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Epstein-Barr virus: the disease and Panbio product training

Epstein-Barr virus: the disease and Panbio product training. EBV: a ubiquitous pathogen. EBV is a member of the herpesviridae - the only human virus in the gamma herpesvirus subfamily (Lymphocryptovirus genus)

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Epstein-Barr virus: the disease and Panbio product training

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  1. Epstein-Barr virus: the disease and Panbio product training

  2. EBV: a ubiquitous pathogen • EBV is a member of the herpesviridae - the only human virus in the gamma herpesvirus subfamily (Lymphocryptovirus genus) • As with all herpesviruses, EBV establishes lifelong persistent infections that may undergo periodic reactivation, particularly in immunosuppressed hosts

  3. EBV: key features • Viron: Spherical, 150-200 nm diameter(icosahedral capsid, 100nm) • Genome: Circular, double-stranded DNA, ~172 kbp • Subtypes: No classification system exists, however different strains have been detected with varying genome structure, antigen expression and biological properties

  4. EBV: infection rates & transmission • It is estimated that over 90% of adults are seropositive for EBV • Infection usually occurs during childhood - many cases are asymptomatic • The virus is transmitted primarily by saliva - transmission by blood transfusion is rare • EBV is often found in the saliva of asymptomatic people

  5. Pathology of EBV infection 1. Primary infection occurs whenEBV encounters a circulating B cell in the oropharynx 2. Virus replication follows and the infected B cell undergoes growth-transformation 3. Chronic virus carrier state is established by the interaction between the EBV-transformed B cells and the T cell-mediated immune response 4. Occasional lytic infection (reactivation) leads to shedding of EBV in the saliva EBV also infects epithelial cells, but the significance of this is unknown - B cells are thought to be the mediator of primary and persistent infection

  6. Rare complications Hepatitis, Myocarditis Thrombocytopenia Clinical outcomes of EBV infection Common symptoms Fever, Sore throat Lymphadenopathy Fatal complications In males with an XLP gene, infectious mononucleosis can be fatal due to liver failure Uncommon symptoms Splenomegaly

  7. EBV & development of malignancies • EBV is linked to the development of several malignant tumours, including B cell neoplasms such as Burkitt's lymphoma, Hodgkin's disease, some forms of T cell lymphoma, undifferentiated nasopharyngeal carcinoma and a proportion of gastric cancers • Lymphoproliferative diseases in immunosuppressed hosts • Immunodeficient patients - e.g. AIDS, post-transplant - are susceptible to EBV-induced lymphoproliferative diseases, including lymphomas (diffuse polyclonal), lymphocytic interstitial pneumonitis and hairy oral leukoplakia of the tongue

  8. EBV & development of malignancies Post-transplant lymphoproliferative disorder: • Following organ transplantation, immunosuppressive therapy can promote the expansion of EBV-infected T cells Is EBV involved in the development of multiplesclerosis? • A recent study suggests a relationship between EBV infection and development of MS1: • Blood samples were collected from over 3 million US military personnel • The 83 patients who developed MS had significantly higher levels of EBV VCA IgG and EBNA antibodies

  9. EBV and development of malignancies • Burkitt's lymphoma • Burkitt's lymphoma is a rare cancer of the jaw, first described in East African children and young adults • Tumours in > 90% of African sufferers contain EBV DNA and EBNA antigen, compared with 20% of sufferers elsewhere • It is thought that EBV plays a causative role in early stages by immortalizing B cells, but it is not known how Burkitt's lymphoma cells escape elimination by the immune system

  10. EBV pathogenesis and serology • Following primary infection with EBV, antibodies to a number of viral antigens appear in the bloodstream

  11. EBV pathogenesis and serology • Viral capsid antigen (VCA) is the primary marker used for the diagnosis of EBV infection • VCA IgM appears within the first week of infection and remains detectable for up to three months • VCA IgG appears within 4-7 days after onset of symptoms and may persist for life • Epstein-Barr nuclear antigen (EBNA) IgM antibodies are detectable 3-6 days after onset of symptoms, often appearing before VCA IgM • EBNA IgG antibodies mark the transition from acute infection to convalescence and may persist for life • Early antigen diffuse (EA-D) IgG antibodies peak at three weeks and decline to undetectable levels by three months

  12. Diagnosis of EBV • Susceptibility: • If VCA antibodies are not found, the patient is susceptible to EBV infection • Primary infection: • VCA IgM present, EBNA IgG absent • Rising or high VCA IgG - antibodies appear 4-7 days after onset of symptoms • 80% of patients with active EBV infection produce EA-D IgG - levels peak at three weeks • Past infection: • EBNA IgG antibodies indicate past infection • In the absence of VCA IgM, VCA IgG may also be a marker of past infection

