the vacuum system 92 to draw a continuous sidestream sample of exhaled breath through the collection device 80 until a sufficient amount of EBC has been collected. The suction rate by the pump of the vacuum system 92 can be set to about 100 mL / min.

  FIGS. 7 and 8 are side cross-sectional schematic and top views of one reaction chamber assembly 120 suitable for use with the harvesting devices 10, 60, 80 of FIGS. 2, 4, and 5, respectively. The reaction chamber assembly 120 is designed to detect and quantify the amount of LPS present in the EBC. The assembly 120 is similar to the general configuration of a sterile ampoule that is commonly used to store medication for injection. The assembly 120 can function as both a reaction chamber and a spectrophotometer cuvette, and measures the percentage of visible light transmitted that is proportional to the concentration of LPS in the sample. Assembly 120 is vacuum sealed and includes reaction chamber 122 and plug 124, retaining ring 126, and protective cap 128. The reaction chamber 122 is preferably made from glass or another transparent material, but is alternatively semi-transparent if the selected color or light changes or phenomena described below can be easily seen therethrough. It can be made from a material (translucent or other non-opaque). The reaction chamber 122 preferably has a volume of about 0.5 to 1.0 mL. As shown, the reaction chamber 122 is circular, but the reaction chamber 122 can be square as well, or have another geometric shape that facilitates insertion into the spectrophotometer, Alternatively, it will be understood that it can be modified so that it can be “modified” into an existing commercial laboratory analysis system. The reaction chamber 122 further includes a threaded male luer lock connector 132 similar to that of the condensate collection device 10, 80 described above. Of course, the luer lock fitting 132 may not be necessary when utilizing the device 60 as disclosed in FIG.

  In order to maintain a sealed, sterile and pyrogen-free environment inside the reaction chamber 122, a plug 124, preferably formed from rubber, can be placed on top of the reaction chamber 122. The plug 124 is retained in the reaction chamber 122 by a retaining ring 126, which is preferably made of aluminum, and an opening that allows exhaled condensate to be pumped into the reaction chamber 122 via the inlet port 130 of the plug 124. In the center. In a preferred embodiment, delivery port 130 is a resealable lid that can be punctured with a hypodermic needle. A protective cap 128, preferably formed from plastic, is temporarily attached to the top of the assembly 120 to prevent damage or contamination of the retaining ring 126 and plug 124.

  In use, the sterile reaction chamber assembly 120 is removed from the reservoir and an EBC sample is taken using one of the disclosed devices 10, 60, 80 (or another equivalent device). The protective cap 128 of the reaction chamber assembly 120 is removed, and then a relatively accurate amount of EBC sample is delivered to the reaction chamber 122 by inserting each hypodermic needle 35 into the delivery port 130 of the plug 124. Where provided, the respective luer lock fittings can be engaged to effectively secure the devices 10, 60, 80 to the reaction chamber assembly 120. The plunger assembly 25 can then be depressed until a sufficient amount of EBC has been fed into the assembly 120. Although a calibration line 134 can be attached to the reaction chamber 122 to indicate the required volume, such a line 134 may not be required if the volume has been previously calibrated with the sampling device 60. During assembly, the reaction chamber 122 is sealed under vacuum to draw a sufficient amount of EBC required for accurate measurement of endotoxin analyte.

  Reaction chamber 122 contains calcium and magnesium salts to maintain a pH of about 7.4 upon hydration of pre-designated dry mass 136 of Factor C, Factor B, proclotting enzyme, and chromogenic substrate, and With phosphate, or other organic compounds such as TRIS aminomethane buffer. The amounts and activities of these enzymes are pre-calibrated so that a predetermined amount of EBC allows detection of clinically relevant ranges of LPS. The amount ranges from 0 to 1.00 corresponding to a concentration of LPS ranging from 0 to about 10 EU / mL in undiluted EBC when measured at a reaction time of 15-30 minutes at about 37 ° C. A range of linear optical density readings is calibrated to result from the reaction. The temperature of the reaction is preferably maintained at least about 34 ° C. More preferably, the temperature of the reaction is maintained at least about 37 ° C.

  The chromogenic substrate can withstand temperatures up to about 43 ° C. without loss of activity. Thus, the reaction chamber can be heated to about 43 ° C. by a controlled exothermic reaction. It is preferred to control the exothermic reaction in order to bring about an even culture at 34-43 ° C. for at least 15 minutes, preferably 25 minutes. Therefore, as described below, it is necessary to take measures to control the rate of the exothermic hydration reaction.

