PATHFINDER RSV 50 TESTS 79674

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1 PATHFINDER RSV 50 TESTS QUALITATIVE ENZYME IMMUNOASSAY (EIA) FOR THE DIRECT DETECTION OF HUMAN RESPIRATORY SYNCYTIAL VIRUS (RSV) ANTIGEN IN NASAL WASH AND NASAL ASPIRATE SPECIMENS IVD For In Vitro Diagnostic Use

2 TABLE OF CONTENTS 1- INTENDED USE SUMMARY AND EXPLANATION PRINCIPLES OF THE PROCEDURE PRODUCT INFORMATION WARNINGS AND PRECAUTIONS SPECIMEN COLLECTION AND PREPARATION PROCEDURE QUALITY CONTROL DETAILS OF CALIBRATION RESULTS LIMITATIONS OF THE PROCEDURE EXPECTED VALUES SPECIFIC PERFORMANCE CHARACTERISTICS REFERENCES

3 1- INTENDED USE The PATHFINDER TM Direct Antigen Detection System for RSV is a qualitative enzyme immunoassay (ELISA) for the direct detection of human respiratory syncytial virus (RSV) antigen in nasal wash and nasal aspirate specimens. 2- SUMMARY AND EXPLANATION Respiratory syncytial virus is a major cause of bronchiolitis and pneumonia in infants and children. The most severe disease occurs during the first year of life (1). Epidemics due to this virus occur nearly every year in the fall, winter, and spring (2,3). RSV causes significant respiratory illness in healthy adults. However, the disease in adults tends to be milder than in young children (4). Rapid diagnosis of RSV infection has gained in importance with the advent of effective therapy. It allows formulation of a prognosis, initiation of treatment, and early action to control spread of the disease. Traditional cell culture methods (5,6,7,8) can take up to 3 weeks, and are highly dependent on the quality of specimen transport (8,14). Immunologic tests offer advantages of rapidity, technical simplicity, and lower costs when compared with viral culture. Immunofluorescent (5,6,7,9,10) and enzyme immunoassay (EIA) (11,12,13) methods have been shown to be effective for the detection of RSV. The PATHFINDER RSV Direct Antigen Detection System uses polyclonal and monoclonal antibodies to detect RSV directly in patient specimens. This assay combines the specificity and reproducibility of monoclonal antibodies with the speed and efficiency of an EIA. Qualitative results are available within 90 minutes of beginning the assay. The PATHFINDER RSV Direct Antigen Detection System can detect nonviable (culture negative) RSV. This test offers a reliable alternative to the timeconsuming cell culture and subjective, labor-intensive fluorescent techniques. 3- PRINCIPLES OF THE PROCEDURE RSV antigens present in the specimen bind to polyclonal anti-rsv antibodies coated on the tube. HRP-conjugated monoclonal antibodies directed against RSV attach to antigen captured on the tube wall. A washing step removes unbound specimen and conjugate. Peroxidase substrate added to the tube will generate a blue color if HRP-conjugated antibodies are present. Visual or spectrophotometric evaluation of the specimen determines the presence or absence of RSV. A specimen displaying blue color or with an absorbance value greater than or equal to the upper Gray Zone Cut-off Value is positive for RSV antigen. A specimen displaying no blue color or with an absorbance value less than or equal to the lower Gray Zone Cut-off Value is negative for RSV antigen. 4- PRODUCT INFORMATION Store all kit reagents at +2-8 C. Avoid freezing reagents. Bring reagents to room temperature (23 ± 3 C) before each use. Promptly return them to +2-8 C after use. All reagents are stable to the expiration date stated on the label when stored as recommended. Do not substitute reagents from other manufacturers or between different kit lot numbers. Do not use reagents beyond expiration dates. All reagents are intended for in vitro diagnostic use. 3

4 R1 Labeling Reagents Presentation RSV antibody tubes Antibody Tubes : rabbit anti-rsv IgG-coated polystyrene tubes R2 Conjugate Conjugate : horseradish peroxidase conjugated murine monoclonal antibodies in buffered saline, ph 7.4, protein stabilizer. Preservative 0.01% Thimerosal R3 R4 R5 Sample diluent Sample diluent : Buffered saline, ph 7.5, with EDTA and surfactant Preservative 0.01% Thimerosal Positive control Positive control : inactivated RSV (Long) in buffered saline, ph 7.5, EDTA, and surfactant Preservative 0.01% Thimerosal Substrate Buffer Substrate Buffer : citrate buffer, ph 4.2, and hydrogen peroxide 1 x 50 tubes 1 x 5 ml 1 x 25 ml 1 x 3 ml 1 x 40 ml R6 Chromogen TMB Chromogen : tetramethylbenzidine dissolved in dilute HCI 1 x 2.5 ml R7 Assay TreatmentAssay Treatment buffer : buffered saline, ph 10.3, with 1 x 2.5 ml buffer EDTA. Preservative 0.01% Thimerosal RSV Antibody Tubes: Store the tubes in the bag. Do not open the tube bag until it reaches room temperature (23±3 C). When ready to use, remove tubes needed for the assay. Return all unused tubes in the bag and carefully close the bag. Once opened, the tubes, stored as recommended and in absence of microbial contamination, are stable for 3 months. Pathfinder TM RSV positive control : 1 x 3 ml NOTE: Do not substitute reagents from other manufacturers or use reagents beyond expiration date Physical and Chemical Indications of Instability A cloudy appearance of any liquid reagent may indicate microbial contamination. 5- WARNINGS AND PRECAUTIONS 1. For in vitro diagnostic use. 2. Patient samples and positive control may be routinely processed with minimum risk using the procedure described. However, handle these products as potentially infectious according to universal precautions and good clinical laboratory practices, regardless of their origin, treatment, or prior certification. Use an appropriate disinfectant for decontamination. Store and dispose of these materials and their containers in accordance with local regulations and guidelines. 3. Reagents containing animal sourced biologicals may be routinely processed with minimum risk. However, handle these products as potentially biohazardous according to universal precautions and good laboratory procedures. 4. Avoid skin and mucous membrane contact with Chromogen and sulfuric acid. If contact occurs, wash with water. Do not ingest. 5. Neutralize any liquid waste containing acid prior to decontamination. 4

