Utilizing lipopolysaccharide in exhaled breath condensate to diagnose gram negative pneumonia

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.

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         43 . A device for collecting exhaled breath condensate from a subject breathing with the assistance of a mechanical ventilation circuit, comprising:
 (a) a mechanical ventilation circuit for facilitating breathing of the subject, wherein the mechanical ventilation circuit includes an expiratory flow tube that serves as a conduit for removing exhaled breath of the subject;   (b) a central chamber having an interior, wherein said central chamber may be cooled to a temperature of about 32 degrees Fahrenheit and below to promote condensation;   (c) a breath input assembly, disposed at one end of the central chamber, in fluid communication with the interior of the central chamber and the expiratory flow tube, whereby the breath input assembly connects the expiratory flow tube and the central chamber;   (d) an exit assembly, disposed at the other end of the central chamber, in fluid communication with the interior of the central chamber; and   (e) a vacuum device connected to the exit assembly for collecting exhaled breath condensate from the central chamber.   
     
     
         44 . A breath condensate collection device, comprising:
 (a) a central chamber having an interior and first and second opposing ends;   (b) a breath input assembly in fluid communication with the interior of the central chamber; and   (c) an exit assembly in fluid communication with the interior of the central chamber, wherein the exit assembly includes a narrow tube, said narrow tube having a fibrous plug disposed therein, said plug being impregnated with a reaction reagent that causes gelation upon exposure to lipopolysaccharide.   
     
     
         45 . The breath condensate collection device of  claim 44 , wherein the plug is a fiber matrix impregnated with enzymes from a limulus amoebocyte. 
     
     
         46 . The breath condensate collection device of  claim 44 , further comprising a plunger assembly having a piston and a handle, wherein the piston is slidably disposed in the interior of the central chamber and wherein the handle extends from the first end of the central chamber so as to permit the piston to be moved within the central chamber, whereby the collected breath condensate may contact the fibrous plug disposed within the narrow tube. 
     
     
         47 . A reaction chamber assembly for measuring the concentration of lipopolysaccharide in exhaled breath condensate, comprising:
 (a) a reaction chamber;   (b) a delivery port for delivery of the exhaled breath condensate into the reaction chamber;   (c) a reaction reagent to react with lipopolysaccharide present in the exhaled breath condensate; and   (d) a viewing portion to allow for visible detection of a physical change in the reaction chamber.   
     
     
         48 . The reaction chamber of  claim 47 , wherein the delivery port is a re-sealable cover that may be punctured with a hypodermic needle for delivery of the exhaled breath condensate. 
     
     
         49 . The reaction chamber of  claim 47 , wherein the viewing portion is a transparent wall. 
     
     
         50 . The reaction chamber of  claim 47 , wherein the reaction chamber includes an interior surface, and wherein a lipopolysaccharide-immobilizing agent is bound to at least a portion of the interior surface of the reaction chamber. 
     
     
         51 . The reaction chamber of  claim 50 , wherein the lipopolysaccharide-immobilizing agent is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, polymyxin antibiotic, lipopolysaccharide-binding protein and limulus anti-lipopolysaccharide factor. 
     
     
         52 . The reaction chamber of  claim 47 , further comprising a chemical disposed within the reaction chamber, wherein the chemical liberates heat upon hydration, whereby a temperature in the range of 34 to 43 degrees Celsius is achieved for a period of 15 to 30 minutes upon hydration of the chemical. 
     
     
         53 . The reaction chamber of  claim 52 , wherein the chemical is a salt in a semipermeable matrix. 
     
     
         54 . The reaction chamber of  claim 52 , wherein the chemical is a salt in crystalline form. 
     
     
         55 . The reaction chamber of  claim 52 , wherein the chemical is sodium thiosulfate pentahydrate. 
     
     
         56 . A test kit cartridge for detecting the presence of lipopolysaccharide in exhaled breath condensate, comprising:
 (a) a housing;   (b) a test module disposed within the housing, said test module utilizing a user-initiated chemical reaction to detect the presence of lipopolysaccharide in the exhaled breath condensate; and   (c) a positive control module disposed within the housing adjacent to the test module, said positive control module having lipopolysaccharide disposed herein for showing a definite positive result for the presence of lipopolysaccharide for comparison with the test module.   
     
     
         57 . The test kit cartridge of  claim 56 , further comprising a chemical disposed in the housing that provides a controlled exothermic reaction, whereby a temperature between 34 and 43 degrees Celsius is achieved for a period of 15 to 30 minutes. 
     
     
         58 . The test kit cartridge of  claim 57 , wherein the chemical is sodium thiosulfate pentahydrate. 
     
     
         59 . The test kit cartridge of  claim 56 , wherein each module of the test kit includes a re-sealable injection port, a reaction well, a fluid connection between the reaction well and the injection port, a first one-way valve disposed in the fluid connection, an exit tube providing an outlet for the reaction well, and a second one-way valve disposed in the exit tube. 
     
     
         60 . The test kit cartridge of  claim 59 , wherein each reaction well includes a clear covering adapted to allow visual determination of physical change. 
     
     
         61 . The test kit cartridge of  claim 59 , wherein each reaction well includes an insoluble polymeric lipopolysaccharide-binding matrix. 
     
     
         62 . The test kit cartridge of  claim 59 , wherein the positive control module reaction well includes lipopolysaccharide from a suitable species of Gram negative bacteria. 
     
     
         63 . The test kit cartridge of  claim 59 , further comprising a chromogenic substrate, reconstituted from a reaction chamber. 
     
     
         64 . The test kit cartridge of  claim 59 , further comprising a color strip, the color strip having color patches of incrementally increasing color intensity, wherein each color patch corresponds to a prescribed range of endotoxin units to allow for visual comparison against a color change in the reaction wells. 
     
     
         65 . The test kit cartridge of  claim 56 , wherein the housing is formed from a polymer. 
     
     
         66 . The test kit cartridge of  claim 65 , wherein the polymer is plastic.