Download Laboratory Diagnostics, Specimen Collection, and Biosafety

Laboratory Diagnostics,
Specimen Collection,
and Biosafety Issues
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Learning Objectives
• Describe avian influenza-related laboratory
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procedures
Define laboratory safety
List elements of specimen collection kit
Explain how to collect & transport specimens
Describe infectious waste disposal
Explain how to manage laboratory data
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Session Overview
• Laboratory diagnosis of human cases of avian
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influenza
Laboratory safety
Specimen collection and transportation
Specimen storage, handling, packaging, and
transportation
Waste disposal and decontamination
Managing and analyzing laboratory data
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Laboratory Diagnosis of
Human Cases of Avian Influenza
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Laboratory Diagnostics for Avian
Influenza
Tests on respiratory samples:
• Virus isolation
• Immunofluorescence
• Rapid antigen detection
Tests on serum:
• Measure specific antibodies
• PCR-based techniques
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H5N1 Virus Found in
Other Human Specimens
• H5N1 virus infection of cerebrospinal fluid
documented in a fatal case with seizures and coma
• H5N1 virus has also been found in:
 Rectal swab specimens and stool of fatal cases with
diarrhea
 Serum and plasma of fatal cases
• All respiratory secretions and bodily fluids of H5N1
patients should be considered potentially infected
with H5N1 virus
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PCR-based Techniques
• Respiratory samples
• Diagnostic assays and sequencing
• Sensitivity depends on:
Particular assay
Influenza strain
Type of the specimen
Quality of the specimen
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PCR: H5N1 Virus Testing
Primary method: detection of H5N1 viral RNA
by reverse-transcription polymerase chain
reaction (RT-PCR)
• Conventional RT-PCR
• Real-time RT-PCR (rt-RT-PCR)
• Highly sensitive and specific
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Interpretation of Real-time
RT-PCR Results
Test reactions
A
H1 H3
H5
B
RNP
Results
Sample 1
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+
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+
A/H5
Sample 2
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+
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+
Inconcl
Sample 3
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+
Neg
Sample 4
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Inconcl
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Virus Isolation of H5N1 Viruses
Gold Standard
• Requires biosafety level 3 (BSL-3)- enhanced
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laboratory
Requires select agent approval from USDA
(United States)
Allows antigenic & genetic characterization
Allows drug susceptibilities tests
Performed at WHO Influenza Centers
Antigenic characterization of viral isolates
Collect specimens for vaccine development
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Immunofluorescence for H5N1
Testing
• Requires H5 monoclonal antibody
• Not part of WHO Reagent kit for seasonal
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influenza isolates
Interpretation difficult
Results:
Presence of influenza virus
Subtype identification
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Indirect IF Staining of Cells From
Tracheal Aspirate
Anti-H5
Anti-H3
Anti-A/NP
Anti-B
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Taken from: World Bank Training by Alexander Klimov, CDC
Rapid Antigen Tests
• Quick results and commercially available
• Low sensitivity
• False negatives and false positives
• Identifies viral type (type A or B) NOT subtype (H5,
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H3, H1)
Clinically useful to guide treatment, but must test for
H5 with another assay
RAPID TESTS NOT RECOMMENDED
For Detection of human H5N1 virus infection 13
Using Serology
Delay for anti-H5
levels to rise
Requires acute and
convalescent sera, 3 weeks
apart
Not useful for clinical
management
Can confirm epidemic
cases, if respiratory
specimens not available
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Influenza Serology Tests
• Micro-neutralization assays
• Western Blot
• Enzyme immunoassays
Require acute and convalescent sera
(serum obtained >21 days from onset)
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Interpreting Serology Tests
Acute
Sample
low
low
high
Convalescent
Sample
Interpretation
low
No evidence of
Infection
high*
Seroconversion
(evidence of infection)
high
Inconclusive; past
exposure to influenza
or previous vaccination
possible
* four-fold or greater increase between acute and convalescent sera
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Serological Specimens
• Paired serum specimens most useful
4-fold rise in antibody titer
• Single convalescent serum specimens:
Useful in outbreak investigations of novel human
influenza viruses
If prevalence unknown, case serum specimen
compared to age-matched control specimen
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How to Avoid Misinterpretation of
Diagnostic Tests
• Use appropriate controls
• Understand
What is test identifying?
What are limitations of assay?
• Use more than one test to confirm results and
use more than one sample from each patient
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Review Question 1
Which of the following is the best technique to
test respiratory samples for avian influenza?
a.
b.
c.
d.
PCR-based techniques
Virus isolation
Immunofluorescence
Rapid antigen detection
Answers: a. PCR-based techniques
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Review Question 2
Which of the following situations is most likely to
lead to the misinterpretation of diagnostic tests?
a.
b.
c.
d.
Use appropriate controls for each assay
Understand what the test is identifying
Understand the limitations of each assay
Use only one test if you are sure that it is correct
Answer: d. Instead, you should use multiple tests to
confirm results.