5 6. This kit contains 0.01% thimerosal ( 0.005% mercury weight per volume). Mercury compounds may be considered reproductive toxicants and environmental polluants by government agencies at certain concentrations/quantities. Please handle appropriately and dispose of according to local, regional, and national regulations. 6- SPECIMEN COLLECTION AND PREPARATION Collect a minimum of 150 µl nasal wash or nasal aspirate using the routine collection and transport procedure for your laboratory. Nasal swabs are not considered as sensitive for detection of RSV (5). Avoid using collection containers with preservatives, metal ions, or detergents, all of which interfere with the assay. Do not use transport media containing sera. If possible, obtain the nasal secretions during the acute phase of the illness when the greatest amount of viral shedding occurs. As the illness progresses the viral shedding decreases, thus decreasing the amount of detectable virus. Store specimens for up to 24 hours at +2-8 C and protect from evaporation and contamination. For longer storage or shipment, freeze specimens at -70 C or colder. Repeated freezing and thawing will cause deterioration of the specimen. Ship specimens in compliance with federal transportation regulations for etiologic agents (42 CRF 72). Samples containing visibly high levels of blood should be avoided to ensure accuracy of results. 7- PROCEDURE A) Materials Provided R1. Anti-RSV Antibody Tubes R2. Anti-RSV HRP Conjugated Monoclonal Antibodies R3. Sample Diluent R4. Positive Control R5. Substrate Buffer R6. Chromogen R7. Assay Treatment Buffer B) Materials Required, But Not Provided 1. Deionized or distilled water for diluting sulfuric acid and washing tubes. 2. 1N Sulfuric Acid H 2 SO 4 to stop reaction prior to spectrophotometric analysis. Use an analytical reagent grade concentrated H 2 SO 4 to make the 1N acid. To prepare 500 ml 1N H 2 SO 4, add 14 ml concentrated H 2 SO 4 to 486 ml distilled or deionized water. Store in a tightly covered glass container at room temperature (23 ± 3 C). 3. Test tube racks capable of holding 12 x 75 mm tubes. 4. Pipettes with disposable tips capable of accurately delivering 100 µl, 300 µl, and 500 µl. 5. Disposable serological pipettes, 10 ml. 6. Disposable 12 x 75 mm glass test tubes. 7. Clean glass tube for preparing working substrate solution (see procedure step 9). 8. Plastic film. 9. Apparatus for washing the tubes which includes rinsing and aspirating devices and appropriate flasks and tubing for trapping the liquid waste (see Diagram 1). a) Rinsing devices: plastic wash bottle, b) Aspirating devices: Aspirator tip and vacuum pump. 10.Vortex mixer. 5

6 11.Spectrophotometer with variable wave lengths within the visible light range and a linear range of absorbance units for reading absorbance at 450 nm. 12.Sodium hypochlorite (bleach) for decontamination. 13.Timer capable of timing 15 and 60 minutes. DIAGRAM 1 Aspirating Equipment Aspiration Pipette Vacuum Trap Vacuum Pump Procedure Comments 1. Bring all reagents and specimens to room temperature (23 ± 3 C) before use. 2. Incubations at temperatures or times other than those specified may give erroneous results. 3. When adding reagents, avoid delays or interruptions. Do not allow the tubes to dry once the assay has been started. 4. CAUTION: Do not pipette by mouth. 5. Always pipette with the pipet tip near the bottom of the tube. Do not deliver reagents down the wall of the tube. Do not touch the pipet tip to the reaction solution. 6. Change pipet tips between samples. 7. Reusable glassware must be washed and thoroughly rinsed free of all detergents. Use deionized or distilled water. 8. All reagents have been optimized for detection of RSV. Dilution or adulteration of reagents may result in loss of sensitivity. C) PROCEDURE Mix all reagents well before use 1. Label one Antibody Tube for the negative control and one Antibody Tube for the positive control. Label one antibody tube for each patient specimen NOTE : A positive and negative control (Sample Diluent) must be run with each assay. 2. Add 1 drop Assay Treatment Buffer to each tube. 3. Add 100 µl HRP-Conjugated Monoclonal Antibody to each tube. 4. Add 300 µl Sample Diluent to the negative control tube; add 300 µl Positive Control to the positive control tube. 5. Add each patient specimen to the appropriately labeled tube according to the following instructions: If the specimen is received in SERUM-FREE transport medium, vortex thoroughly and add 300 µl to the appropriate tube. If the specimen is not in transport medium, dilute with an equal volume of Pathfinder TM Sample Diluent. Add 300 µl diluted specimen to the appropriate tube. Dilution of the specimen more than recommended may result in loss of sensitivity. 6

