Ezmedtest Influenza A&B Ag Rapid Test

INTENDED USE

The Influenza A & B Ag Rapid Test is an in vitro immunochromatographic assay for the qualitative detection of influenza A and B nucleoprotein antigens in nasopharyngeal (NP) swab, nasal swab, and nasal wash/aspirate specimens. It is intended to aid in the rapid differential diagnosis of influenza A and B viral infections.

SUMMARY AND EXPLANATION

  • Influenza is an acute and highly contagious viral infection of the respiratory tract. The causative agents of the disease are immunologically diverse, single-strand RNA virus known as influenza viruses.
  • There are three types of influenza viruses: A, B and C. Type A viruses are the most prevalent and are associated with most serious epidemics, while Type B infection is generally milder. Type C virus have never been associated with a large epidemic of human disease.
  • Both type A and B viruses can circulate simultaneously, but usually one type is dominant during a given season and particular epidemic area. The disease is easily transmitted through coughing and sneezing of aerosolized droplets containing live virus.
  • Influenza outbreaks normally occur each year during fall and winter seasons. Rapid diagnosis of influenza infection will help healthcare professionals to treat patients and control the disease more efficiently and effectively.

PRINCIPLE OF THE TEST

  • The Influenza A&B Ag Rapid Test is an immunochromatographic membrane assay that uses highly sensitive monoclonal antibodies to detect influenza type A and B nucleoprotein antigens in nasopharyngeal swab and nasal aspirate samples.The test strip is composed of the following parts: namely sample pad, reagent pad, reaction membrane, and absorbing pad.
  • The reagent pad contains the colloidal-gold conjugated with the monoclonal antibodies against Influenza virus A and B; the reaction membrane contains the secondary antibodies either for virus A or for B. The whole strip is fixed inside a plastic device. When the sample is added into the sample well, conjugates dried in the reagent pad are dissolved and migrate along with the sample. If influenza A presents in the sample, a complex formed between the anti-influenza A conjugate and the virus will be captured by the specific anti-influenza A monoclonal antibodies coated on the A region (A).
  • If the sample contains influenza B, a complex formed between the anti -influenza B conjugate and the virus will be captured by the specific anti-influenza B monoclonal antibodies coated on the B region (B).

Results appear at 10 minutes in the form of a red line that develops on the membrane. To serve as a procedural control, a red line will always appear in the control region(C) indicating that proper volume of sample has been added and membrane wicking has occurred.

MATERIALS PROVIDED

1.Influenza A&B Ag test devices      2. Buffer     3. Sterile Swabs     4. Extraction tubes      5. Package insert

MATERIALS REQUIRED BUT NOT PROVIDED

1.Clock,timer or stopwatch

WARNINGS AND PRECAUTIONS

  1. For in vitro diagnostic use only.
  2. The test device should remain in the sealed pouch until use.
  3. Do not use kit past its expiration date.
  4. Swabs, tubes and test devices are for single use only.
  5. The extraction buffer contains a solution with a preservative (0.09% sodium azide). If solution comes in contact with the skin or eyes, flush with ample volumes of water.
  6. Solutions that contain sodium azide may react explosively with lead or copper plumbing. Use large quantities of water to flush discarded solutions down a sink.
  7. Do not interchange or mix components from different kit lots.
  8. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+facility is available to receive and culture specimens.
  9. Humidity and temperature can adversely affect results.
  10. Used testing materials should be discarded in accordance with local regulations.

STORAGE AND STABILITY

  1. The kit can be stored at room temperature or refrigerated (2-30℃).
  2. Do not freeze any of the test kit components.
  3. Do not use test device and reagents after expiration date.
  4. Recap the desiccated container immediately after removing a test device.
  5. Test devices that have been outside of the desiccated container for more than 1 hour should be discarded.

SPECIMEN COLLECTION

It is applicable to the diagnosis of the influenza virus A and B from the samples of nasal swabs,throat swabs or nasal aspirates. Use freshly collected samples for optimal test performance.Inadequate sample collection or improper sample handling may yield a false-negative result.

SAMPLE PREPARATION PROCEDURE

Insert the test tube into the paper stand in this product. Make sure that the tube is standing firm and reaches the bottom of the stand.

Add 0.5 ml (about 10 drops) of the sample extraction buffer into the extraction tube .

1) Nasal or Throat Swabs

Insert the swab into the extraction tube which contains 0.5 ml of the extraction buffer. After mixing, squeeze the tube several times with fingers from outside of the tube to immerse the swab. Remove the swab. The extracted solution will be used as test sample.

2) Nasal Aspirate Fluids

Add 0.5 ml of the nasal aspirate fluids into the extraction tube which contains 0.5 ml of the extraction buffer , and mix well to be used as test sample.

