The document discusses the essential protocols for collaborating with laboratories during outbreak investigations, highlighting the importance of communication, specimen collection, and biosafety. It provides guidelines on specimen handling, transportation methods, and quality assurance measures to ensure accurate results and safety for all involved. Key recommendations include developing good rapport with labs, adhering to collection guidelines, and maintaining high biosafety standards.

1. Working with the laboratory during
outbreak investigations
Integrated Disease Surveillance Programme (IDSP)
district surveillance officers (DSO) course

2. 2
Preliminary question to the group
• What is your experience in working with the
laboratory on outbreak investigations?
• If yes, what difficulties did you face?
• What would you like to learn about working
with a laboratory?

3. 3
Outline of this session
1. Communicating with the laboratory
2. Specimen collection, storage and
transportation
3. Biosafety
4. Quality assurance

4. 4
Participation of laboratory specialists
in field investigation
• Presence in the field: The ideal option
 Laboratory specialist provide real time input
 Time consuming, expensive
 Most useful in difficult situations
• Remote participation: The common option
 Involve the laboratory early
 Exchange information
 Most efficient in routine situations
Communication

5. 5
Communicating with the laboratory
• Share initial information at the earliest
about the investigation
 Epidemiological characteristics
 Suspected pathogens (differential diagnosis)
• Organize communication on an ongoing basis
 Identify focal person, obtain contact information
 Generate outbreak number
 Provide updates
 Send the final epidemiological report
Communication

6. 6
General framework to decide
what kind of specimens to take
• What are the suspected pathogens?
• What tests are used to diagnose the
suspected pathogens?
• What is the stage of the illness?
 No virus isolation at a late stage of illness
Communication

7. 7
Elements to consider when
choosing a laboratory
• Location
• Referral protocols
• Capacity
• Biosafety level
• Quality, accreditation or certification
 (e.g., Polio)
• Credibility, track record with your team
• Costs
Communication

8. 8
Whom to sample during
a classical outbreak?
• Typical cases
 Should represent the majority of the specimens
• Untreated patients
 Without antibiotics
• Cases likely to carry the pathogen
 Children
• Atypical cases
 Few specimens
• Healthy contacts
 Few specimens
Collection

9. 9
When to sample?
• Isolation of agent, PCR or antigen:
 At the earliest
 Before anti-microbial administration
• For antibody estimation:
 Ideally two paired specimens
• At earliest
• After 7 - 10 days
 Alternately, one specimen 4-5 weeks after onset
Collection

10. 10
How many specimens to take?
• Ensure sufficient number of specimens (At least 20)
 Avoid sampling error
 Obtain reliable results
• Avoid overwhelming the laboratory with excessive
specimens
• Repeat sampling in some case-patients
 Acute and convalescent sera
 Exploration of chronic carriage
 Intermittent shedding (e.g., Stool microscopy for parasites)
 Unknown etiology
Collection

11. 11
WHO guide
Rule of thumb regarding the number of
specimens to take during a cholera
outbreak
• 10 specimens to confirm the outbreak
• Five specimens per week during the outbreak
• Specimens at the end to confirm that the
outbreak is over
Collection

12. 12
Transport medium
• Allows organisms to survive under adverse
conditions
• Does not allow organisms to proliferate
• Available for bacteria
 e.g., Cary Blair
• Available for viruses
 Virus transport media (VTM)
Collection

13. 13
What is a Viral Transport Medium?
• Sterile buffered solution (Pink coloured) containing
antibiotics for preservation of viruses
• Used in the collection of specimens for viral
isolation and testing
• Save the viruses from drying
 Nutrient, glycerol
• Prevents specimen from drying out
• Prevents bacterial and fungus growth
• Prepared in the lab or commercially obtained
• Storage for short periods at 4 - 8 ºC
Collection

14. 14
To avoiding hemolysis for blood
specimens, avoid:
• Fine needles
• Forced suction of blood with syringe
• Unclean tube (residual detergents)
• Shaking tube vigorously
• Forced expulsion of the blood through
needle
• Freezing / thawing of blood
• High speed centrifugation before complete
clotting
Collection

15. 15
Vacutainers
• Vacuum tube with rubber stopper mounted
on a needle system
 Tubes may be changed for collection of different
tubes for different purposes
• Smooth blood flow, lower risk of hemolysis
• Reduces risk of spillage
Collection