7 6. Mix tubes gently and incubate at room temperature (23 ± 3 C) for 60 ± 10 minutes. 7. Carefully aspirate samples from tube. 8. Wash each tube 6 times with 2 4 ml of deionized or distilled water per wash. Remove excess moisture from the tubes after final wash by inverting the tubes and blotting the rim on paper towels or by thorough aspiration of the tubes. Inadequate washing or washing with impure water may cause erroneous results. 9. Prepare the working color reagent. CAUTION: Do not allow any metal or oxidizing agent to contact chromogen or working color reagent. A)Required amount of Substrate Buffer = 0.5 ml Buffer per assay tube. Required amount of Chromogen = 1 drop Chromogen per ml of Substrate Buffer. Example: For 6 assay tubes The required amount of Substrate Buffer = 3.0 ml ; The required amount of Chromogen = 3 drops. Add the required amount of Substrate Buffer and Chromogen to a clean glass or plastic tube. B) Cover the tube with plastic film and mix contents by inverting tube several times. Use substrate solution within 30 minutes. Minimize exposure to light. Spontaneous blue color generation may be indicative of reagent contamination. The working color reagent must remain colorless before use. 10.Add 0.5 ml working color reagent to each tube. Mix the tubes gently, and incubate at room temperature (23±3 C) for 15 minutes, minimizing exposure to strong light. Do not allow color reaction to exceed 15 minutes. 11.Read the tubes visually or with a spectrophotometer to determine the test results. Visual Determination Do not stop reaction by adding acid. At the end of the incubation period, the negative control tube should be colorless. The positive control should be deep blue. Specimens displaying blue color are positive for RSV antigen. Specimens displaying no blue color are negative for RSV antigen. Note : Visual results for weakly positive specimens should be processed and read per the following Spectrophotometric Determination method. Spectrophotometric Determination A) Add 1 ml 1N H 2 SO 4 to each tube in the same order and approximately at the same time interval that the working color reagent was added to each tube. Vortex the tubes. Remove air bubbles before reading absorbance. After the addition of acid to each tube, the final reaction material is stable for up to 8 hours if protected from light. B) Blank the spectrophotometer with deionized or distilled water at 450 nm. C) Read the absorbance of the negative control (Sample Diluent), positive control and specimen tubes at 450 nm. To distinguish positive samples from negative samples, calculate the Cutoff Value and the Gray Zone, as described in the Results Section. A value greater than or equal to 0.06 absorbance units for the negative control (sample diluent) indicates possible reagent contamination or inadequate washing. 7

8 8- QUALITY CONTROL A positive and negative control must be run with each assay. Negative Control (Sample Diluent) Visual : the Negative Control should be colorless. Spectrophotometric : the absorbance value should be less than 0.06 units. Positive Control Visual : the Positive Control should be deep blue. Spectrophotometric : the absorbance value should be greater than 0.6 units. If controls do not react as expected, results are invalid. 9- DETAILS OF CALIBRATION Spectophotometric Method Only 1. Calculate the Cut-off Value by adding to the negative control (Sample Diluent) absorbance unit value. Example: Negative control value Cut-off Value = Calculate the Gray Zone. The Gray Zone equals ± 10% of the Cut-off Value (or 0.90 to 1.10 times the Cut-off Value). Example: Cut-off Value = Gray Zone = to Cut-off Value x (0.90) = (0.092) x (0.90) = Cut-off Value x (1.10) = (0.092) x (1.10) = RESULTS a) Visual Specimens displaying blue color are positive for RSV antigen. Specimens displaying no blue color are negative for RSV antigen. The negative control should be colorless. The positive control should be deep blue. b) Spectophotometric Specimens with absorbance values less than or equal to the lower Gray Zone Cut-off Value are negative for RSV antigen. A specimen with an absorbance value within the Gray Zone is questionable. The laboratory should repeat the test. If the repeated result, using either the same specimen or a new specimen, shows an absorbance value within the Gray Zone, the results should be reported as indeterminant. Specimens with absorbance values greater than or equal to the upper Gray Zone Cut-off Value are positive for RSV antigen. Strongly positive specimens may form dark particles of precipitate in the tube. 11- LIMITATIONS OF THE PROCEDURE 1. Infection with other respiratory pathogens may be present simultaneously with RSV infection, therefore, additional testing may be indicated in parallel with the PATHFINDER RSV. Refer to the Specific Performance Characteristics section for the bacterial and viral crossreactivity studies. 8

