WO2010036930A1 - Methods and kits for detecting joint infection - Google Patents

Abstract

The invention provides methods, kits, and devices for detecting joint infection in a patient, using detection reagents which are specific for leukocyte markers present in a synovial fluid sample collected from the patient suspected of having the joint infection.

Description

METHODS AND KITS FOR DETECTING JOINT INFECTION

SPECIFICATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Provisional Application Serial No. 61/100,568, filed on September 26, 2008, entitled METHODS AND DEVICES FOR DETECTING PERIPROSTHETIC JOINT INFECTION and whose disclosure is incorporated by reference herein.

BACKGROUND FIELD OF INVENTION

[0002] This invention relates to methods and kits for detecting joint infection and specifically, periprosthetic joint infection. BACKGROUND

[0003] Total joint arthroplasty is routinely performed to alleviate pain and improve quality of life in patients with advanced arthritis. However, like other surgical procedures, complications may arise including periprosthetic joint infection (PJI) , which can hamper the patient's functional outcome and result in excess morbidity. Infection is one of the most devastating causes of prosthetic joint failure, with an incidence of around 2% and an even higher rate after revision procedures. Even though this percentage is relatively small, the large number of arthroplasties performed every year constitutes a huge economical burden on the healthcare system and a considerable source of morbidity.

[0004] The diagnosis of infection after total joint arthroplasty continues to pose a challenge, particularly when presenting as subacute or low grade infection

(Parvizi et al, Journal of Bone and Joint Surgery

(American). 2006 ; 88 : 138-147) . Currently, there is no universally accepted diagnostic test or modality that is accurate or reliable in determination of joint infection. The diagnosis of PJI relies on clinical suspicion and a combined armamentarium of serological and imaging modalities, with isolation of organisms from the intraoperative culture samples constituting the "gold standard" for ultimate diagnosis.

[0005] The first step in development of PJI is for the infecting organism to gain access to the joint and attach itself to a surface of the artificial implant. When such process takes place, the body mounts an immune response that includes recruitment of the white blood cells (leukocytes) to the infected joint to fight the infection. The white blood cells arriving at the site of infection proliferate in number, hence increasing the number of white blood cells in the joint fluid of patients with active infection. The white blood cells fight infection by secreting various enzymes that attempt to punch holes in the wall of the bacteria or infecting organism at the site of infection. Some of these enzymes result in digestion of the bacterial cell wall while others may prevent bacterial proliferation by interfering with each step of proliferation. [0006] The white blood cell count of a bodily fluid (e.g., urine) can provide very useful information in the diagnosis of an infection. The white blood cell count of the bodily fluid may be calculated indirectly from the presence in the bodily fluid of various enzymes produced by the white blood cells. There are tests designed to detect the presence of infection of different organs, and in particular, to diagnose urinary tract infection. However, unlike urine, which is devoid of any proteins or macromolecules unless there is an infection, analysis of the joint fluid for the presence of enzymes is difficult because the joint fluid has numerous constituents which can interfere with the testing and which are difficult to separate or remove. [0007] Accordingly, there remains a need in the art for an easy and reliable test for PJI. SUMMARY

[0008] The present invention fills the foregoing need by providing methods and kits for detecting infection in an artificial joint.

[0009] In one aspect, a method for detecting infection in an artificial joint is provided. The method comprises obtaining a sample of the joint fluid, detecting the amount of three or more leukocyte markers in the sample, and taking a first action based on the amount of three or more leukocyte markers in the sample. The three or more leukocyte markers are selected from the group consisting of cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, gelatinases , IL-lβ, metalloproteinases (MMPs), cathepsins, such as cathepsin A and cathepsin B, phospholipases , such as, for example, phospholipase A and phospholipase B, and combinations thereof. In one embodiment, the sample maybe pre-treated before being tested for the presence of three or more leukocyte factors .

[0010] In another aspect, a kit for detecting infection is provided. The device comprises a housing having one or more wells containing reagents for detecting the amount of one or more leukocyte markers in a sample of the joint fluid disposed inside the housing. These reagents are selected to detect in a sample of the joint fluid three or more of the following enzymes: cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, gelatinases, IL-I beta, metalloproteinases (MMPs), cathepsins, such as cathepsin A and cathepsin B, phospholipases, such as, for example, phospholipase A and phospholipase B, or combinations thereof. Exemplary chemokines and cytokines include, but are not limited to, interleukins, including IL-I alpha and beta, and IL-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, interferon gamma (IFNγ) and other interferons, including interferon alpha, beta and omega, tumor necrosis factor (TNF) and its related ligands and receptors. In one embodiment, the sample maybe pre- treated before being tested for the presence of one or more leukocyte factors.

[0011] The invention provides a kit for detecting joint infection in a patient, said kit comprising (a) at least three detection reagents which are specific for each of at least three leukocyte markers present in a synovial fluid sample collected from the patient suspected of having the joint infection, wherein said at least three detection reagents are adapted to detect a threshold level of each of said at least three leukocyte markers, said threshold level adapted to indicate a presence of joint infection; and (b) instructions for using said at least three detection reagents to evaluate joint infection in the patient. The invention further provides a kit wherein said joint infection is periprosthetic joint infection. The invention further provides the kit wherein said at least three leukocyte markers are selected from the group consisting of leukocyte elastase, leukocyte esterase, gelatinase B, cathepsins, interleukins, and cytokines. The invention further provides the kit wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and gelatinase B, and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample; and wherein said threshold level of leukocyte esterase is at least about 23 pg/ml of leukocyte esterase in said synovial fluid sample,- and wherein said threshold level of gelatinase B is at least about 10 pg/ml of gelatinase B in said synovial fluid sample. The invention further provides the kit wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and IL-lβ and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample; and wherein said threshold level of leukocyte esterase is at least about 23 pg/ml of leukocyte esterase in said synovial fluid sample; and wherein said threshold level of IL-lβ is at least about 2 pg/ml of IL-lβ in said synovial fluid sample. The invention further provides the kit wherein a detection reagent is selected from the group consisting of antigens; haptens; monoclonal and polyclonal antibodies; natural and synthetic mono-, oligo- and polysaccharides; lectins; avidin and streptavidin,- biotin,- growth factors; hormones; receptor molecules; and combinations thereof. The invention further provides the kit wherein the detection reagent is selected from monoclonal and polyclonal antibodies. The invention further provides the kit wherein the detection reagent comprises an antibody or antibody fragment .

[0012] The invention further provides a kit, wherein said kit comprises a detector device having a support in communication with at least one of said at least three detection reagents. The invention further provides the kit wherein said kit comprises a dipstick, a swab, and a container. The invention further provides the kit wherein the detection reagents are attached to a detectable label. The invention further provides the kit further comprising one or both of a positive and a negative control.

