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Hepatitis E Virus(HEV)  and Test Introduction 

Hepatitis E virus (HEV) rapid test uses for detection immunoglobulin G (IgG) and immunoglobulin M (IgM) of past and present infection respectively as shown above picture. This test is a single-use, rapid device intended for the qualitative detection of IgM and IgG classes of antibodies to the hepatitis E virus in serum, plasma, or whole blood samples. It is intended to be used in clinical laboratories for diagnosis of acute hepatitis E and management of patients related to infection with hepatitis E virus. The benefits of this test over reverse transcriptase-polymerase chain reaction (RT-PCR) are simple, cheap and the test can be performed even in a simple set up whereas RT-PCR to detect the hepatitis E virus RNA in blood and/or stool; this assay requires specialized laboratory facilities. This test is particularly needed in areas where hepatitis E is infrequent, and in cases with chronic Hepatitis E virus infection. Hepatitis E virus is a non-enveloped, single-stranded RNA virus identified in 1990. Infection with Hepatitis E virus induces acute or sub-clinical liver diseases similar to hepatitis A. Hepatitis E virus infections, endemic and frequently epidemic in developing countries, is seen also in developed countries in a sporadic form with or without a history of traveling to endemic areas. The overall case fatality is 0.5~3%, and much higher (15~25%) among pregnant women. A hypothesis that Hepatitis E virus infection is a zoonosis was presented in 1995. Then a swine Hepatitis E virus and later an avian Hepatitis E virus were identified and sequenced separately in 1997 and 2001. Since then, Hepatitis E virus infection includes anti-HEV, viremia, and feces excretion of Hepatitis E virus was seen in a wide variety of animals ( swine, rodents, wild monkeys, deer, cow, goats, dogs, and chicken) in both the developing and developed countries. Direct testimony was reported that the consumption of uncooked dear meat infected with HEV led to acute hepatitis E in humans. And Hepatitis E virus genome sequences can be detected in pork livers available in supermarkets in Japan. With the discovery of conformational epitopes in HEV, HEV serology was further explored and understood. The phenomenon of long-lasting and protective antibodies to HEV was observed which greatly enhance the understanding of the diagnosis, epidemiology, zoonosis-related studies, and vaccine development.  The virus has at least 4 different types: genotypes 1, 2, 3, and 4. Among them, 1 and 2 have been found only in humans whereas 3 and 4 circulate in several animals without causing any disease, and occasionally infect humans. The mode of infection is the feco-oral route. The virus is shed in the stools of infected persons and enters the human body through the intestine. It is transmitted mainly through contaminated drinking water. Usually, the infection is self-limiting and resolves within 2–6 weeks. Occasionally a serious disease, known as fulminant hepatitis (acute liver failure) develops, and a proportion of people with this disease can die.

Principle

The One Step HEV IgG/IgM Test is a qualitative membrane cassette-based immunoassay for the detection of Hepatitis E Virus antibodies ( IgG and IgM) in Whole Blood /Serum / Plasma. The test device consists of 1) a burgundy-colored conjugate pad containing Hepatitis E virus recombinant envelope antigens conjugated with Colloid gold (HEV conjugates) and rabbit IgG-gold conjugates,2) a nitrocellulose membrane strip containing two test bands (T1 and T2 bands) and a control band (C band). The T1 band is pre-coated with the antibody for the detection of IgM anti-HEV, the T2 band is coated with the antibody for the detection of IgG anti-HEV, and the C band is pre-coated with goat anti-rabbit IgG. When an adequate volume of the test specimen is dispensed into the sample well of the test cassette, the specimen migrates by capillary action across the cassette. IgG anti-HEV, if present in the specimen, will bind to the HEV conjugates. The immunocomplex is then captured by the reagent pre-coated on the T2 band, forming a burgundy colored T2 band, indicating an HEV IgG positive test result and suggesting a recent or repeat infection. IgM anti-HEV if present in the specimen will bind to the HEV conjugates. The immunocomplex is then captured by the reagent coated on the T1 band, forming a burgundy-colored T1 band, indicating an HEV IgM positive test result and suggesting a fresh infection. The absence of any T bands (T1 and T2) 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 any of the T bands. Otherwise, the test result is invalid and the specimen must
be retested with another device

