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An enzyme immunoassay (EIA) based system for the qualitative detection of Legionella pneumophila serogroup 1 antigen (L. pneumophila serogroup 1 antigen) in human urine as an adjunct to culture for the presumptive diagnosis of past or current Legionnaires' Disease. The majority of Legionnaires' Disease is caused by Legionella pneumophila serogroup 1 and is characterized as an acute febrile respiratory illness that Legionella is common nosocomial infection Legionella is present in water Transmitted via aerosolized droplets Affects immunocompromised or otherwise ill persons Increasingly common in the community setting Empiric therapy may not cover Legionella Treatment of choice: azithromycin A total of 294 patient urine specimens were evaluated using the Binax Legionella Urinary Antigen EIA Test kit and a commercially available method. The results of this correlation are as follows: Sensitivity: 97.7% Specificity: 100 % Agreement: 99.0 % Positive and negative controls (liquid) All necessary reagents Ordering information
851-000 NOW® Legionella Urinary Antigen EIA
2. Correlation of Results A total of 294 patient urine specimens were evaluated using the Binax Legionella Urinary Antigen EIA Test kit and a commercially available method. The results of this correlation are as follows: Legionella Urinary Antigen EIA
Sensitivity: 97.7% (0.951 to 1.00, 95% Confidence Interval) I. PROPRIETARY NAME
Specificity: 100 % Binax Legionella Urinary Antigen EIA, 96 Wells Agreement: 99.0 % II. INTENDED USE
Statement of Cross-ReactivityOf the 164 negative specimens: 76 specimens were bacteremic (other than Legionella sp.) pneumonia patients, 34 had urinary tract infections (UTI), 16 had other pulmonary conditions, 19 FOR IN VITRO DIAGNOSTIC USE had Mycobacterial infections, and 2 were pneumonias caused by transtracheal aspirates (TTA). This kit is an enzyme immunoassay (EIA) based system intended for IN VITRO DIAGNOSTIC USE to qualitatively detect the presence of Legionella pneumophila serogroup 1 antigen (L. pneu-mophila serogroup 1 antigen) in human urine as an adjunct to culture for the presumptive diagnosis of past or current Legionnaires' Disease. 3. Binax Legionella Urinary Antigen EIA Kit Precision III. SUMMARY AND EXPLANATION OF TEST
Within Assay Precision Legionnaires' Disease, named after the outbreak in 1976 at the American Legion convention in Philadelphia, is caused by Legionella pneumophila and is characterized as an acute febrile res- Within Assay Precision was determined on three urine samples containing different antigen concentrations. The samples were run in twenty replicates. Each sample was correctly identified as piratory illness ranging in severity from mild illness to fatal pneumonia (1). Since that time, it has been recognized that the disease occurs in both epidemic and endemic form and that spo- being positive or negative 100% of the time. radic cases are not readily differentiated from other respiratory infections by clinical symptoms. It is estimated that about 25,000 cases of Legionella infections occur annually (2). Known riskfactors include immunosuppression, cigarette smoking, alcohol consumption and concomitant pulmonary disease (2). The resulting mortality rate, which ranges up to 25% in untreated Mean (absorbance) Standard Deviation Coefficient of Variation (%) immunocompetent patients, can be lowered if the disease can be diagnosed rapidly and appropriate antimicrobial therapy is instituted early. Legionella pneumophila is estimated to be respon- sible for 80-85% of reported cases of Legionella infections with the majority of cases being caused by L. pneumophila serogroup 1 alone (3). Current methods for the laboratory detection of pneumonia caused by Legionella pneumophila require a respiratory specimen (e.g. expectorated sputum, bronchial washing, transtracheal aspirate, lung biopsy) or paired sera (acute and convalescent) for accurate diagnosis. These techniques include Legionella culture, direct fluorescent antibody (DFA), DNA probe, and indirect fluorescent antibody (IFA). All of these rely oneither obtaining an adequate respiratory specimen for sufficient sensitivity, or collecting sera at a two to six week interval. Unfortunately, one of the presenting signs of patients with Legionnaires' Between Assay Precision Disease is the relative lack of productive sputum. This necessitates in many patients the use of an invasive procedure to obtain a respiratory specimen. Diagnosis by serological techniques is Between assay precision was determined on three urine samples containing different antigen concentrations. Each sample was analyzed on two lots of reagents in a total of 33 assays. The sam- usually retrospective in nature, and even then, patient compliance in obtaining the necessary samples is poor. ples were correctly identified as being positive or negative 100% of the time It was shown as early as 1979 by Berdal (4), that a specific soluble antigen was present in the urine of patients with Legionnaires' Disease (5,6,7,8). The presence of specific antigen in urine Mean (absorbance) Standard Deviation Coefficient of Variation (%) makes this an ideal specimen for collection, transport and subsequent detection in early, as well as later, stages of the disease (9). The Binax Legionella Urinary Antigen EIA Kit uses microtiter 0.047 0.013 27.7 ELISA methodology for the detection of soluble antigen in urine from patients with Legionella pneumophila serogroup 1 infections. 0.187 0.036 19.5 IV. PRINCIPLES OF PROCEDURE