  13. Diagnosis of EBV • Reactivation: • EBNA IgG and elevated EA-D IgG antibodies suggest reactivated infection • VCA IgM may also be present • EBV reactivation is not associated with a clearly defined clinical syndrome • Chronic EBV infection: • Reliable lab evidence is seldom found in patients with symptoms of four months duration or more

  14. Why test for EBV antibodies? • Patient peace of mind • Rule out other more serious causes of malaise (e.g. cancer) • Determine EBV status prior to organ transplant

  15. Panbio offers 4 EBV ELISAs

  16. Which combinations should I recommend? • The combination of EBV ELISAs used will vary depending on the country. Such factors as the healthcare rebate system may influence the decision. • Panbio recommends the use of a VCA IgG ELISA (gp125 or p18), VCA IgM ELISA and EBNA IgG ELISA to obtain the most information about the patient’s serological profile • However, if only two ELISAs are able to be used, the combination of a VCA IgM ELISA and EBNA IgG ELISA is the most appropriate.

  17. Why offer two VCA IgG ELISAs? • More choice for the customer • p18 is popular in Europe

  18. VCA-p18 v gp125 • VCA-p18 • VCA-p18 is encoded within the open reading frame BFRF3 of EBV and is highly immunogenic in humans 1. • Panbio uses a synthetic VCA-p18 peptide • More sensitive than gp125 • p18 does not appear to have sequence homologues to other human herpesviruses1 and IgG to this antigen is not lost during immunosuppression4 • p18 is recognised by healthy EBV-seropositive persons worldwide therefore making it ideal for EBV VCA serologic testing2. • Tests on the use of p18 have demonstrated sensitivities of >95% for IgG detection 1,3. • VCA gp125 • 125 kD protein • Major component of the VCA complex • Panbio uses an affinity purified VCA gp125

  19. Which VCA IgG kit should I offer? • Inform potential customers of both Panbio VCA IgG ELISAs • Allow the customer to decide • Customer may wish to evaluate both • Panbio data suggests the performance of each is comparable.

  20. Why doesn’t Panbio offer anEA-D IgG ELISA? • EA-D IgG is of limited diagnostic value • EA-D IgG antibody testing is not useful in the diagnosis of IM as anti-EA antibodies, although transient like VCA IgG, only occur in 2/3 of patients (Field and Dwyer 1996). • Detection of EA-D IgG antibodies has been suggested indicative of a reactivated infection, however the presence for EA-D IgG antibodies in the absence of VCA IgM antibodies could also suggest a recent primary infection (Manual of Clinical Microbiology)

  21. EA-D IgG positive in current primary and recent primary. Could be positive or negative in the case of EBV reactivation EA-D IgG response Table taken from the Manual of Clinical Microbiology

  22. Product positioning • The Panbio EBV range provides a convenient, user-friendly alternative to heterophile antibody tests and IFA. • Panbio EBV ELISAs offer superior sensitivity and specificity • Target: • Private pathology laboratories • Hospital pathology laboratories in particular hospitals performing transplants

  23. Panbio EBV ELISAs: superior sensitivity and specificity • Sensitivity of the Panbio EBV ELISA kits is > 95% • Importantly specificity is also very high • The balance between sensitivity and specificity provides excellent performance

  24. Superior F-values equals confidence in your results Superior sensitivity and specificity

  25. Panbio EBV ELISAs • Accurate, meaningful results • Numerous studies confirm Panbio's superior sensitivity and specificity (F-value) 7,8,9 • Reliable performance • Consistent results reduce the need for repeat testing • Easy to use • All liquid reagents are supplied colour-coded and ready to use - reduces errors and preparation time • Compatible with standard 96-well microplate technology • 8-well break-apart strips reduce costs through efficient kit usage • Troubleshooting guides and technical support hotline

  26. Specific serum antibodies combine with EBVantigens attached to the polystyrenesurface of the microwells Washing removes residual serum Peroxidase-conjugated anti-human specific immunoglobulin is added The colourless substrate, tetramethylbenzidine/hydrogen peroxide (TMB / H2O2) is hydrolysed to a blue chromogen Stopping the hydrolytic reaction with acid turns the TMB yellow Colour development indicates the presence EBV antibodies in the test sample

  27. Publications/Posters • Mitchell et al. (1998). Comparison of commercially available Epstein-Barr virus IgG ELISAs utilising viral capsid antigen (VCA) gp125 and p18. Panbio Limited • Dickeson et al. (2001). Evaluation of commercial E.I.A. kits for the detection of Epstein Barr virus viral capsid antigen IgG and IgM. Poster P4.9 Australian Society for Microbiology, Annual Scientific Meeting, Perth, Australia, October 2001. • Cannone, A.M. et al. (2002). A comparison of three commercially available Epstein-Barr virus VCA-p18 IgG ELISAs. Poster presented at the Australian Society for Microbiology Annual Scientific Meeting, Melbourne, Australia, September 29 – October 3, 2002.

