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

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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

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Microscopy

• Light Microscopy – elementary bodies

• Electron Microscopy Rota viral detection

• Florescent Microscopy Direct / Indirect

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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)

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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

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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

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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.

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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.

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Detection of Viral antigen

• Use of Immunofluorescence

Imunoelectrphoresis Radio immunoassay

RIA

ELISA

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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

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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.

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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

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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

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© 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

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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

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Regular Methods in Use

• Egg inoculation Pox virus, Influenza

• Into tissue culture

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Advantages of Molecular Methods with

• To Increases Sensitivity and Specificity.

• With PCR• RT PCR

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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

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