Working with the laboratory

during outbreak investigations

Integrated Disease Surveillance

Programme (IDSP) district surveillance
officers (DSO) course
 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?

2
 Outline of this session

1. Communicating with the laboratory

2. Specimen collection, storage and
transportation
3. Biosafety
4. Quality assurance

3
 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

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Communication
 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

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Communication
 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

6
Communication
 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

7
Communication
 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

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Collection
 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
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Collection
 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
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Collection
 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

WHO guide
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Collection
 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)

12
Collection
 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
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Collection
 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
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Collection
 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

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Collection
 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
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Collection
 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
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Collection
 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

18
Collection
 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
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Collection
 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)
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Collection
 Collecting and handling
cerebrospinal fluid
• Collection
 Lumbar puncture
• Aseptic conditions
• Trained person
 Sterile tubes
• Handling and
transportation
 For bacteria, transport
at ambient temperature
or preferably in trans- Trans-isolate
isolate medium (pre- biphasic medium
warmed to 25-37°C
before inoculation)
 For viruses, transport
at 4-8oC for up to 48
hours or at -70oC for
longer duration
21
Collection
 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)

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Collection
 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
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Collection
 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
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Collection
 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
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Collection
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
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Collection
 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

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Collection
 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
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Collection
 The label of the specimen

• Name: _________
• Age: ______
• ID number: _____
• Specimen type:
______
• Date, time of
collection:________
___
• Place of
collection:________
___
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Collection
 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
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Collection
 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
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Collection
 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

32
Collection
 Biosafety 1/3:
Protect the patient
• Use single use equipment
• Disinfection
• Work in a clean, dedicated area

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Biosafety
 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
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Collection
 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
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Collection
Triple-packaging of specimens:

Two goals
• Protect the environment and the
carrier
• Protect the specimen

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Collection
 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

37
Collection
 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

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Collection
 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

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Collection
 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!
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Collection
 Quality assurance

Quality Internal External

quality control quality
assurance =(Continuous, concurrent+ assessment
control of (Retrospective and
laboratory work) periodic assessment)

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Quality
 Internal quality control

• Test request and specimen

collection
• Test processing
 Temperature
 Reagent
 Maintenance of equipment
• Reporting and using test results

42
Collection
 External quality assessment

• Within the state IDSP system

 L1 by L2
 L2 by L3
• Through external agency
 External quality assurance scheme for
selected tests

43
Collection
 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

44
Collection
 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!
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 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

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Collection