mr-said warsame nur

1. Laboratory Training for Field Epidemiologists
Lecture 12Lecture 12
•Laboratory and
field investigation

2. Laboratory Training for Field Epidemiologists
Learning objectivesLearning objectives
At the end of the presentation, participants should
understand the:
• Procedures, preparation, processing and
transport of specimens

3. Laboratory Training for Field Epidemiologists
Successful laboratorySuccessful laboratory
investigationsinvestigations
 Advance planning
 Collection of adequate and appropriate specimens
 Sufficient documentation
 Biosafety and decontamination
 Correct packaging
 Rapid transport
 Choice of a laboratory that can accurately perform the tests
 Timely communication of results

4. Laboratory Training for Field Epidemiologists
Specimen collection:Specimen collection:
key issueskey issues
Consider differential diagnoses
Decide on test(s) to be conducted
Decide on clinical samples to be collected to conduct
these tests
• consultation between microbiologist, clinicians and
epidemiologist

5. Laboratory Training for Field Epidemiologists
Transport mediumTransport medium
Allows organisms (pathogens and contaminants) to
survive
Non-nutritive - does not allow organisms to proliferate
For bacteria – i.e., Cary Blair
For viruses - virus transport media (VTM)

6. Laboratory Training for Field Epidemiologists
Blood for smearsBlood for smears
Collection
Capillary blood from finger prick
• make smear
• fix with methanol or other fixative
Handling and transport
Transport slides within 24 hours
Do not refrigerate (can alter cell morphology)

7. Laboratory Training for Field Epidemiologists
Blood for culturesBlood for cultures
Collection
Venous blood
• infants: 0.5 – 2 ml
• children: 2 – 5 ml
• adults: 5 – 10 ml
Requires aseptic technique
Collect within 10 minutes of fever
• if suspect bacterial endocarditis: 3 sets of blood culture

8. Laboratory Training for Field Epidemiologists
Blood for culturesBlood for cultures
Handling and Transport
Collect into bottles with infusion broth
• change needle to inoculate the broth
Transport upright with cushion
• prevents hemolysis
Wrap tubes with absorbent cotton
Travel at ambient temperature
Store at 4o
C if can’t reach laboratory in 24h

9. Laboratory Training for Field Epidemiologists
SerumSerum
Collection
Venous blood in sterile test tube
• let clot for 30 minutes at ambient temperature
• glass better than plastic
Handling
Place at 4-8o
C for clot retraction for at least 1-2 hours
Centrifuge at 1 500 RPM for 5-10 min
• separates serum from the clot

10. Laboratory Training for Field Epidemiologists
SerumSerum
Transport
4-8o
C if transport lasts less than 10 days
Freeze at -20o
C if storage for weeks or months
before processing and shipment to reference
laboratory
Avoid repeated freeze-thaw cycles
• destroys IgM
To avoid hemolysis: do not freeze unseparated
blood

11. Laboratory Training for Field Epidemiologists
Collection
Lumbar puncture
Sterile tubes
Aseptic conditions
Trained person
Cerebrospinal fluid (CSF)Cerebrospinal fluid (CSF)

12. Laboratory Training for Field Epidemiologists
CSFCSF
Handling and transportation
Bacteria
• preferably in trans-isolate medium,
pre-warmed to 25-37°C before inoculation
OR
• transport at ambient temperature (relevant pathogens do
not survive at low temperatures)
Viruses
• transport at 4-8o
C (if up to 48hrs or -70o
C for longer
duration)

13. Laboratory Training for Field Epidemiologists
Stool samplesStool samples
Collection:
Freshly passed stool samples
• avoid specimens from a bed pan
Use sterile or clean container
• do not clean with disinfectant
During an outbreak - collect from 10-20 patients

