Viral diagnostic methods aim to (1) confirm infections, (2) define disease processes, (3) monitor epidemics, and (4) educate physicians. Appropriate specimen collection is crucial, obtaining samples early and from sites with high viral loads. Diagnostic methods include direct examination of samples for viral antigens, genomes, or structures under microscopy, as well as indirect examination using cell cultures to isolate viruses and identify them through cytopathic effects. Newer molecular techniques like PCR can rapidly detect viral genomes.
Viral diagnostic methods aim to (1) confirm infections, (2) define disease processes, (3) monitor epidemics, and (4) educate physicians. Appropriate specimen collection is crucial, obtaining samples early and from sites with high viral loads. Diagnostic methods include direct examination of samples for viral antigens, genomes, or structures under microscopy, as well as indirect examination using cell cultures to isolate viruses and identify them through cytopathic effects. Newer molecular techniques like PCR can rapidly detect viral genomes.
Viral diagnostic methods aim to (1) confirm infections, (2) define disease processes, (3) monitor epidemics, and (4) educate physicians. Appropriate specimen collection is crucial, obtaining samples early and from sites with high viral loads. Diagnostic methods include direct examination of samples for viral antigens, genomes, or structures under microscopy, as well as indirect examination using cell cultures to isolate viruses and identify them through cytopathic effects. Newer molecular techniques like PCR can rapidly detect viral genomes.
Viral diagnostic methods aim to (1) confirm infections, (2) define disease processes, (3) monitor epidemics, and (4) educate physicians. Appropriate specimen collection is crucial, obtaining samples early and from sites with high viral loads. Diagnostic methods include direct examination of samples for viral antigens, genomes, or structures under microscopy, as well as indirect examination using cell cultures to isolate viruses and identify them through cytopathic effects. Newer molecular techniques like PCR can rapidly detect viral genomes.
First clues to the diagnosis of a viral decreases dramatically each day infection after the lesion manifests - Patient’s history - Symptoms 2) Go “where the action is” - Aspirated secretions – maximum PURPOSE OF VIRAL LABORATORY recovery STUDIES - Swabs – use cotton, Dacron or 1) To confirm the diagnosis by identifying nylon the viral agent of infection - Ca alginate: toxic to many viruses 2) To define the disease process 3) To monitor the disease 3) Transport epidemiologically - Medium: buffered isotonic 4) To educate physicians and patients solution + some type of protein e.g., albumin CARDINAL RULES IN SPECIMEN - Plus antibiotics COLLECTION - Refrigerated or on ice (not frozen) 1) Appropriate time of collection - Delay of 4-7 days: -70oC (not - 20oC: ice crystals) SPECIMENS FOR VIRAL DIAGNOSIS
PSALM 56:3 When I am afraid, I put my trust in You. 1
THE CHOICE OF LAB METHOD OF ADVANTAGE DIAGNOSIS DEPENDS ON - Result available quickly, usually within - Type of viral disease a few hours. - Capability the lab DISADVANTAGE - Availability of the method DIAGNOSTIC METHODS IN VIROLOGY - Often very much reduced sensitivity compared to cell culture, can be as low 1) Direct Examination as 20%. Specificity often poor as well. 2) Indirect Examination (Virus Isolation) - Requires good specimens. 3) Serology - The procedures involved are often tedious and time-consuming and thus DIRECT EXAMINATION expensive in terms of laboratory time. 1) Antigen Detection RAPID DIAGNOSIS: VIRAL ANTIGEN - immunofluorescence, ELISA etc. DETECTION 2) Electron Microscopy - morphology of virus particles 1) Nasopharyngeal Aspirate - immune electron microscopy - RSV 3) Light Microscopy - Influenza A and B - histological appearance - Parainfluenza - inclusion bodies - Adenovirus 4) Viral Genome Detection 2) Faeces - hybridization with specific nucleic acid - Rotaviruses probes - Adenoviruses - polymerase chain reaction (PCR) - Astrovirus
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3) Skin DISADVANTAGE - HSV - VZV - Expensive equipment 4) Blood - Expensive maintenance - CMV (pp65 antigenaemia test) - Require experienced observer - Sensitivity often low ELECTRON MICROSCOPY CYTOLOGY (LIGHT MICROSCOPY) - 106 virus particles per ml required for visualization, 50,000 - 60,000 - Rapid diagnosis of viral infections that magnification normally used. produce cytopathic effects (CPE) Viruses may be detected in the ◼ Changes in cell morphology following specimens. ◼ Cell lysis ◼ Vacuolation 1) Faeces ◼ Syncytia - Rotavirus, Adenovirus ◼ Inclusion bodies - Norwalk like viruses MOLECULAR METHODS - Astrovirus, Calicivirus 2) Vesicle fluid - Based on the detection of viral - HSV genome - VZV - in practice, the role played in a 3) Skin scrapings routine diagnostic virus laboratory is - papillomavirus, orf still small compared to conventional - molluscum contagiosum methods. - It is certain though that the role of IMMUNE ELECTRON MICROSCOPY molecular methods will increase rapidly in the near future. - The sensitivity and specificity of EM may be enhanced by immune electron CLASSICAL MOLECULAR TECHNIQUES microscopy. There are two variants:- 1) Classical Immune electron They depend on the use of specific microscopy (IEM) - the sample is DNA/RNA probes for hybridization. treated with specific anti-sera - Dot-blot, before being put up for EM. Viral - Southern blot, particles present will be - in-situ hydridization agglutinated and thus congregate - the sensitivity of these techniques is together by the antibody. not better than conventional viral 2) Solid phase immune electron diagnostic methods. microscopy (SPIEM) - the grid is - usually more tedious and expensive coated with specific anti-sera. Virus than conventional techniques, never particles present in the sample will found widespread acceptance be absorbed onto the grid by the antibody.
