Infectious Mononucleosis Tests

Synonyms: Heterophil agglutination test, Paul-Bunnell-Davidsohn test; Forssman antibody test; Monospot® test

Following infection with Epstein-Barr virus (EBV) in infectious mononucleosis (IM), 85-90% of patients produce specific IM heterophile antibodies. These antibodies can be detected by:

• Paul-Bunnell test: sheep red blood cells agglutinate in the presence of heterophile antibodies.
• Monospot® test: horse red cells agglutinate on exposure to heterophile antibodies. The Monospot® uses this in conjunction with the principle of the Davidsohn differential test. Sensitivity and specificity for Monospot are 94% and 98% respectively.¹
• Davidsohn differential test:
  • Agglutination of the sheep or horse red cells is not specific and only determines the presence or absence of heterophile antibodies including non EBV Forssman heterophile antibodies.
  • Guinea pig kidney cells contain the Forssman antigen therefore absorbing serum with cells removes the Forssman antibodies. Serum 1
  • Ox (beef) erythrocytes contain the IM antigen therefore absorbing serum with erythrocytes removes the specific EBV antibodies. Serum 2
  • When the two absorbed serums are mixed with sheep/horse red cells a positive result is indicated by stronger agglutination with serum 1.

Positivity increases during the first 6 weeks of the illness and so the heterophile antibody test results may be negative early in the course of EBV infectious mononucleosis. The titre does not correlate with the severity of the disease. Agglutinins remain in the blood for 4 to 8 weeks but may remain positive in low levels for up to one year.

False positive heterophile antibody tests are rare but may be associated with:

• Rubella
• Cytomegalovirus
• Toxoplasmosis
• HIV
• Herpes simplex virus infection
• Malaria
• Viral hepatitis
• Rheumatoid arthritis
• Systemic lupus erythematosus
• Leukaemia
• Lymphomas
• Burkitt's lymphoma
• Pancreatic cancer

The false-negative rate is as high as 25 percent in the first week, approximately 5 to 10 percent in the second week, and 5 percent in the third week of illness.² Heterophile antibody tests are less sensitive in patients younger than 12 years.²

• Occur in 10% of adults and 50% of children.
• May occur if testing is performed too early in the course of the illness.
• The heterophile test is less useful in children younger than 2 years, in whom the results are frequently negative. One study found fewer than half of children aged under four to have detectable heterophile antibodies at any time.³
• False negative results are also more likely to occur in elderly patients.⁴

• If a false-positive test result is suspected, then specific testing using an EBV-based antibody serological test may be useful.²
• The antibody response to specific EBV serological testing consists of measuring the antibody response to surface and core EBV viral proteins. The most useful EBV-specific antibodies are the VCAs (Viral Capsid antigens) and the EBNA (EBV Nuclear Antigen). Both VCA and EBNA antibodies are usually reported as IgG or IgM antibodies:
  • VCA-IgG and VCA-IgM tests are useful in diagnosing patients who have highly suggestive clinical features but negative heterophile antibody test results. There is a high (90%) agreement between the heterophile antibody tests and the VCA-IgM ELISA, but the VCA IgM ELISA is more sensitive.⁵
  • IgG and IgM antibodies directed against the VCA of EBV are useful in differentiating recent infection from previous infection.
  • Antibody to Epstein-Barr nuclear antigen (EBNA), while typically not detectable until six to eight weeks after the onset of symptoms, can help distinguish between acute and previous infections.
• EBV IgM VCA titres decrease in most patients after 3-6 months but may persist in low titre for up to 1 year.
• EBV IgG VCA antibodies rise later than the IgM VCA antibodies but remain elevated with variable titres for life.
• Persistent IgG does not indicate chronic infectious mononucleosis and is not relevant in the assessment of chronic fatigue syndrome.⁶
• False-positive VCA antibody titre results may occur on the basis of cross-reactivity with other herpes viruses, e.g. CMV, or with unrelated organisms, e.g. Toxoplasma gondii.
• As with the heterophile test, the EBV antibody response may be falsely negative early in the course of the infection. False negativity also may occur in young children (less than 2 years old).