Date post: | 23-Aug-2014 |
Category: |
Health & Medicine |
Upload: | tumalapalli-venkateswara-rao |
View: | 8,546 times |
Download: | 3 times |
DIAGNOSIS OF VIRAL INFECTIONS
basicsDr.T.V.Rao MD
Dr.T.V.Rao MD
Koch’s PostulatesApplicable in many bacterial diseases
1. Organism present only in diseased individuals
2. Organism cultivated in pure culture from diseased individual
3. Organism causes disease when injected into healthy individuals
4. Organism re-isolated from infected individual from point 3.
Koch’s Postulates
Figure 1.15
River’s Postulates• T.M. River, 1937• Modified from Koch’s Postulates (proof of
bacterial diseases)
1. Isolate virus from diseased hosts.2. Cultivation of virus in host cells.3. Proof of filterability.4. Production of a comparable disease when the
cultivated virus is used to infect experimental animals.
5. Reisolation of the same virus from the infected experimental animal.
6. Detection of a specific immune response to the virus.
Methods of StudyMuch more expensive and
difficult to study animal viruses than bacteriophages
• Cultivation in host cells– Living animal– Embryonated chicken
eggs– Cell or tissue culture (= in
vitro)
Laboratory Diagnosis of Viral Diseases
Different from Bacterial Infections
Dr.T.V.Rao MD 6
Viral Diagnostics in the Clinical Laboratory
• Over 60% of all infectious disease cases seen by a physician are due to viral infections.
• Quality of patient specimens and their transport to the laboratory is important.
Storage and Collection of Biological Specimens for Viral Testing
• What types of specimens are collected to diagnose?– Respiratory tract infections: Nasal and bronchial washings, throat and nasal
swabs, sputum– Eye infections: throat and Conjunctival swab/scraping– Gastrointestinal tract infections: stool and rectal swabs– Vesicular rash: vesicle fluid, skin scrapings – Maculopapular rash: throat, stool, and rectal swabs– CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy,
cerebrospinal fluid– Genital infections: vesicle fluid or swab – Urinary tract infections: urine – Blood borne infections: blood
Three General Approaches for Laboratory Diagnosis of Viral Infections
• Direct detection–Microscopy or
staining• Virus Isolation
–PCR• Serology
–Antibodies
Why Difficult
• In the past Growth of Virus was not rapid.• Diagnosis becomes routine today due to
availability of Rapid method.• Important in HBV and HIV infections.• Rubella in pregnant women.• Simple methods – Microscopy and
detection of inclusion bodies.Dr.T.V.Rao MD 10
Diagnostic Methods in Virology
1. Direct Examination
2. Indirect
Examination (Virus
Isolation)
3. Serology
Dr.T.V.Rao MD 11
Microscopy
• Light Microscopy – elementary bodies
• Electron Microscopy Rota viral detection
• Florescent Microscopy Direct / Indirect
Dr.T.V.Rao MD 12
Direct Detection• Electron Microscopy
– Examine specimen for viruses
• Immuno-electron microscopy– Labeled antibody
• Immunofluorescence– Fluorescent tag bound to
Fc region of Ab
Direct Examination
1. Antigen Detection immunofluorescence, ELISA etc.
2. Electron Microscopy morphology of virus particles immune electron microscopy
3. Light Microscopy histological appearance inclusion bodies
4. Viral Genome Detection hybridization with specific nucleic acid probes
polymerase chain reaction (PCR)
Dr.T.V.Rao MD 14
Direct Detection
• Electron Microscopy– Examine specimen for
viruses• Immuno-electron
microscopy– Labeled antibody
• Immunofluorescence– Fluorescent tag bound to
Fc region of Ab
Quantitative Assays• Plaque assays
–Lytic viruses only–Steps
• Serial dilution of virion-containing solution
• Create tissue culture plates• Spread diluted virus• Overlay with agar—
prevents diffusion• Count number of plaques
–Each plaque represents 1 PFU (Plaque Forming Unit)
Figure 5.8: Plaque assays used to quantitate a viral stock.
Courtesy of Teri Shors
Indirect Examination
1. Cell Culture Cytopathic effect (CPE) haemabsorption immunofluorescence
2. Eggs pocks on CAM haemagglutination inclusion bodies
3. Animals disease or death
Dr.T.V.Rao MD 17
Serology
Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.
Classical Techniques Newer Techniques
1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay
Dr.T.V.Rao MD 18
Virus IsolationCell Cultures are most widely used for virus isolation, there are 3 types of cell cultures:
1. Primary cells - Monkey Kidney2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK
Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the range of viruses supported is often limited.