[0013] The invention provides a method of screening for infection in a joint of a patient, said method comprising the steps of (a) extracting a sample comprising synovial fluid from a joint (b) applying the sample to a detector device, wherein the detector device comprises at least three detection reagents which are specific for each of at least three leukocyte markers, wherein said at least three detection reagents are adapted to detect a threshold level of each of said at least three leukocyte markers, said threshold level correlated with a presence of infection; (c) ascertaining the threshold levels of the leukocyte markers present in said sample, wherein if the concentration each of said at least three leukocyte markers exceeds the threshold level, then this is a positive screen for infection of the joint. The invention provides the method wherein the infection is a periprosthetic joint infection. The invention provides the method wherein said at least three leukocyte markers are selected from the group consisting of leukocyte elastase, leukocyte esterase, gelatinase B, cathepsins, interleukins , and cytokines. The invention provides the method wherein said ascertaining is performed by an enzyme-linked immunosorbent assay. The invention provides the method wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and gelatinase B and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample,- and wherein said threshold level of leukocyte esterase is at least about 20 pg/ml of leukocyte esterase in said synovial fluid sample; and wherein said threshold level of gelatinase B is at least about 10 pg/ml of gelatinase B in said synovial fluid sample.

[0014] The invention provides the method wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and IL-lβ and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample; and wherein said threshold level of leukocyte esterase is at least about 23 pg/ml of leukocyte esterase in said synovial fluid sample; and wherein said threshold level of IL- lβ is at least about 2 pg/ml of IL- lβ in said synovial fluid sample. The invention provides the method wherein the detection reagent is selected from the group of species consisting of antigens; haptens; monoclonal and polyclonal antibodies; natural and synthetic mono-, oligo- and polysaccharides; lectins: avidin and streptavidin,- biotin; growth factors,- hormones; receptor molecules; and combinations thereof. The invention provides the method wherein the detection reagent is selected from monoclonal and polyclonal antibodies. The invention provides the method wherein said reagents comprise an antibody or antibody fragment. The invention provides the method wherein said detector device is a dipstick comprising at least three detection reagents which are specific for leukocyte markers present in a synovial fluid sample.

[0015] The invention provides a method for detecting infection in an artificial joint, the method comprising obtaining a sample of the joint fluid; applying the sample to a detector device; testing the sample to detect the amount of one or more leukocyte markers in the sample; taking a first action based on the findings of the test. The invention further provides a method wherein the one or more leukocyte markers is selected from the group consisting of cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, gelatinases, metalloproteinases (MMPs), cathepsins, phospholipases, and combinations thereof. The invention further provides a method wherein the one or more leukocyte markers is leukocyte elastase, leukocyte esterase, and IL-lβ. The invention further provides a method wherein the first action comprises placing a temporary antibiotic spacer. The invention further provides a method further comprising pre-treating the sample. The invention further provides a method further comprising comparing the amount of one or more leukocytes markers in the sample to a threshold value for the one or more leukocyte markers . The invention provides a device for detecting infection in an artificial joint comprising a housing,- reagents for detecting the amount of one or more leukocyte markers in a sample of the joint fluid disposed inside the housing. The invention further provides a device comprising 3 wells into which reagents for detecting at least three different leukocyte markers are placed. The invention further provides a device wherein one or more leukocyte markers are selected from the group consisting of leukocyte elastase, leukocyte esterase, and IL-lβ and combinations thereof. DETAILED DESCRIPTION

[0016] Methods are provided for detecting, evaluating and treating joint infection, preferably periprosthetic joint infection (PJI) in mammals, including humans. The methods comprise obtaining a sample of a joint fluid, detecting the amount of the leukocyte markers in the sample, and taking an action based on the amount of leukocyte markers detected in the sample.

[0017] The term "joint fluid" refers to any fluid in contact with any surface of a joint, preferably, a joint which underwent a surgical procedure and received an implant, for example, an artificial joint including but not limited to an artificial knee joint or an artificial hip joint. Preferably, such fluid is substantially free of blood or tissue of the patient. The joint fluid may be tested in vivo or in vitro. For in vitro testing, a sample may be obtained, for example, by aspiration using a needle and syringe.

[0018] The inventor has determined that the number of white blood cells (leukocytes) and the percentage of neutrophils in the joint fluid has a high positive predictive value to predict infection. The leukocytes present in an infected joint secrete enzymes that may also be detected and used to confirm infection. Such suitable markers include cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, gelatinases, metalloproteinases (MMPs), cathepsins, such as cathepsin A and cathepsin B, phospholipases , such as, for example, phospholipase A or phospholipase B, and combinations thereof. It will be understood that in some embodiments only one marker may be measured, while in other embodiments more markers may be measured, including at least two, at least three, or at least four markers. The level of the markers are independent of leukocyte count, but are indicative of infection.

[0019] Although it is generally known to test bodily fluids, such as urine, for the presence of infection in the body by detecting leukocyte markers, the concept of testing joint fluid for the presence of PJI by detecting leukocyte markers is novel. The joint fluid is considerably different in many aspects from urine and other bodily fluids typically tested for infection. For example, joint fluid contains a large number of macromolecules, such as proteoglycans, which give the joint fluid its viscous characteristics. More importantly, while the presence of any leukocytes in urine is indicative of an infection, joint fluid may contain many leukocytes in the absence of infection. Hence, detection of white blood cells in joint fluid, unlike in urine, does not always imply an infection. [0020] Prompt diagnosis of infection is critical for delivery of appropriate care. Detecting PJI at early stage enables managing infection by less-invasive methods, such as, for example, retaining the prosthetic joint and treating the patient with antibiotics. In contrast, the prosthetic joint would almost certainly have to be removed if PJI is allowed to progress past a certain point . The instant methods may also have value in the operating room. Currently, one of the standard methods to test for infection involves sending a tissue sample from the joint being operated on to the microbiology laboratory to test for the presence of infection or white blood cells. The latter test termed "frozen section or Gram stain" has a 30 to 40 % accuracy for diagnosing infection. It is also time consuming as the sample needs to be transported to the lab, processed, and analyzed. Determining infection during revision surgery is extremely important. If an infection is believed or proven to be present, then the surgeon may abandon placement of an artificial prosthesis and place a temporary antibiotic spacer. The instant methods have enormous value in helping the clinicians determine the best course of action for the patient. First, and foremost, because of the rapidity of the test the clinician can determine the status of the joint with regard to infection in the office or the operating room setting and deliver the appropriate care. Second, the test is rapid, saving many minutes during surgery and days for patients being evaluated in the office setting. Third, it is more accurate and more cost effective than any other test available for diagnosis of PJI. [0021] The inventor discovered that utilizing at least three detection reagents that are specific for selected leukocyte markers taken together with a threshold value which is indicative of infection allows diagnosing infection in a joint fluid with an accuracy of at least 88%. Accordingly, the instant kits and methods differ from prior techniques because the preferred embodiments of the instant methods do not simply determine whether the leukocytes are present or not, but compare the number of leukocytes in the joint fluid sample with a threshold value which is indicative of infection. Whereas previous techniques utilize a test strip that changes color based on the quantity of enzyme present, the result of the preferred embodiments of the instant methods may be all-or-none, i.e., with color or without color or another differentiating medium. In other words, the clinicians using the preferred embodiments of the instant invention will not be left with an uncertainty regarding the possibility of infection, i.e., trying to determine whether the color change was significant, which would enable clinicians to take prompt actions if necessary. [0022] The threshold value may be represented in terms of leukocyte concentration or number, neutrophil differential, or both. Representing the threshold value in terms of both leukocyte concentration or number and neutrophil differential is preferable. The threshold value may be set to between about 1000 to about 3000 leukocytes per microliter and/or a neutrophil differential of between about 60% to about 80%. By way of non- limiting example, the threshold value may be set to about 1100 white blood cells per microliter and a neutrophil differential of about 64% or greater. In such embodiments, test results greater than 1100 cells per microliter and a neutrophil differential greater than 64% would be indicative of PJI. Furthermore, to ensure accuracy of the tests, in the preferred embodiments, the joint fluid sample may be tested for a combination of enzymes. The test values for each enzyme tested may then be compared to a threshold level indicative of infection for each enzyme.

