Working with the laboratory during outbreak investigations

Integrated Disease Surveillance Programme (IDSP) district surveillance

officers (DSO) course

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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?

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Outline of this session

1. Communicating with the laboratory 2. Specimen collection, storage and

transportation3. Biosafety4. Quality assurance

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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• 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 4-8oC for up to 48 hours or at -70oC for longer duration

Collecting and handling cerebrospinal fluid

Trans-isolate biphasic medium

Collection

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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

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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

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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

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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

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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

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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

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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

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The label of the specimen

• Name: _________• Age: ______• ID number: _____• Specimen type: ______

• Date, time of collection:___________

• Place of collection:___________

Collection

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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

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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

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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

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Biosafety 1/3:Protect the patient

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

Biosafety

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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

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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

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Triple-packaging of specimens:

Two goals• Protect the environment and the carrier

• Protect the specimen

Collection

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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

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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

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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

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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 NitrogenVaccine carriers that have been used for specimen

transport must never be reused for carrying vaccines!

Collection

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Quality assurance

Internal quality control

(Continuous, concurrent control of

laboratory work)

External quality assessment

(Retrospective and periodic assessment)

Quality assurance

= +

Quality

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Internal quality control

• Test request and specimen collection

• Test processing Temperature Reagent Maintenance of equipment

• Reporting and using test results

Collection

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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

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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

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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

Collection