Influenza A & B Test Instructions

Influenza A/B Antigen Rapid Test

Instruction for Use 

An immunochromatographic assay for the qualitative detection of influenza type A (including the subtype H1N1) and B nucleoprotein antigen extracted from nasopharyngeal (NP) swab, throat swab, and nasal swab specimens.

 

INTENDED USE

The Influenza A/B Antigen Rapid Test is an immunochromatographic assay for the qualitative detection of influenza type A (including the subtype H1N1) and B nucleoprotein antigen extracted from nasopharyngeal (NP) swab, throat swab, and nasal swab specimens. It is intended to be used to aid in the differential diagnosis of influenza type A and B infection. The test is recommended for professional use only. All results must be interpreted together with other clinical information available to the physician.

 

SUMMARY

Influenza (commonly known as flu) is a highly contagious, acute viral infection of the respiratory tract. It is a communicable disease that is easily transmitted through the coughing and sneezing of aerosolized droplets containing live virus. Influenza outbreaks occur each year during the autumn and winter months. There are three types of influenza viruses: A, B, and C Only influenza A viruses are further classified by subtype on the basis of the two main surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Influenza A subtypes and B viruses are further classified by strains. Humans can be infected with influenza types A, B, and C viruses. Subtypes of influenza A that are currently circulating among people worldwide include H1N1, H1N2, and H3N2 viruses. Influenza B viruses can cause morbidity and mortality among humans, but in general are associated with less severe epidemics than influenza A viruses. Although influenza type B viruses can cause human epidemics, they have not caused pandemics. Influenza type C viruses cause mild illness in humans and do not cause epidemics or pandemics. 

 

PRINCIPLE

Influenza A/B Antigen Rapid Test is a rapid immunochromatographic test for the visual detection of influenza type A and B antigens (nucleoprotein) extracted from the nasal swab specimen. The test adopts double antibody sandwich method. When the extracted specimen is added into the test device, the specimen is absorbed into the device by capillary action, mixes with antibody-dye conjugate, and flows across the pre-coated membrane, in which influenza type A and B monoclonal antibodies are coated respectively. When the influenza type A antigen levels are at or above the target cutoff (the detection limit of the test), type A antigen in the specimen binds to the specific antibody-dye conjugate and are captured by influenza type A monoclonal antibody immobilized in the relative site of test region "A" of the device. This produces a colored test band in the test region "A". When the influenza type A antigen levels are zero or below the target cut off, there is not a visible colored band in the test region "A" of the device. This indicates a negative result for influenza type A. When the influenza type B antigen levels are at or above the target cutoff (the detection limit of the test), type B antigen in the specimen binds to the specific antibody-dye conjugate and are captured by influenza type B monoclonal antibody immobilized in the relative site of test region "B" of the device. This produces a colored test band in the test region "B". When the influenza type B antigen levels are zero or below the target cut off, there is not a visible colored band in the test region "B" of the device. This indicates a negative result for influenza type B. To serve as a procedure control, a colored line will appear at the control region (C), if the test has been performed properly. 

 

PRECAUTIONS 

1. For professional and IN VITRO diagnostic use only.
2. The test should remain in the sealed pouch until use.
3. Do not use the kit if the foil pouch is punctured or not well sealed.
4. Do not reuse or use kits after the expiration date.
5. Do not mix components from kits with different lot number.
6. Avoid microbial contamination of reagents.
7. Wear gloves during the whole process and avoid reagents or specimen spilling-out. Wash hands thoroughly afterwards. 
8. Dispose the used kit after decontamination of all liquids or solid wastes by following the local law or laboratory rule.
9. Avoid using blood samples. 

 

MATERIAL

Material provided:

• 20 X Test device, each cassette is Individual sealed in a foil pouch with a package of desiccant.
• 20 X Extraction tube with Buffer
• 20 X Swab.
• 1 X Instruction for use.

 

Material Required But Not Provided

• Timer

 

STORAGE AND STABILITY

Store the kit in cool and dry places at a temperature between 2-38°C. Do not freeze. The shelf-life of the kit under these storage conditions is 24 months.