  Such an exothermic reaction can be initiated by placing a salt in the reaction chamber that releases heat upon hydration. An example of such a salt includes, but is not limited to, sodium thiosulfate pentahydrate. When the salt is placed in the reaction chamber, an exothermic reaction can be started by introducing the collected condensate. It is preferred to use a powdered salt that promotes immediate complete hydration within a short heating period. For example, the salt can be in a semi-permeable matrix or crystalline form that can generate a reaction temperature of 34-43 ° C. for at least 15 minutes by controlled hydration. Alternatively, the salt can be placed in a user initiated heating jacket surrounding the reaction chamber. In this embodiment, the reaction chamber can be inserted into a double wall jacket composed of a flexible polymer. The jacket can include a dry salt that can release heat upon hydration, and an ampule of water (2-5 mL) that can be easily broken by squeezing with two fingers. The act of breaking the water ampoule initiates hydration of the salt, causing an exothermic reaction, thus heating the reaction chamber to about 43 ° C. for about 15-30 minutes, at which point the color intensity and corresponding endotoxin activity is reduced. As stated, it can be quantified.

  Preliminary data suggest that concentrations of LPS above about 0.20 EU / mL (calibrated linear optical density reading of about 0.20) correspond to a laboratory test positive threshold indicating the presence of gram-negative bacterial infection in the lung Is done. In a preferred embodiment, the chromogenic substrate can be a peptide conjugated to a dye, such as paraaminoanilide, which is cleaved by a clotting enzyme and releases a yellow color into the reaction solution having a peak absorbance of about 405 nm. It will be readily appreciated that other chromogenic substrates can be substituted and that other dye markers can be conjugated to alternatives that can be detected at different isosbestic points. In addition, the marker molecule can be a compound that includes excitation fluorescence properties, whereby the molecule emits light within a narrow wavelength interval in response to stimulation by an incident beam of monochromatic light. One skilled in the art will appreciate that a variety of chromogenic and fluorescent substrates can be used as markers for detecting the presence of LPS.

  Alternatively, the reaction chamber assembly can be designed to capture LPS using a matrix-bound antibody on the side of the well. FIG. 9 is a perspective view of a first alternative reaction chamber assembly 140. In this alternative embodiment, assembly 140 functions as a vertical microtiter well using enzyme-linked immunoassay techniques and has the advantage of being able to detect species-specific LPS molecules 154. The assembly 140, which can function as a cuvette, can include a chamber 142, a plug 144, and a retaining ring or other structure 146. For purposes that will become apparent below, the window 143 is formed in at least a portion of the wall of the chamber 142. Similar to the ring 126 of the previous device 120, the ring 146 includes an opening that exposes the implantation target region 150 in the center of the plug 144.

  10A-10D are partial schematic partial side cross-sectional views of the first alternative reaction chamber assembly 140 of FIG. The Fc portion of each antibody 152 is conjugated or bound to window 143 of assembly 140 using conventional techniques. Thus, the Fab portion of each antibody 152 is oriented toward the center of the assembly 140 and the LPS154 O-polysaccharide portion is better captured. After introduction of the EBC sample, chamber 142 can then be washed with an albumin-containing buffer (not shown) to remove non-specific substances. Then, by adding water without LPS (not shown), a separate ampoule containing a predetermined amount of the chromogenic substrate 156 as described above is reconstituted, and the chromogenic reagent sample is withdrawn by a syringe, as shown in FIG. 10C. As shown in FIG. Chromogenic substrate 156 reacts with LPS 154, thereby releasing dye 158. After about 15-30 minutes, the optical density can be read by one of several methods. One chromogenic substrate 156 suitable for use in a preferred embodiment of the present invention incorporates paranitroanilide, which can be detected at about 405-410 nm when released.

  Binding other molecules with high affinity specific for any part of LPS to the reaction chamber wall to capture LPS, but not antibodies with specific immunogenicity to O antigen Those skilled in the art will understand that These molecules include HA-1A and E5, lipopolysaccharide binding protein (“LPB”), bactericidal / permeability increasing protein (“BPI”), Limulus anti-lipopolysaccharide factor (“LALF”), or polymyxin antibiotic But are not limited thereto.

  A number of embodiments can be utilized to quantify the LPS concentration. In one embodiment, a semi-quantitative decision can be made using a visual comparison with a color strip. The color strip resembles a standard difference with one color patch displayed in the horizontal direction with the lightest shade placed at the leftmost edge, the darkest shade placed at the rightmost edge, and gradually increasing the shade intensity. Yes. Each shadow patch is associated with a specific range of endotoxin unit (EU) reactivity, such that an increase in shadow intensity represents an increase in EU reactivity. The observer holds the transparent reaction chamber assembly 120, 140 next to the plastic or paper color strip in ambient light to compare the shadow of the contents of the reaction chamber assembly 120, 140 with the color strip patch. can do. The observer can then select the color patch that best matches the shade intensity of the color observed in the reaction chamber. A positive response is indicated by a specific color shadow threshold corresponding to about 0.20 EU / mL or determined from additional testing.