16. 16
Collecting and handling blood for smears
• Collection
 Take capillary blood from finger prick (Lancet)
 Make smear on clean glass slide
 Dry and fix with methanol or other fixative
• Handling and transportation
 Transport slides within 24 hours
 Do not refrigerate
• May alter the morphology of the cells
Collection

17. 17
Collecting blood for cultures
• Collect within 10-30 mn of
fever
• Aseptic technique
• Quantity
 0.5 – 2 ml venous blood for
infants
 2 – 5 ml venous blood for
children
 5 – 10 ml venous blood for adults
• Take three sets of blood
culture when suspecting
bacterial endocarditis
Collection

18. 18
Handling and transporting
blood for cultures
• Collect into blood culture bottles with
infusion broth
 Change the needle to inoculate the broth
• Travel at ambient temperature
Collection

19. 19
Collecting serum
• Collect venous blood in a sterile test tube
• Let specimen clot for 30 minutes at ambient
temperature
• Place at 4-8oC for clot retraction for at least
1-2 hours
• Centrifuge at 1500 RPM for 5-10 min
• Separate the serum from the clot with
pipette / micro-pipette
Collection

20. 20
Handling and transporting serum
• Transport at 4-8oC if transport lasts less than
10 days
• Freeze at -20oC if storage for weeks or
months before processing and shipment to
reference laboratory
 Ship frozen
• Avoid repeated freeze-thaw cycles
 Destroy IgM (e.g., Measles diagnosis)
Collection

21. 21
• Collection
 Lumbar puncture
• Aseptic conditions
• Trained person
 Sterile tubes
• Handling and
transportation
 For bacteria, transport at
ambient temperature or
preferably in trans-isolate
medium (pre-warmed to 25-
37°C before inoculation)
 For viruses, transport at 4-
8o
C for up to 48 hours or at
-70o
C for longer duration
Collecting and handling
cerebrospinal fluid
Trans-isolate
biphasic medium
Collection

22. 22
Collecting and handling
stool specimens
• Take freshly passed stool specimen
 Avoid collecting specimen from a bed pan
• Collect specimen in a sterile container (if
available) or clean container (not cleaned
with a disinfectant)
Collection

23. 23
Rectal swabs
• Advantage
 Convenient
 Adapted to small children, debilitated patients
and other situation where voided stool specimen
collection is not feasible
• Drawbacks
 No macroscopic assessment possible
 Less material available
• Not recommended for viruses
Collection

24. 24
Collecting stool specimens
for viruses
• Timing
 Within 48 hours of onset
• Specimen amount and size
 At least 5-10 ml fresh stool from patients (and controls)
• Method
 Fresh stool unmixed with urines in clean, dry and sterile container
• Storage
 Refrigerate at 4oC. Do not freeze
 Store at -15oC for antigen detection and protein chain reaction (PCR)
• Transport
 4oC (Do not freeze)
 Dry ice for antigen detection and PCR
Collection

25. 25
Collecting stool specimens
for bacteria
• Timing
 During active phase
• Specimen amount and size
 Fresh specimens and two swabs from patients, controls and
carriers (if indicated)
• Method
 In Cary-Blair medium
(+ specimen without transport medium
for antigen detection / PCR)
• Storage
 Refrigerate at 4oC if testing within
48 hours, -70oC if longer
Collection

26. 26
Collecting stool specimens for parasites
• Timing
 As soon as possible after onset
• Specimen amount and size
 At least 3 x 5-10 ml fresh stool from patients (and controls)
• Method
 Mixed with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part
preservative
 Unpreserved specimens for antigen detection and PCR
• Storage
 Refrigerate at 4oC
 Store at -15oC for antigen detection and PCR
• Transport
 4oC (Do not freeze)
 Dry ice for antigen detection and PCR
Collection

27. 27
Collecting a sputum
• Instruct patient to take a deep breath and
cough up sputum directly into a wide-mouth
sterile container
• Avoid saliva or postnasal discharge
• Minimum volume should be about 1 ml
Collection

28. 28
Handling and transportation
of respiratory specimens
• All respiratory specimens except sputum are
transported in appropriate media
 Amie’s or Stuart’s transport medium for bacteria
 Viral transport medium for viruses
• 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
Collection

29. 29
The label of the specimen
• Name: _________
• Age: ______
• ID number: _____
• Specimen type: ______
• Date, time of
collection:___________
• Place of
collection:___________
Collection

