Laboratory Diagnostics, Specimen Collection, and Biosafety Issues

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Laboratory Diagnostics, Specimen Collection,and
Biosafety Issues
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Learning Objectives

• Describe avian influenza-related laboratory
  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
  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
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Virus Isolation of H5N1 VirusesGold Standard

• Requires biosafety level 3 (BSL-3)- enhanced
  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
  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
Taken from World Bank Training by Alexander
Klimov, CDC
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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, 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

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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 gt21 days from onset)

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Interpreting Serology Tests
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?
• 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?
• 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 3-enhanced 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
• 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

• Collect specimens / virus isolates of A(H5N1) and
  other 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?
• 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
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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 (gt 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

• 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
• Subjects 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
• lt500mL 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)?
• They can be stored at 4C up to a week
• They can be stored in a standard freezer
• Answer a. Both sera and specimens in VTM can be
  stored for certain periods at 4C 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 needles and syringes
• Disposable or non-reusable protective clothing

• Disposable or non-reusable gloves
• Used laboratory supplies
• Used disinfectants
• Incineration recommended

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Managing Contamination or Accidents

• Contaminated work surface
• Use 5 bleach solution for at least 5 minutes
• Make bleach solution fresh daily
• 70 ethanol, 5 Lysol is also adequate
• 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
  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 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 and organism has a
  unique pattern.

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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

• 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/gui
  delines/avian_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/gui
  delines/humanspecimens/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/disea
  se/avian_influenza/guidelines/handlingspecimens/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/publicatio
  ns/ebola/WHO_EMC_ESR_98_2_EN/en/index.html