Test Requirements

1. HEV IgG/IgM test kit contains the following items to perform the assay.

• HEV IgG/IgM test device foil pouched with a desiccant
•  Disposable dropper capable of delivering 15 µL sample volume (may not be provided)
• Assay diluents
•  Instruction for use

2. Specimen: Serum/Plasma/Whole blood

3. Wastebin

Test Procedure 

1. Allow all test components and specimens to come to room temperature prior to testing.
2. Remove the test device from the foil pouch, and place it on a flat, dry surface.
3. With a disposable dropper, add about 10 μL of serum/ plasma or whole blood specimen into the sample well-marked “S”.
4. Allow about 30 seconds for the specimen to be absorbed totally.
5. Add 3 drops of diluents buffer to the sample well.
6. As the test begins to work, you will see red color move across the result window in the center of the test device.
7. Interpret test results at 15-20 minutes.

Interpretation of Test Results

• Test Negative: The control line is only visible on the test device. No IgG and IgM antibodies were detected.
• IgM Positive: The control line (C) and IgM line (M) are visible on the test device. This is positive for IgM antibodies to HEV
• IgG Positive: The control line (C) and IgG line (G) are visible on the test device. These are positive for IgG antibodies to HEV.
• IgG and IgM Positive:  The control line (C), IgM (M), and IgG line (G) are visible on the test device. This is positive for both IgM and IgG antibodies to HEV.
  Invalid:  The control line fails to appear. Insufficient specimen volume or incorrect procedural techniques are the most likely reasons for control line failure. Repeat the test using a new test device.
• Result: IgG positive as shown above image.

Limitations of the Test

• The One Step HEV IgG/IgM Test is for in vitro diagnostic use only. The test should be used for the detection of Hepatitis E virus antibodies in Whole Blood /Serum / Plasma specimens only. Neither the quantitative value nor the rate of increase in HEV antibodies can be determined by this qualitative test.
• As with all diagnostic tests, all results must be interpreted together with other clinical information available to the
  physician.
•  If the test result is negative and clinical symptoms persist, additional testing using other clinical methods is recommended. A negative result does not at any time preclude the possibility of HEV infection.

Precaution

• The instructions must be followed to obtain accurate results. Anyone performing an assay with this product must be trained in its use and laboratory procedures.
•  Wear protective gloves while handling specimens. Wash hands thoroughly afterward.
•  The sample must be fresh for the test, avoiding freezing repeatedly.
• The test result is invalid over 20 minutes because reading too late can give false results.
• Do not use after the expiration.
•  If the patient, for the first time, is infected in less than 5 days, with no
  detected specific antibody, the result will be negative when testing.
• The parent antibody can be detected from the samples of a yearling baby,
  therefore, it is not appropriate to take this test to evaluate the baby’s history
  of infection and immunization.
• Do not use other kinds of quality control samples to test the reagent.
  Components of different batches cannot be exchanged for use to avoid
  the erroneous result
• For in vitro diagnostic use only. Do not re-use test device
• Do not eat or smoke while handling specimens.
• Avoid splashing or aerosol formation
• Clean up spills thoroughly using an appropriate disinfectant
• Decontaminate and dispose of all specimens, reaction kits, and potentially contaminated materials, as if they were an infectious waste, in a biohazard container.
•  Do not mix with other specimens.

Keynotes

1. Allow the test device controls to equilibrate to room temperature for 30 minutes (20°C -30°C) prior to testing.
2. Do not open the inner packaging until ready, it must be used in one hour if opened.
3. Internal Quality Control: The “Control Line” is used for procedural control. The Control line should always appear if the test procedure is performed properly and the test reagents of the control line are working. It confirms sufficient specimen volume and correct procedural technique. A clear background is also required.
4. The rate of sensitivity and specificity also vary according to from vendor to vendor.

References

1. https://www.who.int/news-room/fact-sheets/detail/hepatitis-e
2. http://img.creative-diagnostics.com/pdf/DTS773,HEV.pdf
3. https://ctkbiotech.com/product/hev-igm-rapid-test
4. http://biogatelab.com/uploads/3/4/4/1/34413460/hev_iggigm_cassette.pdf
5. https://pubmed.ncbi.nlm.nih.gov/26305830/
6. https://pubmed.ncbi.nlm.nih.gov/26930580/