  More accurate quantification can be obtained with a small, specially designed single beam light spectrophotometer configured to accept a post-incubation reaction chamber as described above. The spectrophotometer can include a heating coil that is activated by insertion of a reaction chamber to warm the sample to 37 ° F. The spectrophotometer can alternatively display or print the measured optical density after 25 minutes of incubation.

  FIG. 11 is a perspective view of a test kit 200 incorporating a second alternative reaction chamber device that allows EBC to be delivered to a single use cartridge that can be read visually without the use of a spectrophotometer. FIG. The test kit 200 has two modules 203 and 223 including a test module 203 and a positive control module 223 arranged in the housing 202. 12 is a side cross-sectional view taken along line 12-12 of the inspection module 203 of FIG. The inspection module 203 includes a port 204. Port 204 preferably has a volume of about 100 microliters, EDTA, calcium and magnesium salts that allow the dry mass to maintain a pH of about 7.4 by hydration and form an aqueous reagent, As well as a dry mass 136 of Factor C, Factor B, proclotting enzyme, and chromogenic substrate, along with phosphates or other organic compounds.

  In FIG. 11, the reaction chamber 208, test matrix 212, a pair of microtubules 214, 216, a pair of one-way valves 218, 220, and an outlet 222 are further shown. Reaction chamber 208 preferably has a volume of at least about 250 microliters. Port 204 and reaction chamber 208 are connected by first microtubule 214, while reaction chamber 208 is connected to outlet 222 by second microtubule 216. One skilled in the art will appreciate that the microtubule 214 can include one or more microtubules. In order to facilitate one-way fluid communication from the port 204 through the reaction chamber 208 to the outlet 222, valves 218, 220 are disposed in the first and second microtubules 214, 216, respectively. If microtubule 214 includes multiple microtubules, more than one valve 218, 220 may be required to accommodate multiple microtubules. At the top of the cartridge is a pre-printed color strip 221 arranged in a series of gradually darkening color patches, each gradually darkening color patch configured to correspond to a gradually increasing interval of endotoxin activity. is there.

  The reaction chamber 208 is coated with a transparent material 210 so that the matrix 212 can be observed for color changes that indicate the presence of LPS. A matrix 212 disposed in the reaction chamber 208 is a surface formed from an insoluble polymer or other material that is received and stained with a chromogenic substrate. Suitable materials include, but are not limited to, polymers derived from cellulose, agarose, methacrylate, polystyrene, polyphenyl, polynaphthyl, polybenzyl, nylon, silk, or other fibers. Although the matrix 212 is absorbent, it is densely woven, allowing the fluid to continue to float in the reaction chamber 208 until the fluid moves through the microtubule 216 by gravity and capillary action. As an advantage, the matrix 212 can essentially bind LPS itself, or covalently bind one of LPS binding molecules such as HA-1A and E5, LPB, BPI, LALF, or polymyxin antibiotics or other Can be combined in any way. Thus, the matrix 212 develops color intensity in proportion to the endotoxin concentration in the unknown sample. Visual comparison with the preprinted color strip 221 allows a semi-quantitative determination of endotoxin concentration as described above.

  The inside of the test kit 200 can also include sodium thiosulfate pentahydrate and a sealed ampule of water that breaks when the test kit 200 is squeezed. When hydration of sodium thiosulfate is initiated by the action of breaking the ampule of water, an exothermic reaction is caused, thus heating the entire test kit 200 to about 43 ° C. for about 25 minutes. The materials for the exothermic reaction are placed in the test kit 200, but they are separated from all areas in the test kit 200 where the sample is introduced and some LPS related chemical reaction occurs.

  In operation, the reaction is initiated by injecting the condensate collected by the apparatus 10, 60, 80 into the reaction chamber 204 containing the dry mass 136 described above. Preferably, at least about 0.35 mL of condensate is injected into the reaction chamber 204. The operation of injecting the condensate causes a mixing action of the condensate and dry mass 136 in the reaction chamber 204 and microtubule 214, thereby initiating the LAL reaction. The pressure differential induced by the injection is combined with the capillary action force, so that the reaction mixture flows into the reaction chamber 208 through the microtubule 214 and continues during the chromogenic substrate reaction at a temperature of 34-43 ° C. Stay there. Since endotoxin is bound to matrix 212, colored molecules hydrolyzed from the chromogenic substrate stain matrix 212 to a hue intensity proportional to the activity of endotoxin in the condensate, and the color change is a color strip for quantification. 221 can be compared visually.

