Class 10 - Warszawski Uniwersytet Medyczny
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Class 10 - Warszawski Uniwersytet Medyczny
Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny English Division, 6-year programme Class 10 DNA viruses I. Seminar: General properties, pathogenesis and clinial features of selected DNA viruses from Herpesviridae, Papillomaviridae and Adenoviridae family II. Assays to be performed: 1. Continuation of Class 9: Haemagglutination assay and haemagglutinin titration 2. Laboratory diagnosis of infectious mononucleosis 2a. Paul-Bunnel-Davidsohn assay (detection of non-specific heterophile antibodies) 2b. ELISA assay for the detection of specific humoral response against various antigens of EBV - interpretation 3. ELISA assay for the detection of specific humoral response against various antigens of EBV in cases of EBV reactivation - interpretation 4. Identification of adenovirus based on nucleotide sequence comparison using blastn software 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 Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 1 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny 1. Haemagglutination assay and haemagglutinin titration 1. Mix one drop of hen’s egg allantoic fluid containing virus with one drop of 1% RBC on microscopic slide, along with the controls (positive and negative) 2. Perform hemagglutinin titration: a. Add 50 µl of PBS (phosphate-buffered saline) to the wells 1-7 and to cell control (CC) b. Add 50 µl of virus suspension to first well in the row (1:2) and mix with PBS. Add 50 µl of virus to the virus control well (VC) c. Transfer 50 µl of diluted virus from first well to the second, mix. Repeat for wells 3-7 d. Discard 50 µl of diluted virus from well 7 (1:128) to the jar with disinfectant e. Add 50 µl of 1% RBC to all wells (1-7, CC, VC) f. Mix and incubate for at least 1 hour at room temperature 2. 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/mm3) and appearance of atypical lymphocytes. After initial recognition of infectious mononucleosis, physician decided to order serological diagnostic tests which should confirm his diagnosis. 2a. Monospot test (modified Paul-Bunnel-Davidsohn assay) – non-specific serological test 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. Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 2 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny 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 Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 3 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny 2b. ELISA assay in the diagnosis of infectious mononucleosis – detection of specific anti-EBV antibodies 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 (to be performed: ELISA interpretation) 3. ELISA assay for detection of specific humoral response (IgM, IgG) against EBV antigens in cases of 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 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 antiEBV 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) Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 4 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny TO BE PERFORMED – ELISA tests interpretation: Pattern of calibrators and tested serum samples in ELISA microplate: Please, interprete the results: VCA IgM Patient 1 AB level VCA IgG EA IgM EA IgG EBNA-1 EBNA-1 IgM IgG 78.9 11.7 32.3 26.9 1.0 1.1 32.7 118.7 24.3 2.3 2.3 80.5 43.3 32.0 18.6 0.9 64.1 32.2 2.9 5.3 1.3 0.9 1.0 11.3 Interpretation Patient 2 AB level Interpretation Patient 3 AB level sample a Interpretation Patient 3 AB level sample b Interpretation Analytical interpretation: Positive results for 10 U/ml and more; negative results for less than 10 U/ml Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 5 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny 4. Virus identification based on nucleotide sequence comparison using BLASTN software 1. In the class facility computer: using notebook windows program open the file “virus.fas” (located on desktop) containing sequence of PCR product. Primers used for PCR were specific for human enteroviruses. 2. In Windows notebook select entire sequence and copy it (ctrl+c). 3. Open web browser window. NCBI BLAST (basic local alignment search tool) interface should be visible. 4. In “Basic BLAST” section choose “nucleotide blast” program 5. Paste copied sequence to “Enter accesion number, gi or FASTA sequence” window (ctrl+v) 6. Check if: a. “nucleotide collection (nr/nt)” database is chosen b. “highly similar sequence” option is marked c. “Show results in a new window” box is checked 7. Click “BLAST” button and wait for the results 8. Identify adenovirus type based on its similarity to other sequences in database. Check complementarity of the most similar database sequence (%/number of nucleotide mismatches) Identified adenovirus type: _____________________________________ Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 6 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny 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 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 Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 7 Katedra i Zakład Mikrobiologii Lekarskiej Warszawski Uniwersytet Medyczny 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 Fe2+ in serum and a high level of C-reactive protein Based 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. Course of primary infection with parvovirus B19 *)TAC – transient aplastic crisis Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw 8