9 2. A negative test result does not exclude the possibility of RSV infection. Small quantities of virus, obtaining sample too late in infection, or inadequate and improper sampling techniques may cause a false negative result. 3. This test can detect nonviable (culture negative) RSV. 4. Results of this test should be interpreted in conjunction with information available from the clinical evaluation of the patient and other diagnostic procedures. 5. The results of this test are qualitative and should be considered as either positive or negative for the presence of RSV. 12- EXPECTED VALUES Expected values will vary depending on patient population tested, geographic location, and time of year. Infection with respiratory syncytial virus can occur in all age groups. Disease is most common in infants 6 weeks to 6 months of age with a peak at 2 months (1). The most common clinical diagnoses in these infants include bronchitis, bronchiolitis and pneumonia. RSV infection in adults is common, but tends to be less severe than in infants. Symptoms can include rhinorrhea, pharyngitis, cough, headache, fatigue and fever (4). Elderly patients with RSV infection may develop bronchopneumonia, which may be severe or fatal (17). 13- SPECIFIC PERFORMANCE CHARACTERISTICS Accuracy Two independent investigators tested 241 respiratory specimens from patients ranging in age from newborn to 93 years of age for respiratory syncytial virus by culture, immunofluorescence (IF), PATHFINDER RSV, and Competitor A s RSV EIA. The independent study sites were university hospitals in the East and West of the USA. All of the specimens run by EIA were evaluated visually and spectrophotometrically. Specimens were collected for this study during an epidemic outbreak of RSV. The specimens were cultured for respiratory viruses and smears were made for immunofluorescent staining. The remainder of the specimen was frozen at 70 C for evaluation by EIA at a later date. In one center, the immunofluorescence was performed on the smears at the time the specimen was received. In the other, the smears were frozen at 70 C and were stained at the time of the EIA study. Specimens which were EIA positive and culture negative were confirmed by the IF results or by blocking assay (15,16). The blocking assay was performed by incubating the specimen for 1 hour at room temperature (23 ± 3 C) with and without polyclonal antiserum to RSV. The antiserum would inhibit the reaction of a true positive specimen. The EIA was then performed on these specimens. A specimen was considered blocked and thus a true positive if the signal in the tube with blocking antibody was decreased by at least 50% compared to the tube without blocking antibody. A specimen was considered a true positive if it was positive by culture or could be confirmed as positive by two other methods: EIA and blocking or EIA and IF. A specimen was considered a true negative if it was negative by culture and a positive EIA signal could not be confirmed by either IF or blocking. Note: Low amounts of RSV antigen occasionally show a discrepancy between the visual and spectrophotometric results. Since spectrophotometry is an objective method, it is slightly more accurate than a visual determination. (See Tables 1 and 2). 9

10 Table 1 Comparison of PATHFINDER RSV with Culture. SPECTROPHOTOMETRIC PATHFINDER RSV CONFIRMED Positive Negative Positive Negative VISUAL PATHFINDER RSV CONFIRMED Positive Negative Positive Negative Spectrophotometric Overall agreement = (TP* + TN) / Total number of samples = ( ) / 239** = 96.2 % Sensitivity = TP / (TP + FN) = 119 / ( ) = 93.7 % Specificity = TN / (TN + FP) = 111 / ( ) = 99.1 % * TP = true positive, TN = true negative, FP = false positive, FN = false negative. Visual Overall agreement = (TP* + TN) / Total number of samples) = ( ) / 241 = 93.8 % Sensitivity = TP / (TP + FN) = 118 / ( ) = 91.5 % Specificity = TN / (TN + FP) = 108 / ( ) = 96.4 % * TP = true positive, TN = true negative, FP = false positive, FN = false negative. **Two specimens were indeterminate by spectrophotometric evaluation and are not included in this data. Table 2 Comparison of PATHFINDER RSV with Competitor A s RSV EIA SPECTROPHOTOMETRIC PATHFINDER RSV COMPETITOR A EIA Positive Negative Positive Negative VISUAL PATHFINDER RSV COMPETITOR A EIA Positive Negative Positive Negative

11 Spectrophotometric Of the 238* specimens tested, 94.1% agreement (224/238) was observed between PATHFINDER and Competitor A s EIA. After all discrepant results were resolved, PATHFINDER RSV showed a sensitivity of 94.5% and a specificity of 99.1% by spectrophotometric interpretation. * Three samples were indeterminate by PATHFINDER spectrophotometric evaluation and are not included in this data. One sample was indeterminate by Competitor A s evaluation and is not included in this data. Visual Of the 241 specimens tested visually, 91.3% agreement (220/241) was observed between PATHFINDER and Competitor A s EIA. After all discrepant results were resolved, PATHFINDER RSV showed a sensitivity of 92.9% and a specificity of 96.5% by visual interpretation. PRECISION Intra-assay Variability 15 replicates of the positive control, 15 replicates of a 1:20 dilution of the positive control, and 20 replicates of the negative control were tested in one assay to determine the intra-assay reproducibility. The mean of the optical absorbances was calculated to determine the intra-assay variability. Table 3 Sample Mean (OD 450 nm) SD % C.V. Positive Low Positive Negative Inter-assay Variability: The positive control, a 1:20 dilution of the positive control, and the negative control were assayed in 9 different assays with 10 replicates per assay from one kit lot to determine interassay reproducibility. The mean of the optical absorbances from each assay was calculated to determine the inter-assay variability. Table 4 Sample Mean (OD 450 nm) SD % C.V. Positive Low Positive Negative CROSS-REACTIVITY Study 1 PATHFINDER RSV was tested with organisms that may cause similar clinical presentations or that may be normal respiratory flora. The assay was performed as directed in the package insert substituting the organisms for the patient sample. Each assay was run with 300 µl suspensions containing 0.1 optical density unit (at 620 nm) of each organism in buffered saline except Mycoplasma pneumoniae, which was tested at a concentration of 5 mg wet cell weight per µl, and Chlamydia trachomatis, which was tested at a concentration of 1x10 8 elementary bodies per µl. In addition, the organisms were also tested for interference in the 11

