METHOD AND SYSTEM FOR DIAGNOSIS OF BACTERIAL INFECTIOUS CONDITIONS

Abstract: A rapid and sensitive screening method and system are disclosed for diagnosing and evidencing bacterial infectious conditions in a patient. The diagnostic method and system can be used to differentiate a bacterial from a non-bacterial infection. The diagnostic method and system measure the concentration of at least one cytokine present in the body tissue specimen of a patient. Bacterial infectious conditions are evidenced if the concentration of the cytokine is found to be over a threshold concentration indicative of bacterial infectious conditions.

Description: Description METHOD AND SYSTEM FOR DIAGNOSIS OF BACTERIAL INFECTIOUS CONDITIONS Field of the Invention The present invention relates to a rapid and sensitive screening diagnostic method and system to distinguish bacterial from non-bacterial etiology of infection in a patient, and more particularly to an in vitro diagnostic method and system utilizing the concentration of a cytokine present in the body tissue specimen of a patient as an indication of bacterial infectious conditions in meningitis, arthritis, pneumonia, peritonitis, peritoneal dialysis catheter infection, pyelonephritis, and cystitis. Background of the Invention Early diagnosis and prompt effective treatment of a disease are essential for the well-being of a patient. Diseases can be infectious or non-infectious. Infectious diseases can be caused by, among others, bacteria, viruses, fungi, spirochetes, and mycoplasma. Most bacterial infectious diseases will respond to antibiotic treatment. On the other hand, diseases caused by viruses will not respond to any of the presently available antibiotics although there are antiviral drugs for some of them. Thus, unless properly diagnosed as a treatable bacterial infectious disease, other infections should not be treated with these pharmaceutical agents. Not only are these pharmaceutical agents ineffective in controlling these untreatable infections, but they expose the patient to adverse effects of the drugs. Misuse of antibiotics can cause the emergence of antibiotic-resistant pathogens, which in turn create a need for new drugs. The development of these new drugs have also contributed to the rising costs of medical care. Thus, early and accurate diagnosis of an infectious disease is essential to achieve lowered morbidity and mortality rates and potentially to reduce the cost of medical care to the patient. Among the diseases that require rapid diagnosis to differentiate bacterial infectious conditions from non-bacterial infectious conditions are meningitis, arthritis, pneumonia, peritonitis, pyelonephritis and cystitis. These bacterial diseases share many common gross symptoms with their non-bacterial counterparts. Thus, early diagnosis of their etiology will allow prompt effective treatment of the diseases. For example, children still die or suffer permanent neurologic sequelae as a result of bacterial meningitis. Prompt diagnosis and aggressive management are the goals, but early signs of meningitis are often subtle and nonspecific and, therefore, may be recognized only in retrospect. The physician must identify among the many febrile children seen every day in office practice — most of whom have spontaneously resolving illnesses usually caused by viruses— the few children who have serious bacterial infection requiring early intervention- The following definitions have been widely adopted: (1) meningitis, inflammation of the meninges that is identified by an abnormal number of white blood cells (WBCs) in cerebrospinal fluid (CSF); (2) bacterial meningitis, meningitis and evidence of a bacterial pathogen in CSF; (3) aseptic meningitis, meningitis in the absence of evidence of a bacterial pathogen detectable in CSF by usual laboratory techniques; (4) encephalitis, inflammation of the brain; (5) meningoencephalitis, inflammation of the brain accompanied by meningitis; (6) sepsis or septicemia, a clinical syndrome suggesting invasion of the bloodstream by microorganisms; (7) bacteremia, the presence of a bacterial pathogen in blood; (8) colonization, the presence of a microorganism without an immune response and without clinical signs or symptoms; (9) infection, the presence of a microorganism with an immune response but without clinical signs or symptoms; and (10) disease, the presence of a microorganism with an immune response and with clinical signs or symptoms. J. 0. Klein, R D.. Feigin and G. H. McCracken, Jr. , "Report on the Task Force on Diagnosis and Management of Meningitis, " Pediatrics, Vol. 78, No. 5, Part 2, Supplement, 959-982 (1986). Suppurative arthritis is defined as an inflammatory reaction within the joint space synovial infection caused by a variety of organisms. It is usually manifested by fever and symptoms related to the joint involved, such as pain, limitation of motion, swelling, and presence of effusion- These patients frequently have an elevated ESR and WBC with a predominance of PMN leukocytes. Analysis- of joint fluid shows in many cases a leukocyte count greater than 50-100, 000/mm 3 with a differential count of more than 90% PMN, glucose less than 40 mg/dl and elevated protein concentration However, all these clinical and laboratorial findings are not specific for bacterial arthritis, since some other inflammatory conditions, such as rheumatoid arthritis, rheumatic fever and reactive arthritis can be at times indistinguishable from cases of suppurative arthritis. To help physicians in diagnosing and differentiating a bacterial infectious disease from a non-bacterial infectious one, various kinds of diagnostic tests have been developed. Most of these tests require culturing some body fluid specimen from a patient and monitoring the growth of infecting bacteria m the culture. Culturing an infecting bacteria as an aid to diagnosis purposes can often be inadequate. The process of culturing is too slow. Also, there are many bacterial diseases where a culture of the infecting bacteria is not generally available. More recently, many immunological assays have been developed to attempt to quantify antibody response to infectious diseases. H. E. Carlsson & A. A. Lindberg, "Application of Enzyme Immunoasεay for Diagnosis of Bacterial and Mycotic Infections, " Scand. J. Immunol. 8 (Suppl. 7): 97-110 (1978). These include immunodiffusion, latex agglutination, counter-immunoelectrophoresis, immunofluorescence, and radioimmunoassay. However, a prerequisite of the usefulness of this type of assays is that the miσrobial particles are antigenic and that the antigens are available for binding to the detecting antibody. Moreover, in most of these techniques, phenomena secondary to the primary antigen-antibody interaction are required for the detection of the reaction. S. J. Challacombe, "Application of ELISA to Microbiology, " E ESA and Other Solid Phase Immunoassavs. D. M. Kemeny and S. J. Challacombe (eds. ), John Wiley & Sons, New York, (1988), Chap. 15. As a function of general cell activation, or because of immune function, a great variety of cells produce soluble inflammatory mediators. When produced by monocytes or macrophageε, these soluble mediators are called "monokines." Generalizing further, "cytokines" represent soluble-mediator products of other cell types such as fibroblasts and other cells. Broadly, cytokines include monokines and lymphokines. Cytokine networks may be operating at the cellular level for the potentiation and modulation of the results of immune reactions. Certain cytokines, such as tumor necrosis factor-alpha (TNFQ) and interleukin-1-beta (IL-lβ), present in a body tissue have been implicated as key mediators of host responses to microbial invasion and immunological insults. No diagnostic test presently available has yet been able to replace the clinical acumen of a physician in identifying early signs of bacterial infections in a patient. Nevertheless, there is still a need for improved rapid and sensitive screening diagnostic methods to distinguish bacterial from non-bacterial etiology in a patient. In particular, rapid and sensitive diagnostic methods are needed to distinguish bacterial from non-bacterial conditions in meningitis, arthritis, pneumonia, peritonitis, pyelonephritis and cystitis in a patient. Summary of the Invention It is therefore an object of the invention to provide a rapid and sensitive in vitro method and system for the diagnosis and