[0023] Typically, the joint fluid sample may be tested as is, i.e., without pre-treatment . In other embodiments, the joint fluid sample may be pre-treated as known in the art to increase the test's accuracy. For example, if the tap is bloody and the aspirate is found to contain many red blood cells, then the joint fluid sample may be separated into layers by, for example, centrifugation, so the red blood cells may be discarded. Drops of the supernatant from the processed fluid may then be tested for the presence of leukocyte markers . [0024] Generally, to test the joint fluid sample for the presence of the leukocyte markers, the sample is exposed to a substance that reacts selectively with three or more leukocyte markers in a detectable manner to indicate the presence or absence, and/or amount, of the leukocyte marker in the sample. The term "substance that reacts selectively" "specifically" or "selectively reactant substance" means an organic or inorganic substance which is changed, such as hydrolyzed or cleaved, by the action of one or more leukocyte markers or which selectively binds to a particular leukocyte marker or is otherwise modified in the presence of a particular leukocyte marker.

[0025] Suitable methods for testing the joint fluid sample include for example, radio-immunoassay, immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) , microarray, or Western blot, but are not limited to these detection methods. One skilled in the art is versed in various immunochemical detection methods, such as those described in The Immunoassay Handbook, D. Wild, Ed. (1994) Stockton Press, New York, incorporated herein by reference in its entirety. For rapid detection and to minimize expenses, the instant methods may preferably utilize the ELISA technique for detection of leukocyte markers. [0026] Detection Reagents

[0027] A detection reagent described herein, e.g., a leukocyte marker described herein, such as IL-lβ, leukocyte elastase, and/or leukocyte esterase, can be an antibody specifically reactive with a protein described herein. An antibody can be an antibody or a fragment thereof, e.g., an antigen binding portion thereof. As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH) , and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL) . The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR) . The extent of the framework region and CDR 's has been

precisely defined (see, Kabat et al . , Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia et al . , J. MoI. Biol. 196:901-917, 1987, which are incorporated herein by- reference) . Each VH and VL is composed of three CDR ¹S and four FRs, arranged from amino-terminus to carboxyl- terminus in the following order: FRl, CDRl, FR2 , CDR2 , FR3 , CDR3 , FR4.

[0028] The antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHl, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system. [0029] The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (e.g., a polypeptide encoded by a nucleic acid of Group I or II) . Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains,-

(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region,- (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al . , Nature 341:544-546, 1989), which consists of a VH domain; and

(vi) an isolated complementarity determining region

(CDR) . Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate nucleic acids, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv

(scFv) ,- see e.g., Bird et al . , Science 242:423-426, 1988, and Huston et al . , Proc . Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope. A monoclonal antibody composition thus typically displays a single binding affinity for a particular protein with which it immunoreacts .

[0030] Anti-protein/anti-peptide antisera or monoclonal antibodies can be made as described herein by using standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane, Cold Spring Harbor Press, 1988).

[0031] A protein described herein, e.g., a leukocyte marker described herein, such as IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B can be used as an immunogen to generate antibodies that bind the component using standard techniques for polyclonal and monoclonal antibody preparation. The full-length component protein can be used or, alternatively, antigenic peptide fragments of the component can be used as immunogens .

[0032] Detection reagents that selectively bind leukocyte markers include, but are not limited to, polyclonal antibodies, monoclonal antibodies, synthetic antibodies, chimeric antibodies, antigen fragments thereof or combination thereof. These primary antibodies may be labelled with an analytically-detectable reagent such as an epitome, a radioisotope, a fluorescent molecule or an enzyme. In another embodiment, a secondary antibody may be utilized to detect the primary antibody/enzyme complexes. Antibodies against every one of the leukocyte markers are available commercially or can be produced in the lab using known techniques.

[0033] In other embodiments, detection reagents that selectively binds to one or more leukocyte markers may comprise one or more substances hydrolysable by one or more leukocyte markers, such as a peptide. For example, suitable substrate linkers for detecting leukocyte elastase include, but are not limited to, one or more of the oligopeptidic sequences Lys-Gly-Ala-Ala-Ala-Lys-Ala- Ala-Ala (SEQ ID NO: 1), Ala-Ala-Pro-Val (SEQ ID NO: 2), Ala-Ala-Pro-Leu (SEQ ID NO: 3), Ala-Ala-Pro-Phe (SEQ ID

NO: 4), Ala-Ala-Pro-Ala (SEQ ID NO: 5) or Ala-Tyr-Leu- VaI (SEQ ID NO: 6) . Alternatively, leukocyte esterase catalyzes the hydrolysis of the derivatized pyrrole amino acid ester to liberate 3 -hydroxy-5-phenyl pyrrole. This pyrrole then reacts with a diazonium salt to produce a purple product. The intensity of the purple color developed may be used to assign a value to esterase activity.