  13 is a side cross-sectional view taken along line 13-13 of the positive control module 223 of FIG. The positive control module 223 includes a port 224, a reaction chamber 228, a test matrix 232, an amount of dry LPS combined with a dry mass 136, here designated 244, a pair of microtubules 234, 236, 1 A pair of one-way valves 238, 240 and an outlet 242 are included. The port 224 and the reaction chamber 228 are connected by a first microtubule 234, and the reaction chamber 228 is connected by a second microtubule 236 to the outlet 242. Valves 238 and 240 are disposed in the first and second microtubules 234 and 236, respectively, so as to allow one-way fluid communication from the port 224 through the reaction chamber 228 to the outlet 242. As shown, LPS 244 is located at port 224, but it will be understood that LPS 244 may alternatively or additionally be located within first microtubule 234 or on test matrix 232.

  A second alternative reaction chamber apparatus can also be used as follows. An EBC sample is injected into the test module port 204 from one of the aforementioned breath condensate collection devices 10, 60, 80 and then directed into the test module reaction chamber 208 through the first test module microtubule 214. Can do. In a preferred embodiment, the amount of EBC sample is about 0.35 mL. The amount of sample is preferably calibrated so that the well 208 containing the matrix 212 is full, thus coating the matrix 212 with liquid and initiating the binding of the LPS to the matrix 212. Excess EBC can flow out through the second test module microtubule 216. The one-way valve 218 prevents LPS from entering the ambient air. Absorbent material can be placed at 216 to prevent external fluid leakage.

  After a relatively accurate predetermined time, which can be about 60 seconds, the test module 203 and the positive control module 223 can each be washed with a volume of sterile (endotoxin free) water. In a preferred embodiment, the amount of sterile water injected into each module 203, 223 is about 0.25 mL. Water passes through the respective reaction chambers 208, 228 and is discharged from the respective outlets 222, 242 via the respective second microtubules 216, 236.

  Next, LPS-free water is added to an ampoule such as reaction chamber apparatus 120 of FIGS. 7 and 8 to maintain a pH of about 7.4 by hydration and form EDTA, calcium to form an aqueous reagent. Like reaction chamber apparatus 120 of FIGS. 7 and 8, including dry mass 136 of factor C, factor B, proclotting enzyme, and chromogenic substrate, along with salts of and magnesium, and phosphate or other organic compounds Added to ampoules. Once the reagent is reconstituted, it uses a syringe (often called a “TB syringe”) (not shown) as commonly used to deliver insulin and provide tuberculosis test antigens. Withdrawal can be injected into both the test module port 204 and the positive control module port 224. The test kit 200 can then be left at 23 ° C. for a predetermined time, eg, 10 minutes to develop color, and then visually inspected for changes in color or pattern in the test well 208. A positive control module 223 is included to ensure the activity of the chromogenic substrate enzyme system.

  In a further alternative, allowing gel formation of the coagulation cascade within the tubule, as described in US Pat. No. 4,370,413 (Neeman et al., '413), which is hereby incorporated by reference in its entirety. Can measure the proportion of LPS in the EBC. FIG. 14 is a partial side cross-sectional view of the breath condensate collection device 260 according to the fourth preferred embodiment of the present invention. The device 260 includes a double wall 22, 32, a central chamber 24, a plunger assembly 65 with a piston 26, a rubber gasket 28, and a handle 30, an exhalation input assembly (not shown), and an outlet 68. 4 is similar to that of FIG. 4 in that it includes a syringe 262 having a threaded nozzle 61 and a removable plastic cover 69. Each of these components is generally similar to those of the device 60 of FIG. 4 except that the syringe 262 further includes a manometer 265 that extends from the side of the syringe in fluid communication with the outlet 68. In addition, each device 60, 260 includes a needle 35, 275, the needle 275 of this fourth embodiment serving as a capillary in which the EBC contacts the LAL assay reagent. Thus, needle 275 is occluded by a dry, insoluble and inert fiber substrate 270 pre-filled with LAL assay reagent to gel when LPS is present in EBC, as shown in FIG. .

  The reaction can be initiated by depressing the plunger assembly 65 until the plug 270 is wetted with EBC. The amount of gelation at the outflow needle 275 is proportional to the amount of LPS in the EBC. After a specified time, the plunger assembly 65 can be forced down again and the manometer 265 can be used to measure the extrusion pressure in the central chamber 24. The extrusion pressure is proportional to the LPS concentration in EBC.

  In all preceding embodiments, chemicals that prevent reaction with β-glucan (factor G), including heating the sample to 75 ° C., adding molecules with alkyl glucosides or beta 1,4 glucoside bonds, or It should be understood that the specificity of the reaction can be improved by including methods.

