Talk overview. Case 1. Case Examples 21/07/2017. Epstein Barr Virus (EBV) quantification in health and disease Bit of background Recent case examples
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1 Belfast Pathology 2017 EBV Quantification in health and disease Talk overview Epstein Barr Virus (EBV) quantification in health and disease Bit of background Recent case examples Susan Feeney PhD FRCPath Regional Virus Laboratory Royal Victoria Hospital Belfast Health and Social Care Trust EBV Gamma-herpesvirus Infectious mononucleosis/mono/glandular fever One of oldest and most common human viruses, infecting roughly 95% adults worldwide Tropism B cells and epithelial cells Primary lytic infection (replication) in oropharynx Latency programs I, II or III (B cells) EBV transformation/ immortilisation of B cells Malignancy association, Burkitts lymphoma, Hodgkins lymphoma, EBV positive diffuse large B-cell lymphoma (DLBCL) and Nasopharyngeal Carcinoma (NPC). EBV is a known risk factor for Post-transplant lymphoproliferative disease (PTLD) in solid organ and hematopoietic Known trigger for Hemophagocytic Lymphohistiocytosis (HLH) The EBV life cycle. Thompson M P, Kurzrock R Clin Cancer Res 2004;10: by American Association for Cancer Research Case Examples EBV Virology test requests Case 1 Mononucleosis 17yr male Amateur rugby player Case 1 Query post viral illness, fatigue Enlarged lymph nodes Enlarged spleen US Laboratory received blood sample for query Glandular fever 1
2 EBV in Health and disease Infectious mononucleosis Infectious mononucleosis/mono/glandular fever Self-limiting Childhood- <10yr, Asymptomatic or mild, brief Teenager/adult - Symptomatic fatigue, fever, inflamed throat, swollen lymph nodes, enlarged spleen, swollen liver, rash Severity of primary EBV infection in adults increases with age Infectious mononucleosis- Acute infectious disease accompanied by atypical large peripheral lymphocytes (Downey cells) activated by CD8 T cells responding to EBVinfected B cells Diagnostic complexities in virology: Clinical manifestations (generally include sore throat, fever, lymphadenopathy and malaise) Haematological evidence of lymphocytosis Supportive serological laboratory findings Heterophile antibody titre Paul-Brunnell, monospot Anti-VCA (Antibody to Viral Capsid Antigen) IgM and IgG Anti-EA (Antibody to EBV Early Antigen) diffuse/restricted Anti-EBNA (Antibody to EBV Nuclear Antigen) Molecular laboratory findings EBV DNA PCR Case 1 Results: Tests Results EBV VCA IgG Reactive EBV IgG numerical ( 20 Reactive) EBV VCA IgM Reactive EBV IgM numerical ( 40 Reactive) EBV Quantitative PCR Positive EBV Quantitative PCR CT EBV Quantitative PCR numerical 5.82 x 10 2 (5.82E2 or 582 IU/ml) Kinetics of EBV-specific antibodies and viral load in infectious mononucleosis. PCR POSITIVE Detectable by PCR EBV Laboratory results should not be interpreted in isolation; patient has an enlarged Spleen. Serological evidence of current acute infection, PCR evidence of current EBV infection, although approaching end of early stage. Oludare A. Odumade et al. Clin. Microbiol. Rev. 2011;24: PCR amplification Serological EBV VCA IgG IgM ± PCR = DIAGNOSIS CT- Cycle Threshold Cycles 1-40 Cycles approaching CT 40 indicate very low concentration target material This Case 1 : CT = equates to 582 IU/ml 2
