E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Overview of Microbiology Methods

Investigation strategies and methods

May 2007

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Learning objectivesLearning 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Managing the sample: administrativeManaging the sample: administrative

• When an outbreak sample is received it is assigned:

• laboratory identification number

• an outbreak identification label

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Managing the sample: technicalManaging 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Types of laboratory methodsTypes of laboratory methods

• Direct methods

• look for/detect the agent

• Indirect methods

• detect host response to the agent

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Direct methodsDirect methods

1. Macroscopic evaluation

2. Direct microscopy

3. Electron microscopy

4. Staining

5. Rapid tests

6. Molecular methods

7. Propagate the agent

No propagation required

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

1. Macroscopic evaluation1. Macroscopic evaluation

• Consistency

• rice water stools for Cholera

• Blood

• Visible parasites

• helminths

• segments

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

2. Direct microscopy 2. Direct microscopy

Wet mount technique

• hanging drop

Dark background microscope

• fragile organisms (e.g. spirochetes)

Viability maintained

• mobility may be observed

Observations

• white blood cells (denotes invasion)

• red blood cells

• parasites

• protozoa

• helminths

• eggs

• moving bacteria

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

3. Electron microscopy 3. Electron microscopy

Referral laboratories

Examination of viruses

e.g. Rotavirus in stool sample

Being replaced by antigen detection

Ebola virus

Photo: WHO

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

4. Staining4. 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)

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

5. Rapid tests5. 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

6. Molecular methods6. 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)

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

No Propagation: No Propagation: advantages/disadvantages advantages/disadvantages

• Advantages

• fast (<1 hour)

• inexpensive

• no major laboratory infrastructure needed

• Disadvantages

• limited sensitivity

• high concentrations needed

• limited specificity

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

7. Direct Methods - propagation required7. 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Propagation: advantages/disadvantages 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Types of laboratory methodsTypes of laboratory methods

• Direct methods

• look for/detect the agent

• Indirect methods

• detect host response to the agent

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Indirect methodsIndirect methods

Detect

1. antibodies against the agent

2. T-cell response against the agent

3. interferon

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

1. Detecting antibodies1. Detecting antibodies

• Precipitation

• Agglutination

• Haemagglutination and haemagglutination inhibition

• Viral neutralization test

• Radio-immunoassays

• ELISA

• Immunoflourescence

• Immunoblotting

• Immunochromatography

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Antibody detection: Antibody detection: advantages/disadvantagesadvantages/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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

2. Detection of T-cell response2. Detection of T-cell response

• Intra-dermal injection of antigen (e.g. Tuberculin skin test)

• some don’t consider this a laboratory test

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

T-cell response: T-cell response: advantages/disadvantagesadvantages/disadvantages

• Advantages

• very specific and sensitive assay for tuberculosis

• easy to perform

• Disadvantages

• delayed response (few days)

• patient has to be seen twice

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

SequencingSequencing

• 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

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

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