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DNA PROBE 1) PRIMARY CELL CULTURE - derived from normal adult tissues, - known DNA fragment used to identify ◼ e.g. Monkey Kidney closely related DNA or RNA - can be passed (subcultured) once or sequences 2X - the best cell culture systems available since they support the widest range of viruses. OTHER NEWER MOLECULAR - very expensive TECHNIQUE - often difficult to obtain a reliable supply.
2) SEMI-CONTINUOUS CELL CULTURE - derived from embryonic tissues; ◼ e.g., Human embryonic kidney and skin fibroblasts PCR AND RT-PCR
- DNA or RNA is extracted 3) CONTINUOUS CELL CULTURE
- PCR - derived from malignant tissues - Gel electrophoresis ◼ e.g., HeLa, Vero, Hep2, LLC-MK2, MDCK INDIRECT METHOD (VIRUS ISOLATION - can be subcultured indefinitely AND IDENTIFICATION) - most easy to handle - Disadvantage: the range of viruses VIRUS ISOLATION supported is often limited 1) Cell culture VIRAL DETECTION - cytopathic effect (CPE) - haemabsorption 1) Cytopathic Effect (CPE) – may be - immunofluorescence specific or non-specific 2) Eggs ◼ ballooning of cells - pocks on CAM ◼ syncytia formation - haemagglutination 2) Hemadsorption – cells infected with - inclusion bodies some viruses express hemagglutinin 3) Animals that bind erythrocytes - Disease or death CONFIRMATION OF IDENTITY CELL CULTURE 1) Neutralization test - are most widely used for virus - (Virus + specific Ab) + cell culture isolation. → no CPE 2) Hemagglutination-inhibition (HAI) test
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- (Virus + specific Ab) + rbc → no HA - Many viruses will not grow in cell 3) Immunofluorescence test culture - Virus + specific Ab-fluorescein ◼ e.g. Hepatitis B, diarrheal viruses, → fluorescence parvovirus, papillomavirus. PROBLEMS SEROLOGY
- Long period (up to 4 weeks) required - Detection of rising titres of antibody
for result. between acute and convalescent - Often very poor sensitivity stages of infection, or the detection of - Susceptible to bacterial contamination. IgM in primary infection. - Susceptible to toxic substances which may be present in the specimen.
2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA) 3. Immunofluorescence techniques (IF) 3. Particle agglutination 4. Neutralization tests 4. Western Blot (WB) 5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay
CRITERIA: DIAGNOSING PRIMARY
INFECTION - 4 fold or more increase in titer of IgG or total antibody between acute and convalescent sera - Presence of IgM - Seroconversion - A single high titre of IgG (or total antibody) - very unreliable CRITERIA: DIAGNOSING REINFECTION - 4 fold or more increase in titer of IgG or total antibody between acute and WESTERN BLOT convalescent sera - HIV-1 Western Blot - Absence or slight increase in IgM - Lane1: Positive Control TYPICAL SEROLOGICAL PROFILE AFTER - Lane 2: Negative Control ACUTE INFECTION - Sample A: Negative - Sample B: Indeterminate - Note that during reinfection, IgM may - Sample C: Positive be absent or present at a low level transiently
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- Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and Dengue, may lead to false positive results. - Immunocompromised patients often give a reduced or absent humoral immune response. - Patients with infectious mononucleosis and those with connective tissue USEFULNESS OF SEROLOGICAL TEST diseases such as SLE may react non- specifically giving a false positive DEPENDS ON THE INDIVIDUAL VIRUS result. - For example, rubella and hepatitis A - Patients given blood or blood products viruses may give a false positive result due to ◼ the onset of clinical symptoms the transfer of antibody. coincide with the development of CSF ANTIBODIES antibodies. ◼ The detection of IgM or rising titres - Used mainly for the diagnosis of of IgG in the serum of the patient herpes simplex and VZV encephalitis would indicate active disease - CSF normally contain little or no - However, many viruses (respiratory antibodies and diarrheal viruses) often produce - presence of antibodies suggest clinical disease before the appearance meningitis or meningoencephalitis of antibodies - Diagnosis depends on the presence of ◼ So in this case, any serological an intact blood-brain barrier diagnosis would be retrospective and therefore will not be that useful. - Viruses like HIV and rabies produce clinical disease months or years after seroconversion ◼ the mere presence of antibody is sufficient to make a definitive diagnosis PROBLEMS WITH SEROLOGY - Long period of time required for diagnosis using paired acute and convalescent sera. - Mild local infections such as HSV genitalis may not produce a detectable humoral immune response.
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