English Division, 6-year programme Class 10 DNA viruses I. Seminar: General properties, pathogenesis and clinial features of DNA viruses from Herpesviridae family II. Assays to be performed: 1. Paul-Bunnel-Davidsohn assay (infectious mononucleosis) 2. Detection of specific antibodies against EBV - interpretation - infectious mononucleosis - reactivation in immunocompetent person - reactivation in immunocompromised patient. III. Demonstrations: 1. Laboratory diagnosis of viral meningoencephalitis application of multiplex-pcr method. 2. ELISA: detection of antibodies and viral antigen in patient s serum in suspected infection with parvovirus B19. 3. Viral CPE: adenovirus, herpes simplex virus 1
Serological diagnosis in infectious mononucleosis (IM) Case report (patient 1): 18-year-old male presented to the physician because of sore throat, malaise and high fever. On physical examination enlarged tonsils and neck lymph nodes, splenomegaly and liver tenderness were noticed. Blood picture presented mild leukocytosis (12 500/mm 3 ) and appearance of atypical lymphocytes. After initial recognition of infectious mononucleosis, physician decided to order serological diagnostic tests which should confirm his diagnosis. 1. Monospot test (modified Paul-Bunnel-Davidsohn assay) In the course of infectious mononucleosis, increase of the level of heterophile antibodies often appears in the first week after first manifestations of the infection. To detect heterophile antibodies, agglutination assays are used. In these assays, stabilized horse erythrocytes containing heterophile antigens (Paul-Bunnel-Davidsohn assay PBD, monospot test) or latex particles covered with heterophile antigen are used. Assay is performed with patient s serum. To be performed: On microscope slides check the reaction of standard sera (positive and negative) with erythrocytes. Next, perform the test with patient s serum. Specific humoral response in IM: In the course of infectious mononucleosis, first anti-vca antibodies arise (against viral capsid antigen), a little bit later anti-ea (against early antigen), but it s worth stressing that these antibodies don t appear in all patients with IM symptoms. Finally, anti-ebna antibodies appear in the patients sera (against nuclear antigen). High titer of anti-ebna antibodies persisting for a long time is characteristic for chronic infections and lymphoproliferative changes as a result of EBV infection. Clinical meaning of anti-ebv antibodies VCA IgM: indicative for acute phase of infection, detectable as a first type of specific antibodies in primary infection, and also during latent virus reactivation VCA IgG: indicator of past infection, detectable in high titers for years EA IgM: like VCA IgM, marker of active infection, they arise in course of primary infection, rarely in reactivations. Response in EA IgM is much less often than in VCA IgM EA IgG: characteristic for convalescent phase. Observed in small percent of infected individuals EBNA IgM: meaning is not fully explained. Probably correspond with establishing of viral latency EBNA IgG: they appear relatively late after convalescence (3-4 months after infection), detectable for years 2
Iga Gałat, 2016 2. ELISA assay in the diagnosis of EBV infection detection of specific antibodies Infectious mononucleosis (patient 1) Detection of specific antibodies against EBV antigens is a good method for infection confirmation. Comparison of the results of specific serological tests, clinical symptoms, blood tests and non-specific tests (PBD, latex test) is helpful in making a correct unambiguous diagnosis of infectious mononucleosis. Latent virus reactivation (patients 2 and 3) Besides diagnostics in suspected infectious mononucleosis, which is the most popular form of EBV infection in immunocompetent population, specific serological assays can be helpful in determination of humoral response in patients from risk groups for severe forms of EBV infections. In immunosuppressed patients, individuals with inherited and acquired immunodeficiencies, and in patients with neoplasms, both primary infections and latent virus reactivations may lead to a variety of clinical syndromes: limphoproliferative diseases, chronic active EBV infections, or syndromes resembling mononucleosis, but with more severe symptoms. Patient 2. 37 year-old man reported to a family physician because of general malaise, physical weakness, loss of apetite, fatiguability and increased body temperature (38 39ºC). Symptoms appeared few days ago. Patient demonstrated skin paleness, enlargement of cervical and submandibular lymph nodes and hepatomegaly. Blood examination revealed slightly decreased number of RBCs, thrombocytopaenia and increased number of 3