14. Laboratory Training for Field Epidemiologists
Stool samples for virusesStool samples for viruses
Timing
• within 48 hours of onset
Sample amount
• 5-10 ml fresh stool from patients (and controls)
Methods
• fresh stool unmixed with urine in clean, dry and sterile container
Storage
• refrigerate at 4o
C; do not freeze
• store at -15o
C - for Ag detection,polymerase chain reaction (PCR)
Transport
• 4o
C (do not freeze); dry ice for (Ag detection and PCR)

15. Laboratory Training for Field Epidemiologists
Stool samples for bacteriaStool samples for bacteria
Timing
• during active phase
Sample amount and size
• fresh sample and two swabs from patients,
controls and carriers (if indicated)
Method
• Cary-Blair medium
• For Ag detection/PCR – no transport medium
Storage
• refrigerate at 4o
C if testing within 48 hours, -70o
C if longer;
store at -15o
C for Ag detection and PCR
Transport
• 4o
C (do not freeze); dry ice for Ag, PCR detection

16. Laboratory Training for Field Epidemiologists
Stool samples for parasitesStool samples for parasites
Timing
• as soon as possible after onset
Sample amount and size
• at least 3 x 5-10 ml fresh stool from patients and controls
Method
• mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1
part preservative
• unpreserved samples for Ag detection and PCR
Storage
• refrigerate at 4o
C; store at -15o
C for Ag detection and PCR
Transport
• 4o
C (do not freeze); dry ice for antigen detection and PCR

17. Laboratory Training for Field Epidemiologists
Throat swabThroat swab
(posterior pharyngeal swab)(posterior pharyngeal swab)
Hold tongue away with
tongue depressor
Locate areas of inflammation
and exudate in posterior
pharynx, tonsillar region of
throat behind uvula
Avoid swabbing soft palate;
do not touch tongue
Rub area back and forth with
cotton or Dacron swab
WHO/CDS/EPR/ARO/2006.1

18. Laboratory Training for Field Epidemiologists
Nasopharyngeal swabNasopharyngeal swab
Tilt head backwards
Insert flexible fine-shafted
polyester swab into nostril and
back to nasopharynx
Leave in place a few seconds
Withdraw slowly; rotating
motion
WHO/CDS/EPR/ARO/2006.1

19. Laboratory Training for Field Epidemiologists
Naso-pharyngeal aspirateNaso-pharyngeal aspirate
Tilt head slightly backward
Instill 1-1.5 ml of VTM /sterile
normal saline into one nostril
Use aspiration trap
Insert silicon catheter in nostril
and aspirate the secretion
gently by suction in each
nostril
WHO/CDS/EPR/ARO/2006.1

20. Laboratory Training for Field Epidemiologists
SputumSputum
Collection
Instruct patient to take a deep breath and cough up
sputum directly into a wide-mouth sterile container
• avoid saliva or postnasal discharge
• 1 ml minimum volume

21. Laboratory Training for Field Epidemiologists
Respiratory samplesRespiratory samples
Handling and Transport
All respiratory specimens except sputum are transported in
appropriate media
• bacteria: Amie’s or Stuart’s transport medium
• viruses: viral transport medium (VTM)
Transport as quickly as possible to the laboratory to reduce
overgrowth by oral flora
For transit periods up to 24 hours
• ambient temperature for bacteria
• 4-8°C for viruses

22. Laboratory Training for Field Epidemiologists
Collection
Biopsy relevant tissues
• place in formalin for histopathology
• place in transport medium for microbiological testing
• place in sterile saline for isolation of viral pathogens
Post-mortem samplesPost-mortem samples

23. Laboratory Training for Field Epidemiologists
Post-mortem samplesPost-mortem samples
Handling and transportation
Fixed specimens can be transported at ambient
temperatures
• transport specimens in transport media within 24h at
ambient temperature
• transport specimens in sterile saline at 4-8o
C within 48h

