Micro - 4th Assessment - Laboratory Diagnosis Of Viral Infections - 29 Jan 2007

Laboratory Diagnosis of Viral Infections

Fourth Medical, 2007 Prof. Widad Al-Nakib, FRCPath.

Overview of Diagnostic Methods

• In general, diagnostic tests can be grouped into 3 categories: (1) Direct examination (2) Indirect examination (3) Serology

Contd.. • In direct examination, the clinical specimen is examined directly for the presence of virus particles, virus antigen or viral nucleic acids. • In indirect examination, the specimen is inoculated into cell culture, eggs or animals in an attempt to grow the virus: this is called virus isolation. • In Serology, the diagnosis is based on the detection of antibody and generally constitute by far the bulk of the work of any virology laboratory.

1. Direct Examination of Specimens • •

• •

Light microscopy, histological appearance - e.g. inclusion bodies Electron Microscopy, virus particle are directly seen in clinical specimens, example, stools, vesicular fluids Antigen detection by immunofluorescence, ELISA etc. Molecular techniques for the direct detection of viral genomes

Contd.. • The specimen used for direct detection and virus isolation is very important. • A positive result from the site of disease would be of much greater diagnostic significance than those from other sites. • For example, in the case of herpes simplex encephalitis, a positive result from the CSF or the brain would be much greater significance than a positive result from an oral ulcer, since reactivation of oral herpes is common during times of stress.

2. Indirect Examination •

• •

Cell culture - cytopathic effect (CPE), haemadsorption, confirmation by neutralization, interference, immunofluorescence etc. Eggs pocks on CAM - haemagglutination, inclusion bodies Animals disease or death confirmation by neutralization

Virus Growth in Embryonated Eggs

Virus Cytopathic Effect (CPE) in Cell Cultures

3. Serology

• Detection of rising titres of antibody between acute and convalescent stages of infection • Detection of IgM in primary infection.

Secondary Immune Response

Immune Response After Hepatitis A Virus Infection

“Classical” Serological Techniques 1. Complement-Fixation (CFT) 2. Haemagglutination inhibition tests (HAI) 3. Immunofluorescence techniques (IF) 4. Neutralization tests (NT) 5. Single Radial Haemolysis (SRH)

“Newer” Serological Techniques 1. Radioimmunoassay (RIA) 2. Enzyme Immunoassay (EIA) 3. Particle agglutination 4. Western Blot (WB) 5. Recombinant immunoblot assay (RIBA)

Immunofluorescence •

Light Microscopy • Replicating virus often produce histological changes in infected cells. These changes may be characteristic or non-specific. • Viral inclusion bodies are basically collections of replicating virus particles either in the nucleus or cytoplasm. • Examples of inclusion bodies include the negri bodies and cytomegalic inclusion bodies found in rabies and CMV infections, respectively. • Although not sensitive or specific, histology nevertheless serves as a useful adjunct in the diagnosis of certain viral infections.

Rabies Negri Bodies

Electron Microscopy (EM) • Virus particles are detected and identified on the basis of morphology. You need 1,000,000 particles per ml. to see the virus • A magnification of around 50,000 is normally used. • EM is now mainly used for the diagnosis of viral gastroenteritis by detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses

CONTD.. • Occasionally it may be used for the detection of viruses in vesicles and other skin lesions, such as herpesviruses, papillomaviruses and poxviruses. • The sensitivity and specificity of EM may be enhanced by immune electron microscopy, whereby virus specific antibody is used to agglutinate virus particles together and thus making them easier to recognize, or to capture virus particles onto the EM grid.

Electron Microscopy (EM)

New Molecular Techniques • • • • •

Polymerase chain reaction (PCR) Ligase chain reaction (LCR) Nucleic acid based amplification (NASBA) Branched DNA (bDNA) ALL depend on some form of amplification, either the target nucleic acid, or the signal itself.

Molecular Techniques • PCR is being increasingly used for viral diagnosis, especially as the cost of the assay come down and the availability of closed automated systems that could also perform quantification for example, measurement of Viral Load (Quantitative PCR).

Principle of PCR

Amplification of different enteroviruses M

1

2

3 4 5

6 7

8 9 10 11 12 13

Lane M – 100bp DNA marker

200 -

M 14 15 16 17 18 19 20 21 22 23 24 25

200 -

Lane 1 – Cox A7

Lane 14 – Polio 1 (Sabin)

Lane 2 – Cox A9

Lane 16 – Echo 4

Lane 3 – Cox A16

Lane 17 – Echo 6

Lane 5 – Cox B1

Lane 18 – Echo 9

Lane 6 – Cox B2

Lane 19 – Echo 11

Lane 7 – Cox B3

Lane 20 – Echo 17

Lane 9 – Cox B4

Lane 21 – Echo 30

Lane 10 – Cox B5

Lane 22 – Echo 34

Lane 11 – Cox B6

Lane 23 – negative

Lane 12 – Entero 70

Lane 24 – Cox B5 (old)

Lane 13 – Entero 71

Lane 25 – Echo 9 (old)

Rapid and Direct Virus Diagnosis • With new techniques such as PCR we are now able to detect viral genomes in very small quantities (by amplifying the viral genome) in body tissues ( in situ PCR), body fluids (CSF, pericardial and vesicular fluids), excretions (urine) and blood. Thus, allowing us to establish a direct relationship between the causative virus and the disease, in a very short period of time.

Contd.. Detecting Viral Genome- a direct association. • • • • • •

Blood - viraemia CSF - viral meningitis and encephalitis Pericardial fluids – viral carditis Vesicular fluids – herpetic lesions BAL – viral pneumonia Cells and tissues- chronic and persistent viral infections.

Viral Load Predicting Disease and Survival CMV DNA Load – Independent marker of CMV disease and survival( Patients responding to preemptive therapy with undetectable plasma CMV viral load)

– Lower risk of disease, higher rate of survival HIV RNA Load - Determine severity of infection - Monitor the efficacy of antiviral chemotherapy - Determine drug resistance if no change in viral load

HIV Resistance Testing Genotype • Sequence of nucleotide bases • Specific base changes or mutations causing resistance Phenotype • Traits or behavior resulting from genotype • Antiviral susceptibility