[0034] Preferably, these detection reagents produce a color or change color when they react with one or more leukocyte markers. If such substance does not produce or change color or produce, or change color less dramatically than desired, then the diagnostic device may further include an indicator agent adapted to change color or produce color when the reaction takes place. In an embodiment, the indicator agent may be conjugated to the support by the substance cleavable by one or more leukocyte markers, so the indicator agent may change color or produce color when the linker is hydrolyzed. For example, para-nitrophenyl is colorless when linked to the support, and turns yellow when cleaved. Suitable indicator agents include, but are not limited to, a dye, a radioactive moiety, a spin label, a luminescent moiety or a fluorophore.

[0035] In certain embodiments, the detection reagents that selectively react with leukocyte markers are immobilised on a support, e.g., a solid support material, utilizing for example, avidin-biotin linking, or dialdehyde derivatization of the support material, followed by cross-linking to a peptide binding partner. The support material may be presented in any suitable form, including but not limited, to porous materials such as nylon, nitrocellulose, cellulose acetate, glass fibers, as well as plates, tubes, or beads of polystyrene. The support can be shaped in a variety of forms, such as, for example, a dipstick, a strip, a swab or a coating on a test container. Alternatively, the leukocyte markers present in the joint fluid sample may be immobilized on the surface of the solid support, either non- specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA) , and then reacted with the detection reagents. [0036] leukocyte Elastase

[0037] Leukocyte elastase is a serine protease that is a major component of neutrophil granules and is essential for defense against infection by invading microorganisms (Bode, W. et al . 1989, "Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity and mechanism-based inhibitors," Biochem. 28:1951 1963). Leukocyte elastase activity may be determined by any standard technique. One example is the use of a double antibody sandwich assay, such as described by Crooks et al . , 1995, J. Clin Microbiol., 33:924-929. Another example is the use of fluorogenic substrates as described in U.S. Pat. No. 4,336,186. Chromogenic substrates that can be used include, but are not limited to, methoxysuccinyl-L- alanyl-L-alanyl-L-prolyl-L-valine 7 amino-4 -methyl - coumarin (Shah et al . , 1996, Euro. Respir. J., 9:531- 534; succinyl-L-alanyl-L-alanine-p-nitroanilide (Tsang et al., 2000, Chest, 117:420-426. One method involves the use of a synthetic substrate N-methoxysuccinyl-ala- ala-pro-val p-nitroanilide (Fujita et al . , 1990, Am. Rev. Respir. Dis., 142:57-62). Reaction of elastase with this substrate produces a colored product detected at 405 to 570 nm. Quantitative determination can be made by comparison with a standard curve or a semi -quantitative determination can be made by comparing to a reference standard. Those skilled in the art will recognize that the reference standard may be a control sample run in parallel or a color graded reference strip. [0038] Leukocyte Esterase

[0039] Leukocyte esterase is an enzyme released from white blood cells. Agents useful in detecting the presence of leukocyte esterase having been disclosed, for example, in the following publications: Herlihy et al . , "New and Rapid Method for Detection of Pyuria by Leukocyte Esterase Reaction", Urology, 23 (2), pages 148-149 (1984); U.S. Pat. No. 4,499,185, to Skjold et al . , issued Feb. 12, 1985; U.S. Pat. No. 4,331,760, to Berger et al., issued May 25, 1982; U.S. Pat. No. 4,296,202, to Berger et al . , issued Oct. 20, 1981; European Patent Application Publication No. 158,204, Miles Laboratories Inc., published Oct. 16, 1985; European Patent Application Publication No. 157,326, Miles Laboratories Inc., published Oct. 9, 1985; European Patent Application Publication No. 157,327, Miles Laboratories Inc., published Oct. 9, 1985; European Patent Application Publication No. 157,360, Miles Laboratories Inc., published Oct. 9, 1985; European Patent Application Publication No. 157,361, Miles Laboratories Inc., published Oct. 9, 1985; and European Patent Application Publication No. 157,362, Miles Laboratories Inc., published Oct. 9, 1985. One or more of these agents may be used as a detective reagent for leukocyte esterase in the present invention. [0040] Gelatinase B

[0041] Gelatinase B (MMP-9; type IV collagenase) belongs to a sub-family of MMPs that plays an important role in tissue remodeling in normal and pathological inflammatory processes and is the terminal member of the protease cascade which leads to matrix degradation. It cleaves gelatins and other substrates, such as IFN-β, and is involved in matrix remodeling during embryogenesis, tissue remodeling and development. Gelatinase B is secreted by a variety of leukocytes including neutrophils, macrophages, lymphocytes, and eosinophils. Generally, the expression of gelatinase B is regulated, however, neutrophils store gelatinase B in secretory granules for rapid release. The expression of gelatinase B in cells can be induced by a variety of inflammatory stimuli including interleukin-lβ, tumor necrosis factor- .alpha. , lymphotoxin, interferon beta, and lipopolysaccharides (LPS) , and by other agents stimulating cell migration. For example, gelatinase B is up-regulated in pathological states such as invasion of cancer cells and when leukocytes are released from the bone marrow and migrate toward an inflammatory event. After stimulation by inflammatory cytokines, or upon delivery of bi-directional activation signals following integrin-mediated cell-cell or cell-ECM contact, gelatinase B also can be secreted by lymphocytes and stromal cells.

[0042] Gelatinase B, like other gelatinases and MMPs, is secreted in a latent inactive form and is converted to an active species by other proteolytic enzymes, including other MMPs. For example, activated gelatinase A can activate progelatinase B in a process that is inhibited by TIMP-I and TIMP-2 (Fridman et al, Cancer Research, 55:2548-2555 (1995). Progelatinase B also can be converted to an active form via an interacting protease cascade involving plasmin and stromelysin-1 (MMP- 3) . Plasmin, generated by the endogenous plasminogen activator (uPA) , is not an efficient activator of progelatinase B. Plasmin is able to generate active stromelysin-1 from an inactive proform and the activated stromelysin-1 is itself a potent activator of gelatinase B (Hahn-Dantona et al . , Ann NY Acad. Sci, 878:372-387 (1999). Latent gelatinase B also can be activated by other proteases including cathepsin G, kallikrein, and trypsin or by incubation with p- aminophenylmercuric acetate (APMA) .

