6 DIAGNOSTIC METHODS IN VIROLOGY 3 categories:(1) direct detection,(2) virus isolation,(3) serology.
7 1. Direct Examination of Specimen clinical specimen - examined directly for the presence ofvirus particlesvirus antigenviral nucleic acids.Electron Microscopy morphology / immune electron microscopyLight microscopy histological appearance - e.g. inclusion bodiesAntigen detection immunofluorescence, ELISA etc.Molecular techniques for the direct detection of viral genomes
8 2. Indirect Examination = Virus isolation Cell Culturecytopathic effect,haemadsorption,confirmation by neutralization, interference,immunofluorescence etc.Eggs pocks - haemagglutination, inclusion bodiesAnimals disease or death confirmation by neutralization
9 detection of IgM in primary infection. Classical Techniques 3. Serologyrising titres of antibody between acute and convalescent stages of infection,detection of IgM in primary infection.Classical Techniques1. COMPLEMENT FIXATION TESTS (CFT)2. HAEMAGGLUTINATION INHIBITION TESTS3. IMMUNOFLUORESCENCE TECHNIQUES (IF)4. NEUTRALIZATION TESTS5. SINGLE RADIAL HAEMOLYSISNewer Techniques1. RADIOIMMUNOASSAY (RIA)2. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)3. PARTICLE AGGLUTINATION4. WESTERN BLOT (WB)
10 1. Direct Examination rapid diagnostic Result => the same/next day. Obs:virus isolationserological methodsMay sometimes give a rapid result. !
11 Examples – IF testing of : 1.1. Antigen DetectionExamples – IF testing of :nasopharyngeal aspirates for respiratory virusese.g.. RSV, flu A, flu B, and adenoviruses,detection of rotavirus antigen in faeces,detection of HSV and VZV in skin scrappings,detection of HBsAg in serum.Usually considered as a serological test!
12 1.1. Antigen Detectionmain advantage: rapid (hours)BUT technique - often tedious- result difficult to read and interpret,- sensitivity and specificity poor.quality of the specimen - most important in order for the test to work properly.
14 1.2. Electron Microscopy (EM) basis = morphology.magnification - 50,000 Xmainly used for viral gastroenteritisdetecting viruses in feces:rotavirus, - calicivirusadenovirus, - Norwalk-like virusesastrovirus,Occasionally used for detection of viruses in skin lesions (e.g. vesicles)herpesvirusespapillomaviruses.
15 1.2. Electron Microscopy (EM) sensitivity and specificity enhanced by immune electron microscopy:virus specific antibody used to agglutinate virus particles =>easier to recognize.Problems with EM:expense - purchasing and maintaining the facility.sensitivity - often poor (106 v.particles/ml in sample required for visualization)observer - highly skilled.
16 Electronmicrographs from patients suffering from gastroenteritis Electronmicrographs from patients suffering from gastroenteritis. From left to right: rotavirus, adenovirus, (Courtesy of Linda M. Stannard, University of Cape Town,
17 Animal virus classification: DNA Viruses milyPoxHerpesAdenoPapovaParvoHepadna<---------------------------------------dsDNA-------------------------------------->ssDNAPartial dsDNAGenomeCapsidComplex<---------------------------------------------Icosahedral-------------------------------------------------->symmetryEnvelope<-----------------Yes------------------><-----------------------------No------------------------------>YesVacciniavirusHerpessimplexHumanPapillomaAdeno-AssociatedHepatitisBe.g.virus 2adenovirusMolluscumContagiosum
20 histology serves as an adjunct test. 1.3. Light MicroscopyReplicating virus => histological changes (characteristic or non-specific) in infected cells.Viral inclusion bodies = collections of replicating virus particles (in nucleus or cytoplasm).ExamplesBabeş-Negri bodies in rabiescytomegalic inclusion bodies in CMV infectionshistology serves as an adjunct test.
27 1.4. Viral Genome Detection molecular methodsfuture direction of viral diagnosisClassical molecular techniques(dot-blot and Southern-blot)use of specific DNA/RNA probes for hybridizationspecificity depends on the conditions used for hybridization.quantification of DNA/RNA present in the specimensensitivity of these techniques ~ conventional viral diagnostic methods.
28 1.4. Viral Genome Detection b) Newer molecular techniques:POLYMERASE CHAIN REACTION (PCR)LIGASE CHAIN REACTION (LCR)NUCLEIC ACID BASED AMPLIFICATION (NASBA)BRANCHED DNA (BDNA)PCRextremely sensitive : 1 DNA molecule in a clinical specimen.PCR – problems:contamination,positive PCR result - difficult to interpretlatent viruses such as CMV !