  Once gram-negative bacterial infection is diagnosed, antibiotic therapy can be initiated. One skilled in the art will appreciate that the methods of the invention can be utilized to monitor the effectiveness of antibiotic treatment. Effective antibiotic treatment may cause an initial increase in LPS concentration in the exhaled breath condensate due to bacterial death with cell membrane rupture and increased release of released LPS into the alveolar epithelial fluid One skilled in the art can predict that there will be no. This increase in concentration is expected in the first 24-72 hours after antibiotic administration. Furthermore, effective antibiotic treatment can ultimately be expected to result in a reduction in the concentration of LPS in exhaled breath condensate that is exhaled during the next 3-10 days after antibiotic administration. Thus, it can be inferred to use continuous LPS measurements to monitor the effectiveness of treatment. The absence of an initial increase in LPS content in exhalation or a decrease in LPS content in exhalation over the next few days may indicate resistance of Gram-negative bacteria to treatment.

  The present invention is advantageous because it provides a method and apparatus for diagnosing Gram-negative bacterial infections relatively easily and painlessly. The present application is directed to diagnosing Gram-negative bacterial pneumonia by measuring the concentration of LPS in the breath condensate. However, the present invention can be used for diagnosis of any pulmonary Gram-negative bacterial infection including but not limited to sepsis. One skilled in the art understands that gram-negative bacterial infections can be treated with the same antibiotic treatment, and therefore, any diagnosis of gram-negative bacterial infection is advantageous for subjects with such infections. Will be done. Furthermore, the present invention can be used to identify the specific strain of Gram negative bacteria that caused the infection in situations where this information is useful.

Example

Example 1

  To wake up and diagnose whether a cooperative, spontaneously breathing patient has Gram-negative bacterial pneumonia, LPS in breath condensate samples from such patients was detected according to the following procedure.

  Subjects for treatment were selected according to the following procedure. Subjects (N = 8 per group) were divided into the following three criteria: 1) Diagnosis of pneumonia, 2) Healthy patients actively smoking more than 10 cigarettes a day, and 3) Healthy non- Recruitment based on smokers. In a cough that produces a colored fistula, a measured fever over 101 ° F., leukocytosis demonstrated by a peripheral leukocyte count over 12000 per cubic microliter, and a chest radiograph to obtain subjects diagnosed with pneumonia Subjects diagnosed with a standard treatment background including the presence of moist material were selected. Exclusion criteria for subjects include the use of antimicrobial drug treatment, acute illness or anatomical abnormalities that make breath collection impossible, and / or suspected pulmonary tuberculosis.

  The breath condensing device used for the collected breath sample is as described below. The apparatus consisted of a 1 meter long polyvinyl tube (15 mm inner diameter) attached to a modified glass flask about 50 cm high. The glass flask had a specially designed removable glass stopper on top, but otherwise looked similar to a standard laboratory volumetric flask. The glass plug had two outlets, one for inserting a glass tube that protruded upward and bent at 90 degrees, and the other was able to expel dry exhaled air into the ambient air. One end of the glass tube was connected to a polyvinyl tube that fits snugly into the glass tube. The other end of the glass tube protruded about 25 cm into the inside of the glass bulb of the flask. The entire glass flask was submerged in a cooled slurry of dry ice in ethanol.

  Exhaled breath condensate was collected by the following procedure. The subject held the polyvinyl tube, and exhaled into the platypus mouthpiece attached to the opposite end of the polyvinyl tube. The subject's breath was sent through a polyvinyl tube into the condensation chamber of the flask where the cooled inside of the flask condensed and frozen the subject's breath water vapor and aerosolized droplets. The subject was asked to take about 30 deep breaths, resulting in about 1-3 mL of sample.

  Prior to taking a breath sample from a subject, multiple steps were taken to reduce contamination of the sample with external endotoxin or beta glucan molecules. All components were sterilized by autoclaving at 220 ° C. for a minimum of 4 hours during use and by using a commercial cleaning solution designed to remove endotoxins. The polyvinyl tube was wrapped with sterile foil before autoclaving. The sterile foil was left on during sampling, but was removed before removing the polyvinyl tube. The technician used sterile gloves to handle all components. However, the end 6 cm of the polyvinyl tube could be grasped by a subject who did not wear sterile gloves. After the condensate was dissolved, it was sucked from the condensation chamber with the tip of a pyrogen free pipette and transferred to a cryotube free of pyrogen under a layered hood. To test for background endotoxins, a “mock” standard for glassware and piping was performed according to the following procedure. Two milliliters of endotoxin-free water (Sigma Chemical, St. Louis, MO) was injected into the cleaned and autoclaved condensation chamber, stirred, and then aspirated and stored in the same manner as the condensate sample. Condensate samples and simulated standard water were stored at -57 ° C until analysis.

  The presence of endotoxin was detected according to the following procedure. Analysis was performed on undiluted dissolved condensate using a chromogenic Limulus assay according to manufacturer's specifications (Associates of Cape Cod, Falmouth, Mass., USA). The reaction was carried out in a 96-well microtiter plate (Associates of Cape Cod, pyrochrome microtiter plate) for 30 minutes at 37 ° C., and a color change of 405 nm was read with a microtiter plate spectrophotometer. All samples were run in one batch. A standard curve was achieved using the assay endotoxin standard and was linear at R = 0.997.

