R.S.V.

KGST200
DIMDI Reg.-Nr.: DE/CA22/1116-131-IVD

Rapid Immunochromatographic Test for the detection of Respiratory Syncytial Virus

I. PRINCIPLE
Respiratory syncytial virus (RSV) is the most important cause of pneumonia and bronchiolitis in infants and small children (1,2). Like the other respiratory viruses, RSV causes a range of respiratory illness, the most common being a cold with profuse rhinorrhea.
Between 1 and 2% of infected infants require hospitalisation, the most severe disease occurring in the first year of life and in those with underlying cardiopulmonary disease.
In normal infants and children, the virus is shed for 2 to 3 weeks overall or 1 or 2 weeks after the children appear in the hospital (3). Because of its high infectivity and because hospital staff as well as patients are susceptible, RSV has emerged as the most frequent cause of nosocomial infections on pediatric wards.
The studies of Hall and Taber and their colleagues established that RSV bronchiolitis and pneumonia could be successfully treated by administration of ribavirin aerosol (4,5). This finding has made accurate, rapid identification of cases through laboratory methods an important part of the care of children in hospitals.
Contrary to the classical methods (Immunofluorescence and ELISA) which are laborious and time consuming, Keul-o-test R.S.V. is a fast, simple and highly sensitive test for the rapid and reliable detection of RSV in the respiratory tract.
The method employs a unique combination of monoclonal dye conjugate and polyclonal solid phase antibodies to selectively identify RSV with a high degree of sensitivity and specificity.
After collection and treatment, the sample is added to the sample well of the reaction device.
As the test sample flows through the absorbent device, the labelled antibody-dye conjugate binds to the RSV antigen (when present in the sample) forming an antibody antigen complex. This complex binds to the polyclonal antibody in the positive reaction window producing a rose-pink colored band.
In the absence of RSV, there is no line in the positive reaction zone. The reaction mixture continues flowing through the absorbent device, past the positive reaction zone and control zone.
Unbound conjugate binds to the reagent in the control window producing a rose-pink colored band demonstrating that the reagents are functioning correctly.

II. Keul-o-test R.S.V. COMPONENTS
Each kit contains everything needed to perform the tests.

• Keul-o-test R.S.V. devices
• Filter-rubes (with cap)
• Swabs
• Dropper-bottles of 11 ml extraction solution
• Instructions leaflet
• Positive control (optional): A freeze-dried preparation derived from in-vitro culture is optionally available as a positive control (1x 0.5 ml). This solution is reconstituted with 0.5 ml of extraction solution and produces an assay result equivalent to that produced by borderline positive specimens (i.e. light pink color) and should be kept at 2-8°C after reconstitution.

III. STORAGE AND STABILITY

1. All Keul-o-test R.S.V. kit components are to be stored at room temperature (4° to 30°C).
2. Do not freeze the test kit.
3. Keul-o-test R.S.V. kit is stable until the expiry date stated on the package label.
4. If a positive control is shipped with the kit (optional), the control must be kept at 2-8°C.

IV. PRECAUTIONS

1. This test is designed for in vitro diagnostic use and for professional use only.
2. Read carefully instructions before using the test.
3. Do not use after the expiry date shown on the package label.
4. All reagents and materials coming in contact with potential infectious specimens must be treated with appropriate disinfectants or autoclaved at 121°C for at least one hour.
5. Do not use a test from a damaged protective wrapper.

V. SPECIMEN COLLECTION AND PREPARATION

1) Preliminary notes
RSV is recovered almost exclusively from the respiratory tract. Secretions are ideally obtained as such, either by aspiration through a catheter or by suction into a soft rubber bulb.
Swabs, either nasopharyngeal or throat, may also be used but are somewhat inferior to aspirated secretions (6).
If the sample analysis is not performed immediately, store the swabs in a clean and dry container (not supplied) at 2-8°C and proceed the analysis in the next 24 hours.

Do not use any holding medium.

• After collection using a suitable procedure, add 0.5 ml of nasopharyngeal aspirate and 0.5 ml of extraction solution into the filter tube.
• Using an automatic pipette, mix well by pipetting few times.
• Add the cap on the filter tube and allow the extraction to occur 15 minutes.

