Span Biotech Ltd.

INTENDED USE

The Chikungunya IgG/IgM Rapid Test is a lateral flow chromatographic immunoassay for the qualitative detection of IgG and IgM anti-chikungunya virus （CHIK） in human whole blood, serum or plasma. It is intended to be used as a screening test and as an aid in the diagnosis of infection with CHIK. Any reactive specimen with the Chikungunya IgG/IgM Rapid Test must be confirmed with alternative testing method(s) and clinical findings.

SUMMARY AND EXPLANATION OF THE TEST

Chikungunya is a rare viral infection transmitted by the bite of an infected Aedes aegypti mosquito. It is characterized by a rash, fever, and severe joint pain (arthralgias) that usually lasts for three to seven days. The name is derived from the Makonde word meaning "that which bends up" in reference to the stooped posture developed as a result of the arthritic symptoms of the disease. It occurs during the rainy season in tropical areas of the world, primarily in Africa, South-East Asia, southern India and Pakistan1-2.

The symptoms are most often clinically indistinguishable form those observed in dengue fever. Indeed, dual infection of dengue and chikungunya has been reported in India3. Unlike dengue, hemorrhagic manifestations are relatively rare and most often the disease is a self limiting febrile illness. Therefore it is very important to clinically distinguish dengue from CHIK infection.

CHIK is diagnosed based on serological analysis and viral isolation in mice or tissue culture. An IgM immunoassay is the most practical lab test method4.

The Chikungunya IgG/IgM Rapid Test utilizes recombinant antigens derived from its structure protein5, it detects IgM anti-CHIK in patient serum or plasma within 15 minutes. The test can be performed by untrained or minimally skilled personnel, without cumbersome laboratory equipment. 

TEST PRINCIPLE

The Chikungunya IgG/IgM Rapid Test is a lateral flow chromatographic immunoassay based on the principle of the IgM capture assay. The test cassette consists of: 1) a burgundy colored conjugate pad containing CHIK antigens conjugated with colloid gold (CHIK conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose membrane strip containing a test band (T1 or T2  band) and a control band (C band). The T band is pre-coated with anti-human M antibody, and the C band is pre-coated with goat anti-rabbit IgG.

When an adequate volume of test specimen is dispensed into the sample well of the cassette, the specimen migrates by capillary action across the cassette.  The IgM antibody to CHIK, if present in the specimen will bind to the CHIK conjugates. The immunocomplex is then captured on the membrane by the pre-coated anti-human IgM antibody, forming a burgundy colored T band, indicating a CHIK IgM positive test result.

Absence of the T band suggests a negative result. The test contains an internal control (C band) which should exhibit a burgundy colored band of the immunocomplex of goat anti-rabbit IgG/rabbit IgG-gold conjugate regardless of the color development on the T band. Otherwise, the test result is invalid and the specimen must be retested with another device.

KIT COMPONENTS

MATERIALS REQUIRED BUT NOT PROVIDED

WARNINGS AND PRECAUTIONS

·    For professional in vitro diagnostic use only.

·    Do not use after expiration date indicated on the package. Do not use the test if its foil pouch is damaged. Do not reuse tests.

·    This kit contains products of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not totally guarantee the absence of transmissible pathogenic agents. It is therefore, recommended that these products be treated as potentially infectious, and handled observing the usual safety precautions (do not ingest or inhale).

·    Avoid cross-contamination of specimens by using a new specimen collection container for each specimen obtained.

·    Read the entire procedure carefully prior to performing any tests.

·    Do not eat, drink or smoke in the area where the specimens and kits are handled. Handle all specimens as if they contain infectious agents. Observe established precautions against microbiological hazards throughout the procedure and follow the standard procedures for proper disposal of specimens. Wear protective clothing such as laboratory coats, disposable gloves and eye protection when specimens are assayed.

·    Buffered Saline contains sodium azide which may react with lead or copper plumbing to form potentially explosive metal azides. When disposing of buffered saline or extracted samples, always flush with copious quantities of water to prevent azide build up.

·    Do not interchange or mix reagents from different lots.

·    Humidity and temperature can adversely affect results.

·    The used testing materials should be discarded in accordance with local, state and/or federal regulations.

STORAGE AND STABILITY

Store as packaged in the sealed pouch either at room temperature or refrigerated (2-30°C). The test device is stable through the expiration date printed on the sealed pouch. The test device must remain in the sealed pouch until use. Do not freeze. Do not use beyond the expiration date.

SPECIMEN COLLECTION AND PREPARETION

· The Chikungunya IgG/IgM Rapid Test Device (Whole Blood/Serum/Plasma) can be performed using whole blood (from venipuncture or fingerstick), serum or plasma.

· Separate serum or plasma from blood as soon as possible to avoid hemolysis. Use only clear, non-hemolyzed specimens.