The microtiter wells contained in the kit are coated with polyclonal rabbit antibody specific for L. pneumophila serogroup 1 antigen. Patient urine is added to the rnicrotiter well and any L. XVII. REFERENCES
pneumophila serogroup 1 antigen present is captured by the antibody coated microtiter well (solid phase). Anti-Legionella HRP conjugate added at the same time as the patient specimen binds 1. Frazer, D.W., T.R. Tsai, W. Orenstin, WE. Parkin, H.J. Beecham, R.G Sharrar, J. Harris, G.F. Mallison, S.M. Martin, J.E. McDade, C.C. Shepard, P.S. Bracham, and the Field Investigation Team.
to L. pneumophila serogroup 1 antigen. After an incubation, the wells are decanted and washed to remove any unbound antigen and conjugate. A color developer is added to the wells and 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297: 1189-1197. incubated. The assay reaction is stopped using stop solution and the resultant absorbances of color are read on a microplate reader. Samples with absorbances greater than or equal to 3 times 2. Kohler, R.B. 1984. Antigen detection for the rapid diagnosis of Mycoplasma and Legionella pneumonia. Diagn. Microbiol. Infect. Dis. 4:475-595. the negative control are considered positive. 3. Reingold, A.L., B.M. Thomason, B.J. Brake, L. Thacker, H.W. Wilkinson, and J.N. Kuritsky. 1984. Legionella pneumonia in the United States: the distribution of serogroups and species caus- ing human illness. J. Infect. Dis. 149:819. V. MATERIALS PROVIDED
4. Berdal, B.P., C.E. Farshy, and J.C. Feeley. 1979. Detection of Legionella pneumophila antigen in urine by enzyme-linked immunospecific assay. J. Clin. Microbiol. 9:575-578. 5. Tilton, R.C. 1979. Legionnaires' disease antigen detected by enzyme-linked immunosorbent assay. Ann. Intern. Med. 90:697-698. Microtiter Wells - Each kit contains 96 Microtiter Wells, coated with purified rabbit anti-Legionella pneumophila serogroup 1 IgG. Ready for use. Store at 2-8°C. 6. Kohler, R.B., S.E. Zimmerman, E. Wilson, S.D. Allen, P.H. Edelstein, L.J. Wheat, and A. White. 1981. Rapid radioimmunoassay diagnosis of Legionnaires' Disease. Ann. Intern. Med. 94:601-605.
Wash Concentrate - One (1) vial with 40mL of 0.1 M Phosphate Buffered Saline, detergent and preservative. Dilute the contents to 400 mL with distilled or deionized water for use. Store at 2- 7. Bibb, W.F., P.M. Arnow, L. Thacker, and R.M. McKinney. 1984. Detection of soluble Legionella pneumophila antigens in serum and urine specimens by enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies. J. Clin. Microbiol. 20:478-482. Positive Control Urine - One (1) vial with 2 mL of human urine containing Legionella pneumophila serogroup 1 antigen and preservative. Ready for use. Store frozen at -10 to -20˚C in aliquots 8. Tang, P. W., and S. Toma. 1986. Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens. J. Clin. Microbiol. 24:556-558. or at 2-8°C as supplied. 9. Kohler, R.B., W.C. Winn, Jr., and L.J. Wheat. 1984. Onset and duration of urinary antigen excretion in Legionnaires' disease. J. Clin. Microbiol. 20:605-607. Negative Control Urine - One (1) vial with 2 mL of normal human urine and preservative. Ready for use. Store frozen at -10 to -20°C in aliquots or at 2-8°C as supplied. XVIII. ADDITIONAL BIBLIOGRAPHY
HRP Conjugate - One (1) vial with 15 mL of purified rabbit anti-Legionella pneumophila serogroup 1 IgG conjugated to horseradish peroxidase (HRP) in a Tris buffer with a protein stabiliz- Klaucke, D.N., R.L. Vogt, D. LaRue, L.E. Witherell, L.A. Orciari, K.C. Spitalny, R. Pelletier, W.B. Cherry and L.F. Novick. 1984. Legionnaires' Disease: the epidemiology of two outbreaks er. Contains less than 0.02% thimerosal as a preservative. Ready for use. Store at 2-8°C. in Burlington Vermont. Am. J. Epidemiol. 119:382-391. Color Developer - One (1) vial with 25mL of chromogenic substrate solution containing tetrarnethylbenzidine (TMB) and hydrogen peroxide. Ready for use. Store at 2-8°C. Sathapatayavongs, B., R.B. Kohler, L.J .Wheat, A. White, W. C. Winn Jr., JC. Girod, and P.H. Edelstein. 1982. Rapid diagnosis of Legionnaires' Disease by urinary antigen detection. Am.