confirmation of a bacterial infectious condition in a patient. This infectious condition is characterized in that its gross symptoms are shared by other infectious and non-infectious diseases. The method includes the steps of; Measuring the activity of a cytokine in a body tissue specimen of the patient; comparing the activity so measured to a threshold activity indicative of the bacterial infectious condition; and evidencing the bacterial infectious condition by the presence of the measured cytokine activity over the threshold activity. It is also an object of the invention to provide a rapid and sensitive in vitro method and system, measuring concentrations of more than one cytokine and evaluating the combined results as a whole for the diagnosis of a bacterial infectious condition in a patient. This infectious condition is characterized in that its gross symptoms are shared by other infectious and non-infectious diseases. The method comprises the steps of: Measuring a first activity of a first cytokine in a first body tissue specimen of the patient; comparing the first activity to a first threshold activity suggestive of the bacterial infectious condition; measuring a second activity of a second cytokine in a second body tissue specimen of- the patient; comparing the second activity to a second threshold activity suggestive of the bacterial infectious conditions; and evidencing, with a higher degree of certainty, the bacterial infectious condition by the presence of the first activity over the first threshold activity and by the simultaneous presence of the second activity over the second threshold activity. It is still another object of the present invention to provide a rapid and sensitive in vitro diagnostic method and system capable of differentiating a bacterial from a non-bacterial condition in a patient with greater accuracy. It is yet another object of the present invention to provide a rapid and sensitive in vitro diagnostic method and.- system utilizing the concentration of a -cytokine present in the body fluid specimen of a patient as an- indication of bacterial infectious conditions in meningitis, arthritis, pneumonia, peritonitis, pyelonephritis, and cystitis. It is a further object of the present invention to provide kits for carrying out the diagnostic tests of the invention. Brief Description of the Drawings FIG. 1 is a diagram illustrating the correlation between concentrations.ώθf I -1-beta in CSF and outcome from neonatal Gram- negative bacillary meningitis. One way analysis of variance was performed to determine whether there was a significant difference between outcome groups. FIG. 2 is a diagram illustrating concentrations of IL-1-beta in CSF of infants and children with bacterial and non-bacterial (aseptic) meningitis at the time of diagnosis. FIG. 3 is a diagram illustrating the correlation between the concentrations of IL-1-beta and TNF-alpha in synovial fluid of 24 children with infectious and non-infectious arthritides. Four patients had neither IL-1-beta nor TNF-alpha detected. FIG. 4 is a diagram illustrating the correlation between leukocyte counts (WBC) and concentration of TNF-alpha in synovial fluid of 24 children with infectious and non-infectious arthritides. FIG. 5 is a diagram illustrating the correlation between leukocyte counts (WBC) and concentration of IL-1-beta in synovial fluid of 24 children with infectious and non-infectious arthritideε. Description of the Invention The present invention is directed to a rapid and sensitive in vitro screening diagnostic method performed upon a body tissue specimen or sample taken from a patient to distinguish, and to evidence or indicate, a bacterial from a non-bacterial condition in the patient. As used herein, "evidence" means "indicate," "demonstrate" or "signal," but may or may not be as conclusive as "confirm. " It is an in vitro diagnostic method utilizing the concentration or activity of at least one cytokine present in the body tissue specimen of a patient as a guide to confirm bacterial infectious conditions in meningitis, arthritis, pneumonia, peritonitis, peritoneal dialysis catheter infection, pyelonephritis, and cystitis. The concentration of the cytokine in the body tissue is measured and compared to a threshold concentration indicative of the bacterial infectious conditions. Bacterial infectious conditions are evidenced or indicated by the presence of the cytokine concentration over the threshold concentration, or activity. In an alternative embodiment of the invention, the concentrations of two or more different cytokines from one or more body tissues are measured and compared with two or more threshold concentrations, each suggestive of bacterial infectious conditions. Again, bacterial infectious conditions are evidenced, with a high- degree of certainty, if each of the measured concentrations of cytokines is higher than each of the threshold concentrations suggestive of bacterial infection. As used herein, the terms "concentration" and "activity" are interchangeable. The preferred body tissue as used herein is a body fluid, but other body tissues which can also be used include packed red blood cells, packed white blood cells, platelets, cryo precipitate from blood plasma, other plasma proteins, skin, cornea, and other tissue from a human. The preferred body fluids for assay include cerebrospinal fluid (CSF) , synovial or j oint fluid, pleura! fluid, peritoneal fluid, urine, fluid within bullae, pericardial effusion, and saliva. Other body fluids include blood, plasma, s erum, fluids containing s aid components , fluids from plasmapheresis, plasma fibrinogen, cryo-poor plasma, albumin, gamma globulins, and semen. For example, detection of high concentrations of certain cytokines in CSF is useful for the diagnosis and evidencing of bacterial meningitis. High activities of certain cytokines in j oint, or synovial, fluid can confirm the suppurative arthritis. Further, high concentrations of certain cytokines in pleural fluid can evidence bacterial infectious conditions in a patient infected with pneumonia. Similarly, quantitation of certain cytokines in peritoneal fluid can aid in the support of diagnosis of bacterial peritonitis. Still further, a high concentration of a certain cytokine in urine indicates bacterial pyelonephritis and cystitis. As discussed above, cytokines include soluble mediators produced by fibroblasts and other cells. Among the cytokines useful for this invention are various kinds of interleukins (IL) , tumor necrosis factors (TNF) , platelet activating factors (PAF) , and certain prostaglandins (PGE) . Included in the interleukins are interleukin- 1 beta (IL-1B) and other interleukins. Interleukin 1 (IL-1 ) is a pluripotent, hormone-like peptide that affects many different cell types and organ systems. IL-1, formerly designated as " lymphocyte activating factor, " is a cytokine which was, at one - - time, strictly defined to be produced by ononuclear phagocytes and to stimulate T lymphocytes. More recent studies have shown that: ( 1 ) IL-1 molecules are produced by cells other than mononuclear phagocytes, such as epithelial cells, astrocytes; and ( 2 ) IL-1, or very similar molecules, can affect a variety of nonlymphoid cells such as fibroblasts, hepatocytes, or hypothalamic cells. Moreover, IL- 1 was shown to participate in the stimulation of B-lymphocytes as well. I. Gery and J. A. Schmidt, " Human Interleukin 1, " Method in Enzymology. Vol. 116, 456-467 ( 1985 ) . TNF is a substance that is found in the serum of animals sensitized to bacilli calmette-guerin (BCG) , or certain other immunopotentiators, and challenged later with endotoxin and that causes the necrosis of some tumors when passively transferred to tumor-bearing animals. TNF lacks species specificity and is able to discriminate between normal and certain tumor cells in vitro. M. __ Ruff and G. E. Gifford, "Tumor Necrosis Factor, " Lymohokines, Vol. 2, E. Pick (Ed. ), (1981), Academic Press, New York, 235-272. List of Abbreviations CSF Cerebrospinal fluid CSF CSF sample taken on admission CSF 2 CSF sample taken 18 to 30 hours after admission IL-1B Interleukin-IB TNFCT Tumor necrosis factor (cachectin) ELISA Enzyme linked immunosorbent assay pg picogram ml illiliter Applicable assays, or measuring methods, for a cytokine include radioimmunoassay, bioassay, immuno-enzymatic assay, immunofluorescent assay, and latex agglutination assay. Different cytokines can be assayed by a similar assay or measuring method. Fluorescent antibody techniques used in diagnostic bacteriology have been described. R H. Page, G. L. Caldroney and C. S. Stulberg, Immunofluorescence in Diagnostic Bacteriology, Am. J. Dis. Child., 101: 47/155 - 51/159 (1961). See also R C. Nairn, Fluorescent Protein Tracing, 4th ed. , Longman, Inc. , New York; J. W. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, New York, 1983, Chap. 7. Fluorescent antibodies against a cytokine can be prepared according to the general procedures described in the literature. L. Wofsy, C. Henry, J. Kimura, and J. North, "Modification and Use of Antibodies to Label Cell Surface Antigens, " Selected Methods in Cellular Immunology. B. B. Mishell and S. M. Shingi (Eds. ), (1980), W. H. Freeman and Company, San Francisco, pp. 287-304; and J. W. Goding, Conjugation of Antibodies With Fluorochromes: Modification to the Standard Methods, J. Immunol. Meth. 13: 215 (1976). Broadly, a fluorochrome^ such as fluorescein isothiocyanate ("FITC"), is coupled to the protein (about 10-20 mg/ml) by dialyzing the protein solution against a solution of FITC (about 0.5 mg/ml of protein) in 0.05 M bicarbonate- buffered saline, pH = 9. 5, at room temperature for two hours in the dark with slight agitation. Alternatively, the mixture could be dialyzed at 4 °C for about 12-14 hours. The unlabelled ' FITC is dialyzed and the resultant mixture is centrifuged ( 10, 000 x g, 10 minutes ) to remove any precipitate. Fluorochrome: protein ratio in the FITC conjugates are determined by spectrophotometric analysis. The optical densities (O. D. ) at 280 nm and 493 nm are measured for the determination of the concentration of FITC-protein, in mg/ml, as described in the literature. Fluorescent staining and visualization can be performed as described by the literature cited above. See also Fluorescent Antibody Techniques and Their Applications , 2nd Ed, A. Kawamura, Jr. (Ed. ) , University Park Press, Baltimore-London- Tokyo, ( 1977 ) , Chaps. 3-5. Assay of Interleukin- IB ELISA Assays The activity of IL-I B was determined using an enzyme-linked immunosorbent assay (ELISA) commercially available (Ciεtron Biotechnology, Code #03-0096, Pine Brook, NJ) . See also, E. W. Gaffney, C-W Chu, S. E. Lingenfelter, P. J. Li si . and G. A. Koch, Enzyme Linked Immunoassay with Monoclonal Antibody for Human Interleukin- IB, BioTechnique, 5: 652-657 ( 1987 ) . Broadly, the measurement of IL-IB in biological fluids by use of a four-stage test were carried out in a microtitration well that had been coated with a monoclonal antibody specific for IL-IB (referred to as the solid phase) . Stage 1: Biological fluid was added to antibody coated test wells and incubated for a sufficient length of time to allow IL-IB present in the sample to bind with the solid phase. Stage 2: A second antibody, polyclonal rabbit anti -IL-I B was added to test wells and incubated. The wells were then washed to remove any unbound polyclonal. The polyclonal bound only to IL-IB which had been bound to the solid phase monoclonal. Stage 3: Goat anti-rabbit antibody which had been conjugated to a detection enzyme (horseradish peroxidase) was added next, incubated, and washed. Any of the second antibody which had been bound to the solid phase bound IL-IB will bind to the conjugate. Stage 4: A substrate for the conjugate enzyme was then added and the color reaction was measured at 490 nm. The color reaction produced will be in proportion to the amount of IL-IB in the sample. Calculations: By construction of a calibration curve containing known amounts of standards, unknown sample concentrations were determined from the curve. The lower limit of sensitivity is 20 pg/ml IL-IB. Radioimmunoassays The principal of this assay is based on the competition of unlabeled IL-IB with radioac ' tively labeled IL-IB for a limited number of binding sites on a very specific anti-IL-lβ antibody. The higher the concentration of IL-IB in a sample, the fewer binding sites will be available to the radioactive IL-IB and, therefore, the lower the counts. Principles of Immunologic Diagnosis in Medicine, F. Milgrom, C. J. Abeyounis, and K. Kano (Eds. ), Lea and Febiges, Philadelphia (1981). Separation of unbound radioactive IL-IB from bound radioactive IL-IB is accomplished by addition of polyethylene glycol containing anti-rabbit immunoglobulin serum. After centrifugation, bound radioactive IL-IB is collected and placed in a gamma counter where counting rate is correlated with concentration by a standard, curve. Bioassays General bioassays for the detection of interleukin 1 have been described. A. J. H. Gearing, C. R Bird, A. Bristow, S. Poole, and S. Thorpe, A Simple Sensitive Bioassay for Interleukin-1 Which is Unresponsive to 10 3 U/ml of Interleukin-2, J. Immunol. Methods, 99: 7-11 (1987); P. J. Conlon, A Rapid Biologic Assay for the Detection of Interleukin 1, J. Immunol., 131: 1280-1282 (1983). Thy ocyte Stimulation Assay Measurement of bioactive IL-1 relies on its ability to stimulate proliferation of mice thymus cells (thymocytes) or very sensitive and specific cell lines (i. e. , D10 G4.1, Dinarrillo, Boston, Mass. ) in the presence of minute levels of mitogenic plant hormone (lectin). This proliferation is measured by uptake of added radioactive thymidine by proliferating DNA. Detection is 20 pg/assay of IL-1. IL-2 "Conversion" Assays This assay relies on the ability of IL-1 to cause lectin co- stimulated T-cell lines (i. e. , LBRM33, EL4, American Type Culture Collection, Rockville, MD) to produce IL-2. IL-2 quantitation can then easily be achieved by using a cell line whose proliferation is dependent on IL-2 only (i. e. , CTLL, American Type Culture Collection). Proliferation of CTLL cell is measured by uptake of added radioactive thymidine into DNA or by uptake of MTT dye. Assay of TNF-Alpha ■ Bioassays Cytolytic Assay For Tumor Necrosis Factor-Cf Bioassays for the detection of TNF-Cf have been reviewed. A. Meager, H. Leung and J. Woolley, Assays for Tumor Necrosis Factor and Related Cytokines, J. Immunol. Methods, 116: 1-17 (1989). TNFQf activity was determined by modification of a previously described cytotoxic assay. D. A. Flick and G. E. Figgard, Comparison of an In Vitro Cell Cytotoxic Assay for Tumor Necrosis Factor, J. Immunol. Methods, 68: 167-175 (1984). Briefly, L929 cells (CCL 1. America Type Culture Collection, Rockville, MD) were maintained in Dulbecco' s modified eagle medium supplemented with 10% fetal bovine serum and 4% penicillin and streptomycin. Confluent cultures were rinsed with sterile phosphate buffer saline, then briefly with 0.05% trypsin, resuspended in fresh medium and added to 96-well plates (7 x 10^ cells/well). After 2-3 hours in culture, 50 ul of cycloheximide (0.3 mg/ml) and 15 ul of CSF samples were added to each well and the plates were incubated overnight (5% CO2, 37°C). Following microscopic evaluation, the medium was decanted, and the wells were filled with 100 ul of 0.5% crystal violet in 25% methanol for 5 minutes, washed thoroughly with normal saline and dried. The dye was solubilized with 100 ul of 50% acetic acid. The degree of cytotoxicity was quantified spectrophotometrically (490 nm) using a computerized automated ELISA plate reader (Biorad Model 2550 EIA reader, Hercules, CA). Equivalent concentrations of recombinant human TNFO were deter-mined for experimental samples by interpolation of the recombi-nant human TNFCf standard curve run simultaneously (0.1 pg/ml - 1 ug/ml). Using this assay, it was possible to reliably detect as little as 10 pg/ml of human TNFCf The samples were assayed in quadruplicate, and a standard deviation within 10% of the mean was observed. Eight samples from patients with bacterial meningitis with the highest TNFCf concentrations were reassayed in the presence and absence of anti-TNFCf monoclonal antibodies. ELISA Assays EIISA Assay - Method 1 The activity of TNFCf can be determined by an ELISA method commercially available (Cistron Biotechnology, Code #04-0096, Pine Brook, NJ). Broadly, measurement of TNFCf in biological fluids is by use of a four stage test carried out in a microtitration well that has been coated with a monoclonal antibody specific for TNFCf Stage 1: A biological sample is added to the test well containing the specific monoclonal antibody to TNFCf incubated and washed with buffer. Any TNFCfthat is present will bind to the monoclonal while other sample components will be