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Laboratory Safety
Dr. Terrence Tumpey examines reconstructed 1918 Pandemic
Influenza Virus inside a specimen vial in a Biosafety Level 3enhanced laboratory setting. Courtesy CDC/James Gathany.
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Biosafety Laboratory Levels (BSL)
• Risk of acquiring infections exists in laboratory
• Laboratory safety guidelines evolved
• Safe work sites result from:
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Engineering controls
Management policies
Work practices and procedures
Medical interventions (occasional)
Increasing biosafety levels = increasing levels of
personnel & environmental protection
http://www.cdc.gov/OD/ohs/symp5/
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BSL-2 and BSL-3
• BSL-2
Good microbiological technique
Protective clothing
Biohazard sign
• BSL-3 =
BSL-2 plus:
Controlled access
Directional air flow
• BSL-3 enhanced = BSL-3 plus:
Respiratory protection and clothing change
Shower out of facility
HEPA filters
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BSL Requirements for Potential
H5N1 Influenza Specimens
• BSL2 lab with BSL3 work practices needed for:
 Aliquoting human specimens
 Nucleic acid extractions
 All Diagnostic testing except viral culture
• BSL3+ safety measures and guidelines necessary to
culture H5N1
If laboratory does not meet BSL2 requirements, ship
appropriately packaged specimen to reference
laboratory
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WHO H5 Reference Laboratories
1. Collect specimens / virus isolates of A(H5N1) and other
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influenza subtypes
Provide timely surveillance data
Conduct antigenic and genetic analyses
Assist in developing methods for global surveillance and
diagnosis of A(H5N1)
Participate in selecting, developing, and distributing candidate
A(H5N1) vaccines
Develop and distribute reagents
Monitor susceptibility of emerging subtypes
Provide expertise and laboratory support to Member States
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Review Question 3
What Biosafety Level needs to be in place in
order to culture H5N1?
a.
b.
c.
d.
BSL-1
BSL-2
BSL-3
BSL-3 enhanced
Answer: d. BSL-3enhanced safety measures and
guidelines should be used for culturing potential H5N1
viruses
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Specimen Collection
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Specimen Collection Kit
• Personal protective
equipment (PPE)
• Viral transport medium
(VTM) collection vials
• Swabs: throat, nasal, &
nasopharyngeal
• Tongue depressors
• Nasal Wash Equipment
• Transfer pipettes
• Secondary container
• Ice packs
• Items for blood
collection
• Field collection forms
• Labels and pen or
marker
Store kit in a dry, cool place
Keep kit accessible for after hours
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Viral Transport Medium (VTM)
• Used to store & transport specimens
• Isolates & maintains virus integrity
• Prevents bacteria and fungi growth
• Can be made in a lab or purchased
• Different types of VTM:
Animal specimen collection
Viral isolation of human specimens
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Storing VTM
• Sterile collection vials
containing 1-3 ml of VTM
• VTM can be stored in a
freezer at -20ºC to -40ºC until
use
• VTM can be stored for short
periods of time
at 4 - 8 ºC
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Polyester Fiber-Tipped Applicator
• Should ideally be dacron,
rayon, or polyester-fiber
swabs
Remember!
Use throat swabs for avian influenza
and
nasopharyngeal swabs for seasonal
influenza
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Personal Protective Equipment for
Caring for H5N1 Patients
• Masks (N-95 or N/P/R-100)
• Gloves
• Protective eye wear (goggles)
• Hair covers
• Boot or shoe covers
• Protective clothing (gown or apron)
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Clinical Specimen Sources
Prepare to collect specimens before you leave
for the field
•Persons meeting trigger criteria
Includes WHO suspected and probable cases
•Symptomatic contacts
Symptomatic people living/working with suspected
cases
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What to Collect
From an Ambulatory patient
• Throat swab (priority) and
• Nasopharyngeal swab
• If necessary, collect into same VTM
From an Intubated patient
• Tracheal aspirate
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When to Collect Specimens
When to Collect
Respiratory Specimens
• As soon as possible after symptoms begin
• Ideally before antiviral medicine administered
• For multiple days
• Sample multiple types of specimens
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When to collect Serum Specimens
Acute specimen
As soon as possible, ideally within 7-10 days
after symptom onset
Convalescent specimen
2-3 weeks after the acute sample (> 21 days
after symptom onset)
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Oropharyngeal (Throat) Swab
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Nasopharyngeal Swab
http://www.nlm.nih.gov/medlineplus/ency/imagepages/9687.htm
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Nasopharyngeal Aspirate
(Nasal Wash)
Collection Process
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Attach mucus trap to vacuum source
Place catheter into nostril parallel to palate
Apply vacuum
Slowly remove catheter while slightly rotating it
Repeat with other nostril using same catheter
After collection, flush catheter with 3 ml VTM
and return VTM to a plastic vial
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Labeling Specimens
Use pre-printed barcode labels:
On specimen container
On field data collection form
In log book
Label each specimen with:
Subject’s unique identification
number
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Field Data Collection Forms
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Review Question 4
True or False: Are nasopharyngeal swabs best
for seasonal or avian influenza?
