Diagnosis of Viral Infections basics

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Diagnosis of Viral Infections basics


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Koch’s Postulates Applicable in many bacterial diseases 1. Organism present only in diseased individuals 2. Organism cultivated in pure culture from diseased individual

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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:

River’s Postulates T.M. River, 1937 Modified from Koch’s Postulates (proof of bacterial diseases) Isolate virus from diseased hosts. Cultivation of virus in host cells. Proof of filterability. Production of a comparable disease when the cultivated virus is used to infect experimental animals. Reisolation of the same virus from the infected experimental animal. Detection of a specific immune response to the virus.

Methods of Study:

Methods of Study Much 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:

Laboratory Diagnosis of Viral Diseases Different from Bacterial Infections Dr.T.V.Rao MD 6

Viral Diagnostics in the Clinical Laboratory:

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:

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:

Three General Approaches for Laboratory Diagnosis of Viral Infections Direct detection Microscopy or staining Virus Isolation PCR Serology Antibodies

Why Difficult:

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:

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:

Direct Detection Electron Microscopy Examine specimen for viruses Immuno-electron microscopy Labeled antibody Immunofluorescence Fluorescent tag bound to Fc region of Ab

Direct Examination:

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:

Direct Detection Electron Microscopy Examine specimen for viruses Immuno-electron microscopy Labeled antibody Immunofluorescence Fluorescent tag bound to Fc region of Ab

Quantitative Assays:

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:

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. Dr.T.V.Rao MD 18

Virus Isolation:

Virus Isolation Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures: 1. Primary cells - Monkey Kidney 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts 3. 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:

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:

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:

Detection of Viral antigen Use of Immunofluorescence Imunoelectrphoresis Radio immunoassay RIA ELISA Dr.T.V.Rao MD 22

Serology :

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 :

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:

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:

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:

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:

ELISA for HIV antibody Micro plate ELISA for HIV antibody: colored wells indicate reactivity Dr.T.V.Rao MD 28

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© Hank Morgan/Science Photo Library/Photo Researchers, Inc. HIV ELISA test.

ELISA Procedures:

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:

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

PowerPoint Presentation:

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)

PowerPoint Presentation:

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:

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 :

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

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Schematic of PCR Each cycle doubles the copy number of the target Dr.T.V.Rao MD 36

Growing Technologies:

Growing Technologies Molecular methods Probes Polymerase chain reaction Can produce Rapid Highly scientific Specific Dr.T.V.Rao MD 37

Regular Methods in Use:

Regular Methods in Use Egg inoculation Pox virus, Influenza Into tissue culture Dr.T.V.Rao MD 38

Advantages of Molecular Methods with:

Advantages of Molecular Methods with To Increases Sensitivity and Specificity. With PCR RT PCR Dr.T.V.Rao MD 39

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Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Professionals in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 40

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