12 detection of RSV by combining 12 µl of Positive Control (Long) with 300 µl of each suspension. The results in Table 5 show that no cross-reactivity or interference occurred with the organisms. This is evidenced by no positive absorbance in the assay by the bacteria alone and the absence of a greater than 50% drop in signal by the bacteria and RSV compared to the Positive Control. Table 5 Microorganisms Tested For Cross-Reactivity Microorganism Strain Absorbance (organism alone) Absorbance (organism+rsv) Streptococcus Group A Staphylococcus aureus, Cowan Staphylococcus aureus Staphylococcus epidermis Escherichia coli Candida albicans Mycoplasma pneumoniae P Chlamydia trachomatis L Negative Control (Sample Diluent) Positive Control Long Study 2 The viruses listed in Table 6 were tested for cross-reactivity in the PATHFINDER RSV. This study used 300 µl suspensions containing 1 x 10 5 TCID 50 per ml in Sample Diluent for each of the viruses except the influenza viruses and Herpes Simplex Type 2. Influenza A was tested at 1 x 10 8 plaque forming units per ml and Influenza B was tested at 1 x 10 7 plaque forming units per ml. HSV Type 2 was tested at 6.8 x 10 7 plaque forming units per ml. Viral interference in the detection of RSV was also tested by combining 12 µl of Positive Control (Long) with 300 µl of each suspension. No crossreactivity or interference occurred in the PATHFINDER RSV with these viruses. This is evidenced by no positive absorbance in the assay by the viruses alone and the absence of a greater than 50% drop in signal by the viruses and RSV compared to the Positive Control. Table 6 Viruses Tested For Cross-Reactivity Virus Virus Strain Absorbance (Virus alone) Absorbance (Virus+RSV) Rhinovirus Type 2 Type 6 Type 8 Type 9 Type 15 Type 19 Type 30 Type 59 HGP Thompson MRH F 611CV

13 Coxsackie Virus Type A-9 Type B-3 Type B-4 Echovirus Type 8 Type 11 Type 19 Parainfluenza Type 1 Type 2 Type 3 Adenovirus Type 2 Type 4 Virus Strain PB Bozek Nancy JVB Berschoten Bryson Gregory Burke C35 Greer C243 Adenoid6 Rl67 Absorbance (Virus alone) Absorbance (Virus+RSV) Cytomegalovirus AD Herpes Simplex Type 1 Type 2 Maclntyre MS Varicella Zoster OKA Influenza A WSN Influenza B B/Lee/ Negative Control N/A (Sample Diluent) Positive Control Long REFERENCES 1. PARROTT, R.H., KIM, H.W., ARROBIO, J.O., HODES, D.S., MURPHY, B.R., BRANDT, C.D., CAMARGO, E., CHANOCK, R.M Epidemiology of respiratory syncytial virus infection in Washington D.C. II. Infection and disease with respect to age, immunologic status, race, and sex. Am J Epidemiol KIM, H.W., ARROBIO, J.O., BRANDT, C.D., JEFFRIES, B.C., PYLES, G., REID, J.L., CHANOCK, R.M., PARROTT, R.H Epidemiology of respiratory syncytial virus infection in Washington D.C. I. Importance of the Virus in Different Respiratory Tract Disease Syndromes and Temporal Distribution of Infection. Am J Epidemiol. 98, MUFSON, M.A., LEVINE, H.D., WASIT, R.E., MOCEGA-GONZALEZ, H.E., KRAUSE, H.E Epidemiology of respiratory syncytial virus infection among infants and children in Chicago. Am J Epidemiol. 98, HALL, W.J., HALL, C.B., SPEERS, D.M Respiratory syncytial virus infections in adults: clinical, virologic, and serial pulmonary function studies. Ann Int Med. 88, MCLNTOSH, K., CLARK, J.C Parainfluenza and respiratory syncytial viruses. In Manual of Clinical Microbiology Fourth Edition, , Edited by Lennette E.H., Balons A., Hauser W.J., Shadony H.J. Washington, D.C. American Society for Microbiology, 13