3) Swab sample

• Use swabs with rayon or dacron tips.
  Do not use swabs with cotton or calcium alginate tips, with wooden shafts, or impregnated with charcoal or transport media containing agar or gelatin.
• Collect the sample, either nasopharyngeal or throat, using an appropriate swab.
• Immediately after collection, immerse the swab in a filter tube containing 1 ml of extraction solution and swirl the swab vigorously for 10 seconds to ensure adequate mixing.
• Allow the extraction to occur for 15 minutes.
• At the end of the extraction time, thoroughly remove liquid from the swab by pressing it against the extraction tube wall.
• Discard the swab as per the guidelines for handling infectious agents (see section precautions).
• Add the cap on the filter-tube.

VI. ASSAY PROCEDURE

a) Samples

1. Bring all reagents at room temperature.
2. Remove the test device from the pouch.
3. Using the dropper filter-tube, add 6-7 drops (200 µl) of the extracted solution into the sample well.
4. Read the results of the test 10 minutes after addition of the sample to the device.

b) Positive control (optional)

1. Remove the test device from the pouch.
2. Add 200 µl of the positive control into the sample well on reaction device.
3. Read results of the test between 10 minutes after addition of sample on the device.

VII. READING TEST RESULTS

C. Inconclusive
If there is no distinct colored band visible in both the test and control window, the test is inconclusive. Repeat the test.

VIII. PERFORMANCE CHARACTERISTICS

1) Accuracy
A study was performed on 79 positive nasopharyngeal aspirate samples obtained from children suffering from RSV type bronchiolitis (RSVA, RSVB and non typed RSV) and on 48 other nasopharyngeal aspirate free of virus.
The Keul-o-test R.S.V. results were compared with Immunofluorescence results obtained using the IMAGEN RSV kit (OXOID/DAKO).

The data are summarized in Tables 1 and  2 :

Table 1 : Evaluation results of Keul-o-test R.S.V. versus IF with positive samples.

Table 2 : Evaluation results of Keul-o-test R.S.V. versus IF with negative samples.

From this study, it appears that Keul-o-test R.S.V. has a sensitivity of 92.41% (73/79) and a specificity of 91.66% (44/48).

2) Cross-reactions
Nasopharyngeal aspirates containing either parainfluenza virus (4 samples), Rhinovirus (7 samples) or Adenovirus (2 samples) have been tested using Keul-o-test R.S.V. and showed repeatedly negative results.

IX. LIMITATIONS
Keul-o-test R.S.V. is specifically designed to detect respiratory syncytial virus in nasopharyngeal aspirate or nasopharyngeal samples.
As it is the case with any diagnostic procedure, the physician should confirm the data obtained using this test by other clinical methods.

X. BIBLIOGRAPHY

1. Chanock, R.M., and L. Findberg. 1957. Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (CCA). II. Epidemiologic aspects of infection in infants and young children. Am. J. Hyg.66: 291-300.
2. Chanock, R.M., H.W. Kim, A.J. Vargosko, A. Deleva, K.M. Johnson, C.Cumming, and R.H. Parrott. 1961. Respiratory syncytial virus. I. Virus recovery and other observations during 1960 outbreak of bronchiolitis, pneumonia, and minor respiratory diseases in children. J. Am. Med. Assoc. 176: 647-653.
3. Hall, C,B, R.G. Douglas, and J.M. Geiman. 1976. Respiratory syncytial virus infections in infants: quantitation and duration of shedding. J. Pediatr. 89: 1443-1447.
4. Hall, C,B,J.T. McBride, E.E. Walsh, D.M. Bell, C.L. Gala, S.Hildreth, L.G. Teneyck, and WW.J. Hall. 1983. Aerosolized ribavirin treatment of infants with respiratory syncytial virus infection. N.Engl. J. Med. 308: 1443-1447.
5. Taber, L.H.V, Knight, B.E. Gilbert, H.W. McClung, S.Z. Wilson, H.J. Norton, J.M. Thurson, W.H. Gordon, R.L. Atmar and W.R. Schlaudt. 1983. Ribavirin aerosol treatment of bronchiolitis associated with respiratory syncytial virus infection in infants. Pediatrics 72:613-618.
6. Ahluwalia, G.J. Embree, P. McNicol, B.Law, and G.W. Hammond. 1987. Comparison of nasopharyngeal aspirate and nasopharyngeal swab specimens for respiratory syncytial virus diagnosis by cell culture, indirect immunofluorescence assay, and enzyme-linked immunoabsorbent assay. J.Clin. Microbiol. 257: 763-767.

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