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Laboratory Diagnostics, Specimen Collection, and Biosafety Issues

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Title: Laboratory Diagnostics, Specimen Collection, and Biosafety Issues


1
Laboratory Diagnostics, Specimen Collection,and
Biosafety Issues
2
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

3
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

4
Laboratory Diagnosis of Human Cases of Avian
Influenza
5
Laboratory Diagnostics for Avian Influenza
  • Tests on respiratory samples
  • Virus isolation
  • Immunofluorescence
  • Rapid antigen detection
  • Tests on serum
  • Measure specific antibodies
  • PCR-based techniques

6
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

7
PCR-based Techniques
  • Respiratory samples
  • Diagnostic assays and sequencing
  • Sensitivity depends on
  • Particular assay
  • Influenza strain
  • Type of the specimen
  • Quality of the specimen

8
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

9
Interpretation of Real-time RT-PCR Results
Test reactions
10
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

11
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

12
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
13
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

14
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
15
Influenza Serology Tests
  • Micro-neutralization assays
  • Western Blot
  • Enzyme immunoassays
  • Require acute and convalescent sera
  • (serum obtained gt21 days from onset)

16
Interpreting Serology Tests
four-fold or greater increase between acute and
convalescent sera
17
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

18
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

19
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

20
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.

21
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.
22
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/

23
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

24
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

25
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

26
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

27
Specimen Collection
28
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
29
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

30
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

31
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
32
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)

33
(No Transcript)
34
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

35
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

36
When to Collect Specimens
37
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

38
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)

39
Oropharyngeal (Throat) Swab
40
Nasopharyngeal Swab
http//www.nlm.nih.gov/medlineplus/ency/imagepages
/9687.htm
41
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

42
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

43
Field Data Collection Forms
44
Review Question 4
  • True or False Are nasopharyngeal swabs best for
    seasonal or avian influenza?
  • Answer Seasonal

45
Specimen Storage, Handling, Packaging and
Transportation
46
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

47
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

48
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

49
Transporting Specimens
  • WHO guidelines for the safe transport
  • http//www.who.int/csr/emc97_3.pdf
  • Follow local regulations
  • Coordinate with the laboratory

50
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

51
Packaging a Specimen for Shipment Demonstration
52
Transporting Specimens from Field to Lab
53
Waste Disposal and Decontamination
54
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

55
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

56
Managing and Analyzing Laboratory Data
57
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

58
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

59
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

60
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

61
Summary
  • If needed, ship specimens to national or regional
    laboratory
  • Share findings with local health officials and
    the WHO Global Influenza Program

62
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.

63
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.

64
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
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