14 6. LAUER, B.A Comparison of virus culturing and immunofluorescence for rapid detection of respiratory syncytial virus in nasopharyngeal secretions: sensitivity and specificity. J Clin Microbiol. 16, MINNICH, L., RAY, C.G Comparison of direct immunofluorescent staining of clinical specimens for respiratory virus antigens with conventional isolation techniques. J Clin Microbiol. 12, HALL, C.B., DOUGLAS JR., R.G Clinically useful method for the isolation of respiratory syncytial virus. J Infec Dis. 131, BELL, D.M., WALSH, E.E., HRUSKA, J.F., SCHNABEL, K.C., HALL, C.B Rapid detection of respiratory syncytial virus with a monoclonal antibody. J Clin Microbiol. 17, KAO, C., MCLNTOSH, K., FERNIE, B., TALIS, A., PIERIK, L., ANDERSON, L Monoclonal antibodies for the rapid diagnosis of respiratory syncytial virus infection by immunofluorescence. Diag Microbiol Infect Diseas. 2, HENDRY, R.M., FERNIE, B.F., ANDERSON, L.J., GODFREY, E., MCLNTOSH, K Monoclonal capture antibody ELISA for respiratory syncytial virus: detection of individual viral antigens and determination of monoclonal antibody specificities. J Immunol Methods. 77, HENDRY, R.M., MCLNTOSH, K Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: development and description. J Clin Microbiol. 16, SARKKINEN, H.K., HALONEN, P.E., ARSTILA, P.P., SALMI, A.A Detection of respiratory syncytial, parainfluenza type 2, and adenovirus antigens by radioimmunoassay and enzyme immunoassay on nasopharyngeal specimens from children with acute respiratory disease. J Clin Microbiol. 13, HAMBLING, M.H Survival of the respiratory syncytial virus during storage under various conditions. Brit J Exp Pathol. 45, HORNSLETH, A., BRENOE, E., FRIIS, B., KNUDSEN, F.U., ULDALL, P Detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay. J Clin Microbiol. 14, MCLNTOSH, K., HENDRY, R.M., FAHNESTOCK, M.L., PIERIK, L.T Enzyme-linked immunosorbent assay for detection of respiratory syncytial virus infection: application to clinical samples. J Clin Microbiol. 16, GARVIE, D.C., GRAY, J Outbreak of respiratory syncytial virus infection in the elderly. Brit Med J. 281, BIO-RAD warrants these products to perform as described in the labeling and literature supplied. BIO-RAD disclaims any implied warranty of merchantability or fitness for any other purpose. In no event shall BIO-RAD be liable for any consequential damages arising out of the aforesaid express warranty. 14

15 PATHFINDER RSV 50 TESTS DETECTION DIRECTE DES ANTIGENES DU VIRUS RESPIRATOIRE SYNCYTIAL (RSV) DANS LES LIQUIDES DE LAVAGE OU D ASPIRATION NASALE PAR METHODE IMMUNOENZYMATIQUE IVD

16 TABLE DES MATIERES 1- OBJECTIF DU TEST RESUME ET EXPLICATIONS PRINCIPE DE LA METHODE INFORMATIONS SUR LE PRODUIT MISES EN GARDE ET PRECAUTIONS D EMPLOI PRELEVEMENT ET PREPARATION DE L ECHANTILLON PROCEDURE CONTROLE QUALITE DETAILS DE L ETALONNAGE RÉSULTATS LIMITES DE LA PROCEDURE VALEURS ATTENDUES CARACTERISTIQUES DE PERFORMANCE SPECIFIQUES REFERENCES

17 1- OBJECTIF DU TEST PATHFINDER TM RSV est un test immunoenzymatique (ELISA) qualitatif pour la détection directe du virus respiratoire syncytial (RSV) dans les liquides de lavage ou d aspiration nasale. 2- RÉSUMÉ ET EXPLICATIONS Le virus respiratoire syncytial est une des causes principales de bronchiolite et de pneumonie chez le nourrisson et l enfant. L affection est la plus sévère au cours de la première année (1). Les épidémies dues à ce virus surviennent pratiquement chaque année en automne, hiver et printemps (2,3). Le RSV est responsable d une affection respiratoire importante chez l adulte sain. Cependant, l affection chez l adulte tend à être moins forte que chez le jeune enfant (4). Un diagnostic rapide d une infection à RSV a d autant plus d importance aujourd hui que l on a mis au point un traitement efficace. Il permet de poser un diagnostic, de débuter le traitement et de contrôler précocement la propagation de l affection. Les méthodes de culture cellulaire traditionnelles (5, 6, 7, 8) peuvent prendre jusqu à 3 semaines et dépendent fortement de la qualité du transport de l échantillon (8, 14). Les tests immunologiques ont l avantage d être rapides, techniquement simples et d un coût moins élevé que les cultures à isolement de virus. Les méthodes d immunofluorescence (5, 6, 7, 9, 10) et de dosage immunoenzymatique (EIA) (11, 12, 13) ont montré leur efficacité dans la détection du RSV. Le PATHFINDER TM RSV utilise des anticorps polyclonaux et monoclonaux pour la détection du RSV directement dans des échantillons de patients. Cette méthode de dosage allie la spécificité et la reproductibilité des anticorps monoclonaux à la rapidité et l efficacité d un EIA. Des résultats qualitatifs sont disponibles en 90 minutes après le début du dosage. Le PATHFINDER TM RSV peut détecter le RSV non viable (culture négative). Cette méthode d analyse représente une alternative fiable par rapport aux cultures cellulaires qui nécessitent beaucoup de temps et aux techniques de fluorescences subjectives qui demandent beaucoup de travail. 3- PRINCIPE DE LA MÉTHODE Les antigènes RSV présents dans l échantillon se fixent aux anticorps polyclonaux anti-rsv capté sur le fond du tube. Les anticorps monoclonaux conjugués à la peroxydase de raifort (HRP) dirigés contre le RSV se fixent à l antigène lui-même fixé sur la paroi du tube. Une étape de lavage permet l élimination de l échantillon et du conjugué non fixés. Le substrat de peroxydase ajouté au tube entraînera une coloration bleue si des anticorps conjugués à la HRP sont présents. Un examen visuel par spectrophotométrie de l échantillon détermine la présence ou l absence de RSV. Un échantillon présentant une coloration bleue ou dont la valeur d absorbance est supérieure ou égale à la Valeur Seuil supérieure de la Zone d Incertitude est positif en antigène RSV. Un échantillon ne montrant aucune coloration bleue ou dont la valeur d absorbance est inférieure ou égale à la Valeur Seuil inférieure de la Zone d Incertitude est négatif en antigène RSV. 4- INFORMATIONS SUR LE PRODUIT Conserver tous les réactifs du kit à +2-8 C. Eviter de congeler les réactifs. Laisser les réactifs revenir à température ambiante (23 ± 3 C) avant chaque utilisation. Après utilisation, les replacer immédiatement à +2-8 C. Tous les réactifs sont stables jusqu à la date de péremption telle que spécifiée sur l étiquette du réactif s ils sont conservés selon les recommandations du fabricant. Ne pas substituer les réactifs par des réactifs d autres fabricants ou provenant de kits portant un numéro de lot différent. Ne pas utiliser les réactifs après leur date de péremption. Tous les réactifs sont destinés à être utilisés dans le cadre d un diagnostic in vitro. 17