removed during subsequent washing. Stage 2: A second antibody, polyclonal rabbit anti-TNFCf is added to the test well and incubated a specified length of time. The polyclonal will bind only to TNFCf already- previously bound to the monoclonal antibody. Any polyclonal- not bound will be removed during subsequent washing. Stage 3: Goat anti-rabbit antibody conjugated to horseradish peroxidase enzyme is added. The goat antibody will bind specifically to bound rabbit polyclonal and any unbound goat antibody will be washed away. Stage 4: The last step involves adding a substrate for the bound enzyme to oxidize, resulting in a measurable color change, proportional to the amount of TNFCf in the original sample. Sensitivity is 20 pg/ml TNFCf ELISA Assay - Method 2 This commercial method (T-Cell Sciences Cat. No. BK1060, Cambridge, MA) for measuring TNFCf involves a sandwich-enzyme immunoassay. A microtitration well is coated with a monoclonal antibody specific for TNFCf to which the unknown TNF-containing sample is applied. Any TNFCfwill bind to the antibody, while other sample components are removed during subsequent washing of the well. Next, an enzyme conjugated-monoclonal antibody is added, which is specific for the bound TNFCf completing the sandwich effect. The well is washed to remove any unbound conjugated antibody and enzyme substrate is added. A colored reaction occurs, which is proportional to the amount of TNFCf present in the sample. Absorbance at 490 nm is measured and a calibration curve is constructed from known standards from which unknown values are determined. Sensitivity is 10 pg/ml TNFCf Latex Agglutination For Detection of IL-IB And TNFCf Polystyrene latex particles or beads (diameter 0.8 microns, Dow Chemical Co., Midland, MI) are coated with antibodies of IL-16, or TNFCf or both (IL-IB available from Cistron Biotechnology, Pine Brook, NJ; and TNF favailable from Genzyme Corp., Boston, MA). Coating of the latex beads involves preparing a 10% latex suspension (W V) in a 1: 500 dilution of antibodies of IL-IB, or TNFCf or both, in glycine buffered saline (GBS) (0.054 M) at pH 8.2, incubate at 37'C for 2 hours, followed by centrifugation for 10 minutes (12,000 G). These beads can be used as diagnostic kits. The suspension of latex beads are washed with glycine-saline (40 ml, 0.054 M) and re-σentrifuged for 10 minuteε (12,000 G) before agglutination is performed. Agglutination is performed by adding 0.05 ml aliquots of CSF, serum or other body fluid to 0.01 ml of antibody-coated latex suspension on a serologic slide with ceramic rings. The slide is rotated in a humidified chamber at 37°C and reaction is examined visually in one hour by illuminating the slide from the side, against a dark background. Agglutination is rated on a scale of 1 to 4, with 4 showing the greatest amount of granularity. Thus, a scale of 4 is recorded when all particles agglutinate into large clumps and the background is clear; a scale of 3 is recorded when all particles agglutinate into large and small clumps and the background is clear; a scale of 2 is recorded when all particles agglutinate into small but definite clumps and the background is cloudy; and a scale of 1 is recorded when particles agglutinate into fine granular agglutination against a milky background. R B. Newman, R W. Stevens and H. A. Gaafar, Latex Agglutination Test for the Diagnosis of Haemophilus Influenzae Meningitis, J. Lab. Clin. Med. , 76: 107-113 (1970). Alternatively, Particle Counting Immunoassay Equipment (PACIA, Technicon) can be used to quantitate the degree of agglutination of the latex beads. EXAMPLES STATISTICAL METHODS Meningitis Studies Comparisons between data from patients with aseptic meningitis who had negative viral cultures and those with proved enteroviral meningitis were performed with the Student t test for independent groups. Mann-Whitney U test was utilized to compare data from patients with bacterial and aseptic meningitis. Finally, the strength of the relationship between CSF IL-IB concentrations and other indices of meningeal inflammation was assessed with Spearman' s correlation coefficients. Arthritis Studies Comparison of means between independent groups was performed using the two-tailed t test. Two-tailed Fisher' s exact test was used to compare proportions between groups. Spearman' s correlation coefficients between TNFCf and IL-IB concentrations and those of other synovial inflammatory indices were calculated to investigate the strength of relationship between pairs. The level of significance was set at p value of <.0.05. STUDY GROUP 1 (BACTERIAL MENINGITIS) One hundred and six patients (57 boys and 49 girls, age 3 months to 5 years) with bacterial meningitis were prospectively evaluated. Aliquots of CSF obtained at the time of diagnosis and again 18 to 30 hours later were available from these patients. The CSF samples were immediately centrifuged and frozen at -70'C within 30 minutes of the lumbar puncture. The sera were obtained at the time CSF was collected, approximately 24 hours after initiation of therapy. Both TNFCf and IL-IB are stable for long periods when stored at -70'C. In this series, H, influenzae type b accounted for 91 (86%) of 106 cases of bacterial meningitis, Streptococcus pneumon ae for 7 cases (7%), Neisseria meningitis for 6 cases (5%), and group B streptococcus for 2 cases (2%). Thirty patients were treated with ceftriaxone and 76 patients with cefuroxime. Dexamethasone was administered at diagnosis to 47 patients and placebo to 59 patients. The number of patients with pathogens recovered from blood and CSF cultures was similar in both groups, and the percentage of patients with positive CSF cultures after 24 hours of therapy was similar for those who received cefuroxime or ceftriaxone and those treated with dexamethasone or placebo. IL-IB Concentrations In CSF The IL-IB assay was performed on 204 CSF samples for 102 patients. At the time of diagnosis, 97 patients (95%) had detectable IL-IB activity in CSF. The mean IL-IB concentration was 944 ± 1293 pg/ml (range <20 to 7700 pg/ml) (Table 1). Concentrations of IL-IB correlated significantly with protein, lactate, and TNFCf concentrations (P <0.05) in the first CSF samples (CSF j ). Patients with CSF } IL-IB concentrations 2500 pg/ml were more likely (P = 0.01) to develop neurologic sequelae (i.e. bilateral moderate to profound hearing impairment, hemiparesis, ataxia, seizures after admission, cerebral infarcts, cerebral atrophy, and hydrocephalus). There was no significant correlation between IL-IB concentrations and either age of the patient or duration of illness before diagnosis. In CSF samples obtain 18 to 30 hours after admissions (CSF 2 ), IL-IB activity was still detected in 67 (66%) of 102 samples. The mean IL-IB concentration was 135 ± 343 pg/ml (range >20 to 2900 pg/ml), and the values correlated directly with CSF 2 leukocyte, protein, and TNFCf concentrations, and inversely with glucose concentrations (P <0.001). The mean CSF 2 IL-IB concentration was significantly higher in patients with abnormal neurologic outcome compared with that in patients with normal neurologic outcome (274 ± 313 vs 70 ± 151; P = 0.036). Compared with CSF 2 samples that had detectable IL-IB activity, those samples with no detectable IL-IB activity had higher CSF glucose concentrations (64.5 ± 22.3 vs 39.8 ± 26.4, P - 0.0001) and lower lactate concentrations (25.8 ± 5.1 vs 50 ± 15, P = 0.0001), and the patients from whom the samples had been obtained had a shorter duration of fever (1.5 ± 1.8 vs 4.6 ± 4.5 days, P = 0.0002) " and a lower incidence of neurologic abnormalities (P = 0.002). The IL-IB concentrations in the 30 sera (16 from dexamethasone-treated patients and 14 from placebo-treated patients) obtained approximately 24 hours after the start of therapy did not correlate with CSF IL-IB concentrations. TNFCfwas not detected in any of these sera. TNFCfConcentrations in CSF The TNFCf cytotoxic assay was performed on 212 CSF samples from 106 patients (Table 1). At the time of diagnosis, 79 (75%) patients had detectable TNFCf activity in CSF. The mean TNFCf concentration was 787 ± 3358 pg/ml (range <10 to 26,000 pg/ml), and these values correlated significantly with CSF protein concentrations. There was no significant correction between TNFCf concentrations and either the age of the patients, duration of illnesε before diagnosis, or outcome of disease. Preincubation of the eight samples with