Unit 5 bacteria and viruses

Laboratory Training for Field Epidemiologists
Viral cultures
Investigation strategies and methods
May 2007
Laboratory Training for Field Epidemiologists
Learning objectives
Learning objectives
At the end of the presentation, participants should:
•
Understand the principle of cultivating viruses
•
Understand the methods and problems with cultivating viruses
Laboratory Training for Field Epidemiologists
Techniques to identify viruses
Techniques to identify viruses
It can take a few hours to weeks to identify a virus
Techniques include:
•
PCR (single round) or nested/semi-nested PCR
•
Real-time PCR
•
Direct electronic microscopy
•
Antigen capture
•
Isolation
–
Long process
–
Gold standard for viruses that can be cultured

Laboratory Training for Field Epidemiologists
Virus culture
Virus culture
Is based upon amplification of potentially infectious pathogens
Implies intracellular replication of viruses in the cytoplasm or
in the nucleus
Is controlled by regulations (i.e. bio-safety level 2, 3 or 4)
Allows for:
•
Identification
•
Further studies
(e.g., Pathogenicity, antiviral sensitivity, research)
Laboratory Training for Field Epidemiologists
Virus culture
Virus culture
Long process
•
Not always possible for front-line diagnosis
Primary objective for the diagnosis of an unknown disease
No generic protocol
Laboratory Training for Field Epidemiologists
How to go about virus culture?
How to go about virus culture?
Obtain suitable specimens
•
Identified specimens with suitable information
Evaluate of chances of success of the process before start
Make sure transportation used cold chain
•

4°C
•
-20°C
•
Dry ice (-79°C)
Use suitable culture protocol
•
In vitro/in vivo cell cultures
Laboratory Training for Field Epidemiologists
Culture procedure
Culture procedure
Use of a variety of cell sources and techniques
Treatment of the specimen prior to inoculation
Follow-up
Viral detection:
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Non specific
–
Cytopathogenic effect (microscope)
–
Electronic microscopy identification (morphology)
•
Specific
–
Immunological detection: antigen detection, PCR, IFA…
Viral load estimation (titration, plaque assay)
Laboratory Training for Field Epidemiologists
Limitations of cultures to
Limitations of cultures to
identify viruses

identify viruses
Absence of detection system for the agent
Inappropriate culture systems
Viruses that cannot be cultured
A negative viral culture results does not mean that the
agent is absent
•
Need of other tests
•
PCR can detect the viral genome in absence of the
complete virus
Laboratory Training for Field Epidemiologists
Specimens used to culture viruses
Specimens used to culture viruses
Blood specimens
•
EDTA
•
Heparin
•
Serum
Stool
Throat swabs
Naso-paryngeal aspirates
Stools, rectal swabs
Urine
Saliva
Cerebro-spinal fluid
Biopsy
•

Skin (filoviridae)
•
Organs (fixation with
formaldehyde 10%)
Laboratory Training for Field Epidemiologists
Potentially infectious specimen forms
Potentially infectious specimen forms
Laboratory Training for Field Epidemiologists
Sequencing
Sequencing
Analysis of sequence of nucleic acid fragment after
PCR amplification
Comparison of the alignment of nucleotides with other
sequences present in different data bases for the
identification of an agent
Confirmatory analysis
•
Final DNA fingerprint is molecular signature of the micro-organism
Laboratory Training for Field Epidemiologists
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
Investigation strategies and methods