  FIG. 16 is a scatter plot showing the endotoxin concentration measured for patients in each of the three test groups. Mean (± SD) endotoxin concentrations for normal, smokers, and pneumonia patients were 0.096 ± 0.050, 0.091 ± 0.040, and 0.234 ± 0.130 EU / mL, respectively. The graph suggests that 0.20 EU / mL may be a useful limit to distinguish between subjects with Gram-negative bacterial pneumonia and subjects who are not Gram-negative bacterial pneumonia.

  Two subjects with pneumonia and test values less than 0.20 EU / mL were labeled “A” and “B”. Prior clinical trials confirmed that these subjects were not Gram-negative bacterial pneumonia. Patient A was positive for streptococcal pneumonia as a result of blood culture and was clinically estimated to be the bacterial cause of the patient's pneumonia. Patient B had a negative blood culture, no other specimens were submitted for analysis, and influenza (viral) pneumonia was clinically suspected.

  Two of the water samples from the simulated standard had values of 0.10 and 0.14 EU / mL (average was 0.12 EU / mL). Therefore, 0.12 EU / mL was considered to be the background “noise” of the system, and this value was subtracted from the 0.20 EU / mL limit determined on the uncorrected sample. Using this analysis, samples of breath condensate containing values above 0.08 EU / mL are considered abnormal test results. It should be understood that this background correction value may be different if different condensers are used in different laboratories.

Example 2

  To diagnose whether a subject breathing with the aid of a ventilator has Gram-negative bacterial pneumonia, LPS was detected in breath condensate samples from such subjects according to the following procedure: .

  Six subjects participated in the study. Four of the subjects receiving mechanical ventilation showed clinical evidence of pneumonia and were treated with antibiotic therapy. Two of the subjects receiving mechanical ventilation showed no clinical evidence of pneumonia and were used as controls.

  A breath condensate sample was obtained according to the following procedure. Analytes were obtained from exhaled breath condensate accumulated in the outflow piping attached to the endotracheal tube of the artificial respiration system. The analyte appeared clear and non-turbid by visual inspection.

  The presence of endotoxin was detected according to the following procedure. Analyzes were performed on undiluted and diluted condensates using a chromogenic Limulus assay according to the manufacturer's specifications (Associates of Cape Cod, Falmouth, Mass., USA). Dilutions included 1:10, 1: 100, and 1: 1000 dilutions in endotoxin free water. The reaction was carried out in a 96-well microtiter plate (Associates of Cape Cod, pyrochrome microtiter plate) for 30 minutes at 37 ° C., and a color change of 405 nm was read with a microtiter plate spectrophotometer. All samples were run in one batch. The upper limit of detection of the assay was 0.25 EU / mL.

  The results of the analysis are as follows. All six samples were above the detection range for undiluted samples (ie, higher than 0.25 EU / mL). The average endotoxin concentration in all 1:10 diluted samples of pneumonia subjects was above the limit of linear detection, but the average endotoxin concentration of the 1:10 dilution of two artificially ventilated patients without pneumonia was 0. Less than 0.05 EU / mL. These data show that when the concentration in the sample of the condensate that appears clear and non-turbid by visual inspection in the outflow piping of the artificial respiration circuit is higher than 0.25 EU / mL, the bronchi that becomes positive for Gram-negative bacterial pneumonia This suggests that the presence of alveolar lavage samples is expected.

Example 3

  LPS in the breath condensate was detected according to the following procedure. A commercially available glass flask with a volume of 1 liter was prepared to collect a breath condensate sample according to the following procedure. The glass was heated to 400 ° C. for 3 hours so that its surface was free of LPS. A sterilized flexible 11 mm inner diameter polyvinyl tube is placed in fluid communication with the branch of the flask so that when the patient exhales into the tube, the patient's exhalation passes through the glass flask and out of the outlet. Set up. To facilitate capture of exhaled breath condensate in the flask, the flask was partially submerged in a dry ice and ethanol slurry mixture as coolant. To prevent capture of aerosolized saliva, the tubes and flasks were arranged so that the condensation flasks were maintained above the patient's height.

  Exhaled air was collected according to the following procedure. Eight subjects with varying health conditions breathed into a tube connected to the flask. Each subject took 100 deep breaths into the flask using a hand counter to track breathing. An average amount of condensate equal to 11 ± 4 mL was obtained from this method.

  LPS in breath condensate was detected using a commercial chromogenic LAL assay (Cape Cod, Mass.) According to the following procedure. Duplicate 50 μL aliquots from each subject's condensate sample were transferred to two wells of a standard 96-well ELISA plate. Reagents necessary for color development LAL assay were added, and the reagents were incubated at 37 ° C. for 30 minutes. An optical density of 405 nm was read with a commercially available plate well reader. Concentrations were determined by comparison with a standard curve using known amounts of LPS obtained from FDA approved sources.

