Guidelines on Standard Operating Procedures for MICROBIOLOGY

Chapter 25- Viral Hepatitis

Viral hepatitis is a systemic disease primarly involving the liver. The viruses which are recognised until now and for which tests are available are:

     Hepatitis A virus (HAV)

     Hepatitis B virus (HBV)

     Hepatitis C virus (HCV)

     Hepatitis D virus (HDV)

     Hepatitis E virus (HEV)

Of these HAV and HEV are transmitted faeco-orally while the remaining three viruses are blood borne.Large scale outbreaks of water-borne jaundice are usually due to hepatitis E virus and rarely caused by hepatitis A virus.

     Collection and transportation of specimens

The role to be played by most of the peripheral and intermediate laboratories in the diagnosis of viral hepatitis may only be that of collection of blood sample and its transportation to a reference laboratory.

     Collection of blood

     Collect blood sample aseptically using disposable/sterile needle and syringe.

     Draw a minimum of 3-5 ml of venous blood.

     Collect the blood in a sterile, dry and labelled vial.

     Separate the serum and store it in a refrigerator, if facilities for deep freezer do not exist.

     In outbreak situation, specimen should be collected from patients with disease of different duration.

At most of the intermediate laboratories, HBsAg detection can be undertaken by RPHA and latex agglutination tests. However, these tests are not very dependable. As far as possible, blood specimens should be screened by ELISA.

     Latex agglutination test for detection of HBsAg

Commercial kits for this test are available. These contain the following reagents and accessories:

All the reagents are stable and active, till the expiry date mentioned, provided they are stored in a refrigerator at -2 to 8^oC.Do not freeze the reagents.

Latex agglutination test is performed on serum harvested from the patient/donor’s blood. Do not inactivate the test or the control serum samples.

     Test procedure

     Allow the reagents to attain room temperature.

     Shake the vials gently to make sure that the latex reagent is completely in suspension.

     Place one drop (50 mL) of undiluted serum in one of the circles on the slide. Fill more circles if more than one test serum samples are to be tested. Use separate droppers for each specimen.

     Add one drop (50 mL) of latex reagent onto each specimen drop in circles, using a disposable dropper.

     Mix the contents of each circle using separate disposable applicator sticks for each circle, and spread the mixture uniformly over the entire area of the circle.

     Rock the slide gently, to and fro, for 5 minutes, and watch for agglutination.

     Interpretation

No agglutination HBsAg negative

Visible agglutination within 5 minutes HBsAg positive

However, the results must be interpreted with caution since latex agglutination tests fail to detect about 10% of HBsAg positive blood donations.

The specific aetiological diagnosis of viral hepatitis is cumbersome and is possible in few selected laboratories where infrastructure and technology are available to detect a variety of antigens and antibodies that are generated by different viruses. Various markers of viral hepatitis and their importance is:

(A blood sample having HBsAg indicates that the specimen is infectious and if it contains HBeAg it will be considered as highly infectious. HBsAg positive blood samples are not transfused and hence the test to detect this antigen is a screening test to ensure safety of blood before transfusion)

The testing of these markers is required for:

     Diagnosis

     Prognosis

     Safety in blood transfusion

     Assessment of exposure

     Detection of carrier state

     Seroconversion following immunization

      Reporting of results

     Report the results of markers tested alongwith interpretation mentioning the test method used.

     Report negative result for the marker tested.

     Report should specify if any other tests are required.

     Quality assurance        

     Collect 3-5 ml blood aseptically, prevent haemolysis, label properly and transport to the laboratory under recommended conditions.

     All the factors related to analytical components such as use of SOPM, good quality reagents, instruments and training and skills of laboratory technical staff are given importance.

     Use positive and negative controls on tests wherever indicated.

     The results should be interpreted and communicated to the user. Mere writing the presence or absence of various markers is not recommended.

     Along with the laboratory results, always mention the type of tests performed while reporting.

     Biosafety           

Viral hepatitis spreads by faeco-oral (A & E) and parenteral route (B,C and D) and hence the biosafety measures are to be focussed on both the aspects. Hepatitis B has emerged as the commonest laboratory acquired infection. The biosafety steps recommended are as follows:

     Biosafety level 2 (BSL-2) practices and facilities are recommended while handling clinical material from hepatitis cases.

     All personnel concerned with handling the clinical material should be screened for HBsAg and if negative must be vaccinated against hepatitis B.

     Mouth pipetting, smoking, eating or drinking in the laboratory should be strictly forbidden.

     Needles/syringes used in the laboratory should be autoclaved before being discarded.

     Work areas should be decontaminated with 0.5% sodium hypochlorite prepared fresh each month.

     Disposable gloves are worn when working with known infectious materials.

     Referral  

     In outbreak situations

     For confirmation of diagnosis

     As a part of quality assurance programme

     Further reading

1.      Guidelines for preventing HIV, HBV and other infections in the health care setting. WHO-SEARO, New Delhi 1996.

2.      Lennette H Edwin, Albert Balows, WJ Hansler and HJ Shadomy Hepatitis viruses In Manual of Clinical Microbiology 4^th Ed, ASM, Washington, 813-836, 1985.