3 CASE 2 Mononucleosis and the rest Case 2 25yr old male Persistent dry cough Loss appetite Poor sleep Flu like, aching all over, sore throat Contacts GP OOH March 2017: SpNo Specimen Arrival Date Request(s) Result Detail Reactive IgM V Serum 09-Mar-17 EBV Monospot PCR Neg OOH GP contacted SpNo Specimen Arrival Date Request (s) Result Action BHSCT Virology Laboratory investigation of Infectious Mononucleosis (IM): Triad of fever, pharyngitis, and lymphadenopathy OOH GP contacted: Flu-like headache Moderate-severe symptoms High risk of patient Pregnant V Serum 09-Mar-17EBV Monospot Reactive IgM , PCR Negative This comment attached to result of EBV VCA IgM Positive/PCR neg prompted GP to call and add on HIV test Comment: IgM pos not confirmed by PCR. Result would suggest a) Specific IgM where virus is no longer detectable b) Unrelated acute infection can result in the nonspecific polyclonal activation of memory cells and the release of EBV VCA IgM. Consider CMV or HIV testing c) If results not acute this result may represent subacute EBV infection as IgM can persist for up to 3 months Investigate IM EBV Serology: VCA IgM, VCA IgG IgM Positive ± IgG Positive qpcr EBV qpcr EBV +ve FBC, Lymphocytosis Screen qpcr EBV -ve IgM negative IgG Positive IgM & IgG Negative HIV, CMV Toxoplasmosis Recode V Serum 15-Mar-17 Blood HIV HIV Positive Not known to be high risk other than foreign travel PCR confirmed EBV IM # EBV IM unconfirmed by PCR may be Non specific, consider other causes Negative Positive Other possible diagnosis, eg Viral Hepatitis Lymphoma Acute Leukaemia CASE 3 EBV driven HLH 37yr old male Irish traveller Case 3 1 yr hx PUO, intermittent night sweats, h-ache, joint pains, non prod cough, LOA, nausea No wt loss, no foreign travel Emergency admission in Jan for 1 week with night sweats Low WCC, low platelets, CRP 216, Hb 13.2, DLFTS No lymphadenopathy CT chest- small node RUL- insignificant size CT abdo- enlarged spleen, no nodes suitable for bx Feb/Mar 3 separate admissions RVH 3
4 Case 3 April: admitted A&E RVH PUO, pancytopenia, hyponatraemic, acute liver, renal and haematological failure- for monitoring and fluid balance Reviewed by: Immunology- Neg antoimmunity Rheumatology-?Stills-unlikely Haematology- BM haemophagocytosis ID- initially no obvious infective process,?hlh Fulfilled most criteria for EBV assoc HLH Definition HLH aka Haemophagocytic LymphoHistiocytosis A syndrome Impaired/absent NK cell and CTL function Uncontrolled immune activation Cellular damage and MOF HLH diagnostic guidelines Molecular test consistent with HLH: Perforin PRF mutations, SAP mutations, MUNC13-4 mutations Common that the 1 st BM examination does not reveal haemophagocytosis Fulfil one of following criteria: 5/8 of: Fever Splenomegaly Cytopenia (2 cell lines) Hypertriglyceridaemia Haemophagocytosisin BM, spleen, LN w/o lymphoma Low/absent NK cell cytotoxicity Hyper ferritinaemia >500ng/ml Elevated soluble CD25 Case th April: chemo-immunotherapy: Dexamethasone Aciclovir Etoposide Cyclosporine IV Ig Rituximab 19/04/10 tx withdrawn, deceased Case 3 Virology 30/Dec GP Mono Neg 18/Jan DHH HBV, HCV, HIV, Mono, CMV IgM, HAV Neg 05/Feb 2F Mono, CMV Neg 27/Feb 6D Flu, RSV Neg 08/Mar 6E Q fever Neg 08/Mar 6F B19 (BM) Neg EBV PCR 228 IU/ml 14/April 7A Mono, CMV, Hep B,C Neg 16/April ICU HSV, VZV, B19 Neg EBV (EDTA) 2.84x10 5 IU/ml EBV (serum) IU/ml EBV IgG Present BM bx MØ with phagocytosed RBCs 4