highest TNFCf activity with excess human TNFCf monoclonal antibodies completely inhibited cytotoxic activity. In CSF 2 , the TNFCf activity was detectable in 53 samples with a mean TNFCf concentration of 21 ± 65 pg/ml (range < 10 to 500 pg/ml). The CSF TNFCf concentrations correlated significantly with IL-IB concentrations (P >0.0001) but did not correlate with any of the indexes of meningeal inflammation nor with outcome of disease. Table 1. Concentrations of IL-IB and TNFCf in CSF of Infants and Children With Bacterial Meningitis: Study population CSF concentrations CSF concentrations at diagnosis lδ-30 hr later Cytokine (No. of patients ) (No. of patients ) IL-IB Mean ± SD (pg/ml ) 944 ± 1293 (102) 135 ± 343 (101) Range <20-7700 <20-2900 TNFCf Mean ± SD (pg/ml ) 787 ± 3358 (106) 21 ± 65 (106) Range <10-26, 000 <10-500 Numbers in parentheses represent the number of patients studied. Effects of Dexamethasone Treatment Of 106 patients, 47 received dexamethasone therapy intravenously (0.6 mg/kg/day in four divided doses for 4 days) and 59 received placebo beginning at diagnosis. The initial CSF IL-IB concentrations were similar for patients who received dexamethasone and those given placebo (Table 2). By contrast, there was a significantly lower mean IL-IB concentration in CSF sampleε obtained 18 to 30 hours after admission in patients treated with dexamethasone compared with those given placebo (28 ± 74 vε 221 ± 442; P = 0.0002). Only 28% of dexa ethasone-treated patients had detectable IL-IB activity in CSF 2 compared with 98% of placebo-treated patientε (P < 0.0001). Dexamethasone-treated patients had a shorter duration of fever (1.2 ± 1 days) compared with those who received placebo (5.5 ± 4.6 days) (P = 0.0001) and a lower incidence of bilateral moderate to profound hearing impairment (P = 0.018). There was no significant effect of dexamethasone treatment on CSF 2 TNFCfactivity (Table 2). The mean CSF } glucose, protein, and lactate concentrations and the mean leukocyte count did not significantly differ in dexamethasone-treated compared with placebo-treated patients. After 18 to 30 hours of treatment, however, CSF 2 glucose concentrations were significantly higher and CSF 2 lactate concentrations were εignificantly lower in dexamethasone-treated compared with placebo- treated patients (P = 0. 0001 ) . Table 2. Concentrations of IL-IB and TNFCf in CSF of Infants and Children With Bacterial Meningitis: Dexamethasone and Placebo Groups CSF concentrations CSF concentrations at diagnosis 18-30 hr later Cytokine (No. of patients ) (No. of patients ) Dexamethasone Placebo Dexamethasone Placebo IL-IB Mean ± SD (pg/ml)* 828 ± 1206(46) 1040 ± 1363(56) 28 ± 74(46) 225 ± 442(55) Median 478 569 0 43 No. (%) with detect¬ able activity 41 (89) 56 (100) 13 (28) 54 f98) TNFCf Mean ± SD (pg/ml)* 841 ± 3014(47) 744 ± 2974(59) 12 ± 22(47) 28 ± 85(59) Median 12 45 0' 10 No. (%) with detect¬ able activity 30 (64) 49 (83) 20 (43) 33 (56) NS Not significant. * Numbers in parentheses represent the number of patients studied. + Lower limits of detectable activity were 20 (pg/ml) for IL-IB and 10 (pg/ml) for TNFCf From this study, it is seen that the percentage of patients with positive CSF cultures and the degree of CSF inflammation was similar for the two antibiotic treatment groups, and the cell wall constituents of both H. influenzae type b and S_. pneumoniae induce 5 IL-IB in CSF in experimental meningitis. The majority of infants and children with bacterial meningitis in this study had detectable concentrations of both IL-IB and TNFC in CSF obtained at the time of diagnosis. The exact source of IL-IB and TNFCf in the CSF of patients with bacterial meningitis is 10 unknown. In CSF } , IL-IB was detected in 95% of samples; the mean (± 1 SD) concentration was 944 ± 1293 pg/ml. Patients with CSF } IL-IB concentrations 500 pg/ml were more likely to have neurologic sequelae (P = 0.001). TNFCfwaε present in 75% of CSF } samples; the 15 mean concentration was 787 ± 3358 pg/ml. In CSF 2 the mean IL-IB concentration was 135 ± 343 pg/ml. The IL-IB concentrations correlated significantly with CSF 2 leukocyte count, with glucose, lactate, protein, and concentrations, and with neurologic sequelae. The TNFCf was detected in CSF 2 specimens of 53 of 106 patients, with a mean concentration of 21 ± 65 pg/ml. Of the 106 patients, 47 received dexamethasone therapy at the time of diagnosis. These patients had significantly lower concentrations of IL-I B and higher glucose and lower lactate concentrations in CSF 2 , and they had a significantly shorter duration of fever compared with the values in patients not treated with steroidε (P <0. 002 ) . The data εuggest a possible role of IL-I B and TNFCf as mediators of meningeal inflammation in patients with bacterial meningitis. STUDY GROUP 2 (BACTERIAL MENINGITIS) Multiple CSF specimens from each patient were available from 42 infants enrolled originally in the first Neonatal Meningitis Cooperative Study. The etiologic agents in these 42 infants were: E. coli Kl strains in 34 ( 80% ) ; E. coli non-Kl strains in 3; Salmonella species and Enterobacter species in 2 each; and Serratia in 1 neonate. The patients were grouped into three outcome categories. Eighteen subj ects were alive and well on long term follow-up examination (Group I ) , 13 had mild to moderate neurologic sequelae (Group II ) and 11 died during hospitalization (Group III ) . No child was considered to have severe neurologic sequelae characterized as failure to develop beyond the age at which disease occurred or by the requirement for custodial care. There were 199 CSF specimens available from these 42 patients (mean, 4 specimens; range, 2 to 10 per patient) . For 78 specimens an insufficient volume of fluid was available to assay for IL-I B and TNFCf Accordingly the bovine serum albumin-phosphate buffer diluent for the IL-IB assay was added to the specimens precluding their use for TNFCf measurement because the diluent interfered with the cachectin bioassay. IL-I B concentrations from <20 to 2616 pg/ml were found in CSF from the 42 patients. Specimens obtained at the time of diagnosis from 40 ( 95% ) infants had detectable IL-IB concentrations (220 pg/ml ) . The analysis of variance performed on the IL-I B concentrations of the subj ectε in the 3 groups was significant overall (P = 0.0074). Multiple comparisons demonstrated that the 11 infants who died had a significantly larger mean peak CSF concentration of IL-IB (1230 pg/ml; range, 302 to 2524 pg/ml) compared with those in Group I (446 pg/ml; range, <20 to 1717 pg/ml) and in Group II (575 pg/ml; range, <20 to 2616 pg/ml) (Table 3). IL-IB concentrations of >200 pg/ml and of >500 pg/ml were observed in 100 and 82%, respectively, of infants who died compared with significantly smaller percentages of infants who survived and are well or have mild or moderate neurologic sequelae on follow-up (FIG. 1). Further analyseε of variance demonstrated significant group differences for the number of days of CSF IL-IB concentrations >200 pg/ml (P = 0.0022), the number of days that endotoxin (P = 0323) and Kl antigen (P = 0.0001) were detected in CSF and the number of days that the CSF cultures were positive (P = 0.0263). For each measurement those infants who died (Group III) were significantly different from those who survived (Table 3). IL-IB concentrations were measured in brain and meninges of three infants who died. The values in brain were 1246, 1260 and 1705 pg/ml and in meninges they were 1661, 750 and 1716 pg/ml, respectively. Table 3. Correlation of Cerebrospinal Fluid IL-IB Concentrations and Other Indices With Outcome in 42 Neonateε With Meningitiε Caused by Gram-negative Enteric Bacilli Statistically significant differences (P 0.05) among outcome groups (one way analysis of variance) . b IL-IB expressed in pg/ml; Kl antigen in mg/ml. c Mean ± SE. NS, not significant. The concentrations and duration of IL-IB in CSF of the 42 infants were correlated using Spearman correlation coefficients with the other CSF values (Table 4). The highest concentrations of IL-IB in CSF correlated significantly with the highest CSF Kl antigen concentrations. The number of days on which IL-IB was detected at concentrations of >200 pg/ml and 0 pg/ml correlated significantly with days of positive CSF cultures and of Kl and endotoxin detection (Table 4). Table 4. Statistically Significant Correlations Between Cerebrospinal Fluid IL-IB Concentrations