18 R1 Labeling Reagents Presentation RSV antibody tubes Tubes d anticorps : Tubes en polystyrène sensibilisés par des IgG de lapin anti-rsv R2 Conjugate Conjugué : Anticorps monoclonaux de souris anti-rsv-hrp conjugués à la peroxydase de Raifort en tampon salin à ph 7,4 avec stabilisateur de protéine Conservateur 0,01% Thimerosal R3 R4 R5 R6 R7 Sample diluent Positive control Substrate Buffer Chromogen TMB Diluant pour échantillon : Tampon salin, ph 7,5, EDTA et surfactant. Conservateur Thimérosal 0,01% Contrôle Positif : RSV inactivé (Long) dans un tampon salin, ph 7,5, EDTA et surfactant Conservateur Thimerosal 0,01% Tampon Substrat : Tampon citrate, ph 4,2 et peroxyde hydrogène Chromogène : tétra-méthyl-benzidine dissout dans du HCI dilué Assay Treatment Tampon de Traitement de l Echantillon : salin, ph 10,3 et buffer EDTA. Conservateur Thimerosal 0,01% 1 x 50 tubes 1 x 5 ml 1 x 25 ml 1 x 3 ml 1 x 40 ml 1 x 2,5 ml 1 x 2,5 ml Tubes d Anticorps RSV : Conserver les tubes dans le sachet. Ne pas ouvrir le sachet contenant les tubes avant qu il n atteigne une température ambiante (23 ± 3 C). Une fois que les tubes sont prêts pour l utilisation, retirer les tubes nécessaires pour le dosage. Replacer tous les tubes inutilisés dans le sachet avec de refermer celui-ci soigneusement. Une fois ouverts, les tubes conservés tel qu il est recommandé et en l absence de contamination microbienne sont stables pendant 3 mois. Pathfinder TM RSV positive control : 1 x 3 ml NOTE: : Ne pas substituer les réactifs par des réactifs d autres fabricants. Ne pas utiliser les réactifs après leur date de péremption. Signes Physiques et Chimiques d Instabilité Un trouble chez un quelconque réactif liquide peut être le signe d une contamination microbienne. 5- MISES EN GARDE ET PRÉCAUTIONS D EMPLOI 1. Destiné à l utilisation diagnostique in vitro. 2. Les échantillons de patients et le contrôle positif peuvent être traités de manière routinière et avec un risque minimum si la procédure décrite est suivie. Néanmoins, il est nécessaire de manipuler ces produits comme des produits potentiellement infectieux selon les précautions universelles et les bonnes pratiques de laboratoire, quelle que soit leur origine, leur traitement ou une détermination antérieure. Utiliser un désinfectant approprié pour la décontamination. Conserver et éliminer ces produits et leurs récipients selon les directives et réglementation en vigueur. 3. Les réactifs contenant des agents biologiques de source animale peuvent être traités de manière routinière et avec un risque minimum. Néanmoins, il est nécessaire de manipuler ces produits comme des produits potentiellement bio-dangereux selon les précautions universelles et les bonnes pratiques de laboratoire. 4. Eviter le contact entre la peau et les muqueuses et le Chromogène et l acide sulfurique. En cas de contact, rincer à l eau. Ne pas ingérer. 5. Neutraliser tout résidu liquide contenant de l acide avant de procéder à la décontamination. 18