[0043] Gelatinase B cleaves a variety of substrates including collagen type II, human myelin basis protein, insulin, and interferon beta. The substrate recognition specificity of gelatinase B has been studied using a phage display library of random hexamers (Kridel et al . , (2001), J. Biol. Chem. 276:20572-20578) and has been empirically assessed on a variety of substrates (Descamps, F J et al . , (2003) FASEB, 17 (8) : 887-9; Van Den Steen et al . , (2002) FASEB, 16: 379-389; and Nelissen et al . (2003) Brain 126: 1371-1381). Gelatinase B typically has a preference for hydrophobic residues at the Pl' position (the position before which cleavage occurs) , such as for example Leucine (L) . Other amino acid residues that have been recognized as preferentially cleaved by gelatinase B include Phenylalanine (F) , Glutamic Acid (E) , Tyrosine (Y) , and Glutamine (Q) . In some cases, protein glycosylation can affect gelatinase B cleavage. For example, proteolysis is more pronounced with IFN-β-lb than with IFN-β. [0044] IL- lβ

[0045] Interleukin-1 beta (IL-lβ) , a cytokine secreted by cells such as monocytes, macrophages and dendritic cells, mediates a wide range of immune and inflammatory responses. One can modulate IL-lβ production to treat a variety of disorders, such as rheumatoid arthritis, hematosepsis, periodontal disease, chronic heart failure, polymyositis/dermatomyositis , acute pancreatitis, chronic obstructive pulmonary disease, Alzheimer's disease, osteoarthritis, bacterial infections, multiple myeloma, myelodysplastic syndrome, uveitis, central nervous system injury, viral respiratory disease, asthma, depression, and scleroderma. See, e.g., Taylor P. C. et al Curr Pharm Des. 2003; 9(14): 1095-106; Dellinger R. P. et al Clin Infect Dis. 2003; 36(10): 1259-65; Takashiba S. et al J Periodontol. 2003; 74(1): 103-10; Diwan A. et al Curr MoI Med. 2003; 3(2): 161-82; Lundberg I. E. et al Rheum Dis Clin North Am. 2002; 28(4): 799-822; Makhija R. et al J Hepatobiliary Pancreat Surg. 2002; 9(4): 401-10; Chung K. F. et al Eur Respir J Suppl . 2001; 34:50s-59s,- Hallegua D. S. et al Ann Rheum Dis. 2002; 61(11) : 960-7; Goldring M. B. et al Expert Opin Biol Ther. 2001; 1(5): 817-29; Mrak R. E. et al Neurobiol Aging. 2001; 22(6): 903-8; Brady M. et al Baillieres Best Pract Res Clin Gastroenterol. 1999; 13(2): 265-89; Van der Meer J. W. et al Ann N Y Acad Sci. 1998; 856:243-51; Rameshwar P. et al Acta Haematol. 2003; 109(1): 1-10; de Kozak Y et al Int Rev Immunol. 2002; 21(2-3): 231-53; Wang C. X. et al Prog Neurobiol. 2002; 67(2): 161-72; Van Reeth K. et al Vet Immunol Immunopathol . 2002; 87(3-4): 161-8; Stirling R. G et al Br Med Bull. 2000; 56(4): 1037-53; Leonard B. E. et al Int J Dev Neurosci. 2001; 19(3): 305-12; Allan S. M. et al Ann N Y Acad Sci. 2000; 917:84-93; and Cafagna D. et al Minerva Med. 1998; 89(5) : 153-61.

[0046] Detective reagents can further include so called "direct labels" for visualizing binding with leukocyte markers. Direct labels such as gold sols and dye sols, are already known per se . They can be used to produce an instant analytical result without the need to add further reagents in order to develop a detectable signal. They are robust and stable and can therefore be used readily in a analytical device which is stored in the dry state. Their release on contact with an aqueous sample can be modulated, for example by the use of soluble glazes as described in described in U.S. Pat. No. 5,656,503.

[0047] Further modifications to the method and kit of the present invention for detecting the infection in a joint liquid can be made by adapting the methods and kits known in the art for the rapid detection of proteins and ligands in a biological sample. Examples of methods and kits that can be adapted to the present invention are described in U.S. Pat. No. 6,500,627, issued to 0 ' Conner et al. on Dec. 31, 2002, U.S. Pat. No. 4,870,007, issued to Smith-Lewis on Sep. 26, 1989, U.S. Pat. No. 5,273,743, issued to Ahlem et al . on Dec. 28, 1993, and U.S. Pat. No. 4,632,901, issued to Valkers et al . on Dec. 30, 1986, all such references being hereby incorporated by reference. [0048] Assays

[0049] The presence, level, expression, or activity of a leukocyte marker protein described herein, e.g., IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B can be evaluated in a variety of ways well known in the art, such as immunoassay, e.g., immunoprecipitation, Western blot analysis (immunoblotting) , ELISA, fluorescence-activated cell sorting (FACS), and bead-based detection assays, such as the Luminex^® detection technology provided by the MultiAnalyte Profiling Kit (Luminex Corporation, Austin, Tex.) . Typically, the level of protein and/or activity, e.g., IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B protein or activity, in a subject sample is compared to the level and/or activity in a control, e.g., the level and/or activity in a tissue from a non-disease subject.

[0050] Various types of immunoassays are known in the art. One example of an immunoassay is a "sandwich" type assay, in which a target analyte(s) such as IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B is "sandwiched" between a labeled antibody and an antibody immobilized onto a solid support. The assay is read by observing the presence and amount of antigen- labeled antibody complex bound to the immobilized antibody. Another immunoassay useful in the methods and kits described herein is a "competition" type immunoassay, wherein an antibody bound to a solid surface is contacted with a sample (e.g., a synovial fluid sample) containing both an unknown quantity of antigen analyte and with labeled antigen of the same type. The amount of labeled antigen bound on the solid surface is then determined to provide an indirect measure of the amount of antigen analyte in the sample. Such immunoassays are readily performed in a "dipstick" or other test device format (e.g., a flow-through or migratory dipstick or other test device design) for convenient use, e.g., home use or use by a health care provider. For example, numerous types of dipstick immunoassays assays are described in U.S. Pat. No. 5,656,448.

[0051] A test device, e.g., a "dipstick", refers to a substrate, preferably a substrate that is insoluble in aqueous solution, e.g., synovial fluid sample, onto which an agent described herein, e.g., an agent that detects a protein described herein, e.g., an antibody described herein, is immobilized. The agent can be applied as a layer on the substrate, or can also penetrate into the substrate. A test device can be a substrate, e.g., a membrane, e.g., a membrane strip, onto which an agent described herein is immobilized. A test device can include a housing for the substrate, e.g., a membrane, e.g., a membrane strip, onto which an agent described herein is immobilized. In one embodiment, the substrate is a substrate other than glass, and is preferably flexible.

[0052] In one embodiment, the substrate, e.g., the membrane, e.g., the membrane strip, is, e.g., between about 0.1 and 0.5 inches in width, e.g., between about 0.2 and 0.4, e.g., between about 0.25 and 0.3 inches in width, and is, e.g., between about 1 and 4 inches in length, e.g., between about 2 and 3 inches in length. In one embodiment, an agent described herein, e.g., an antibody described herein, covers an area of the substrate, e.g., the membrane, e.g., the membrane strip, that is greater than about 0.01 cm², e.g., greater than about 0.1, 0.5, or 1 cm².