and Other Variables in 42 Infants With Meningitis Caused by Gram- . negative Enteric Bacilli Correlation Concentration/Variables Coefficients P I. Days IL-IB >200 pg/ml and Days CSF cultures positive Days endotoxin detected Days Kl antigen detected II. Days IL-IB 220 pg/ml and Days CSF cultures positive Days endotoxin detected Days Kl antigen detected III. Peak IL-IB concentration and 0.4519 0.0: peak Kl antigen concentration * Spearman correlation coefficients. Spearman correlations for three additional pairs of measurements were calculated. The number of days on which endotoxin was detected in CSF correlated significantly with days of poεitive CSF cultureε (0.6456, P = 0.0001) and with dayε of CSF Kl detection (0.8078, P = 0.0001). Similarly the days of positive CSF cultures correlated with days of Kl antigen detection (0.7368, P = 0.0001). There were 108 undiluted CSF specimens from 27 patients available for measurement of TNFCf TNFCf activity was detected in CSF of 25 of these patients but data from 7 could not be interpreted because CSF specimens collected in the first several days of treatment or intermittently throughout the . illnesε were either unavailable or had been diluted with bovine εerum albumin-phoεphate buffer. It was impossible therefore to determine the maximum concentration or duration of TNFC activity in those 7 patients. In the remaining 20 infants maximum concentrations ranged from <10 (2 infants had undetectable activity) to 6200 pg/ml; specimens from 2 patients had maximum concentrations of 1000 (Group I) and 6200 (Group III) pg/ml, respectively, whereas those from the other 18 infants ranged from <10 to 326 pg/ml (mean, 72 pg/ml). There was no correlation between outcome and CSF TNFCf concentrations in those 18 patients. In this study 95% of the infants had IL-IB detected in CSF at time of diagnosis and the peak concentration and persistence of this cytokine in CSF correlated significantly with outcome from neonatal meningitis caused by Gram-negative enteric bacilli. These data illustrate the important role of IL-IB as a mediator of meningeal inflammation and potentially of brain injury. Although TNFCf activity was detected in more than 90% of patients studied, there was insufficient data in this study to determine the pathogenetic role of this cytokine in neonatal meningitis. STUDY GROUP C (NON-BACTERIAL MENINGITIS) Thirty-six infants and children, aged 1 month to 14 years, with a diagnosis of aseptic meningitis were prospectively evaluated. The following criteria were required to be enrolled in the study: (1) CSF leukocyte count > 10/cu mm; (2) Negative bacteriologic studies including CSF and blood cultures, CSF .-Gram stained smear, and conventional antigen detection tests, and (3) No antibiotic therapy in the previous 2 weeks. CSF specimens were routinely cultured for bacteria and analyzed for cell count and differential and for glucose and protein concentrations. Viral cultures were performed on all samples; 0.2-0.3 ml CSF aliquots were inoculated directly into human embryonic lung (HEL), A549, and Rhesus monkey kidney (RhMK) cell lines. Occasionally Buffalo green monkey kidney (BGMK) and MRC-5 cells were also included. These cultures were observed for a minimum of AA days. A positive result was reported when definite cytopathic effect (CPE) waε noted. Isolates producing typical enteroviral CPE were identified by neutralization using the intersecting virus antiserum pools supplied by the Centers for Disease Control, Atlanta, GA. The remaining CSF was stored at -70°C until assayed for TNFCfand IL-IB. A second group of CSF samples, considered as controls, were obtained from 14 patients admitted without suspected infection and whose attending .physicians requested cerebrospinal fluid examination All these specimens had < 5 WBC/cu mm, normal glucose and protein concentrations, and negative bacteriologic studies. These samples were also kept at -70'C until assayed for cytokines. Thirteen (36%) of 35 patients with non-bacterial meningitis evaluated had a positive viral culture in RhMK cells. All isolates were identified as enteroviruses and nine have been typed aε follows: Echovirus 9 (3 isolates); echovirus 11 (2); coxsackivirus B-2; echovirus (4) (1); echovirus 5 (1); and echovirus 31 (1). Because there were no significant differences m the CSF leukocyte count and glucoεe and protein concentrationε for patients with and without a viral isolate, they were considered as one group for the analysis (Table 5). Table 5. CSF Findings and IL-IB and TNFCf Concentrations in Patients with Non-bacterial Meningitis *Mean ± SEM ND - not detectable (< 10 pg/ml) TNFCfConcentrations in CSF None of the 36 samples from patients with aseptic meningitis and from the 14 controls had detectable TNFCf activity. By contrast, 75% of CSF specimens obtained at diagnosis from patients with bacterial meningitis had measurable TNFCf (787 ± 326 pg/ml). IL-IB Concentrations in CSF Thirty-two (88%) of 36 patients with aseptic meningitis had detectable IL-IB concentrations; the range of values was from < 20 p /ml to 400 pg/ml. There were no significant differences betwee patients with positive viral culture (73.8 _ 30.0 pg/ml) and those without a viral isolate (33. 8 ± 4. 4 pg/ml; P = 0. 54; Table 6 ) . Two of 14 controls had IL-IB concentrations ( 42 pg/ml and 21 pg/ml, respectively) that were near the lower limit of sensitivity for the assay. Correlations between CSF IL-IB and other indices of CSF inflammation in patients with aseptic meningitis were also investigated. There was a significant correlation between CSF IL-I B and CSF WBC count (Spearman' s coefficient = 0. 48, P = 0. 003 ) . No significant correlations were found for IL-IB and protein (r = 0. 25, P = 0. 09) and glucose (r = -0. 009, P = 1. 0 ) concentrations in CSF. Concentrations of CSF IL-IB from patients with non-bacterial were compared to those with bacterial meningitis. For this purpose values from 36 patients with aseptic meningitis and from 106 patients with bacterial meningitis reported in Study Group 1 (discussed earlier) were analyzed. The difference between IL-IB concentrations in these two groups of patients, 944 ± 128 - pg/ml m patients with bacterial meningitiε vs. 48 ± 11 pg/ml in patients with aseptic meningitis, was highly significant (P < 0. 0000001 ) (Figure 2 ) . Because these two patient groups had very different CSF WBC counts ( 5233 ± 1058 and 288 ± 62 in patients with bacterial and aseptic meningitis, respectively) and because it is known that there is a εtrong correlation between the number of white blood cellε and IL-IB concentrations in the CSF, the concentrations of IL-I B in those patients of the two groups who had CSF WBC counts < 500 per cu mm were evaluated ' (Table 6 ) . Although the CSF WBC counts were comparable for the two groups, the CSF IL-I B concentrations were εignificantly larger ( 1. 180 ± 61 pg/ml vε. 48 + 11 pg/ml, P = 0. 003 ) in patients with bacterial infection. Table 6. CSF IL-IB Concentrations in Meningitis Patients with CSF WBC Count < 500 per cu mm Bacterial (n=1_T Non-bacterial (n=3β ' ι P-value " WBC 295 ± 43* 288 ± 62 0.199 per cu mm IL-IB 1, 180 ± 61 48 ± 11 0.003 (pg/ml) *Mean ± SEM WPC, white blood count The potential role of measuring CSF concentrations of these two cytokines for diagnostic purposes to distinguish bacterial from non- bacterial meningitis was then assessed (Table 7) . For this analysis, two groups were included: 106 patients with bacterial meningitis and 36 with aseptic meningitis. When a cut-off of 10 pg/ml or greater of IL-IB in CSF was used the sensitivity was 84. 3%, the specificity was 94. 4% and the positive predictive value was 97. 