  The following results were obtained. All four normal volunteers were found to have LPS concentrations below 100 pg / mL, while two otherwise healthy smokers clearly showed concentrations below 200 pg / mL became. Two smokers with clinical pneumonia were found to have EBC LPS concentrations in excess of 800 pg / mL. From this experiment, it was concluded that the LPS content of the EBC sample can vary based on the lung health of the subject providing the sample. These data also suggest that EBC can be sampled without contamination from the oral flora of healthy subjects.

  Based on the above information, those skilled in the art will readily understand that the present invention has a wide range of practicalities and uses. Many embodiments and adaptations of the invention other than those specifically described herein may be used in this invention, as well as many variations, modifications, and equivalent arrangements, without departing from the spirit or scope of the invention. It is clear and reasonably suggested from the invention and its above description. Thus, while this invention has been described herein with reference to preferred embodiments thereof, this disclosure is merely illustrative and exemplary of the invention and provides a disclosure that allows the full and practice of the invention. It should be understood that this was done only for the purpose. The above disclosure is not intended to limit the present invention or to exclude such other embodiments, adaptations, modifications, changes, or equivalent arrangements. The present invention is limited only by the claims appended hereto and their equivalents. Although specific terms are used herein, they are used in a general and descriptive sense rather than for purposes of limitation.

Claims (35)