24. Laboratory Training for Field Epidemiologists
Specimen
Transport
media
Storage condition
Purpose/ Lab
investigationTransport Pending test
Throat swab VTM 2-8 0
C -20 0
C Isolation
NPA/ swab VTM 2-8 0
C -20 0
C Isolation
CSF No 2-8 0
C -20 0
C Isolation,
serology
Stool No 2-8 0
C -20 0
C Isolation
Urine No 2-8 0
C -20 0
C Isolation
Serum/
Clotted blood
No 2-8 0
C -20 0
C
2-8 0
C
Isolation,
serology
Whole blood No 2-8 0
C 2-8 0
C Isolation,
serology
Virologic InvestigationsVirologic Investigations

25. Laboratory Training for Field Epidemiologists
Labeling specimensLabeling specimens
Patient’s name
Clinical specimen
Unique ID number
(Research/Outbreak)
Specimen type
Date, time and place of collection
Name/ initials of collector

26. Laboratory Training for Field Epidemiologists
Glass slides for microscopyGlass slides for microscopy
Label slides individually
• use glass marking pencil
• ensure markings don’t interfere with staining process
Each slide should bear:
• patient name
• unique identification number
• date of collection

27. Laboratory Training for Field Epidemiologists
Case investigation formCase investigation form
Epidemiologist sends:
Patient information
• age (or date of birth), sex, complete address
Clinical information
• date of onset of symptoms, clinical and immunization
history, risk factors or contact history where relevant,
anti-microbial drugs taken prior to specimen collection
Laboratory information
• acute or convalescent specimen
• other specimens from the same patient
Line listing – if large number of patients

28. Laboratory Training for Field Epidemiologists
Case investigation formCase investigation form
Receiving laboratory records:
Date and time when specimen was received
Name and initials of the person receiving specimen
Record of specimen quality

29. Laboratory Training for Field Epidemiologists

30. Laboratory Training for Field Epidemiologists
Biosafety: protect the patientBiosafety: protect the patient
Use single use equipment
Disinfect
Work in a clean, dedicated area

31. Laboratory Training for Field Epidemiologists
Biosafety: protect yourselfBiosafety: protect yourself
Use personal protective equipment
• disposable gloves
• laboratory coats / gown
• mask
• protective eyewear / face shields if procedure is likely to
generate aerosols
If no sharps container: collect sharps immediately to prevent
needle-stick injury
Have first aid kit readily accessible
Do not reuse contaminated equipment

32. Laboratory Training for Field Epidemiologists
Biosafety: protect others, theBiosafety: protect others, the
environmentenvironment
Package samples appropriately for transport
Decontaminate spills - 10% bleach after wiping the surface clean
Disinfect working areas for future use - 1% household bleach daily
Soak contaminated non-disposable equipment/material in 1%
household bleach for 5 minutes
• wash in soapy water before re-use, sterilize if necessary
Place waste in leak-proof biohazard bags - ensure safe final
management of waste
Protect cleaning/decontamination personnel with protective coat,
thick rubber gloves

33. Laboratory Training for Field Epidemiologists
Infection control precautionsInfection control precautions
Precautions Use Requirements
Contact
precautions
Patients known or suspected to have
serious illnesses easily transmitted
by direct patient contact or by contact
with items in the patient's
environment
•Gloves
•Gown
Droplet
precautions
Barrier to stop infections spread by
large (>5 microns), moist droplets
produced by people when they
cough, sneeze or speak
•Contact precautions
•Well-fitting mask
•Eye protection
Airborne
precautions
Patients known or suspected to have
serious illnesses transmitted by
airborne droplet nuclei
•Contact precautions
•Droplet precautions
• N95 mask
•Isolation room
(In hospital)

34. Laboratory Training for Field Epidemiologists
WHO reference materialsWHO reference materials
Guidelines for the collection of clinical specimens during field
investigation of outbreaks, WHO, 2000
The role of laboratories and blood banks in disaster situations,
WHO publication, 2001
Sampling during avian influenza investigations (2006)
IDSR guidelines for specimen collection (2003)
Laboratory Needs for Emergency Situations (2003)
Overview of Laboratory Structure and Operational Needs for
the Iraqi Crisis (2003)
Costing for sampling materials and diagnostic reagents for the
Iraq crisis (2003)