5 EBV associated HLH HLH often infectious trigger EBV HLH has worst prognosis Cytokine storm worse in EBV HLH EBV poor response to aciclovir However, Etoposide - inhibits EBV nuclear Ag in EBV infected cells (+toxic to mφ) Case 4 PTLD 62 yr old male Case 4 Living donor transplant Sept 2016, membranous glomerulonephritis Donor son Admitted 2 month post transplant Dec 2016 Pancytopenia acute graft dysfunction, pyrexic, wt loss High viral load EBV Lymphoproliferative disorders Molecular: SpNo Specimen Arrival Date Codes Result V EDTA 05-Dec-16 EBV qpcr 4.6 x 10 6 V EDTA 08-Dec-16 EBV qpcr 6.6 x 10 6 V EDTA 12-Dec-16 EBV qpcr >15 x 10 6 V EDTA 12-Dec-16 EBV qpcr >15 x 10 6 V EDTA 12-Dec-16 EBV qpcr >15 x 10 6 V EDTA 15-Dec-16 EBV qpcr >15 x 10 6 V Serum 15-Dec-16 EBV qpcr >15 x 10 6 V Serum 19-Dec-16 EBV qpcr 2.0 x 10 6 V EDTA 22-Dec-16 EBV qpcr 0.9 x 10 6 V EDTA 28-Dec-16 EBV qpcr 0.05 x 10 6 V Serum 28-Dec-16 EBV qpcr 0.5 x 10 6 Lymphoproliferative disorders Serology: SpNo Specimen Arrival Date Codes Result V Serum 04-Mar-15 EBV IgG 10 V Serum 06-Aug-15 EBV IgG 10 V Serum 19-Sep-16 EBV IgG <10 Transplant Sept 2016 V EDTA 08-Dec-16 EBV IgG <10 V EDTA 12-Dec-16 EBV IgG <10 V Serum 13-Dec-16 EBV IgG <10 V EDTA 15-Dec-16 EBV IgG 302 post IVIG V Serum 15-Dec-16 EBV IgG 286 post IVIG V Serum 19-Dec-16 EBV IgG 326 post IVIG V Serum 28-Dec-16 EBV IgG 134 post IVIG Case 4 Transfer ICU, acute kidney injury, liver injury, splenomegaly Fulminating EBV Deceased 29 Dec 2016 PM: multiple abscesses heart due to Candida albicans septicaemia Systemic histological findings of fungi forming microabscesses Multi-organ polymorphic B cell Lymphoproliferative Disorder PTLD 5
6 Lymphoproliferative disorders Post mortem samples: Histology L SpNo Specimen Arrival Date Codes Result V Lung 30-Dec-16 EBV qpcr 1.5 x 10 6 V Liver 30-Dec-16 EBV qpcr >1.5 x 10 6 V Kidney 30-Dec-16 EBV qpcr >1.5 x 10 6 G Deceased , ICU, mof, acute liver injury, d-lfts, thrombocytopenic/dic PM: Lymphoreticularpathology review concluded Polymorphic B Cell Lymphoproliferative Disorder PTLD This is an image of the kidney taken at post mortem showing a glomerulus (G) and dense lymphoid infiltrate (L) Courtesy Dr Brian Herron Histology EBV mismatch kidney transplant Unusual EBV IgG seronegative in adulthood (only 3-5% IgG Negative) Living transplant son EBV IgG Positive The infiltrate stains strongly positive for EBV with immunohistochemistry EBV positive post-transplant with no demonstrable light chain restriction on immunohistochemistry Courtesy Dr Brian Herron EBV in the Health and disease Infectious mononucleosis is, with rare exceptions, asymptomatic or self-limited disease. Complications uncommon and rare Haematologic:, haemophagocytic syndrome (HLH) Immunologic: lymphoproliferative syndromes (PTLD), X-linked LPD Neurological; cardiac, respiratory, renal, oncological... Essential to request correct tests, serological or molecular, in clinical context Regional Virus Lab Molecular suite - automated interfaced technology Quantification in form qpcr essential in EBV health and disease serological results for GF/mono whereas Molecular Quantification essential in HLH and PTLD 6
7 Pathology Modernisation Quantitative Realtime PCR WHO Standardised calibration Thank you RVL: Prof Peter Coyle, Dr Conall McCaughey Dr Tanya Curran, Dr A Watt and all the laboratory staff Dr Brian Herron, Consultant Pathologist Dr Chris Hill, Renal Consultant 7
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