SPECIMEN TRANSPORT AND STORAGE

Specimens should be tested as soon as possible after collection. If transport of the samples is required, the following transport media are recommended and have been tested and shown not to interfere with the performance of the test: Hank’s Balanced salt solution, M5 Media, or saline. Alternatively, samples may be stored refrigerated (2-8℃), or at room temperature(15-30℃), in a clean, dry, closed container for up to eight hours prior to testing. Nasal wash/aspirate specimens may also be stored frozen(-70℃ or colder) for up to one month.

TEST PROCEDURE

Allow the test device, test sample and buffer to equilibrate to room temperature (15-30°C) prior to testing.

  1. Remove test device from the sealed pouch just prior to the testing and lay flat on work bench.
  2. Insert a nozzle with filter into the sample extraction tube tightly.
  3. Reverse the sample extraction tube, and add 4 drops (about 120 -150 μl) of test sample by squeezing the extracted solution tube into the sample window.
  4. Wait for the colored band(s) to appear. The result should be read in10 minutes. Do not interpret the result after 10 minutes.

    Flu A&B (+ ) (+) (+ ) ()
           
      C C C C
      B B B B
    C A A A A
  FluB        
  B Flu A&B Flu B Flu A Neg
  A& A
           
S ABC          
      C C C C
      B B B B
    S A A A A
           

 

Influenza A&B Ag Rapid Test (Cassette)

INTERPRETATION OF RESULTS

1. POSITIVE:

1.1 Flu A Positive:

The presence of two lines as control line (C) and A test line within the result window indicates a positive result for Influenza A viral antigen.

1.2 Flu B Positive:

The presence of two lines as control line (C) and B test line within the result window indicates a pos itive result for Influenza B viral antigen.

1.3 Flu A+B Positive:

The presence of three lines as control line (C), A test line and B test line within the result window indicates a positive result for Influenza A and Influenza B viral antigen.

2. NEGATIVE:

The presence of only control band (C) within the result window indicates a negative result.

3. INVALID:

If the control band (C) is not visible within the result window after performing the test, the result is considered invalid. Some causes of invalid results are because of not following the directions correctly or the test may have deteriorated beyond the expiration date. It is recommended that the specimen be re-tested using a new test.

NOTE:

1.The intensity of color in the test region (A/B) may vary depending on the concentration of analyses present in the specimen. Therefore, any shade of color in the test region (A/B) should be considered positive. Please note that this is a qualitative test only, and cannot determine the concentration of analytes in the specimen.

2.Insufficient specimen volume, incorrect operating procedure or expired tests are the most likely reasons for control band failure.

QUALITY CONTROL

A procedural control is included in the test. A red line appearing in the control region (C) is the internal procedural control. It confirms sufficient specimen volume and correct procedural technique. Control standards are not supplied with this test. However, it is recommended that positive and negative controls are sourced from a local competent authority and tested as a good laboratory practice, to confirm the test procedure and verify the test performance.

LIMITATIONS

  1. The Influenza A&B Ag Rapid Test is for professional in vitro diagnostic use, and should only be used for the qualitative detection of influenza A and/or B.
  2. The etiology of respiratory infection caused by microorganisms other than influenza A or B virus will not be established with this test. The Influenza A&B Ag Rapid Test is capable of detecting both viable and non-viable influenza particles. The performance of the Influenza A&B Ag Rapid Test depends on antigen load and may not correlate with cell culture performed on the same specimen.
  3. If the test result is negative and clinical symptoms persist, additional testing using other clinical methods is recommended. A negative result does not at anytime rule out the presence of influenza A and/or B viral antigens in specimen, as they may be present below the minimum detection level of the test. As with all diagnostic tests, a confirmed diagnosis should only be made by a physician after all clinical and laboratory findings have been evaluated.
  4. The validity of Influenza A&B Ag Rapid Test has not been proven for identification or confirmation of cell culture isolates.
  5. Inadequate or inappropriate specimen collection, storage, and transport may yield false negative test result.
  6. Although this test has been shown to detect cultured avian influenza viruses, including avian influenza A subtype H5N1 virus, the performance characteristics of this test with specimens from humans infected with H5N1 or other avian influenza viruses are unknown.
  7. Performance characteristics for influenza A were established when influenza A/H3 and A/H1 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary.
  8. Children tend to shed virus for longer periods of time than adults, which may result in differences in sensitivity between adults and children.
  9. Positive and negative predictive values are highly dependent on prevalence. False positive test results are more likely during periods of low influenza activity when prevalence is moderate to low.

PERFORMANCE CHARACTERISTICS

  1. Analytical Sensitivity

The minimum detection limit is 1.5 x 104 TCID50/test for the Influenza A virus antigen and is 1.5 x 105 TCID50/test for the Influenza B virus antigen.

  1. Analytical Reactivity

Analytical sensitivity was established using a total of 15 human epidemic strains of influenza viruses: 10 influenza A and 5 influenza B.