7%. With a cut-off of 10 pg/ml or greater of TNFCf in CSF, the sensitivity was 75%, and the specificity and positive predictive values were 100%. There were 27 patients with bacterial meningitis who had undetectable TNFCf activity; of those patients 18 had IL-IB concentrations of greater than 100 pg/ml and would have been identified using this assay. Table 7. Diagnoεtic Value of CSF IL-IB and TNFCf Concentrations to Diεtinguish Bacterial from Non-bacterial Meningitis The TNFCf activity was documented in 79 (75%) of 106 CSF specimens obtained at diagnosis from infants and children with meningitiε cauεed by Haemophiius influenzae, Streptococcus pneumoniae, Neisseria meningitides and group B Streptococcus, (Study Group 1), and from 25 (92%) of 27 neonates with Gram-negative enteric bacillary meningitis (Study Group 2). By contrast none of 36 patients with aεeptic meningitis evaluated in the present study had detectable TNFCf activity. Thus, detectable concentration of about or above 10 pg/ml of TNFCf in CSF is a εtrong suggestion, perhaps an indication of bacterial meningitiε in a patient. IL-IB was detected in CSF in 32 (88%) (mean ± SEM, 48 ± ' ll pg/ml) of 36 patients with aseptic meningitis. This cytokine was found in 97 (95%) of 102 infants and children with bacterial meningitis. (Study Group 1). Although the percentage of patients whose CSF samples contained measurable amounts of IL-IB did not substantially differ, the concentrations detected in patients with bacterial meningitis were on the average 20-fold greater than those in patients with aseptic meningitis (Figure 2), This was also true when only the patients with bacterial meningitis who had fewer than 500 WBC per cu mm in CSF were compared with those with aseptic meningitis (Table 6). There was a significant correlation between IL-IB concentrations and WBC in CSF (Spearman's coefficient = 0.48; P = 0.003) despite the relatively small number of patients with aseptic meningitis evaluated. Only 2 of the 14 control patients had detectable CSF IL-lβ concentrations of 21 and 42 pg/ml. Thus, concentrations of about 100 pg/ml, or greater, of IL-IB in CSF is suggestive of bacterial meningitis. This concentration iε a leεs likely, or less probable, indication of bacterial meningitis in a patient. Bacterial meningitis, however, can be diagnosed with more certainty and greater accuracy if the CSF concentration of each of ' the IL-IB and the TNFCf is found to be above the respective threshold level, each suggestive of bacterial infection. STUDY GROUP 4 (SUPPURATIVE ARTHRITIS) Twenty-four infants and children, aged 2 months to 15 years, with diagnoses of any kind of arthritis requiring diagnoεtic aspiration of synovial fluid were enrolled in this ' Study Group. Of those, 16 patients were classified as having suppurative arthritis and 8 as other kinds of arthritiε. The clinical and laboratory characteriεtics of these patients are summarized in Table 8. Table 8. General Characteristics of Patients With Suppurative Arthritis and With Non-Infectious Synovial Effusions Characteristic Age (months median (range) Male sex Duration of symptoms before arthrocentesis mean ± S. E. M. (days) Single joint involved Fever (> 38°C) ESR (> 50 mm/h) Blood WBC (> 10, 000/mm 3' ) 10 (62) 6 (63) a ^ Number of cases (percent) b No significant differences were observed between both groups (Two-tailed Fisher's exact test and Student t test). Synovial fluid was collected from joint arthrocentesis performed for diagnostic purposes by orthopedic surgeons. A blood sample was obtained simultaneously in these patients. Samples were centrifuged-..at 1500g for 10 minuteε to remove cells and then stored at -70 ° C until assayed. In patients with ' bacterial infection, Haemophilus influenzae type b was recovered from synovial fluid in 5 cases. Staphylococcus aureuε in 4, Streptococcus pneumoniae in 2, Streptococcus pyrogenes in 1, Salmonella group B in 1, and Kingella kingae in 1. In two patients, no organism waε isolated from joint fluid; one of them had concomitant osteomyelitis caused by S, aureuε, and in the other one the diagnosis was based on clinical grounds, high erythrocyte sedimentation rate (ESR 120 mm/h), significant changes in synovial inflammatory indices (WBC: 135, 000/mm; glucose: 10 mg/dl; protein: 4 g/dl), and rapid response to antimicrobial treatment. Two patientε had received previouε oral antimicrobial therapy but organisms were recovered from joint fluid in both cases. Blood cultures were positive for H. influenzae type b in two patients. In the group of patients with non-infectious arthritides, 3 were considered to have traumatic effusions, 3 reactive arthritiε, and 2 juvenile rheumatoid arthritis. The diagnosis of reactive arthritis was based on the presence of one or more characteristics, such as multiple joint involvement, previous epiεode of diarrheal illness, positive HLA-B27 marker, and failure to isolate microorganisms from blood, bone or synovial fluid. The synovial fluid analysis results of all patients are shown in Table 9. There were significant differences between both groups regarding leukocyte and PMN cell countε, and cytokine concentrations. Table 9. Synovial Fluid Analysis of Patientε With Suppurative Arthritiε and With Non-Infectious Arthritides Suppurative Non-infectious Characteristic Arthritis Arthritides p value c (n = 16) fn - 8) Leukocytes/mm 3 92,190 ± 13, 125 a 33,080 ± 12,151 p < 0.05 Polymorphonuclear cells/mm 3 83,893 ± 2,766 25,141 ± 5,637 p < 0.01 Glucose (mg/dl) 32.1 ± 7.1 46.8 ± 4.3 NS (p > 0.10) Protein (mg/dl) 3,386 ± 186 2,283 ± 630 NS (p > 0.10) TNFCf (> 10 pg/ml) 12 (75) b 0 < 0.001 IL-IB (> 200 pg/ml) 15 (94) 0 < 0.001 ^Mean ± S. E. M. ■ '-'Number of cases (percent) c Fisher' s .exact test and Student t test; NS: not significant TNFCf was not detected in the group of patients with non- infectiouε arthritides. In contrast, three-quarters of patients with suppurative arthritis had detectable TNFCf in synovial fluid with a mean ± SEM of 18,628 ± 7,762 pg/ml (range: 200-85,000). TNFCf was detected in all 7 patients with suppurative arthritis caused by Gram-negative organisms (39,000 ± 15,934 pg/ml), whereas it was found in 4 of 7 patients with Gram-positive organisms (2,300 ± 1,371 pg/ml) (P = 0.07). TNFCf was also present in one patient with presumed suppurative arthritis but sterile joint fluid (6,500 pg/ml). IL-18 waε detected in all but one patient with suppurative arthritiε (8,418 ± 2,198 pg/ml, range: 400-26,000 pg/ml), and in 5 of 8 (63%) patients with non-infectious etiology (79 ± 8.1 pg/ml, range: 45-120 pg/ml) (P < 0.01). In this latter group IL-IB was detected in all 3 patients with JRA, in 2 of 3 with reactive arthritis, and in none of 3 patients with traumatic effusions. In the patient with suppurative arthritis who did not have detectable IL-IB S_. aureus was recovered from synovial fluid and from bone aspirate. This patient did not have detectable TNFCf either. In a group of 6 patients with suppurative arthritis who had less than 50,000 leukocytes /mm 3 in synovial fluid (38,200 + 8, 608/mm 3 ) and comparable counts to those with non-infectious non- traumatic effusions (42,600 ± 12, 150/mm 3 ), the mean IL-IB concentrations were 27 timeε greater (2, 133 ± 530 vε 79 ± 8.1 pg/ml, respectively) (P < 0.05). The mean IL-IB concentration in those patients with Gram-negative infection was 14, 071 ± 3, 314 pg/ml compared with 4, 556 ± 2658 pg/ml in those with Gram-positive infection (P > 0/10). TNFCf and IL-IB concentrations correlated significantly (P < 0.0001) and each correlated with leukocyte counts in synovial fluid (P = 0.026 and P < 0.0001, respectively) (Figures 3, 4 and 5). There was no correlation between cytokines and other inflammatory indices. Serum samples obtained simultaneously were negative for both cytokines. Thus, detectable concentration of about or above 10 pg/ml, of TNFCf in synovial, or joint, fluid is indicative of bacterial suppurative arthritis in a patient. Concentration of about or above 200 pg/ml of IL-IB in synovial fluid is suggestive, or a less certain indication, of bacterial εuppurative arthritis in a patient. The reεultε presented above demonstrate that the diagnosis of a patient with bacterial infectious conditions can be supported by comparing the concentration of a cytokine present in the body tisεue specimen of the patient with a threshold concentration indicative of the bacterial infectious conditions. The bacterial infectious conditions can also be evidenced, with even higher certainty, if concentrations of two or more different cytokineε preεent in one or more body tissue specimens of a patient are higher than two or more respective threshold concentrations, each suggeεtive of bacterial infectiouε conditions. Each and every publication, patent or otherwiεe, specifically identified in this specification represents a teaching of the understanding of those skilled in the art at the time this invention was made and is herein individually incorporated by reference to the same extent as if it had been physically reproduced in the location and for the purpose as identified by the context in which it is found. The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as defined in the following claims.

