Utilizing lipopolysaccharide in exhaled breath condensate to diagnose gram negative pneumonia

This application claims the benefit of, and claims priority to provisional U.S. Patent Application Ser. No. 60/577,641, filed on Jun. 7, 2004, which is incorporated herein by reference in its entirety.

This application is also a continuation-in-part of U.S. patent application Ser. No. 10/742,721 filed Dec. 19, 2003, which claims the benefit of provisional U.S. Patent Application Ser. No. 60/434,916 filed Dec. 20, 2002 and provisional U.S. Patent Application Ser. No. 60/447,581 filed Feb. 14, 2003. The entirety of each of the aforementioned applications is incorporated herein by reference.

In addition, this is a continuation-in-part of U.S. patent application Ser. No. 10/778,477 filed Feb. 13, 2004, which claims the benefit of provisional U.S. Patent Application Ser. No. 60/447,581 filed Feb. 14, 2003. The entirety of each of the aforementioned applications is likewise incorporated herein by reference.

1. Field of the Present Invention

The present invention relates generally to a method and devices for diagnosing gram negative bacterial pneumonia and, in particular, to diagnosing gram negative bacterial pneumonia by detecting the presence of lipopolysaccharide in exhaled breath condensate.

2. Background

Pneumonia represents a common disease with significant morbidity and mortality. Pneumonia is the number one cause of death by infectious disease and the sixth most common cause of death in the United States. The National Hospital Ambulatory Medical Care Survey found that in 2001, 1.48 million emergency department visits were related to a diagnosis of pneumonia. The National Hospital Discharge Survey found that in 1998, 1.32 million patients were discharged after having been treated for pneumonia.

Pneumonia can be caused by lung infection of many types of microorganisms, including viruses, chlamydia, mycoplasma, protozoa, fungi, and bacteria. For a patient with suspected pneumonia, the clinician has a duty to determine the exact cause of infection because the identity of the infectious agent dictates the choice of antimicrobial treatment. The most common cause in Western society is bacterial pneumonia, and when bacterial pneumonia is suspected, clinicians generally seek to categorize the cause of bacterial pneumonia as Gram positive, Gram negative or anaerobic.

In particular, clinicians are motivated to identify the presence of Gram negative bacterial infection because Gram negative lung infections are aggressive and are associated with higher rates of complications and death. The Gram negative feature of bacteria refers to the color of the bacteria after a staining protocol that will be well understood by those skilled in the art. Gram negative bacterial infections, including gram negative bacterial pneumonia, require specific antimicrobial therapy, which is different from treatment for other types of bacterial infections, and warrant an elevated level of financial reimbursement from third party payors such as Medicare.

In current clinical practice, pneumonia is diagnosed by combining clinical, laboratory and radiographic information. In general, features such as the patient\'s complaint, the patient\'s vital signs, the peripheral white blood count, and the results of chest radiography are used to determine the presence or absence of pneumonia. When these sources of data fit a typical pattern, the diagnosis can be made with reasonable clinical certainty, and antimicrobial therapy can be initiated prior to the results of bacteriological cultures. Although nonspecific clinical data is often used to initiate antibiotic treatment for Gram negative infection, common practice dictates that the final diagnosis of Gram negative pneumonia requires more specific evidence of Gram negative bacterial lung infection.

Toward this goal, a Gram\'s stain can be performed immediately on sputum that is coughed from the lower airways, and microscopic analysis may reveal bacteria with morphology and color suggesting Gram negative infection. However, useful sputum samples are notoriously difficult to obtain from humans with pneumonia. When blood specimen cultures from a patient who has a clinical pattern consistent with pneumonia grow a Gram negative bacterium, this provides a specific indication of Gram negative pneumonia. Unfortunately, more often than not, the blood is sterile in a patient with Gram negative pneumonia.