29 Specimen - inoculated into cell culture, eggs or animals 2. Virus IsolationSpecimen - inoculated into cell culture, eggs or animalsEggs and animals - difficult to handlemost viral diagnostic laboratories - cell culture only.3 types of cell cultures:Primary cellsSemi-continuous cellsContinuous cells
30 Primary cells culture - e.g. Monkey Kidney. 2.1. Types of cell culturesPrimary cells culture - e.g. Monkey Kidney.= normal cells obtained from freshly killed adult animals.(+)best cell culture systems availablesupport the widest range of viruses(-)can only be passaged once or twice.very expensivedifficult to obtain a reliable supply.
31 b. Semi-continuous cells 2.1. Types of cell culturesb. Semi-continuous cellsHuman embryonic kidneyskin fibroblasts.cells taken from embryonic tissuepassaged up to 50 times.c. Continuous cellsHeLa, Vero, Hep2, LLC-MK2, BGM.immortalized cells i.e. tumour cell linespassaged indefinitely.most easy to handlerange of viruses supported is often limited.
32 2.2. Identification of growing virus a. Cytopathic Effect (CPE) - specific or non-specificHSV and CMV produces a specific CPE,enteroviruses do not produces a specific CPE.b. Haemadsorptioncells acquire the ability to stick to mammalian red blood cells.mainly used for the detection ofinfluenzaparainfluenzaviruses.
33 Left to Right: Cytopathic effect of HSV, enterovirus 71 (ballooning), and RSV in cell culture (syncytia formation) (Linda Stannard. University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)
34 2.3 Problems with cell culture long period for a result (up to 4 weeks)sensitivity - often poor and depends on many factors,condition of the specimen,condition of the cell sheet.very susceptible tobacterial contaminationtoxic substances in the specimen.many viruses - not grow in cell culture at all :Hepatitis B and C,Diarrhoeal viruses,parvovirus etc.
37 2.4 Rapid Culture Techniques viral antigens - detected 2 to 4 days after inoculation.Examples: shell vial cultures and the CMV DEAFF test.CMV DEAFF testcell sheet grown on individual cover slips in a plastic bottle.then bottle is spun at a low speed for one hour (to speed up the adsorption of the virus)incubated for 2 to 4 days.cover slip taken outexamined for the presence of CMV early antigens by IF (immunofluorescence)
38 Left: Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus. Also note the presence of syncytia (indistinguishable from that of RSV) (Courtesy of Linda Stannard, University of Cape Town). Right: Positive CMV DEAFF test. (Virology Laboratory, Yale-New Haven Hospital)
40 3. Serology = the mainstay of viral diagnosis IgM - first antibody to appear,IgG – follow, much higher titerTechniques:EIA and RIAspecifically for IgM or IgG,most sensitive tests availableCFT, HAI => detect total antibody (comprises mainly IgG)EIAsbetter sensitivity, specificity and reproducibility than classical techniques ( CFT and HAI. )
41 3. Serology 3.1. Criteria for Primary Infection a. Significant rise in titre of IgG/total antibodyacute - convalescent seraCFT and HAI: 4 X or greaterProblem: dg. = retrospectiveb. Presence of IgM- EIA, RIA, and IFrapidProblems:interference by rheumatoid factor,re-infection by the virus,unexplained persistenceSeroconversion = changing from a previously antibody negative state to a positive state.HIV following a needle-stick injury,antirubella following contact with a known case.
42 3.2. Criteria for diagnosing re-infection/re-activation difficult to differentiate re-infection/re-activationimportant under certain situationsrubella or toxoplasma infectionfirst trimester of pregnancy:primary infection - high risk of fetal damagere-infection not associated with a high risk of fetal damagesharp large rise in antibody titres found in re-infectionIgM usually low or absent in cases of re-infection/re-activation.
43 Serological events in primary infection and reinfection. Reinfection: IgM absent or only present transiently at a low level.
44 Rubella and hepatitis A: 3.3. LimitationsRubella and hepatitis A:onset of clinical symptoms - development of antibodies=> detection of IgM or rising titres of IgG = active disease.Many viruses (respiratory and diarrhoeal):clinical disease before the appearance of antibodies => serological diagnosis = retrospectiveSome viruses (HIV and rabies):clinical disease months or years after seroconversion => the mere presence of antibody is sufficient to make a definitive diagnosis.
45 CFT in Microtiter Plate. Rows 1 and 2: acute and convalescent phase serum specimens, respectively.The observed 4-fold increase is significant and indicates infection.
46 Microplate ELISA: coloured wells indicate reactivity darker the colour, higher the reactivity
47 healthy person - little or no antibodies in CSF. 3.4. Antibody in the CSFhealthy person - little or no antibodies in CSF.viral meningitis or encephalitis,antibodies - produced by lymphocytesfinding of antibodies in the CSFsignificant when ratio between the titre of antibody in the serum and that in the CSF is less than 100 (depend on an intact blood-brain barrier !)