Investigation strategies and methods
Overview of
Microbiology Methods
May 2007
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Laboratory Training for FieldEEpidemiologists
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Learning objectives
At the end of the presentation, participants should:
• Understand what the laboratory does with samples
that arrive
• Have an understanding of the range of test methods
used to analyse samples
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Managing the sample: administrative
•
When an outbreak sample is received it is assigned:
• laboratory identification number
• an outbreak identification label
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Managing the sample: technical
•
Macroscopic evaluation
•
Split sample for different laboratory disciplines
•
Two possible approaches:
• perform only those tests requested by sender
• perform diagnostics for syndromes/clinical description
(laboratory initiative)
•
Storage of samples
• refrigerator or freezer
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Types of laboratory methods
•
Direct methods
• look for/detect the agent
•
Indirect methods
• detect host response to the agent
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Direct methods
1.
Macroscopic evaluation
2.
Direct microscopy
3.
Electron microscopy
4.
Staining
5.
Rapid tests
6.
Molecular methods
7.
Propagate the agent
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No propagation
required
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1. Macroscopic evaluation
•
Consistency
• rice water stools for Cholera
•
Blood
•
Visible parasites
• helminths
• segments
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2. Direct microscopy
Wet mount technique
Observations
• white blood cells
(denotes invasion)
• hanging drop
Dark background microscope
• red blood cells
• fragile organisms
(e.g. spirochetes)
• parasites
• protozoa
Viability maintained
• helminths
• mobility may be observed
• eggs
• moving bacteria
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3. Electron microscopy
Referral laboratories
Examination of viruses
e.g. Rotavirus in stool
sample
Being replaced by
antigen detection
Ebola virus
Photo: WHO
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4. Staining
•
Aspecific staining
• Gram staining
•
Specific staining with chemicals
• Ziehl Neelsen staining (Mycobacteria)
• Modified Ziehl Neelsen staining (Cryptosporidium)
•
Specific staining with labelled antibodies
• Immunofluorescence - used when gram stain cannot
help in diagnosis (e.g. Legionella too small to be visible
in a Gram stain)
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5. Rapid tests
•
Goals
• bacterial, viral or parasite antigen
(surface antigen, soluble antigen)
• toxin in biological fluids
(e.g. cerebrospinal fluid, blood, urine)
•
Main techniques
• direct agglutination: slides, cards
• latex agglutination: slides, cards
• immuno-chromatography: dipsticks
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6. Molecular methods
•
Direct blotting
• no amplification (enough DNA)
• DNA of the agent is released
– gets spotted onto a membrane and fixed
– is recognized by labelled probes (hybridization)
» radio-labelling
» electro-luminescent labelling
•
Polymerase chain reaction (PCR)
• amplification (not enough DNA)
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No Propagation:
advantages/disadvantages
•
Advantages
• fast (<1 hour)
• inexpensive
• no major laboratory infrastructure needed
•
Disadvantages
• limited sensitivity
• high concentrations needed
• limited specificity
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7. Direct Methods - propagation required
•
Bacteriology and mycology
• most agents can be propagated on culture media
•
Virology
• most agents can be propagated on cells
•
Parasitology
• monocellular organisms can be propagated in culture
media
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Propagation:
advantages/disadvantages
•
Advantages
• allows anti-microbial susceptibility testing
• allows typing of the micro-organism
• allows storage of the strain
•
Disadvantages
• depends upon the viability/condition of the agent
• takes time
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Types of laboratory methods
•
Direct methods
• look for/detect the agent
•
Indirect methods
• detect host response to the agent
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Indirect methods
Detect
1. antibodies against the agent
2. T-cell response against the agent
3. interferon
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1. Detecting antibodies
•
Precipitation
•
Agglutination
•
Haemagglutination and haemagglutination inhibition
•
Viral neutralization test
•
Radio-immunoassays
•
ELISA
•
Immunoflourescence
•
Immunoblotting
•
Immunochromatography
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Antibody detection:
advantages/disadvantages
•
Advantages
• inexpensive
• easy to perform
• allows identification of
• IgM (acute infection)
• IgG (past infection)
•
Disadvantages
• delayed response
(false negative results during sero-conversion window)
• time of infection not always clear
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2. Detection of T-cell response
•
Intra-dermal injection of antigen (e.g. Tuberculin skin
test)
• some don’t consider this a laboratory test
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T-cell response:
advantages/disadvantages
Advantages
•
•
very specific and sensitive assay for tuberculosis
•
easy to perform
Disadvantages
•
•
delayed response (few days)
•
patient has to be seen twice
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Sequencing
•
Sequence analysis of nucleic acid fragment after
PCR amplification
•
Compares alignment of nucleotides with other
sequences present in different data bases for the
identification of an agent
•
Confirmatory analysis
• the final DNA fingerprint is molecular signature of the
micro-organism
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Investigation strategies and methods
Developed by the Department of Epidemic and
Pandemic Alert and Response of the World Health
Organization with assistance from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
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