1. Laboratory Training for Field Epidemiologists Overview of Microbiology Methods Investigation strategies and methods May 2007

2. Laboratory Training for Field Epidemiologists 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

3. Laboratory Training for Field Epidemiologists Managing the sample: administrative When an outbreak sample is received it is assigned: laboratory identification number an outbreak identification label

4. Laboratory Training for Field Epidemiologists 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

5. Laboratory Training for Field Epidemiologists Types of laboratory methods Direct methods look for/detect the agent Indirect methods detect host response to the agent

6. Laboratory Training for Field Epidemiologists Direct 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

7. Laboratory Training for Field Epidemiologists 1. Macroscopic evaluation Consistency rice water stools for Cholera Blood Visible parasites helminths segments

8. Laboratory Training for Field Epidemiologists 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

9. Laboratory Training for Field Epidemiologists 3. Electron microscopy Referral laboratories Examination of viruses e.g. Rotavirus in stool sample Being replaced by antigen detection Ebola virus Photo: WHO

10. Laboratory Training for Field Epidemiologists 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)

11. Laboratory Training for Field Epidemiologists 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

12. Laboratory Training for Field Epidemiologists 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)

13. Laboratory Training for Field Epidemiologists No Propagation: advantages/disadvantages Advantages fast (<1 hour) inexpensive no major laboratory infrastructure needed Disadvantages limited sensitivity high concentrations needed limited specificity

14. Laboratory Training for Field Epidemiologists 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

15. Laboratory Training for Field Epidemiologists 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

16. Laboratory Training for Field Epidemiologists Types of laboratory methods Direct methods look for/detect the agent Indirect methods detect host response to the agent

17. Laboratory Training for Field Epidemiologists Indirect methods Detect 1.antibodies against the agent 2.T-cell response against the agent 3.interferon

18. Laboratory Training for Field Epidemiologists 1. Detecting antibodies Precipitation Agglutination Haemagglutination and haemagglutination inhibition Viral neutralization test Radio-immunoassays ELISA Immunoflourescence Immunoblotting Immunochromatography

19. Laboratory Training for Field Epidemiologists 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

20. Laboratory Training for Field Epidemiologists 2. Detection of T-cell response Intra-dermal injection of antigen (e.g. Tuberculin skin test) some don’t consider this a laboratory test

21. Laboratory Training for Field Epidemiologists 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

22. Laboratory Training for Field EpidemiologistsSequencing 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

23. 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