· Testing should be performed immediately after specimen collection. Do not leave the specimens at room temperature for prolonged periods. Serum and plasma specimens may be stored at 2-8°C for up to 3 days. For long-term storage, specimens should be kept below -20°C. Whole blood collected by venipuncture should be stored at 2-8°C if the test is to be run within 2 days of collection. Do not freeze whole blood specimens.

· Bring specimens to room temperature prior to testing. Frozen specimens must be completely thawed and mixed well prior to testing. Specimens should not be frozen and thawed repeatedly.

· If specimens are to be shipped, they should be packed in compliance with local regulations covering the transportation of etiologic agents.

ASSAY PROCEDURE

Allow test device, specimen, buffer and/or controls to reach room temperature (15‑30°C) prior to testing.

1.Bring the pouch to room temperature before opening it. Remove the test device from the sealed pouch and use it as soon as possible.

2.Place the test device on a clean and level surface. Hold the dropper vertically and transfer 2 drops of whole blood (approximately 80 µL) or 1 drop of plasma/serum (approximately 40 µL) to the specimen well (S) of the test device, then add 2 drops of buffer and start the timer.

3.Wait for the colored line(s) to appear. Read results at 10 minutes. Do not interpret results after 20 minutes.

INTERPRETATION OF ASSAY RESULT

POSITIVE RESULT:

IgM Positive:* The colored line in the control line region (C) appears and a colored line appears in test line region 1 (T1). The result is positive for Chikungunya specific-IgM antibodies and is indicative of primary Chikungunya infection.

IgG Positive:* The colored line in the control line region (C) appears and a colored line appears in test line region 2 (T2). The result is positive for Chikungunya virus specific-IgG and is probably indicative of secondary Chikungunya infection.

IgG and IgM Positive:* The colored line in the control line region (C) appears and two colored lines should appear in test line regions 1 and 2 (T1 and T2). The color intensities of the lines do not have to match. The result is positive for IgG & IgM antibodies and is indicative of secondary Chikungunya infection.

*NOTE: The intensity of the color in the test line region(s) (T1 and/or T2) will vary depending on the concentration of Chikungunya antibodies in the specimen. Therefore, any shade of color in the test line region(s) (T1 and/or T2) should be considered positive.

NEGATIVE RESULT:

The colored line in the control line region (C) appears. No line appears in test line regions 1 or 2 (T1 or T2).

INVALID RESULT:  

Control line (C) falls to appear. Insufficient buffer volume or incorrect procedural techniques are the most likely reasons for control line failure. Review the procedure and repeat the procedure with a new test device. If the problem persists, discontinue using the test kit immediately and contact your local distributor.

Note:

1. The appearance of any burgundy color in the T band, regardless of intensity, must be considered as presence of the band.

2. Samples with positive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made.

QUALITY CONTROL

Using individual Chikungunya IgG/IgM Rapid Test device as described in the Assay Procedure above, run 1 Positive Control and 1 Negative Control (provided upon request) under the following circumstances to monitor test performance:

1.     A new operator uses the kit, prior to performing testing of specimens.

2.     A new test kit is used.

3.     A new shipment of kits is used.

4.     The temperature used during storage of the kit falls outside of 2°C -30°C.

5.     The temperature of the test area falls outside of 15°C-30°C.

PERFORMANCE CHARACTERISTIC

1. Comparison of the Chikungunya IgG/IgM Rapid Test Device with MAC-ELISA

An evaluation study was carried out at Unite de virologie , Institute de Medecine Tropicale de Service de Sante des Armees,  Ministere De la Defense, France.

The evaluation specimen panel consisted of 72 recently infected specimens diagnosed by MAC-ELISA and 21 specimens containing 10 from other arbovirus infection, 3 from O’Nyong nyong infection, and 8 negative for all the tests. The evaluation data are showed in the following table.

Relative Sensitivity:  90.3%, Relative Specificity:  100%, Overall Agreement:   92.4%

LIMITATIONS OF TEST

1.The Assay Procedure and the Assay Result Interpretation must be followed closely when testing the presence of IgM anti-CHIK in serum or plasma from individual subjects. Failure to follow the procedure may give inaccurate results.

2.The Chikungunya IgG/IgM Rapid Test is limited to the qualitative detection of IgM anti-CHIK in human serum or plasma. The intensity of the test band does not have the linear correlation with the antibody titer in the specimen.

3.A negative result for an individual subject indicates absence of detectable IgM anti-CHIK. However, a negative test result does not preclude the possibility of exposure to or infection with CHIK

4.A negative result can occur if the quantity of IgM anti-CHIK present in the specimen is below the detection limits of the assay, or the antibodies that are detected are not present during the stage of disease in which a sample is collected.

5.Some specimens containing unusually high titer of heterophile antibodies or rheumatoid factor may affect expected results.

6.The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings.