Overview of Microbiology Methods

1. Investigation strategies and methods Overview of Microbiology Methods May 2007

2. 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. Managing the sample: administrative • When an outbreak sample is received it is assigned: • laboratory identification number • an outbreak identification label

4. 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. Types of laboratory methods • Direct methods • look for/detect the agent • Indirect methods • detect host response to the agent

6. Direct methods • Macroscopic evaluation • Direct microscopy • Electron microscopy • Staining • Rapid tests • Molecular methods • Propagate the agent No propagation required

7. 1. Macroscopic evaluation • Consistency • rice water stools for Cholera • Blood • Visible parasites • helminths • segments

8. 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. 3. Electron microscopy Referral laboratories Examination of viruses e.g. Rotavirus in stool sample Being replaced by antigen detection Ebola virus Photo: WHO

10. 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. 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. 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. No Propagation: advantages/disadvantages • Advantages • fast (<1 hour) • inexpensive • no major laboratory infrastructure needed • Disadvantages • limited sensitivity • high concentrations needed • limited specificity

14. 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. 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. Types of laboratory methods • Direct methods • look for/detect the agent • Indirect methods • detect host response to the agent

17. Indirect methods Detect • antibodies against the agent • T-cell response against the agent • interferon

18. 1. Detecting antibodies • Precipitation • Agglutination • Haemagglutination and haemagglutination inhibition • Viral neutralization test • Radio-immunoassays • ELISA • Immunoflourescence • Immunoblotting • Immunochromatography

19. 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. 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. 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. 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

23. 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