Viral Strain Viral Type Minimum Detectable Level (pfu/mL)
Hong Kong A 6.60 x 10-1
Duck/England A 6.70 x 100
Puerto Rico/8/34 A 2.60 x 102
Tokyo/3/6 A 3.40 x 102
NWS/33 A 1.00 x 103
Texas/77 A 3.30 x 103

 

Aichi     A 3.20 x 103
Sydney     A 2.00 x 104
Bangkok     A 3.30 x 104
Equine/Miami     A 1.70 x 105
Panama     B 1.00 x 100
Lee     B 6.60 x 102
Hong Kong     B 7.00 x 102
Maryland     B 6.60 x 103
Stockholm     B 3.30 x 105
3. Clinical Study Data Summary        
The Influenza A&B Ag Rapid Test performance vs. Cell Culture  
Kind of samples Type Sensitivity(%) Specificity(%) Accuracy(%)
Nasal Swab   A 92.6 (25/27) 96.4 (81/84) 95.5 (106/111)
  B 90.0 (27/30) 95.8 (91/95) 94.4 (118/125)
   
Throat Swab   A 83.3 (20/24) 95.2 (59/62) 91.9 (79/86)
  B 82.6 (19/23) 91. 8 (67/73) 89. 6 (86/96)
   
Nasal Aspirate   A 88.9 (48/54) 93.3 (125/134) 92.0 (173/188)
  B 91.2 (52/57) 95.4 (98/103) 93.8 (150/160)
   
Nasal discharge/ A 80.7 (46/57) 94.9 (93/98) 89.7 (139/155)
Nasal mucus   B 89.6 (62/69) 94.6 (87/92) 92.5 (149/161)
  1. Analytical Specificity And Cross-reactivity

The Influenza A&B Ag Rapid Test was evaluated with a total of 30 bacterial and viral isolates. Bacterial isolates were evaluated at a concentration between 107 and 10 9 org/mL. Viral isolates were evaluated at a concentration of at least 104–108 TCID50/mL. Adenovirus 18 and Parainfluenza virus 3 were tested at 102 TCID50/mL. None of the organisms or viruses listed below gave a positive result in the Influenza A&B Ag Rapid Test.

Bacterial Panel:  
Acinetobacter calcoaceticus Bacteroides fragilis
Neisseria gonorrhoeae Neisseria meningitidis
Pseudomonas aeruginosa Staphylococcus aureus
Streptococcus pneumoniae Streptococcus sanguis
Proteus vulgaris Streptococcus sp. Gp. B
Streptococcus sp. Gp. C Streptococcus sp. Gp. G
Mycobacterium tuberculosis Mycoplasma orale
Viral Panel:    
Human Adenovirus B Human Rhinovirus 2
Human Adenovirus C Human Rhinovirus 14
Adenovirus type 10 Human Rhinovirus 16
Adenovirus type 18 Measles
Human Coronavirus OC43 Mumps
Human Coxsackievirus A9 Sendai virus
Coxsackievirus B5 Parainfluenza virus 2
Human herpesvirus2 Parainfl uenza virus 3
 

5. Interfering Substances

Whole blood, and several over-the-counter (OTC) products and common chemicals were evaluated and did not interfere with the Influenza A&B Ag Rapid Test at the levels tested: whole blood (2%); three OTC mouthwashes (25%); three OTC throat drops (25%); three OTC nasal sprays (10%); 4-Acetamidophenol (10 mg/mL); Acetylsalicylic Acid (20 mg/mL); Chlorpheniramine (5 mg/mL); Dextromethorphan (10 mg/mL); Diphenhydramine (5 mg/mL); Ephedrine (20 mg/mL); Guaiacol glyceryl ether (20 mg/mL); Oxymetazoline (10 mg/mL); Phenylephrine (100 mg/mL); and Phenylpropanolamine (20 mg/mL).

REFERENCES

1.Williams, KM, Jackson MA, Hamilton M. (2002) Rapid Diagnostic Testing for URIs in Children: Impact on Physician Decision Making and Cost. Infect. Med. 19(3): 109-111.

2. Dowdle, W.R, Kendal, A.P., and Noble, G.R. (1980). Influenza Virus, p 836-844. Manual of Clinical Microbiology, 3rd edition, in Lennette, et. al (ed.). American Society for Microbiology, Washington, D.C.

3.“Key Facts about Avian Influenza (Bird Flu) and Avian Influenza A (H5N1) Virus” CDC Publication, May 24, 2005. http://www.cdc.gov/flu/avian/gen-info/facts.htm

4.“Avian Influenza Infection in Humans” CDC Publication, May 24, 2005. http://www.cdc.gov/flu/avian/gen-info/avian-flu-humans.htm

5.“Updated Interim Guidance for Laboratory Testing of Persons with Suspected Infection with Avian Influenza A (H5N1) Virus in the United States” CDC Health Alert, June 7, 2006. http://www.phppo.cdc.gov/HAN/ArchiveSys/ViewMsgV.asp?AlertNum=00246