[0053] In one embodiment, the test device includes a marker indicating how far to "dip" the substrate into a biological sample, e.g., synovial fluid.

[0054] In one embodiment, a method described herein employs a dipstick or other test device format to measure the presence, level, expression or activity of a protein described herein, e.g., IL-lβ, leukocyte elastase, and/or leukocyte esterase. A dipstick or other test device assay can, for example, provide a color indication for an increased risk for accelerated PJI based upon the levels of a protein described herein, such as IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B, in a sample, e.g., a synovial fluid sample. In one scenario, the dipstick or other test device can react to produce one color if a level of a first protein is exceeded, a different color if a level of a second protein is exceeded, a third color if a level of a third protein is exceeded, and when three levels are exceeded, the three colors will combine to yield a separate color that is easily distinguishable from the others. A dipstick or other test device-based assay optionally includes an internal negative or positive control.

[0055] A dipstick or other test device-based assay could find use in a clinical setting by quickly and reliably indicating a heightened risk for PJI. This could save valuable time by allowing the physician to initiate treatment sooner, thereby minimizing the harmful effects of the disease .

[0056] In another embodiment, the method of the present invention may be utilized in combination with a densitometer or generally a device for measuring light intensity, transmittance, reflection or refraction, or for measuring the wavelength of light as a measure of assay result. Such a device can be used in a setting such as a doctor's office, a clinic or a hospital. The densitometer or other device can provide rapid measurement of the optical density of dipstick or other test device strips that have been contacted with a bodily fluid or tissue.

[0057] In a preferred embodiment, a change in color, density, or other parameter can be read by the naked eye .

[0058] In a preferred embodiment, the assay can be read without the addition of a reagent not already on the substrate . [0059] Another possible approach to a diagnostic assay includes the use of electrochemical sensor strips, such as those used for home glucose testing, onto which a sample is placed, and which strips include reagents for initiating a reaction when wetted by the sample. The sensor strip is inserted into a meter that measures, e.g., diffusion-limited current of a reaction species indicative of the analyte of interest, such as IL-lβ, leukocyte elastase, and/or leukocyte esterase. The meter then yields a display indicative of the concentration of analyte in the sample.

[0060] Tom et al . , U.S. Pat. No. 4,366,241, and Zuk, EP-A 0 143 574 describe migration type assays in which a membrane is impregnated with the reagents needed to perform the assay. An analyte detection zone is provided in which labeled analyte is bound and assay indicia are read.

[0061] Bernstein, U.S. Pat. No. 4,770,853, May et al . , WO 88/08534, and Ching et al . , EP-A 0 299 428 describe migration assay devices that incorporate within them reagents that have been attached to colored direct labels, thereby permitting visible detection of the assay results without addition of further substances. [0062] Valkirs et al . , U.S. Pat. No. 4,632,901, disclose a flow-through type immunoassay device comprising antibody (specific to a target antigen analyte) bound to a porous membrane or filter to which is added a liquid sample. As the liquid flows through the membrane, target analyte binds to the antibody. The addition of sample is followed by addition of labeled antibody. The visual detection of labeled antibody provides an indication of the presence of target antigen analyte in the sample. [0063] Korom et al . , EP-A 0 299 359, discloses a variation in the flow-through device in which the labeled antibody is incorporated into a membrane that acts as a reagent delivery system.

[0064] Baxter et al . , EP-A 0 125 118, disclose a sandwich type dipstick immunoassay in which immunochemical components such as antibodies are bound to a solid phase. The assay device is "dipped" for incubation into a sample suspected of containing unknown antigen analyte. Enzyme-labeled antibody is then added, either simultaneously or after an incubation period. The device next is washed and then inserted into a second solution containing a substrate for the enzyme. The enzyme-label, if present, interacts with the substrate, causing the formation of colored products that either deposit as a precipitate onto the solid phase or produce a visible color change in the substrate solution.

[0065] Kali et al . , EP-A 0 282 192, disclose a dipstick device for use in competition type assays.

[0066] Rounds in U.S. Pat. No. 4,786,589 describes a dipstick immunoassay device in which the antibodies have been labeled with formazan. [0067] Detection Device and Kits

[0068] In another aspect, a kit is provided for detecting leukocyte markers in a joint fluid sample. In some embodiments, the kit comprises a housing having a chamber consisting of one or more test wells disposed inside the housing and having an inlet above each well to allow the introduction of the joint fluid samples. Each test well may contain the detection reagents, such as, for example, antibodies, for the specific leukocyte markers. In the preferred embodiment, the housing may include at least 3 wells having detection reagents for detecting at least three leukocyte markers. [0069] The housing may be at least partially transparent, or may have windows provided therein, for observation of an indicator region that undergoes a color or fluorescence change. The kit may be designed to contain a dipstick, a lateral flow device, or migration-type device. Preferably, the instant device is a dipstick as described in the International Patent Application Publication WO/2002/033413. In other embodiments, the device is a lateral flow device of the type disclosed, for example, in U.S. Patents No. 7,297,529,- 7,344,893; and 7,090,803.

[0070] Typically, a lateral flow device may comprise a housing having an inlet for the sample and side walls defining a fluid lateral flow path extending from the inlet. By "lateral flow", it is meant liquid flow in which the dissolved or dispersed components of the sample are carried, suitably at substantially equal rates, and with relatively unimpaired flow, laterally through the carrier. Preferably, the fluid flow path contains one or more porous carrier materials that are hydrophilic, but preferably do not absorb water. Suitable examples include, but are not limited to, paper, cellulose, nitrocelloluse, pressed fibers, including glass fibers, sintered glass, ceramic or plastic materials, such as nylon, polyethylene or polyester. The porous carrier materials may be in fluid communication along substantially the whole fluid flow path so as to assist transfer of fluid along the path by capillary action. The porous carrier materials may function as solid substrates for attachment of reagents or indicator moieties. In some embodiment, the device may have a control substance that interacts with the sample of the joint fluid to improve the accuracy of the test .

[0071] The size and shape of the carrier are not critical and may vary. The carrier defines a lateral flow path and may be presented in the form of one or more elongated strips or columns. In certain embodiments, the porous carrier is one or more elongated strips of sheet material, or a plurality of sheets making up in combination an elongate strip. One or more reaction zones and detection zones would then normally be spaced apart along the long axis of the strip. However, in some embodiments the porous carrier could, for example be in other sheet forms, such as a disk. In these cases the reaction zones and detection zones would normally be arranged concentrically around the center of the sheet, with a sample application zone in the center of the sheet. In yet other embodiments, the carrier is formed of carrier beads, for example beads made from any of the materials described above. The beads may suitably be sized from about 1 micrometer to about 1 mm. The beads may be packed into the flow path inside the housing, or may be captured or supported on a suitable porous substrate such as a glass fiber pad.

