Diagnosis and treatment of Hepatitis B

Introduction

Viral hepatitis is a systemic disease with primary inflammation in the liver. Major viral hepatitis clinically resembles each other but infection caused by hepatitis B is most severe and fetal. Most cases of acute viral hepatitis in children and adults are caused by hepatitis A, B and C. The viruses cause acute inflammation of the liver, resulting in the clinical symptoms like fever anorexia, malaise followed by nausea, vomiting, and liver tenderness. Hepatitis B virus is more structurally complex than hepatitis A virus. The outer surface or envelope contains hepatitis B surface antigen (HBS antigen). It is made up of lipid, protein, and carbohydrate. The hepatitis B virus genome consists of 3.2-kilo base pair molecule of circular ds DNA. Hepatitis B virus is present in the blood and in the body fluids such semen, vaginal secretion, menstrual discharge, saliva, colostrums and breast milk. Less than 1 ul of blood contaminating a syringe and needle can readily transmit hepatitis B from one individual to another. Hepatitis B virus can be transmitted from carrier mother to her infant and also by means of blood sucking insects and bed bugs.

Hepacard is one step immunoassay based on the antigen capture or sandwich ELISA principle. The method uses monoclonal antibody conjugated to colloidal gold and a polyclonal antibody immobilized on a nitrocellulose strip in a thin line. The test sample is introduced to and flows laterally through an absorbent pad where it mixes with a signal reagent. If the sample contains hepatitis B antigen, the colloidal gold antibody conjugate binds to the antigen, forming an antigen- antibody colloidal gold complex. The complex then migrates through the nitrocellulose strip by capillary action. When the complex meets the line of immobilized antibody (test line) ‘T’, the complex is trapped forming an antibody- antigen- antibody-gold complex. This forms a pink band indicating the sample is reactive for hepatitis B antigen. To serve as a procedural control, an additional line of antimouse antibody (control line) ‘C’ has been immobilized at a distance from the test line on the strip. If the test is performed correctly, this will result in the formation of the pink band upon contact with the conjugate.

Clinical pictures

• A different course in adult and children both differing from that among infants.
• The difference in the clinical course id due to a difference in the bodily defense mechanism that every individual bears serious liver disease.
• Hepatocellular carcinoma.

Diagnosis

A serological test is used for diagnosis of acute and chronic hepatitis B infection.

• HBsAg: Present in chronic HBV infection.

: It is used as a general marker of HBV infection.

• HBsAb: Present during the convalescent phase of acute HBV infection

: These are used to document recovery or immunity to HBV infection

• Anti- HBcIgM is a marker of acute infection.
• Anti- HBeIgG indicates past or chronic infection.
• HBeAg indicates active replication of a virus.
• Anti- HBe: detection of anti-HBe antibodies indicates the virus is no longer replicating. However, the patient can still be positive for HBsAg.

For all these serological procedures ELISA is used. PCR assays are useful to detect viral DNA.

Laboratory diagnosis of HBV

Specimen types/handing

Serum or plasma should be separated from whole blood within 24 hrs for serology. These samples may be stored for 4-5 days at 2-8C or frozen indefinitely at -70C. Collection tubes should not contain heparin, as this can interfere with the performance of the assay. Repeated freeze/thaw cycles should be avoided.

For nucleic acid assays (viral levels, genotype, resistance testing), blood samples should be collected in tubes containing EDTA (lavender top) or citrate dextrose (yellow top). Processing should be done within 6 hrs, testing should be performed within 24 hrs or the sample should be frozen at -70C.

Direct Examination

Microscopy

• HBV detection by electron microscopy is expensive and laborious and is therefore only performed in a research setting.
• Three distinct morphologic entities can be visualized in the spherical surface antigens, the filamentous form of surface antigens and the double-shelled spherical particle containing the HBV virion, known as Dane particle.

Antigen detection

• HBV antigens are detection by ELISA or EIAs.
• The surface antigen (HBsAg) is detected in both as acute and chronic infections.
• The e antigen (HBeAg) is indicative of acute replication and has been associated with a higher risk of hepatocellular carcinoma (HCC).

Nucleic acid detection

• There are 3 commonly used methods for detecting HBV DNA: PCR branched chain DNA amplification (bDNA) and hybrid capture.
• Real-time PCR Quantitation of HBV DNA is highly sensitive and simple compared to bDNA
• Using the Taq Man platform, real- time PCR Quantitation is sensitive and linear from a range of 1.7-8.0 log 10 IU/ ml and is equally efficacious for genotypes A-H.

Serology

• A serological test is routinely used for the diagnoses of acute or chronic hepatitis B infection.
• The fetal core antibody (HBcAb) is indicative of past or current infection.
• Core TgM is produced in acute infections and the thus marker of acute infection. Core IgG indicates past exposure to HBV.
• Approximately 10 weeks after exposure, HBcIgM typically becomes positive.
• EIA, ELISA or chemiluminescence techniques are employed.

Point to remember:

• HBsAg is the first serologic marker of infection, appearing as early as 1-week post-exposure, but usually between 6 to 10 weeks.
• Shortly, therefore, the HBeAg is found in the blood several weeks after the HBeAg.

Interpretation of HBV serological tests is given below:

Treatment

• Interferon
• It is given for HBeAg-positive carriers with chronic active hepatitis.
• Response rate is 30-40%
• Alfa- interferon 2b ad alpha- interferon 2a ARE GIVEN
• Lamivudine
• It is nucleoside analog, which inhibits reverse transcriptase enzymes.
• However, the resistant mutants have been approved for this drug.
• Adefovir
• It is nucleoside analog reverse transcriptase inhibitor.
• Used for treatment of chronic Hep B in adults with evidence of active viral replication.
• It is more expensive and toxic.
• Entecavir
• Most powerful antiviral drug.
• It is similar to adefovir but less toxic.

Successful response to treatment will result in the disappearance of HBsAg, HBV DNA, and Seroconversion to HBeAg.

Prevention and control

• Vaccination
• The rDNA yeast derived vaccine
• Plasma-derived vaccine
• Hep B immunoglobulin providing immediate protection.
• Screening of blood donors for HBV infection.
• Encouragement of blood voluntary blood donation programs.
• Sterilization of instrument used in the high-risk occupational environment.
• Awareness program on carrier states and the risk of transmission.
• Media description of the disease and the transmission risk along with the measures of prevention.
• School level education on the disease and causative agent.
• Levels of HBsAb> 10 IU/Ml are considered protective.

REFERENCE

Cheesbrough, M.Medical Laboratory Manual for Tropical Countries. Vol 2. ELBS London, 2007.

Tille, P.Diagnostic Microbiology.13th. Elsevier, 2014.

Grenwood D, Slack RCB, and Peutherer J.Medical Microbiology.Dunclude Livingstone: ELBS, 2001.