Answer: Seasonal
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Specimen Storage, Handling,
Packaging and Transportation
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How to Store Specimens
For specimens in VTM:
•Transport to laboratory as soon as possible
•Within 48 hours store at 4 °C to 8 °C
•Beyond 48 hours store at -70 °C to -80 °C
•Do not use standard freezer; keep on ice or in
refrigerator
 Avoid freeze-thaw cycles
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How to Store Specimens
For sera:
•Store specimen at
 4 °C to 8 °C for short periods of time
 -20 °C to - 40 °C for long term storage
•Avoid freeze-thaw cycles
•Centrifuge blood and aliquot serum to another
container before shipping and before freezing
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Packing Specimens for Transportation
Goal: protect specimens during transportation
•Use three packaging layers
•Use water tight first layer
•Use absorbent material in all layers
•<500mL of liquid in specimen collection container
•If transporting specimens a long distance, send on
dry ice. If transporting a short distance, ice is
acceptable
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Transporting Specimens
• WHO guidelines for the safe transport
http://www.who.int/csr/emc97_3.pdf
• Follow local regulations
• Coordinate with the laboratory
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Review Question 5
Which of the following is true about storing clinical
specimens (sera or specimens in VTM)?
a.They can be stored at 4°C up to a week
b.They can be stored in a standard freezer
Answer: a. Both sera and specimens in VTM can be
stored for certain periods at 4°C for up to a week.
Neither of these samples should be stored in a standard
freezer, because the freeze-thaw cycle will destroy the
virus
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Packaging a Specimen for Shipment
Demonstration
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Transporting Specimens from Field
to Lab
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Waste Disposal and
Decontamination
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Items Requiring Disposal
• Infectious blood, body
fluids
• Disposable or nonreusable gloves
• Disposable needles and • Used laboratory
syringes*
• Disposable or nonreusable protective
clothing*
supplies*
• Used disinfectants
* Incineration recommended
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Managing Contamination
or Accidents
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Contaminated work surface:
Use 5% bleach solution for at least 5 minutes
Make bleach solution fresh daily
70% ethanol, 5% Lysol is also adequate
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Exposed laboratory worker:
Remove infected clothing
Wash any exposed areas
Give post-exposure prophylaxis according to the
established emergency procedure
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Managing and Analyzing
Laboratory Data
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Specimen Tracking System
Maintain a database to track:
• Identification number
 same ID # as on epidemiologic data collection forms
• Subject information
 Age, sex, exposures
• Specimen collection date
• Specimen collection location
• Diagnostic test results
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Data Management Rules
• Double check data entry accuracy
• Include unique identification numbers
• Keep subject names confidential
• Track testing dates and results
• Back up the database
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Summary
• Maintain stocked specimen collection kits and
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store them properly
Throat swabs are the most important
specimens to collect for human H5N1
detection
Nasopharyngeal swabs are best for detecting
seasonal influenza viruses
Collect multiple specimens on multiple days
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Summary
• Proper specimen storage, handling, and
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shipping is vital for laboratory test success
Track specimen data in database or logbook
Use safety precautions when handling
infectious materials in the laboratory or in the
field
Properly dispose of any infectious material
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Summary
• If needed, ship specimens to national or
regional laboratory
• Share findings with local health officials and
the WHO Global Influenza Program
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Glossary
Aliquot
A portion of a total amount of a solution
Centrifuge
A machine that uses high-speed rotation to separate materials
with different densities.
Culture
Growing of microorganisms in a nutrient-rich medium.
Nucleic acid
Component genetic material such as DNA or RNA found in all
cells in humans, animals, bacteria, and viruses. Every species
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and organism has a unique pattern.
Glossary
Pipette
A glass or plastic tube used to measure or transfer small
amounts of liquid.
Saline
A liquid solution made of salt and water.
Viral Transport Medium (VTM)
The preservative liquid in which specimens are stored until
they are tested.
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References and Resources
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Recommended laboratory tests to identify avian influenza A
virus in specimens from humans. World Health Organization,
June, 2005.
http://www.who.int/csr/disease/avian_influenza/guidelines/avi
an_labtests2.pdf
WHO guidelines for the collection of human specimens for
laboratory diagnosis of avian influenza infection, 12 Jan 2005.
http://www.who.int/csr/disease/avian_influenza/guidelines/hu
manspecimens/en/index.html
WHO laboratory biosafety guidelines for handling specimens
suspected of containing avian influenza A virus. 12 Jan 2005.
http://www.who.int/csr/disease/avian_influenza/guidelines/han
dlingspecimens/en/
Infection control for viral haemorrhagic fevers in the African
health care setting. WHO/EMC/ESR/98.2 Section 6: Dispose
of Waste Safely
http://www.who.int/csr/resources/publications/ebola/WHO_E
MC_ESR_98_2_EN/en/index.html
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