[0072] It will be appreciated that the devices in the apparatus according to the present invention may be adapted to detect more than one marker. This can be done by the use of several different reagents in a single reaction zone, or suitably by the provision in a single device of a plurality of lateral flow paths each adapted for detecting a different leukocyte marker. In certain embodiments, the plurality of lateral flow paths are defined as separate fluid flow paths in the housing, for example the plurality of lateral flow paths may be radially distributed around a sample receiving port. In other embodiments, the plurality of fluid flow paths are physically separated by the housing. In yet other embodiments, multiple lateral flow paths (lanes) can be defined in a single lateral flow membrane by depositing lines of wax or similar hydrophobic material between the lanes .

[0073] An absorbent element may also be included in the instant devices. The absorbent element is a means for drawing the whole sample through the device by capillary attraction. Generally, the absorbent element will consist of a hydrophilic absorbent material such as a woven or nonwoven textile material, a filter paper or a glass fiber filter.

[0074] The device may further comprise at least one filtration element to remove impurities from the sample before the sample undergoes analysis. The filtration device may for example comprise a microporous filtration sheet for removal of cells and other particulate debris from the sample. The filtration device is typically provided upstream of the sample application zone of the fluid flow path, for example in the inlet of the housing or in the housing upstream of the inlet.

[0075] In certain embodiments, the instant devices may also include a control moiety in a control zone of the device, wherein the control moiety can interact with a component of the joint fluid sample to improve the accuracy of the device. Suitably, the control zone is adapted to reduce false positive or false negative results. A false negative result could arise for various reasons, including (1) the sample is too dilute, or (2) the sample was too small to start with. In order to address false negative mechanism, the control zone may further comprise a reference assay element for determining the total leukocyte marker content of the sample, that is to say for establishing that the total leukocyte marker content or the total protein content of the sample is higher than a predetermined minimum. It is possible to indicate the presence of protein by the use of tetrabromophenol blue, which changes from colorless to blue depending on the concentration of protein present .

[0076] In addition, the instant device may further comprise a sampling device for collecting a sample of a joint fluid. To that end, the instant device may include a connector for attaching a needle to the device and a plunger for extracting a sample of the joint fluid from the body of the patient through the needle into the housing of the device. Additional elements of the instant device may include, but are not limited to, a color chart for interpreting the output of the diagnostic device, a wash liquid for carrying a sample of fluid through the device, and a pretreatment solution containing a reagent for pretreatment of the fluid sample .

[0077] In another embodiment, the kit includes a syringe for obtaining a sample of joint fluid wherein, the barrel of the syringe is coated with a mix of detection reagents and an appropriate carrier such that the detection occurs in the barrel once the sample of joint liquid is drawn or within the time necessary for the detection reagents to react with leukocyte markers. [0078] The invention also includes kits for detecting the presence of a protein described herein, e.g., a leukocyte marker (e.g., IL-lβ, leukocyte esterase, gelatinase B and/or leukocyte elastase) , in a biological sample. For example, the kit can include a compound or agent capable of detecting protein (e.g., an antibody) or mRNA (e.g., a nucleic acid probe) of a protein described herein in a biological sample; and a standard. The agent can be coupled to a detectable label, such as a colored, absorbent or fluorescent label. The kit can also include a positive and a negative control, e.g., a reagent that contains a protein described herein, e.g., one or more of IL-lβ, leukocyte esterase, and/or leukocyte elastase.

[0079] The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to evaluate a subject, e.g., for PJI. [0080] Another embodiment of the present invention is a dipstick or other test device-based kit, suitable for home testing. Such a screening test would provide convenience, privacy and eliminate the necessity and cost of visiting a physician for a screening test, although the dipstick or other test device kit could also be used in a clinical setting. The dipstick or other test device kit could be similar to a home pregnancy kit, known to those of skill in the art, and could provide a color indication for an increased risk for accelerated PJI based upon the levels of a protein described herein, e.g., a leukocyte marker described herein, e.g., IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B, in the sample. Such a dipstick or other test device-based kit could be provided with a small plastic cup for collecting and retaining the sample and for conducting the test. In one scenario, the dipstick or other test device can react to produce one color if a level of a first protein, , a different color if a level of a second protein is exceeded, and when both levels are exceeded, the two colors will combine to yield a third color that is easily distinguishable from the others. [0081] In one embodiment, the kit includes at least 1, e.g., at least 2, 5, 10, 20, 30, or 50, test devices, e.g., dipsticks, e.g., membranes, e.g., membrane strips described herein. In one embodiment, the kit contains a container suitable for collecting a synovial fluid sample .

[0082] A dipstick or other test device-based assay, similar to that described above, could find use in a clinical setting by quickly and reliably indicating an heightened risk for PJI. This could save valuable time by allowing the physician to initiate treatment sooner, thereby minimizing the harmful effects of the disease. [0083] In another embodiment, the method of the present invention may be utilized in combination with a densitometer in a device for use in a setting such as a doctor's office, a clinic or a hospital. The densitometer can provide rapid measurement of the optical density of dipstick or other test device strips that have been contacted with a bodily fluid or tissue. [0084] Other possible approaches include the use of electrochemical sensor strips, such as those used for home glucose testing, onto which a sample is placed, and which strips include reagents for initiating a reaction when wetted by the sample . The sensor strip is inserted into a meter that measures, e.g., diffusion-limited current of a reaction species indicative of the analyte of interest, e.g., IL-lβ, leukocyte esterase, and/or leukocyte elastase. The meter then yields a display indicative of the concentration of analyte in the sample .

[0085] The kit can also contain a device to obtain a tissue sample, such as a cotton swab or wooden swab. [0086] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agent that modulates a protein described herein, e.g., a leukocyte marker described herein (e.g., IL-lβ, leukocyte elastase, leukocyte esterase, and/or gelatinase B) to treat PJI . [0087] EXAMPLES

[0088] Example 1 - Intraoperative aspiration was performed on 92 consecutive patients undergoing revision total knee arthroplasty. 1-2 cc of synovial fluid was removed from the joint of all patients recruited into this study and subjected to ELISA analysis. The remainder of the fluid was sent for leukocyte cell count and culture for correlation. Intraoperative tissue samples were also obtained and sent for culture. Knees were aspirated after superficial dissection but before arthrotomy to minimize the incidence of bloody samples.