J. Medicine.72:576-582. Stop Solution - One (1) vial with 10 mL of 1N H2S04. Store at 2-8°C. Kohler, R.B., M.L.V. French, L.J. Wheat, P.L. Meenhorst, W.C. Winn, Jr., and P.H. Edelstein. 1985. Cross-reactive urinary antigens among patients infected with Legionella pneumophilaserogroups 1 and 4 and the Leiden 1 strain. J. of Inf. Diseases. 152:1007-1011. Kohler, R.B., L.J Wheat. 1982. Rapid diagnosis of pneumonia due to Legionella pneumophila serogroup 1. J. of Inf. Diseases. 146:144. Safety Considerations Sathapatayavongs, B., R.B. Kohler, L.J. Wheat, A. White and W.C. Winn, Jr. 1983. Rapid diagnosis of Legionnaires' Disease by latex agglutination. Am. Rev. Respir. Dis. 127:559. HANDLE URINE SPECIMENS AND POSITIVE AND NEGATIVE CONTROL URINES AS IF CAPABLE OF TRANSMITTING AN INFECTIOUS AGENT. XIX. TECHNICAL ASSISTANCE
Control urines supplied in the kit have been autoclaved for a minimum of 15 minutes at 121°C, filtered through 0.2µm membrane filter and contain 0.8% boric acid as preservative. Althoughprocessed by these accepted methods of microorganism inactivation, it should not be assumed that this will provide full inactivation. For further information contact Technical Services at 1-800-323-3199 or 1-207- 772-3988. Do not pipet by mouth. Do not eat, smoke or drink in areas in which specimens or kit reagents are handled. Wear disposable gloves throughout the test procedure. Dispose of gloves in bio- XX. ORDERING
hazard waste. Thoroughly wash hands afterwards. Avoid contact with skin or mucous membranes. 851-000 Binax Legionella Urinary Antigen EIA 96 Test Kit Stop Solution contains sulfuric acid. Contact with skin or eyes may result in severe burns. Wear eye protection and impermeable gloves. If exposure occurs, wash with large quantities of water. Manufactured by: Color Developer contains N,N-Dimethylformamide as part of a mixture. May be harmful if swallowed, inhaled or absorbed through the skin.
217 Read Street Portland, Maine 04103 USA HRP Conjugate contains an ingredient listed under California Chapter 3, Safe Drinking Water and Toxic Enforcement Act of 1986, Section 12000, Chemicals Known to Cause Cancer or TEL: 207-772-3988 or 800-323-3199 Reproductive Toxicity. The listed ingredient is thimerosal, known to the State of California to be a reproductive toxicant. FAX: 207-761-2074 Do not use kit components beyond expiration date. The date is printed on individual component labels and on the kit box. Do not substitute reagents from other kit lots. Reagents are kit specific. Each kit lot has been optimized to detect Legionella pneumophila serogroup 1 antigen directly from clinical specimens.