30. 30
Labeling glass slides for microscopy
• Label slides individually
• Use glass marking pencil
• Make sure procedure will not interfere with
the staining process
• Each slide should bear:
 Patient' name
 Unique identification number
 Date of collection
Collection

31. 31
The case investigation form:
What the 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 of patients
Collection

32. 32
The case investigation form:
What the receiving laboratory records
• Date and time when specimen was received
• Name and initials of the person receiving
specimen
• Record of specimen quality
Collection

33. 33
Biosafety 1/3:
Protect the patient
• Use single use equipment
• Disinfection
• Work in a clean, dedicated area
Biosafety

34. 34
Biosafety 2/3:
Protect yourself
• Use personal protective equipment
 Disposable gloves
 Laboratory coats / gown
 Mask
 Protective eyewear / face shields if procedure is likely to
generate aerosols
• Collect sharps immediately, in the absence of recapping
in sharps container to prevent needle-stick injury
• Have first aid kit readily accessible
• Do not reuse contaminated equipment / supplies such
as gloves
• Do not leave the specimen on the request form
Collection

35. 35
Biosafety 3/3:
Protect others and the environment
• Package specimens 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 or materials in
1% household bleach for 5 minutes. Wash in soapy water
before use and sterilize if necessary
• Place waste in leak-proof biohazard bags
• Ensure safe final management of waste
• Protect personnel in charge of cleaning or decontamination
with protective coat and thick rubber gloves
Collection

36. 36
Triple-packaging of specimens:
Two goals
• Protect the environment and the carrier
• Protect the specimen
Collection

37. 37
The basic triple packaging system:
1/3: The primary receptacle
• Sealed specimen container to be placed in a suitably
sized plastic bag/ ziploc bag
• Packaged with sufficient absorbent material to
absorb the entire content of the primary receptacle
in case of breakage
• Specimens from different patients should never be
sealed in the same bag
• Two or more sealed specimens of the same patient
may be put in a larger plastic bag and sealed
Collection

38. 38
The basic triple packaging system:
2/3: The secondary receptacle
• Leak-proof secondary plastic container with
screw capped lids
• Enclose and protect the primary
receptacle(s)
 Place the sealed bags containing the specimens
inside secondary plastic containers
 Specimens from several patients may be packed
inside the same secondary plastic container
• Sufficient additional absorbent material used
to absorb all fluid in case of breakage
Collection

39. 39
The basic triple packaging system:
3/3: The outer packaging
• Secondary packaging(s) are placed in outer shipping
packaging with suitable cushioning material
• Outer packaging protect their contents from outside
influences, such as physical damage, while in transit
• Resistant, high density external cover (metal, wood,
fiberboard)
• Smallest overall external dimension
 10x10 cm
Collection

40. 40
Refrigeration methods to obtain
different temperatures
• 2-8 °C/4 °C
 Wet ice/ice packs/domestic refrigerator
• -8/ -10 °C
 Freezer of domestic refrigerator
• -20 °C
 Freezer cabinet
• -70 °C
 Deep freezer/dry ice
• -170/ -196 °C
 Liquid Nitrogen
Vaccine carriers that have been used for specimen transport must
never be reused for carrying vaccines!
Collection

41. 41
Quality
assurance
Internal
quality control
(Continuous, concurrent
control of
laboratory work)
External
quality
assessment
(Retrospective and
periodic assessment)
Quality assurance
= +
Quality

42. 42
Internal quality control
• Test request and specimen collection
• Test processing
 Temperature
 Reagent
 Maintenance of equipment
• Reporting and using test results
Collection

43. 43
External quality assessment
• Within the state IDSP system
 L1 by L2
 L2 by L3
• Through external agency
 External quality assurance scheme for selected
tests
Collection

44. 44
Criteria for rejection of specimens
• Mismatch of information on the label and the
request
• Inappropriate transport temperature
• Inappropriate transport medium
• Insufficient quantity
• Leakage
• Excessive delay in transportation
• Specimen received in a fixative
• Dry specimen
• Specimen with questionable relevance
Collection

45. 45
Take home messages
1. Develop rapport with the laboratory
2. Collect specimen according to the
guidelines and access on-line resources if
needed
3. Protect the patient, yourself and others
4. You can contribute to quality assurance!

46. 46
Additional reading
• WHO Guidelines for the collection of clinical
specimens during field investigation of
outbreaks
• IDSP bio safety manual
• Section 6 of operations manual
• Module 6 of training manual
Collection