A method for determining and monitoring an indicator of pulmonary Gram-negative bacterial infection in an air-breathing vertebrate subject comprising:
(A) providing a reaction chamber having an internal surface, the reaction chamber having a material that binds to a lipopolysaccharide disposed therein;
(B) sending at least a portion of exhaled breath condensate collected from an air-breathing vertebrate subject into the reaction chamber;
(C) washing the reaction chamber with a buffer;
(D) sending a reaction reagent into the reaction chamber;
(E) determining whether the subject has an intrapulmonary gram-negative bacterial infection based on a physical change that occurs when at least a portion of the collected breath condensate is pumped into the reaction chamber And wherein the physical change is caused by the presence of lipopolysaccharide in the exhaled breath condensate;
(F) identifying a specific strain of intrapulmonary gram-negative bacteria;
Only contains specific to process, based on the determination of O- antigen of lipopolysaccharide molecules, the method comprising identifying a particular strain of intrapulmonary Gram negative bacteria, binding material disposed in a reaction chamber a matrix Method embedded in the surface . The method of claim 1 , wherein the reaction reagent comprises a chromogenic substrate or a fluorescent substrate . 2. The method of claim 1 , wherein the binding material is an antibody that binds to a specific species of lipopolysaccharide. 4. The method of claim 3 , wherein the antibody is disposed on the internal surface of the reaction chamber. The method of claim 1 , wherein the binding material is an insoluble polymer. The method further includes the step of visually matching the color change of the reaction chamber with a standard, wherein the standard includes a print strip of color patches of increasing hue intensity, each color patch corresponding to an increasing concentration of lipopolysaccharide. The method of claim 1 . 2. The method of claim 1 , wherein a hue intensity corresponding to a concentration of at least 0.2 endotoxin units per milliliter indicates the presence of an intrapulmonary gram-negative bacterial infection. 2. The method of claim 1 , wherein the air-breathing vertebrate subject is a subject that spontaneously breathes or a subject that undergoes mechanical ventilation . The method of claim 1 , wherein feeding at least a portion of the exhaled breath condensate comprises injecting at least a portion of the exhaled breath condensate into the reaction chamber via a hypodermic needle. A method for determining and monitoring an indicator of pulmonary Gram-negative bacterial infection in an air-breathing vertebrate subject comprising:
(A) preparing a reaction chamber in which a reaction reagent is disposed;
(B) sending at least a portion of exhaled breath condensate collected from an air-breathing vertebrate subject into the reaction chamber;
(C) determining whether the subject has an intrapulmonary gram-negative bacterial infection based on physical changes that occur when at least a portion of the collected exhaled breath condensate is pumped into the reaction chamber And wherein the physical change is caused by the presence of lipopolysaccharide in the exhaled breath condensate;
(D) identifying a specific strain of gram-negative bacteria in the lung;
Only including the step of identifying, based on the determination of O- antigen of lipopolysaccharide molecules involves identifying the specific strain intrapulmonary Gram-negative bacteria, method. 11. The method of claim 10 , wherein the reaction reagent comprises a Limulus deformed cell lysate. The method of claim 10 , wherein the reaction reagent comprises a chromogenic substrate or a fluorescent substrate . The method of claim 10 , wherein the reaction reagent comprises a chemical that releases heat through an exothermic reaction by feeding at least a portion of the breath condensate. The method of claim 10 , wherein the physical change is the formation of a gel. The method further includes the step of visually matching the color change of the reaction chamber with a standard, wherein the standard includes a print strip of color patches of increasing hue intensity, each color patch corresponding to an increasing concentration of lipopolysaccharide. The method according to claim 10 . 16. The method of claim 15 , wherein a hue intensity corresponding to a concentration of at least 0.2 endotoxin units per milliliter indicates the presence of an intrapulmonary gram negative bacterial infection. 11. The method of claim 10 , wherein the air-breathing vertebrate subject is a subject that spontaneously breathes or undergoes mechanical ventilation . 11. The method of claim 10 , wherein feeding at least a portion of the exhaled breath condensate comprises injecting at least a portion of the exhaled breath condensate into the reaction chamber via a hypodermic needle. A method for determining and monitoring an indicator of pulmonary Gram-negative bacterial infection in an air-breathing vertebrate subject comprising:
(A) preparing a reaction chamber in which a reaction reagent is disposed;
(B) sending at least a portion of exhaled breath condensate collected from an air-breathing vertebrate subject into the reaction chamber;
(C) determining whether the subject has an intrapulmonary gram-negative bacterial infection based on physical changes that occur when at least a portion of the collected exhaled breath condensate is pumped into the reaction chamber And wherein the physical change is caused by the presence of lipopolysaccharide in the exhaled breath condensate;
(D) a step of visually matching the color change of the reaction chamber with a standard, wherein the standard includes a print strip of a color patch of increasing hue intensity, each color patch having an increasing concentration of lipopolysaccharide Corresponding to each process,
(E) identifying a specific strain of pulmonary Gram-negative bacteria;
Only including the step of identifying, based on the determination of O- antigen of lipopolysaccharide molecules involves identifying the specific strain intrapulmonary Gram-negative bacteria, method. 20. The method of claim 19 , wherein the reaction reagent comprises a Limulus deformed cell lysate. The method according to claim 19 , wherein the reaction reagent comprises a chromogenic substrate or a fluorescent substrate . 20. The method of claim 19 , wherein the reaction reagent comprises a chemical that releases heat through an exothermic reaction by feeding at least a portion of the breath condensate. 20. The method of claim 19 , wherein the physical change is gel formation. 20. The method of claim 19 , wherein a hue intensity corresponding to a concentration of at least 0.2 endotoxin units per milliliter indicates the presence of an intrapulmonary gram negative bacterial infection. 20. The method of claim 19 , wherein the air-breathing vertebrate subject is a subject that spontaneously breathes or undergoes mechanical ventilation . 20. The method of claim 19 , wherein feeding at least a portion of the exhaled breath condensate comprises injecting at least a portion of the exhaled breath condensate into the reaction chamber via a hypodermic needle. A method for determining and monitoring an indicator of pulmonary Gram-negative bacterial infection in an air-breathing vertebrate subject comprising:
(A) providing a reaction chamber having an internal surface, the reaction chamber containing a material that binds to a lipopolysaccharide disposed therein;
(B) sending at least a portion of exhaled breath condensate collected from an air-breathing vertebrate subject into the reaction chamber;
(C) washing the reaction chamber with a buffer;
(D) sending a reaction reagent into the reaction chamber;
(E) determining whether the subject has an intrapulmonary gram-negative bacterial infection based on a physical change that occurs when at least a portion of the collected breath condensate is pumped into the reaction chamber And wherein the physical change is caused by the presence of lipopolysaccharide in the exhaled breath condensate;
(F) identifying a specific strain of intrapulmonary gram-negative bacteria;
And the step of identifying comprises identifying a particular strain of pulmonary Gram-negative bacteria based on the determination of the O-antigen portion of the lipopolysaccharide molecule. 28. The method of claim 27, wherein the reaction reagent comprises a chromogenic substrate or a fluorescent substrate. 28. The method of claim 27, wherein the binding material is an antibody that binds to a specific species of lipopolysaccharide. 30. The method of claim 29, wherein the antibody is disposed on the interior surface of the reaction chamber. 28. The method of claim 27, wherein the binding material is an insoluble polymer. The method further includes the step of visually matching the color change of the reaction chamber with a standard, wherein the standard includes a print strip of color patches of increasing hue intensity, each color patch corresponding to an increasing concentration of lipopolysaccharide. The method of claim 27. 28. The method of claim 27, wherein a hue intensity corresponding to a concentration of at least 0.2 endotoxin units per milliliter indicates the presence of an intrapulmonary gram negative bacterial infection. 28. The method of claim 27, wherein the air-breathing vertebrate subject is a subject that spontaneously breathes or undergoes mechanical ventilation. 28. The method of claim 27, wherein delivering at least a portion of the exhaled breath condensate comprises injecting at least a portion of the exhaled breath condensate into the reaction chamber via a hypodermic needle.

Images