Dr.T.V.Rao MD 19
Cytopathic Effects• Visible results of viral infection• Cell death by
– Multiplying viruses– Inhibition of DNA, RNA or protein synthesis– Effects on permeability of membrane
• Cytopathic effects (CPEs) of infected cells can be observed with inverted light microscopes
• Rounding/detachment from plastic flask• Syncytia/fusion
– Fusion of cells• Shrinkage• Increased refractivity• Aggregation• Loss of adherence• Cell lysis/death
• Common observations of CPEs– Inclusion body formation
• Intracellular virus parts (replication or assembly)– Hemadsorption assays
Cell Cultures
Growing virus may produce
1. Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or non-specific.
2. Haemabsorption - cells acquire the ability to stick to mammalian red blood cells.
Confirmation of the identity of the virus may be carried out using neutralization, haemabsorption-inhibition or immunofluorescence tests.
Dr.T.V.Rao MD 21
Detection of Viral antigen
• Use of Immunofluorescence
Imunoelectrphoresis Radio immunoassay
RIA
ELISA
Dr.T.V.Rao MD 22
Serology • Since the isolation and identification of viruses is
not commonly done in the clinical laboratory, the clinical picture and serology plays a greater role in the diagnosis of viral disease. The major types of antibodies that are assayed for are neutralizing, haemagglutination inhibiting and complement fixing antibodies. Complement fixing antibodies follow the kinetics of IgM and are most useful in indicating a current or recent infection
Dr.T.V.Rao MD 23
Serology • The development of
antibodies to different components of the virus is used in staging the disease. For example in hepatitis B and HIV infections this approach is used.
Dr.T.V.Rao MD 24
Viral Serology• Indirect
– Primary and secondary responses to viral infections• IgM (1st exposure)• IgG (2nd exposure)
Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.
Serological Diagnosis
• Detection of Immunologlublins Ig G. Ig M Ig A
• Raise of titers Ist sample later sample (convalescent sample) tested after 10 – 14 days Raise of titer is diagnostic
Dr.T.V.Rao MD 26
Viral Serology• Enzyme-Linked Immuno-Sorbant
Assays (ELISAs)–Enzyme reacts with substrate to
produce colored product–Very sensitive
• HIV test– If positive twice, Western Blotting is performed
next
ELISA for HIV antibody
Micro plate ELISA for HIV antibody: colored wells indicate reactivity
Dr.T.V.Rao MD 28
© Hank Morgan/Science Photo Library/Photo Researchers, Inc.
HIV ELISA test.
ELISA Procedures
Figure 5-19a
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition . ASM Press, 2000.
Figure 5-19b
Western Blot
HIV-1 Western Blot• Lane1: Positive Control• Lane 2: Negative Control• Sample A: Negative• Sample B: Indeterminate• Sample C: Positive
Dr.T.V.Rao MD 31
Figure 5.21b: The structure of HIV-1.
Image courtesy of Bio-Rad Laboratories
Figure 5.21c: The typical results of a Western blot testing patient serum for HIV-1 antibodies.
(c)(b)
Figure 5.21a: The basic principles behind the Western blotting procedure. Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third Edition. ASM Press, 2000.
Virus Isolation– Nucleic acid methods
• PCR (DNA), RT-PCR (RNA)
• Can be used to detect viruses that are noncultivatable
• Rapid identification (e.g. RT-PCR—4 Corners outbreak of hantavirus or FRET in the field)
• Can be used to manage patients (e.g. HIV viral load)
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
Polymerase Chain Reaction • Advantages of PCR:
– Extremely high sensitivity, may detect down to one viral genome per sample volume
– Easy to set up– Fast turnaround time
• Disadvantages of PCR– Extremely liable to contamination– High degree of operator skill required– Not easy to set up a quantitative assay.– A positive result may be difficult to interpret, especially with latent
viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.
• . Dr.T.V.Rao MD 35
Schematic of PCR
Each cycle doubles the copy number of the targetDr.T.V.Rao MD 36
Growing Technologies
• Molecular methods
• Probes • Polymerase
chain reaction Can produce Rapid Highly scientific Specific
Dr.T.V.Rao MD 37
Regular Methods in Use
• Egg inoculation Pox virus, Influenza
• Into tissue culture
Dr.T.V.Rao MD 38
Advantages of Molecular Methods with
• To Increases Sensitivity and Specificity.
• With PCR• RT PCR
Dr.T.V.Rao MD 39
• Programme Created by Dr.T.V.Rao MD for Medical and Paramedical
Professionals in the Developing World• Email
Dr.T.V.Rao MD 40