Working with the laboratory during outbreak investigations

1
Working with the laboratory during outbreak
investigations

• Integrated Disease Surveillance Programme (IDSP)
  district surveillance officers (DSO) course

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
Outline of this session

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

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
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
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
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
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
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
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
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
Collection
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
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
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
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
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
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
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
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
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
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-isolate medium (pre-warmed to
  25-37C before inoculation)
• For viruses, transport at 4-8oC for up to 48
  hours or at -70oC for longer duration

Trans-isolate biphasic medium
Collection
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
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
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
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
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
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
Handling and transportation of respiratory
specimens

• All respiratory specimens except sputum are
  transported in appropriate media
• Amies or Stuarts 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
• 4-8C for viruses

Collection
29
The label of the specimen

• Name _________
• Age ______
• ID number _____
• Specimen type ______
• Date, time of collection___________
• Place of collection___________

Collection
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
The case investigation formWhat 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
The case investigation formWhat 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
Biosafety 1/3Protect the patient

• Use single use equipment
• Disinfection
• Work in a clean, dedicated area

Biosafety
34
Biosafety 2/3Protect 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
Biosafety 3/3Protect 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
Triple-packaging of specimens Two goals

• Protect the environment and the carrier
• Protect the specimen

Collection
37
The basic triple packaging system1/3 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
The basic triple packaging system2/3 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
The basic triple packaging system3/3 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
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
Quality assurance
Quality assurance
Internal quality control (Continuous,
concurrent control of laboratory work)
External quality assessment(Retrospective and
periodic assessment)


Quality
42
Internal quality control

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

Collection
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
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
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
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