Dilution and alteration of reagents may result in loss of sensitivity. Avoid microbial contamination of reagents. Microbial contamination may interfere with the sensitivity of the assay. XIII. QUALITY CONTROL
Do not interchange vial or bottle caps and stoppers; this will lead to cross contamination of reagents. The Positive and Negative Control Urine must be included with each run of patient specimens in order to calculate patient test results and to monitor test performance. These control samplesare ready to use as supplied, and must be tested in the same manner as patient specimens. Additionally, known positive and negative urine specimens may be run as controls. Incubation times or temperatures other than those specified may give erroneous results. The average absorbance of the Negative Control Urine should be less than or equal to 0.100. The average absorbance of the Positive Control Urine must be greater than or equal to three (3) Color developer is light sensitive. Store in amber bottle. If using reagent boats, dispense only a quantity appropriate for run size, approximately 2mL for each microtiter strip. times that of the Negative Control Urine. Store diluted Wash Solution in a clean container to avoid contamination. Do not discard unused diluted Wash Solution until all Microtiter Wells have been utilized or passed expiration date.
Results of each replicate of the duplicate wells from patient samples, Positive Control Urine and Negative Control Urine must correspond (both score as either positive or negative in the assay).
The wash step is critical. Insufficient washing can cause erroneous results. Use of an automated washing apparatus may require additional washes. If the negative control absorbance is over Otherwise, the test result is not valid and should be repeated. 0.100 absorbance units, increase the number of washes until the negative control absorbance is in the acceptable range (< 0.100 absorbance units). Do not report patient test results if controls do not meet above criteria for acceptability*. Cross contamination of patient samples could cause false results. * Quality Control References: Control urines, samples, and reagent solutions must be pipetted directly to the bottom of each well.
1. Federal Register, Vol. 57, No.40, February 28, 1992 (CLIA '88 Final Rule) VII. PREPARATION OF REAGENTS 2. "Draft FDA Guidance to Manufacturers of In Vitro Analytical Test Systems for Preparation of Premarket Submissions Implementing CLIA", December 17, 1992. All reagents must equilibrate to room temperature before use. 3. NCCLS Document C24-A, "Internal Quality Control Testing: Principles and Definitions", 1991. Wash Solution - Dilute the entire volume (40 mL) of the Wash Concentrate to 400 mL with distilled or deionized water. Mix well. Do not discard unused diluted Wash Solution until the kit lot XIV. CALCULATION OF RESULTS
expires or is used up. NOTE: Upon storage at 2-8°C, wash concentrate may form a crystalline precipitate. Before use, warm to room temperature and be sure all of the crystals have dissolved. The absorbance of the well A1 (substrate blank) should be subtracted from the absorbance of each well, to obtain a corrected absorbance for use in the calculation of patient results. Positive and Negative Control Urines - Control Urines which have been stored frozen must be thawed and mixed sufficiently before use. Calculation of Mean Absorbance All other reagents are supplied ready to use. Determine the mean absorbance of the duplicate Positive Control Urine, Negative Control Urine and patient urine by: VIII. STORAGE INSTRUCTIONS
MEAN Absorbance = Absorbance (1) + Absorbance (2) Positive and Negative Control urines may be stored at -10 to -20°C if stored in aliquots or at 2-8°C as supplied. All other kit components should be stored at 2-8°C. Calculation of RATIO to Negative Color developer is light sensitive and must be stored in an amber bottle at 2-8°C. Diluted Wash Solution may be stored at 2-30°C until the expiration of the specific kit lot in which it was a reagent. Determine the ratio of Positive Control Urine absorbance to Negative Control Urine absorbance and of patient urine absorbance to Negative Control Urine absorbance in the following manner: IX. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS
RATIO = MEAN Positive Control Urine or Patient Urine Absorbance Changes in the physical appearance of the reagents supplied may indicate instability or deterioration of these materials. Do not use reagents which are visibly turbid. MEAN Negative Control Absorbance Reproducible decrease in Positive Control Urine ratio results below 3.0 may indicate instability of reagents or coated microtiter wells A typical positive result might be 0.300 Absorbance = 7.5 RATIO 0.040 Absorbance X. SPECIMEN COLLECTION AND PROCESSING
Interpretation of Results Urine specimens should be collected in standard sterile containers, stored at room temperature or refrigerated (2-8°C), and assayed within 24 hours of collection. Alternatively, specimens maybe stored at 2-8°C for up to 14 days or at -10 to -20°C for longer periods before testing. Long term storage conditions have not been determined. Boric acid may be used as a preservative.