 

SPECIMEN COLLECTION

1. Nasal swab Specimen: Use the nasal swab supplied in the kit. Prior to collecting the nasal swab, blow your nose before sampling. To collect a nasal swab sample, insert the entire absorbent tip of the nasal swab (usually % to 1 of an inch (1.5 to 2.5cm) inside the nostril and firmly sample the nasal wall by rotating the swab in a circular path against the nasal wall at least 5 times. Take approximately 15 seconds to collect the sample per nostril. Be sure to collect any nasal drainage that may be present on the swab. Sample both nostrils with the same swab before testing. Do not return the Nasal swab to the original paper packaging.
2. Throat swab Specimen: Insert a sterilized swab into pharynx and collect muco-epidermis mainly wiping flare region of post-pharyngeal wall and palatine tonsil several times, and be careful not to make saliva attach to the swab. Nasal swabbing: Insert the swab 2-2.5cm into the nostril. Rub the swab around the inside of each nostril in a circular motion at least 5 times. Then repeat to take samples from the other nostril with the same swab. Patient samples perform best if tested immediately after collection. If immediate testing is not possible, the swab should be placed in a dry, sterile plastic tube (not provided) and stored at 2°C-4°C for up to 8 hours.
3. Nasopharyngeal swab Specimen: Insert swab through the nostril parallel to the palate (not upwards) until resistance is encountered or the distance is equivalent to that from the ear to the nostril of the patient, indicating contact with the nasopharynx. Swab should reach depth equal to distance from nostrils to outer opening of the ear. Gently rub and roll the swab. Leave swab in place for several seconds to absorb secretions. Slowly remove swab while rotating it. Specimens can be collected from both sides using the same swab, but it is not necessary to collect specimens from both sides if the swab is saturated with fluid from the first collection. If a deviated septum or blockage create difficulty in obtaining the specimen from one nostril, use the same swab to obtain the specimen from the other nostril.

 

TEST PREPARATION

Before testing, open the package and equilibrate the test cassette, extraction solution and specimens to room temperature, and shake the extraction solution gently before use. The most suitable temperature condition to perform the test is room temperature (15~30 C). If the test kit is stored at room temperature, it can be opened and used immediately.

 

TEST PROCEDURE

1. Tear off the sealing film of the extraction solution tube.
2. Place the swab in the extraction solution tube, rotate the swab for about 10 times and no less than 15 seconds, and press the swab tip against the tube wall to release the antigen in the swab.
3. Squeeze the swab over the swab tip to withdraw the swab so as to extract as much liquid as possible from the swab. Dispose of used swabs according to biohazard waste disposal method and local regulations.
4. Install the dripper of the tube and shake well the tube.
5. Take out the test cassette from sealed foil pouch and place on a dry, clean and level surface. Add two drops of extracted specimen to each of the specimen wellS) on the test card, and start the timer. Notes: Applying sufficient amount of extracted specimen is essential for a valid test result. If migration (the wicking of membrane) is not observed in the test window after one minute, add one more drop to specimen well.
6. Read the results at 10 minutes, and the result after 20 minutes is no longer valid.

 

INTERPRETATION OF RESULTS

*Read results of each test individually.

Negative: One red line appears in the control region "C", and no red line appears in the region "A" or the region "B".

Positive:

FLU A positive: One red line appears in the control region "C", and the  other red line appears in the region "A".

FLU B positive: One red line appears in the control region "C", and the other red line appears in the region "B".

FLU A&B positive: One red line appears in the control region "C", and the other red lines appear in both region "A" and "B"

Note: A faint test line ("A") or ("B") may appear. In this case, it shall be considered as positive result. Samples with positive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made.

Invalid: No red line appears in the control region "C", regardless if the red line appears or not in the region "A" or the region "B". Review the procedure and repeat the test with a new test device. If the problem persists, contact your local distributor.

 

QUALITY CONTROL

A procedural control is included in the test. A colored line appearing in the control region (C) is considered an internal procedural control. It confirms sufficient specimen volume, adequate membrane wicking and correct procedural technique. Good laboratory practice recommends the use of the control materials. Users should follow the appropriate federal state, and local guidelines concerning the frequency of assaying external quality control materials.

