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The one-step solution for the diagnosis of Infectious Mononucleosis



About IM

 

Introduction

 

Infectious mononucleosis (IM) was first described in 1889 by Pfeiffer, when it was known as glandular fever1. The disease is characterised by a triad of symptoms; fever, pharyngitis and cervical lymphodenopathy for which treatment is largely supportive with enforced bed rest and analgesics to control the pain2. The name infectious mononucleosis refers to the appearance of infected white blood cells, as they appear to have a grossly distorted single nucleus, together with an increase in the number of monocytes1. However, diagnosis of the disease cannot be made on cell appearance alone as other disease states can produce cells of similar appearance such as malignant lymphoma3. Diagnosis of IM has therefore relied upon the demonstration of IgM antibodies directed against the causative agent, the Epstein-Barr virus4.

 

Epstein-Barr Virus

 

The Epstein-Barr virus (EBV) was first described in 1964. It is a member of the herpes virus group having the ability to immortalise human B lymphocytes. The virus is ubiquitous, through genital and oropharyngeal secretions, and as such, in most populations, over 95% of individuals show some level of EBV antibody5.

 

The only known target cells of EBV are B lymphocytes and epithelial cells. During the lytic phase of the disease, the virus enters epithelial cells via the calls CD21 surface cell receptor. Once inside the cell it inserts itself into the nuclear genome. The virus utilises the cell genome for synthesis of its own proteins and for replication into virons. Replication causes a proliferation of B lymphocytes. Latency is characterised by an increase in the number of circulating B lymphocytes which correlates with the severity of the disease. EBV Nucleic Antigen (EBNA) produced during latency allows T lymphocyte cells to seek out and destroy the virus. Cytokines are produced by the T cells in response to increased production of B lymphocytes and it is thought that the cytokines contribute to the symptomology of the disease5. The T cell response is so efficient in eliminating the disease that serious complications only arise in those with depleted immunity, and the majority of patients seroconvert with few symptoms. A small number of infected B lymphocytes circulate following primary infection, leading to a permanent carrier state. Unlike other herpes viruses, EBV does not lead to re-infection except in the immuno-compromised6.

 

Epidemiology

 

Infectious mononucleosis can occur at any age, but peak incidence occurs between 15 and 19 years of age with 345-671 cases per 100,000 per year2. Although young children may become infected with EBV, the majority seroconvert with few or no symptoms, whereas infection of adolescents or young adults results in the disease state in 30-75% of cases6. In adulthood, the disease can produce severe symptoms, although by this age the majority of the population would have already been in contact with the virus with little or no consequence, and so development of the disease in this age group is less common2. EBV infection results in permanent carrier state, but differs from other herpes viruses in that during the latent phase of the disease there is no resurgence of viral activity. However, transplant patients and the immuno-compromised may show some viral reactivity following primary infection6.

 

In childhood, EBV infection is associated with low socio-economic status, poor hygiene and crowding. For this reason, IM is less common in developing countries as the majority of the population would have already acquired immunity to the EBV virus during childhood. In affluent populations, improvements in living conditions over the last few decades has led to shift in the peak age of EBV infection, with many more individuals not coming into contact with the virus until adolescence, which has led to an increased incidence of infectious mononucleosis7.

 

Transmission of EBV is primarily though oropharyngeal secretions from carriers and infected individuals and as such IM is commonly referred to as the ‘kissing disease’. The rate of shedding of EBV varies from individual to individual, although it follows that the immuno-suppressed shed EBV at a far greater rate. Of previously infected individuals, 20% intermittently shed virus in saliva, and can be recovered from infected individuals in 15-20% of attempts. The requirement for salivary contact explains how the disease is easily transmitted to partners but the incidence in the home schools, hospitals, college dormitories or military barracks is relatively low6.

 

Diagnosis

 

Although IM can develop severe symptoms such as splenic rupture, and haemolytic anaemia, many IM patients exhibit vague symptoms such as a fever and sore throat that are common to  many other disease states such as that caused by cytomegalovirus, lymphoproliferative disorders or even a common cold. In addition to the symptoms, many of the immune responses observed in IM are also found in other disease states. As EBV establishes life long infection in the host, identification of IM cannot be confirmed with the presence of EBV alone. This presents a diagnostic challenge to the physician. Diagnosis of IM therefore relies on a combination of haematological, serological and symptomatic assessment of the patient in order to decide on the best course of treatment for the illness6.

 

Haematological indicators of IM are the rise in white blood cell count to approximately 10-15,000 cells per mm2 in the first 2 to 3 weeks of the disease. This leads to lymphocytosis in approximately 70% of cases. Both B and T lymphocytes contribute to 10-30% of the characteristic increase in atypical lymphocytes but in older patients this increase is not as marked. Compared with normal lymphocytes, these atypical cells are generally larger with a distorted shaped nucleus. However, the lymphocytes are not exclusive to IM and may be associated with other disease states such as that caused by cytomegalovirus, viral hepatitis, measles, rubella, and drug reactions. In addition to lymphocytosis, more than 50% of patients develop mild thromobocytopenia6.

 

The most effective serology test for IM is the demonstration of IM heterophile antibody. Heterophile antibodies have the capacity to react with antigens that are unrelated to the one producing the antibody response. IM heterophile antibody will react with sheep, bovine and horse erythrocytes but does not react with EBV specific antigens. Other heterophile antibodies such as Forssman and serum sickness antibodies are produced by a variety of other diseases, and these must be differentiated from IM heterophile antibody for accurate diagnosis8.