  28. Mitchell et al., 1998 • The purpose of this study is to compare the Panbio EBV VCA P-18 ELISA which utilises recombinant VCAp18 against the PANBIO VCA IgG ELISA kit which is based on gp125. • A total of 143 samples submitted for routine EBV analysis were simultaneously tested on the PANBIO VCA IgG ELISA (gp125), and the PANBIO VCA-p18 IgG ELISA. • Although there was good agreement between the two VCA IgG ELISAs (88%), it appears that the p18 kit is more sensitive than the gp125. • The VCA-p18 IgG ELISA is a sensitive method for the detection of IgG antibodies to VCA and is useful to assist in the diagnosis of EBV infection and determination of EBV immune status.

  29. Dickeson et al., 2001 • EBV ELISAs from Biotec, Diesse, Gen-Bio, Meridian, Panbio, Zeus, Trinity and IBL were compared using a panel of serum samples • The Panbio and Biotec VCA IgG kits performed the best using IFA as the reference method • The Panbio VCA IgG kit was one of the kits recommended by th investigators and appeared to be the most suitable for the laboratory with positive and negative predictive values above 95%.

  30. Cannone et al., 2002 • Study evaluating three VCA p-18 ELISA kits from Panbio Limited, DiaSorin and Trinity Biotech. • The results of this study indicate that the DiaSorin kit, while having high sensitivity for past infection, has the lowest specificity of the 3 kits. In contrast the Trinity Biotech kit has excellent specificity but poor sensitivity. The Panbio kit demonstrates specificity and sensitivity for past infection of greater than 90%. • In this study the Panbio EBV VCA-p18 IgG ELISA has performed the best in terms of combined specificity and sensitivity for past infection and is suitable for both routine diagnosis and pre-transplantation screening.

  31. Ordering information

  32. Ordering information

  33. References • van Grunsven, W.M.J., W.J.M. Spaan and J.M. Middeldorp. (1994) Localization and diagnostic application of immunodominant domains of the BFRF3-encoded Epstein-Barr virus capsid protein. • van Grunsven, W.M.J., A. Nabbe and J.M. Middeldorp. (1993) Identification and molecular characterization of two diagnostically relevant market proteins of the Epstein-Barr virus capsid antigen. J. Med. Virol. 40:161-169. • Hinderer, W., D. Lang, M. Rothe, R. Vornhagen, H.H. Sonneborn and H. Wolf. (1999) Serodiagnosis of Epstein-Barr virus infection by using recombinant viral capsid antigen fragments and autologous gene fusion. J. Clin. Microbiol. 37:3239-3244. • Bauer, G. (2001) Simplicity through complexity: immunoblot with recombinant antigens as the new gold standard in Epstein-Barr virus serology. Clin. Path. 47:223-230. • Field, P.R. and D. E. Dwyer. (1996) Difficulties with the serological diagnosis of infectious mononucleosis: a review of the RCPA quality assurance programs. Pathology. 28:270-276. • Levette, ET. Epstein-Barr virus. p912-918. In Murray (ed.) Manual of Clinical Microbiology, 7th edition. American Society for Microbiology, Washington. • Dickeson, D.J., Chan, S., Patel, D.B., (2001), Evaluation of Commercial E.I.A. Kits for the Detection of Epstein Barr Virus Viral Capsid Antigen IgG and IgM,. Poster P4.9 ASM Annual Scientific Meeting, Perth, Australia. • Gibb, R. and McGoldrick, C. (2000) Serological assays for EBV: comparison of the Gull and Panbio EBV VCA IgG, EBV VCA IgM and EBNA IgG assays. Panbio News, Issue 17:5. • Cannone, A.M., Gardam, M.J., Gould, S.N. (2002) A comparison of three commercially available EBV VCA-p18 IgG ELISAs. Poster presented at the Australian Society for Miocrobiology Annual Scientific Meeting, Melbourne, Australia.

  34. www.panbio.com

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