[0089] According to clinical, serological and operative criteria, 14 of those knees were deemed to be infected and were treated accordingly. The mean level of leukocyte esterase, elastase and gelatinase were significantly elevated in the infected patients compared to non-infected knees (p<0.0001) (sensitivity 100%) . This allowed determining a threshold level for each marker. The analysis was conducted to determine whether the level of each marker was above the threshold in the infected and below the threshold in the non- infected group. This was true in 12 of infected patients and 78 of the non-infected patients (88% specificity) . However, when the threshold for all three enzymes were considered together, there were no non- infected knees in which the level of these three enzymes were above the threshold. This allowed us to improve the specificity to 100% also.

[0090] Note that the specifics embodiments are described in an exemplary manner and are not intended to limit the invention. In particular, infusion devices and needles manufactured of any acceptable material are contemplated to be within the scope of the invention, as are infusion devices and needles having varying design configurations and numbers of chambers and lumens . The scope of the invention is therefore defined in the claims which follow.

Claims

What is claimed is:

1. A kit for detecting joint infection in a patient, said kit comprising:

(a) at least three detection reagents which are specific for each of at least three leukocyte markers present in a synovial fluid sample collected from the patient suspected of having the joint infection, wherein said at least three detection reagents are adapted to detect a threshold level of each of said at least three leukocyte markers, said threshold level adapted to indicate a presence of joint infection; and

(b) instructions for using said at least three detection reagents to evaluate joint infection in the patient .

2. The kit according to claim 1, wherein said joint infection is periprosthetic joint infection.

3. The kit according to claim 1, wherein said at least three leukocyte markers are selected from the group consisting of leukocyte elastase, leukocyte esterase, gelatinase B, cathepsins, interleukins, and cytokines.

4. The kit according to claim 3, wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and gelatinase B, and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample; and wherein said threshold level of leukocyte esterase is at least about 23 pg/ml of leukocyte esterase in said synovial fluid sample,- and wherein said threshold level of gelatinase B is at least about 10 pg/ml of gelatinase B in said synovial fluid sample.

5. The kit according to claim 3, wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and IL- lβ and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample; and wherein said threshold level of leukocyte esterase is at least about 23 pg/ml of leukocyte esterase in said synovial fluid sample,- and wherein said threshold level of IL-lβ is at least about 2 pg/ml of IL-lβ in said synovial fluid sample.

6. The kit according to claim 3, wherein a detection reagent is selected from the group consisting of antigens; haptens; monoclonal and polyclonal antibodies; natural and synthetic mono-, oligo- and polysaccharides; lectins; avidin and streptavidin,- biotin,- growth factors; hormones; receptor molecules; and combinations thereof .

7. The kit according to claim 6, wherein the detection reagent is selected from monoclonal and polyclonal antibodies .

8. A kit according to claim 7, wherein the detection reagent comprises an antibody or antibody fragment.

9. A kit according to claim 1, wherein said kit comprises a detector device having a support in communication with at least one of said at least three detection reagents.

10. A kit according to claim 1, wherein said kit comprises a dipstick, a swab, and a container.

11. The kit of claim 1, wherein the detection reagents are attached to a detectable label .

12. The kit of claim 1, further comprising one or both of a positive and a negative control.

13. A method of screening for infection in a joint of a patient, said method comprising the steps of:

(a) extracting a sample comprising synovial fluid from a joint (b) applying the sample to a detector device, wherein the detector device comprises at least three detection reagents which are specific for each of at least three leukocyte markers, wherein said at least three detection reagents are adapted to detect a threshold level of each of said at least three leukocyte markers, said threshold level correlated with a presence of infection;

(c) ascertaining the threshold levels of the leukocyte markers present in said sample, wherein if the concentration each of said at least three leukocyte markers exceeds the threshold level, then this is a positive screen for infection of the joint.

14. The method of claim 13, wherein the infection is a periprosthetic joint infection.

15. The method of claim 13, wherein said at least three leukocyte markers are selected from the group consisting of leukocyte elastase, leukocyte esterase, gelatinase B, cathepsins, interleukins, and cytokines.

16. The method of claim 13, wherein said ascertaining is performed by an enzyme-linked immunosorbent assay.

17. The method of claim 13, wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and gelatinase B and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample,- and wherein said threshold level of leukocyte esterase is at least about 20 pg/ml of leukocyte esterase in said synovial fluid sample; and wherein said threshold level of gelatinase B is at least about 10 pg/ml of gelatinase B in said synovial fluid sample.

18. The method of claim 13, wherein said at least three leukocyte markers are leukocyte elastase, leukocyte esterase, and IL- lβ and wherein said threshold level of leukocyte elastase is at least about 12 pg/ml of leukocyte elastase in said synovial fluid sample; and wherein said threshold level of leukocyte esterase is at least about 23 pg/ml of leukocyte esterase in said synovial fluid sample,- and wherein said threshold level of ILl-β is at least about 2 pg/ml of IL- lβ in said synovial fluid sample.

19. The method of claim 13, wherein the detection reagent is selected from the group of species consisting of antigens; haptens,- monoclonal and polyclonal antibodies; natural and synthetic mono-, oligo- and polysaccharides; lectins: avidin and streptavidin,- biotin,- growth factors,- hormones; receptor molecules; and combinations thereof.

20. The method of claim 19, wherein the detection reagent is selected from monoclonal and polyclonal antibodies .

21. The method of claim 20, wherein said reagents comprise an antibody or antibody fragment.

22. The method of claim 13, wherein said detector device is a dipstick comprising at least three detection reagents which are specific for leukocyte markers present in a synovial fluid sample.

23. A method for detecting infection in an artificial joint, the method comprising: obtaining a sample of the joint fluid; applying the sample to a detector device,- testing the sample to detect the amount of one or more leukocyte markers in the sample; taking a first action based on the findings of the test.

24. The method of claim 23 wherein the one or more leukocyte markers is selected from the group consisting of cytokines, chemokines, oxygen and nitrogen radicals, leukocyte elastase, leukocyte esterase, gelatinases, metalloproteinases (MMPs), cathepsins, phospholipases, and combinations thereof.

25. The method of claim 24 wherein the one or more leukocyte markers is leukocyte elastase, leukocyte esterase, and IL- lβ.

26. The method of claim 23 wherein the first action comprises placing a temporary antibiotic spacer.

27. The method of claim 23 further comprising pre- treating the sample.

28. The method of claim 23 further comprising comparing the amount of one or more leukocytes markers in the sample to a threshold value for the one or more leukocyte markers .

29. The device for detecting infection in an artificial joint comprising: a housing; reagents for detecting the amount of one or more leukocyte markers in a sample of the joint fluid disposed inside the housing.

30. The device of claim 29 comprising 3 wells into which reagents for detecting at least three different leukocyte markers are placed.

31. The device of claim 29 wherein one or more leukocyte markers are selected from the group consisting of leukocyte elastase, leukocyte esterase, and IL-lβ and combinations thereof.