In addition, a patient\'s blood may be examined for endotoxin concentration using chemical assays for the endotoxin molecule. However, endotoxin concentrations have been found to be an inaccurate predictor of either the cause or severity of the more general sepsis syndrome. Endotoxin concentrations in the blood may fluctuate widely over short time periods. Further, certain disease states, including liver disease, polytrauma, hypertension, and hematological malignancies are associated with chronically elevated endotoxin concentrations in the absence of clinically significant infection. No study has examined whether circulating endotoxin concentrations can predict a gram negative source of pneumonia.

A sensitive and specific method to diagnose Gram negative lung infection is to perform bronchalveolar lavage and to perform a bacteriological culture on the lavage fluid. Another method is to chemically assay for lipopolysaccharide content in bronchoalveolar lavage samples. Investigators using this method found that high concentrations of lipopolysaccharide are associated with concomitant growth of gram negative bacteria in cultures of the bronchalveolar fluid. A complete description of this method may be found in Flanagan, P. G., Jackson, S. K., Findlay, G., “Diagnosis of Gram negative, ventilator associated pneumonia by assaying endotoxin in bronchial lavage fluid”, J. Clin. Pathol. 2001, 54:107-110 and Pugin, J., Auckenthaler, R. and Delaspre, O., “Rapid Diagnosis of gram-negative pneumonia by assay of endotoxin in bronchoalveolar lavage fluid”, Thorax 1992, 47:547-549. Both methods have the drawbacks that special endoscopic equipment and subspecialty expertise are required and that they are relatively invasive and uncomfortable procedures. Moreover, known culture methods require at least 24 hours to obtain results. As such, a patient may wait for up to at least 24 hours before receiving an effective antibiotic treatment.

The cell wall of Gram negative bacteria comprises endotoxins. Endotoxins are toxic materials released by bacterium on bacterial lysis. While endotoxins were first recognized for their ability to induce fever, they are now known to have a broad spectrum of biologic activities. On bacteriolysis, endotoxins consisting of aggregates of lipopolysaccharides and protein and lipids, are released from the bacterium into surrounding medium. Endotoxins consist primarily of lipopolysaccharide (“LPS”) with various amounts of protein and lipid. Since almost all of the biologic activities usually attributable to bacterial endotoxins can also be elicited with isolated chemically pure lipopolysaccharide, the terms “endotoxin” and “lipopolysaccharide” are used interchangeably.

From a pathogenic standpoint, the presence of lipopolysaccharide is one of the most important implications of a Gram negative infection. As such, detecting lipopolysaccharide in a patient\'s bodily fluid is an indicator of Gram negative bacterial infection. More specifically, the presence of lipopolysaccharide in a patient\'s bodily fluid is an important potential indicator of Gram negative pneumonia.

The present invention overcomes the above-described clinical disadvantages of diagnosing Gram negative bacterial pneumonia by performing an assay for lipopolysaccharide on the liquid derived from condensation of exhaled breath. Using novel devices and methods described herein, exhaled breath condensate may be obtained from a spontaneously breathing subject via a mouthpiece, facemask or other similar means or from a subject breathing with the assistance of a mechanical ventilator via a connection to the expiratory tubing of the mechanical ventilator. An additional advantage of the present invention is the shorter time period for diagnosing a Gram negative bacterial infection, such as pneumonia, than is required for other diagnostic methods.

Alternative devices for collecting exhaled breath condensate are also known. These devices include those disclosed in Gaston et al., U.S. Pat. Nos. 6,033,368 and 6,419,634; Hunt et al., U.S. Pat. No. 6,585,661; Baddour, U.S. application Ser. No. 10/257,912; and EU Patent No. 0,759,169 B1 to Winsel et al., all of which are incorporated by reference herein in their entireties. In addition, Kline, U.S. application Ser. Nos. 10/742,721 and 10/778,477, two commonly-assigned non-provisional patent applications, disclose devices for collecting exhaled breath and are incorporated by reference herein in their entireties.

Exhaled condensate is known to contain many molecules that can serve as markers of many lung diseases, as reviewed by Kharitinov et al. in 2001 (Biomarkers 7 (1):1-32, 2002.). However, the concept of measuring lipopolysaccharide in exhaled breath condensate for the purpose of diagnosing Gram negative pneumonia or other Gram negative bacterial infections has not been disclosed previously.