Urine samples that have a RATIO value greater than or equal to 3 are to be considered positive for the presence of Legionella pneumophila serogroup 1 antigen. Positive specimens may be Other preservatives have not been evaluated. retested to confirm results. Whenever possible, urine specimens should be shipped in leakproof containers at 2-8°C or frozen. Urine samples that have a RATIO value less than 3 are considered negative. A negative test result does not rule out the possibility of Legionella infection due to other serogroups or species of Urine specimens stored at -20 to 8°C which contain excess urates, phosphates or other dissolved salts may be found with salt crystals after storage. It is acceptable to warm these specimens to Legionella. 37˚C in order to facilitate dissolving these crystals before assay. Results Reporting Urine specimens exhibiting large particulates should be filtered before running in the assay, as this may indicate contamination of the specimen. RATIO Recommended XI. MATERIALS REQUIRED BUT NOT PROVIDED
≥ 3.0 Presumptive positive for the presence of L. pneumophila serogroup 1 antigen in urine, suggesting current or past infection. Pipettors and/or pipets that can accurately and precisely deliver 50, 100, 200, 250 µL volumes. Presumptive negative for L. pneumophila serogroup 1 antigen in urine, suggesting no recent or current infection. Legionnaires' disease cannot be ruled out since other serogroupsand species may also cause disease. Microtiter plate reader at 450 nm. Graduated cylinder to dilute Wash Concentrate to 400 mL. XV. LIMITATIONS OF THE TEST
Stock bottle to store diluted Wash Solution. This assay has been validated using urine samples only. Other samples (e.g., plasma, serum or body fluids) that may contain Legionella antigen have not been tested. Distilled or deionized water to dilute Wash Concentrate. The Legionella Urinary Antigen EIA test will not detect infections caused by other serogroups and L. micdadei or L. longbeachae; culture is recommended for suspected pneumonia to detect XII. ASSAY PROCEDURE
causative agents other than L. pneumophila serogroup 1 and to confirm infection. Reserve well A1 for substrate blank.
The diagnosis of Legionnaires' Disease cannot be based on clinical or radiological evidence alone. There is no single satisfactory laboratory test for Legionnaires' Disease. Culture results, serol- Pipet 100 µL of well-mixed Positive Control, Negative Control and patient specimens into the appropriately labelled duplicate wells.
ogy and antigen detection methods should all be used along with clinical findings for diagnosis. Pipet 100 µL of rabbit anti-Legionella HRP Conjugate into all previously pipetted wells, except A1.
Excretion of Legionella antigen in urine may vary depending on the individual patient and their underlying illness or treatment. Some individuals have been shown to excrete antigen for an Gently tap the plate to mix reagents within wells, being careful to avoid splashing and cross-contamination of wells.
extended period of time, so that a history of a recent respiratory illness compatible with Legionnaires' Disease should be sought. Early treatment with appropriate antibiotics may also decreaseantigen excretion in some individuals. Antigen excretion may begin as early as 3 days after onset of symptoms and persist for up to 1 year afterwards (9). Incubate at room temperature (20-25°C) for 2 hours.
Antigenuria may persist for prolonged periods and may therefore indicate previous infection rather than current infection. After incubation, decant or aspirate sample and conjugate mixture from wells. If decanting, blot strips on absorbent toweling.
Add 250 µL per well of Wash Solution. Decant or aspirate. Repeat wash step for a minimum of three washes (see Section VI, Precautions: Performance
Testing diuretic urine may affect the ability of the test to detect antigen. XVI. EXPECTED VALUES AND PERFORMANCE CHARACTERISTICS
Pipet 200 µL of Color Developer to all previously pipetted wells, including A1 (substrate blank).
1. Expected Values Incubate in the dark at room temperature (20-25°C) for 15 minutes. Covering the plate is sufficient.
10. After incubation, stop color development by pipetting 50 µL of stop solution to each previously pipetted well, including A1.
One hundred twenty-five randomly selected, presumed negative, uncharacterized urine specimens were tested using the Legionella Urinary Antigen EIA kit. One hundred twenty-one specimenstested negative for the presence of Legionella urinary antigen. The four presumed negative samples giving positive results were tested twice more. In one assay, two of the four samples tested 11. Gently tap plate to mix reagents. NOTE: All the wells should appear yellow, not green. Green color indicates insufficient mixing.
low positive and two tested negative, and in the second assay, all samples tested negative. 12. Read absorbances immediately at 450 nm on a microtiter plate reader, blanking against well A1 (substrate blank).
Distribution of Expected Values Ratio to Negative Control Number of Samples

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