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
• PCR-based techniques
• 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 - - - A/H5
Sample - - - - Inconcl
Sample - - - - - Neg
Sample - - - - - - Inconcl
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Virus IsolationGold Standard

• Biosafety level 3 (BSL-3) laboratory
• Antigenic genetic characterization
• Drug susceptibilities tests
• WHO Influenza Centers
• Antigenic characterization of viral isolates
• Collect specimens for vaccine development

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Immunofluorescence

• Requires H5 monoclonal antibody
• Not part of WHO Reagent kit
• 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

• Fast 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 H5N1 virus

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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
Acute Sample Convalescent Sample Interpretation
low low No evidence of Infection
low high Seroconversion (evidence of infection)
high high Inconclusive past exposure to influenza 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 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 multiple tests to confirm results

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Review Question 1

• Name two ways to test respiratory samples for
  avian influenza.
• Possible Answers
• PCR-based techniques
• Virus isolation
• Immunofluorescence
• Rapid antigen detection

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Review Question 2

• Which of the following is NOT a way to avoid
  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
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Biosafety Laboratory Levels (BSL)

• Risk of acquiring infections
• 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 () BSL-3 plus
• Respiratory protection and clothing change
• HEPA filters

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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 specimen to reference laboratory

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WHO H5N1 Laboratory Network

• Reference laboratories responsible for
  surveillance and vaccine preparation
• Perform detailed antigenic and genetic
  characterization
• Report positive samples to WHO
• Submit positive samples to WHO laboratory for
  characterization
• Include background clinical information with the
  shipment
• WHO results shared with original laboratory

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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
• Answer c. BSL-3 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 2-3 ml of VTM
• Vials can be stored in a freezer at -20ºC to
  -40ºC until use
• Vials 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

• 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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(No Transcript)
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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 Do I Collect Respiratory Specimens?

• As soon as possible after symptoms begin
• Before antiviral medicine administered
• For multiple days
• Sample multiple types of specimens

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When to collect Serum Specimens

• Acute specimen
• Within 7 days after symptom onset
• Convalescent specimen
• 2-3 weeks after the acute sample (gt 21 days
  weeks 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

1. Attach mucus trap to vacuum source
2. Place catheter into nostril parallel to palate
3. Apply vacuum
4. Slowly remove catheter while slightly rotating it
5. Repeat with other nostril using same catheter
6. 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 If you collect a respiratory
  specimen (throat swab, nasopharyngeal swab), you
  should collect acute and convalescent specimens.
• Answer False. Acute and convalescent specimens
  are only needed for serological tests. However,
  you should collect respiratory specimens on
  multiple days.

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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 before and
  during transportation
• 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
• Keep on ice for a week instead of freezer

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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

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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
  

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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 4-8 C
• They can be stored in a standard freezer
• Answer a. Both sera and specimens in VTM can be
  stored for certain periods at 4-8 C. 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, leftover
  biological samples
• 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
• 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
• 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 January 2005.
  http//www.who.int/csr/disease/avian_influenza/gui
  delines/humanspecimens/en/index.html
• 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