 

The presence of IM heterophile antibodies was first demonstrated by Paul and Bunnell in 1932 after they demonstrated the agglutination of IM heterophile antibodies to sheep erthrocytes9. Following from this, Davidsohn and Beer showed the need for differential absorption of sera to remove other non-specific heterophile antibodies10,11. Fletcher and Woolfolk showed that antigens derived from bovine erythrocytes were more specific to the IM heterophile antibody than erythrocytes produced from sheep or horses12.

 

During the acute phase of the illness, IM heterophile antibodies are produced in 80-90% of cases. The IM heterophile antibodies are usually demonstrable one week after onset of the illness, peaking at 2-4 weeks, and declining to lower levels after 12 weeks2. One year after the onset of the illness, IM heterophile antibodies have been detected in patient's serum, although this is not common4,8. If testing is carried out before sufficient antibody is present then a false negative result can be obtained, hence further testing is required at a later date.

 

In addition to this, 10-20% of adults and 50% children fail to produce the IM heterophile antibody. Diagnosis of IM based purely on the presence of IM heterophile antibodies can therefore lead to false negative results. EBV specific serology testing is therefore required in patient's whose clinical symptoms suggest IM but no heterophile antibody has been demonstrated2. However, these tests are more costly than the demonstration of the IM heterophile antibody.

 

EBV Serology

 

Infection with EBV is characterised by the development of specific antibodies to antigenic components of the virus. These antigens appear at different stages of infection and differ in lytic versus latent infection. Antibodies to EBV antigens measured for clinical purposes are those to viral capsid antigen (VCA), early antigens (EA), and Epstein-Barr nuclear antigen (EBNA). EA are expressed early in the lytic cycle, while VCA and membrane antigens are structural proteins expressed late in the lytic cycle. EBNA is expressed in cells that are latently infected. Antibodies to these proteins are measured by enzyme immunoassays, indirect immunofluorescence assays, and immunoblot assays13,14.

 

VCA-IgM represents the most useful EBV serology test in the diagnosis of acute IM. VCA-IgM is usually measurable at symptom onset, peaks at 2-3 weeks, then declines and becomes undetectable by 3-4 months; hence it is a good indicator of primary infection. VCA-IgG rises shortly after symptom onset, peaks at 2-3 months, then drops slightly but persists for life. Antibodies to EBNA appear during convalescence and remain present for life. Antibodies to EA appear transiently for up to 3 months during the acute phase of IM in 85% of patients. However, a diagnosis of chronic EBV should not be based on the presence of antibodies to EA since elevated anti-EA titres may also be found in patient's with other diseases as well as healthy individuals with past EBV infections13,14.

 

The most efficient and cost effective method of diagnosing IM in the early stages of the disease is therefore through demonstrating the presence of IM heterophile antibodies. Clearview IM uses a glycoprotein from bovine erythrocytes and is therefore highly specific, requiring no pre-treatment of the specimen and producing clear, unambiguous results.

 

 

References

 

  1. Davidsohn I. (1937) Serological Diagnosis of Infectious Mononucleosis: JAMA. 108(4): 289-295.
  2. Bailey R.E. (1994) Diagnosis and treatment of infectious mononucleosis. Am Fam Physician. 49(4), 879-88.
  3. Plumbley J.A., Fan H., Eagan P.A., Ehsan A., Schnitzer B. & Gulley M.L. (2002) Lymphoid tissues from patients with infectious mononucleosis lack monoclonal B and T cells. J Mol Diagn. 4(1): 37-43.
  4. Elgh F. & Linderholm M. (1996) Evaluation of 6 commercially available kits using heterophile anigen for the rapid diagnosis of Infectious Mononucleosis compared with Epstein-Barr virus specific serology. Clin. Diag. Virol. 7, 17-21.
  5. Andersson J. (2000) An Overview of Epstein-Barr Virus from Discovery to Future Directions for Treatment and Prevention: Herpes. 7(3): 76-82.
  6. Moffat L.E. (2001) Infectious mononucleosis. Prim Care Update Ob/Gyns. 8: 73-77
  7. Morris M.C. & Edmunds W.J. (2002) The Changing Epidemiology of Infectious Mononucelosis. J Infect. 45, 107-132.
  8. Gray J.J., Caldwell J. & Sills M. (1991) The rapid serological diagnosis of infectious mononucleosis. J Infect. 25, 39-46.
  9. Paul J.R. & Burnell W.W. (1932) The Presence of Heterophile Antibodies in Infectious Mononucleosis. Am. J. Med. Sci. 183, 90-104.
  10. Davidsohn I. (1937) Seriological Diagnosis of Infectious Mononecleosis. JAMA. 108, 289-295.
  11. Beer P. (1936) The Heterophile Antibodies in Infectious Mononucleosis & After the Injection of Serum. J. Clin. Invest. 15, 591-599.
  12. Fletcher M.A., Woolfolk B.J. (1971) Immunochemical Studies of Infectious Mononucleosis: 1 Isolation & Characterisation of Heterophile Antigens from Haemoglobin-Free Stroma. J. Immunol. 107, 842-853.
  13. Field P. R. & Dwyer D. E. (1996) Difficulties with the serological diagnosis of infectious mononucelosis: a review of the RCPA Quality Assurance Programs. Pathology. 28, 270-276.
  14. NIH Conference (1993) Epstein-Barr virus infection: biology, pathogenesis and management. Annals of Internal Medicine. 118, 45-58.
 


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