leukocytes. Liver aminotransferases and billirubin were at increased levels. Patient was redirected to a hospital for further diagnosis and treatment. After admission, examination for specific EBV antibodies was performed, as well as serological testing for CMV and toxoplasma infection. (to be performed: ELISA interpretation) Patient 3 Woman, 43 y.o., was hospitalised because of acute lymphoblastic leukemia. At day 42 after allogeneic umbillical cord blood stem cells transplantation, patient developed high fever. Among series of microbiological examinations, PCR for detection of EBV DNA in whole blood gave positive result. Detection of specific anti- EBV antibodies was performed with serum sample obtained at the same day (ELISA plate: sample 3-a). Introduced treatment (vidarabine, cytostatics and steroids) lead to viraemia negativization after 11 days. Next 10 days later, another examination of anti-ebv antibodies was performed (sample 3-b) (to be performed: ELISA interpretation) TO BE PERFORMED ELISA interpretation: Pattern of calibrators and tested serum samples in ELISA microplate: 4
On the basis of absorbance reading chart (page 8), please fill the table and interprete the results: Patient 1 Patient 2 Patient 3 sample a Patient 3 sample b Titer Interpretation Titer Interpretation Titer Interpretation Titer Interpretation VCA IgM VCA IgG EA IgM EA IgG EBNA-1 IgM EBNA-1 IgG Analytical interpretation: Positive results for 10 U/ml and more; negative results for less than 10 U/ml Demonstrations: 1. Diagnosis of viral meningoencephalitis application of multiplex PCR technique Case report: A 37-year-old male with temperature of 39ºC, chills, intense headaches, nausea and vomits was transported to the admission room of a regional hospital. Physical examination revealed confusion, dyssynergia, balance disorders, slight hemiparesis and tachycardia. Blood and cerebro-spinal fluid were taken for microscopic, biochemical and microbiological examinations. Laboratory examination of cerebro-spinal fluid revealed mild pleocytosis, glucose and protein levels normal. Based on clinical examination and laboratory results, multidirectional microbiological examination was performed, including virological testing of CSF using multiplex-pcr technique for herpes simplex viruses. Test procedure: I. DNA extraction from the clinical sample (200 µl of cerebro-spinal fuid) using alkaline lysis method II. Evaluation of quantity and purity of obtained DNA III. DNA amplification a. Preparation of reaction mixture and working mixtures from sample and control b. Samples/controls placing in the thermocycler, setting the reaction conditions, amplification IV. Detection of amplified products a. Preparation of agarose gel (with ethidium bromide) in proper electrophoretic buffer b. Moving the samples to the gel c. Electrophoresis of the PCR products d. Visualization in UV light 5
Picture of photographed gel: Legend: 100 bp M: product length marker; 100 basepairs K (-): negative control HSV 1 TK-, HSV 2 TK-: standard viral strains with altered TK gene, resistant to acyclovir HSV 1 TK+, HSV 2 TK+: standard viral strains with normal TK gene, sensitive to acyclovir CSF: examined cerebro-spinal fluid Products length : HSV-1: 469 pz HSV-2: 390 pz Thymidine kinase: 1200 pz 2. ELISA assay in the diagnosis of aplastic crisis bound to the parvovirus B19 infection Case report:a mother decided to take her 8-year-old boy to paediatric clinic, because of reported episodes of nose bleeding in he past couple of days. Patient s medical history revealed in the past two weeks progressing decrease of motoric activity, drowsiness, lose of appetite and sustained elevation of body temperature (37,5 C). On physical examination, skin paleness and tiny haemorrhagic effusions on mucosa of oral cavity and throat were observed.the results of laboratory blood tests were as follows: significantly decreased number of reticulocytes, normocytic anaemia, granulocytopaenia with relative lymphocytosis and thrombocytopaenia as well as prolonged bleeding time, elevated level of Fe 2+ in serum and a high level of C-reactive proteinbased on the results of clinical and laboratory examination, decreased haemopoietic ability of the bone marrow was diagnosed. As one from possible reasons, infection with parvovirus B19 was recognized. Physician commited serological assays: detection of anti-b19 antibodies in IgM and IgG classes and detection of viral antigen in patient s serum. 6
Course of primary infection with parvovirus B19 *)TAC transient aplastic crisis 7
[Page 1 ] Test 17) EBV IgG/IgM Modified: 2015.11.15 10:07:52 Current: 2016.04.05 07:36:41 Read: Point To Point 8 Well Lot Number: Exp. Date: User: WAVELENGTHS=450/630nm Curve Axis:Y=Abs, X=Conc Calibrator 1=1.0 IU/mL 0.007 Abs Calibrator 2=10.0 IU/mL 0.653 Abs Calibrator 3=50.0 IU/mL 1.679 Abs Calibrator 4=200.0 IU/mL 3.442 Abs Interpretation Criteria Positive >= 10.00; Negative < 10.00 Running New Standard Curve Well Sample ID Abs IU/mL Interpret A-1 Calibrator 1 0.013 1.0 B-2 Calibrator 2 0.583 10.0 C-3 Calibrator 3 1.353 50.0 D-4 Calibrator 4 2.872 200.0 Well Sample ID Abs IU/mL Interpret A-2 5 1.689 78.9 Positive B-2 6 0.869 32.7 Negative C-2 7 1.357 50.3 Positive D-2 8 0.112 2.9 Negative A-3 13 0.601 11.7 Positive B-3 14 2.152 118.7 Positive C-3 15 1.008 32.0 Positive D-3 16 0.389 5.3 Positive A-4 21 1.197 42.3 Negative B-4 22 0.767 24.3 Positive C-4 23 0.671 14.6 Positive D-4 24 0.034 1.3 Negative A-5 29 0.909 26.9 Positive B-5 30 0.098 2.3 Negative C-5 31 0.012 0.9 Negative D-5 32 0.009 0.9 Negative A-6 37 0.020 1.1 Negative B-6 38 0.100 2.3 Negative C-6 39 1.392 64.1 Positive D-6 40 0.015 1.0 Negative A-7 45 0.020 1.1 Negative B-7 46 1.708 80.5 Positive C-7 47 1.010 32.2 Positive D-7 48 0.486 9.9 Positive End of Run Test Ended 8