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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
  • PCR-based techniques
  • 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
A H1 H3 H5 B RNP Results
Sample - - - A/H5
Sample - - - - Inconcl
Sample - - - - - Neg
Sample - - - - - - Inconcl
10
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

11
Immunofluorescence
  • Requires H5 monoclonal antibody
  • Not part of WHO Reagent kit
  • 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
  • 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

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
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
17
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

18
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

19
Review Question 1
  • Name two ways to test respiratory samples for
    avian influenza.
  • Possible Answers
  • PCR-based techniques
  • Virus isolation
  • Immunofluorescence
  • Rapid antigen detection

20
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.

21
Laboratory Safety
22
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/

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 () BSL-3 plus
  • Respiratory protection and clothing change
  • HEPA filters

24
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

25
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

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
  • Answer c. BSL-3 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 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

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
  • 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 Do I Collect Respiratory Specimens?
  • As soon as possible after symptoms begin
  • Before antiviral medicine administered
  • For multiple days
  • Sample multiple types of specimens

38
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)

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

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 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.

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 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

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

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

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 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

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, 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

55
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

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
  • 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 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
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