Claims: We claim: 1. An in vitro method for the diagnosis and the differentiation of bacterial from non-bacterial meningitis in a patient, said method comprises the steps of: measuring a concentration of a cytokine in a sample of body fluid of said patient; comparing said concentration to a threshold concentration indicative of bacterial meningitis; and evidencing said bacterial meningitis by the presence of said concentration of said cytokine over said threshold concentration 2. The method of claim 1 wherein said body fluid is cerebrospinal fluid. 3. The method of claim 1 wherein εaid cytokine is tumor necroεis factor-alpha. 4. An in vitro method for the diagnosis and the di fferentiation of bacterial suppurative from non-bacterial arthritis in a patient, said method comprises the steps of: measuring a concentration of a cytokine in a sample of a body fluid of said patient; comparing s aid concentration to a threshold concentration indicative of bacterial suppurative arthritis; and confirming said diagnosis of said bacterial suppurative arthritiε by the presence of said concentration of said cytokine over said threshold concentration. 5. The method of claim 4 wherein said body fluid is synovial fluid. 6. The method of claim 4 wherein said cytokine is tumor necrosis factor-alpha. 7. An in vitro method for the diagnosis of a bacterial infectious condition in a patient, said condition being characterized in that its grosε symptoms are shared by other infectious and non-infectious diseases, said method comprises the steps of: measuring a first activity of a first cytokine in a first body tissue specimen of said patient; comparing said first activity to a first threεhold activity suggestive of said bacterial infectious condition; measuring a second activity of a second cytokine in a second body tissue specimen of said patient; comparing said second activity to a second threshold activity suggestive of said bacterial infectious conditions; and evidencing said bacterial infectious condition by the presence of said first activity over said first threshold activity and by the presence of said εecond activity over εaid εecond threshold activity. 8. The method of claim 7 wherein said bacterial infectious condition is εelected from a group selected "from the group consisting of meningitis, arthritis, pneumonia, peritonitis, pyelonephritis, and cystitis. 9. The method of claim 7 wherein said first body tissue specimen and said second body tissue specimen are the same. 10. The method of claim 7 wherein εaid first body tissue specimen is selected from a group consisting of cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, and urine. 11. The method of claim 7 wherein said second body tiεεue specimen is selected from a group consisting of cerebrospinal fluid, synovial fluid, pleural fluid, peritoneal fluid, and urine. 12. The method of claim 7 wherein said first cytokine is selected from a group consisting of interleukin, tumor necrosis factor and platelet activating factor. 13. The method of claim 7 wherein said second cytokine is selected from a group consisting of interleukin, tumor necrosis factor and platelet activating factor. 14. The method of claim 7 wherein said first activity is measured by a method selected from a group consisting of immuno- enzymatic assay, radioimmunoassay, bioassay, immunofluorescent assay, and latex agglutination. 15. The method of claim 7 wherein said second activity is measured by a method selected from a group consisting of immuno- enzymatic assay, radioimmunoassay, bioassay, immunofluorescent assay, and latex agglutination. 16. An in vitro method for the diagnosis and the differentiation of bacterial from non-bacterial meningitis in " a patient, said method comprises the stepε of: measuring a first activity of a first cytokine in a first body tissue specimen of said patient; comparing said first activity to a first threshold activity suggestive of bacterial meningitiε; measuring a second activity of a second cytokine in a second body tissue specimen of said patient; comparing said second activity to a second threshold activity suggestive of bacterial meningitis; and evidencing said bacterial meningitis by the presence - of said first cytokine activity over said first threshold activity and by the presence of said second cytokine activity over said second threεhold activity. 17. " The method of claim 16 wherein both said first body tissue specimen and said second body tissue specimen are cerebrospinal fluid. 18. An in vitro method for the diagnosis and the di fferenti ation of bacterial suppurative from non-bacterial arthritis in a patient, said method comprises the steps of: measuring a first activity of a first cytokine in a first body tissue specimen of said patient; ; . f- : ■ comparing said first activity to a first threshold activity suggestive of bacterial suppurative arthritis; measuring a second activity of a second cytokine in a second body tissue specimen of said patient; comparing εaid second activity to a second threshold activity suggestive of bacterial suppurative arthritis; and evidencing said bacterial suppurative arthritis by the presence of said first cytokine activity over said first threεhold activity and by the preεence of said second cytokine activity over said second threshold activity. 19. The method of claim 18 wherein both said first body tiεεue specimen and said second body tissue specimen are synovial fluid. 20. A diagnostic kit for the detection of the presence or absence of a human cytokine indicative or suggestive of a bacterial infectious condition, said condition being characterized in that its grosε symptomε are shared by other infectious and non-infectious diseases, said kit comprises: an antibody of said cytokine; and means for detecting the formation of immune complexes between said cytokine and said antibody, wherein said antibody and said means are present in an amount sufficient to perform said detection. 21. The diagnoεtic kit of claim 20 wherein εaid bacterial infectiouε condition iε selected from a group consisting of meningitis, arthritis, pneumonia, peritonitis, pyelonephritis, and cystitis. 22. The diagnostic kit of claim 20 wherein said cytokine is selected from the group consisting of interleukin, tumor necrosis factor and platelet activating factor. 23. The diagnostic kit of claim 20 wherein said means for detecting the formation of the immune complexes are immunological assay means selected from the group consisting of radioimmunoasεay, bioassay, immuno-enzymatic and immunofluorescent aεsay means. 24. A diagnostic kit for measuring the activity of a cytokine present in a body fluid specimen of a patient said activity being suggestive or indicative of a bacterial infectious condition, εaid condition being characterized in that its groεs symptoms are shared by other infectious and non-infectious diseases, said kit compriseε: latex bead; and said latex bead being coated with an antibody or antigen to said cytokine, said coated latex bead being characterized in that it formε agglutination upon incubation with a suspension of said body fluid specimen containing said cytokine, wherein said bead and said coating of antibody or antigen are present in an amount sufficient to perform said agglutination. 25. The diagnostic kit of claim 24 wherein said bacterial infectious condition is selected from a group consiεting of meningitis, arthritis, pneumonia, peritonitis, pyelonephritis, and cystitis. 26. The diagnostic kit of claim 24 wherein said cytokine iε selected from the group consisting of interleukin, tumor necrosis factor and platelet activating factor.

Inventors: MCCRACKEN GEORGE H JR, MUSTAFA MAHMOUD, NELSON JOHN D, RAMILO OCTAVIO, SAEZ-LLORENS XAVIER, MCCRACKEN, GEORGE, H., JR., MUSTAFA, MAHMOUD, NELSON, JOHN, D., RAMILO, OCTAVIO, SAEZ-LLORENS, XAVIER
Applicants: UNIV TEXAS, UNIVERSITY OF TEXAS SYSTEM BOARD OF REGENTS
Application year: 1990
Main classification: G01N33
IPC codes: G01N33