 

LIMITATIONS OF ASSAY

1. As it is with any diagnostic procedure, a confirmed diagnosis should only be made after all clinical and laboratory findings have been evaluated.
2. A negative test result may occur if the level of antigen in a sample is below the detection limit of the test, or from improper sample collection.
3. Negative test results are not intended to rule-out other non-influenza viral infections.
4. Positive test results do not rule out co-infections with other pathogens and does not identify specific influenza A virus subtypes.
5. Performance of this test has not been established for monitoring antiviral treatment of influenza.

 

MINIMAL DETECTION LIMIT

For Flu A: 3.5× 104TCID50/ml

For Flu B: 1.5x105TCID50/ml

 

ACCURACY
A comparison study of Influenza A/B Antigen Rapid Test and method of cell culture was carried out. Compare the sensitivity and specificity between the two methods.
The results for detection of influenza A are summarized in
Table 1.1, 1.2 and the results for detection of influenza B are summarized in Table 2.1, 2.2.

Table 1.1: Results of Sensitivity for Influenza A
Sample         +/+     -/+     %Sens
NP Swab        18      1       94.7
Throat Swab    9       4       69.2
Nasal Swab     52      7       88.1
Overall        79      12      86.8

Table 1.2: Results of Specificity for Influenza A
Sample         -/-     +/-     %Spec
NP Swab        83      4       95.4
Throat Swab    63      4       94.0
Nasal Swab     106     8       93.0
Overall        252     16      94.0

Table 2.1: Results of Sensitivity for Influenza B
Sample         +/+     -/+     %Sens
NP Swab        17      4       81.0
Throat Swab    10      1       90.9
Nasal Swab     50      2       96.2
Overall        77      7       91.7

Table 2.2: Results of Specificity for Influenza B
Sample         -/-     +/-     %Spec
NP Swab        84      1       98.8
Throat Swab    66      3       95.7
Nasal Swab     118     3       97.5
Overall        268     7       97.5

 

ANALYTICAL REACTIVITY

Influenza A/B Antigen Rapid Test was tested with the following influenza A and B viral strains listed in Table 3. All showed positive results. Although the specific influenza strains causing infection in humans can vary year to year, all contain the conserved nucleoproteins targeted by the Influenza A/B Antigen Rapid Test.

Table 3: Influenza A and B Viral Strains

Flu A: Subtype of H1N1: 5 Strains, H2N2: 3 Strains, H3N2: 7 Strains
Flu B: B/1715, B/1704, B/179, B/668, B/427, B/424, B/180, B/5, B/39

 

CROSS REACTION

1. No cross reaction with following pathogens: Adenovirus Type 1-8,11,19,37; Coxackie virus Type A 16, B1-5, Cytomegalovirus, Echovirus Type 3,6,9,11,14,18,30; Enteroviru Type 71; HSV-1; Mumps virus; Parainfluenza virus Type 1-3; Poliovirus Type 1~3; Respiratory syncytical virus; Rhinovirus Type 1A, 13, 14.
2. No cross reaction with Chlamydia Pneumoniae, Chlamydia psittaci, Chlamydia Trachomatis, Mycoplasma Pneumoniae.
3. No cross reaction with following bacteria: Acinetobacter baumannii, Bordetella pertussis, Bacteroides fragilis, Candida glabrata, Candida albicans, Eikenella corrodens, Cardiobacterium hominis, Escherichia coli, Enterococcus gallinarum, Haemophilus influenzae, Haemophilus paraphrophilus, Haemophilus parainfluenzae, Kingella kingae, Listeria monocytogenes, Moraxella catarrhalis, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus sp. Group C, G, F, Streptococcus mutans.

 

BIBLIOGRAPHY

1. Ruef C.: Diagnosing influenza-clinical assessment and/or rapid antigen testing. Infection 2007; 35: 49-50 
2. Lode H: Respiratory treat infection: when is antibodies therapy indicated Chin Ther 1991;13;149-156
3. P. Pothier, G. A. Denoyel etc.: Use of Monoclonal Antibodies for Rapid Detection of Influenza A Virus in Nasopharyngeal Secretions. Eur. J.
Clin. Microbiol., June 1986, p. 336-339.