19 6. Ce kit contient du Thimerosal 0.01 % ( % de mercure poids par volume). Les composés de mercure peuvent être considérés à certaines concentrations/quantités comme des substances toxiques pour la reproduction et comme polluants de l environnement par les agences gouvernementales. Veuillez les manipuler de manière appropriée et les éliminer selon la réglementation locale, régionale et nationale. 6- PRÉLÈVEMENT ET PRÉPARATION DE L ÉCHANTILLON Prélever un minimum de 150 µl d échantillon nasal par lavage ou aspiration suivant les méthodes de prélèvement et de transport de routine de votre laboratoire. Les prélèvements nasaux à l aide d un coton-tige ne sont pas considérés comme assez sensibles pour la détection du RSV (5). Eviter l utilisation de récipients pour échantillons contenant des conservateurs, des ions métalliques ou des détergents, ceux-ci pouvant interagir avec le dosage. Ne pas utiliser de milieux de transport contenant des sérums. Si possible, obtenir les sécrétions nasales au cours d une phase aiguë de l affection lorsque l excrétion du virus est à son maximum. Plus l affection progresse, plus l excrétion du virus diminue, ce qui diminue également la quantité de virus détectable. Conserver les échantillons jusqu à 24 heures à +2-8 C et les garder à l abri de l évaporation et de la contamination. Pour une conservation plus longue ou un envoi, congeler les échantillons à 70 C ou à une température inférieure. Des congélations-décongélations répétées peuvent détériorer l échantillon. Transporter les échantillons selon la réglementation en vigueur dans votre pays pour le transport d agents étiologiques. Les échantillons contenant des quantités visiblement élevées de sang doivent être évités afin de s assurer de l exactitude des résultats. 7- PROCÉDURE A) MATÉRIEL FOURNI R1. Tubes d Anticorps Anti-RSV R2. Anticorps Monoclonaux anti-rsv conjugués à la peroxydase de raifort R3. Diluant pour Echantillon R4. Contrôle Positif R5. Tampon Substrat R6. Chromogène R7. Tampon de Traitement de l Echantillon B) MATÉRIEL NÉCESSAIRE MAIS NON FOURNI 1. Eau désionisée ou distillée pour dilution de l acide sulfurique et lavage des tubes. 2. Acide Sulfurique H 2 SO 4 1N pour arrêter la réaction avant de procéder à l analyse par spectrophotométrie. Utiliser un réactif analytique H 2 SO 4 concentré de qualité supérieure pour obtenir l acide 1 N. Pour la préparation de 500 ml d H 2 SO 4 1 N, ajouter 14 ml de H 2 SO 4 concentré à 486 ml d eau distillée ou désionisée. Conserver à température ambiante (23 ± 3 C) dans un récipient en verre recouvert hermétiquement. 3. Des supports pour tubes à essai capables de tenir 12 tubes de 75 mm. 4. Des pipettes avec embouts jetables capables de distribuer de manière précise 100 µl, 300 µl, et 500 µl. 5. Des pipettes sérologiques jetables de 10 ml tubes à essai en verre jetables de 75mm 7. Tube en verre propre pour la préparation de la solution substrat de travail (voir étape n 9 de la procédure). 19

20 8. Film plastique. 9. Système pour le lavage des tubes comprenant des dispositifs de rinçage et d aspiration, ainsi que des ballons et système de tube adéquats pour la récupération des résidus liquides (voir Diagramme 1). a) Dispositifs de rinçage : bouteille de lavage en plastique, b) Dispositifs d aspiration : embout d aspiration et pompe à vide. 10.Agitateur Vortex 11.Spectrophotomètre avec longueurs d onde variables au sein d une amplitude lumineuse visible et d une amplitude linéaire de 0-2,0 unités d absorbance pour permettre une lecture de l absorbance à 450 nm. 12.Hypochlorite de sodium (agent désinfectant) pour la décontamination. 13.Minuteur capable de chronométrer 15 et 60 minutes. DIAGRAMME 1 Equipement d Aspiration Pipette d aspiration Piège à vide Pompe à vide Commentaires sur la méthode 1. Avant utilisation, laisser tous les réactifs et échantillons revenir à température ambiante (23 ± 3 C). 2. Des incubations à des températures ou pendant des durées différentes de celles spécifiées peuvent engendrer des résultats erronés. 3. Lors de l addition des réactifs, éviter tout retard ou interruption. Ne pas laisser les tubes sécher à l air une fois que le dosage a débuté. 4. ATTENTION : Ne pas pipeter à la bouche. 5. Toujours pipeter à l aide de l embout de la pipette et à proximité du fond du tube. Ne pas pipeter le long de la paroi du tube. Ne pas mettre l embout de la pipette en contact avec la solution de réaction. 6. Changer les embouts de pipettes entre chaque échantillon. 7. Les instruments en verre réutilisables doivent être lavés et rincés abondamment sans aucun détergent. Utiliser de l eau désionisée ou distillée. 8. Tous les réactifs ont été optimisés pour procéder à l identification du RSV. La dilution ou la dénaturation des réactifs peut entraîner une perte de sensibilité. C) PROCÉDURE Bien mélanger tous les réactifs avant utilisation. 1. Etiqueter un Tube d anticorps pour le contrôle négatif et un autre pour le contrôle positif. Etiqueter un tube d anticorps pour chaque échantillon de patient. NOTE : Un contrôle positif et un contrôle négatif (Diluant pour Echantillon) doivent être conduits pour chacun des dosages. 20

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