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Laboratory Diagnostics, Specimen Collection, and Biosafety Issues. 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

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learning objectives
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
session overview
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
laboratory diagnostics for avian influenza
Laboratory Diagnostics for Avian Influenza
  • Tests on respiratory samples:
  • Virus isolation
  • Immunofluorescence
  • Rapid antigen detection
  • Tests on serum:
  • Measure specific antibodies
  • PCR-based techniques
h5n1 virus found in other human specimens
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
pcr based techniques
PCR-based Techniques
  • Respiratory samples
  • Diagnostic assays and sequencing
  • Sensitivity depends on:
    • Particular assay
    • Influenza strain
    • Type of the specimen
    • Quality of the specimen
pcr h5n1 virus testing
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

Interpretation of Real-time

RT-PCR Results

Test reactions

virus isolation of h5n1 viruses gold standard
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
immunofluorescence for h5n1 testing
Immunofluorescence for H5N1 Testing
  • Requires H5 monoclonal antibody
  • Not part of WHO Reagentkit for seasonal influenza isolates
  • Interpretation difficult
  • Results:
    • Presence of influenza virus
    • Subtype identification
indirect if staining of cells from tracheal aspirate
Indirect IF Staining of Cells From Tracheal Aspirate





Taken from: World Bank Training by Alexander Klimov, CDC

rapid antigen tests
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


For Detection of human H5N1 virus infection

using serology
Using Serology

Delay for anti-H5

levels to rise

Requires acute and convalescent sera, 3 weeks apart

Not useful for clinical


Can confirm epidemic cases, if respiratory specimens not available

influenza serology tests
Influenza Serology Tests
  • Micro-neutralization assays
  • Western Blot
  • Enzyme immunoassays

Require acute and convalescent sera

(serum obtained >21 days from onset)

interpreting serology tests
Interpreting Serology Tests

* four-fold or greater increase between acute and convalescent sera

serological specimens
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
how to avoid misinterpretation of diagnostic tests
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
review question 1
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

review question 2
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.

laboratory safety

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.

biosafety laboratory levels bsl
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

bsl 2 and bsl 3
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
bsl requirements for potential h5n1 influenza specimens
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

who h5 reference laboratories
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
review question 3
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

specimen collection kit
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

viral transport medium vtm
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
storing vtm
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

polyester fiber tipped applicator
Polyester Fiber-Tipped Applicator
  • Should ideally be dacron, rayon, or polyester-fiber swabs


Use throat swabs for avian influenza and

nasopharyngeal swabs for seasonal influenza

personal protective equipment for caring for h5n1 patients
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)
clinical specimen sources
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
what to collect
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
when to collect respiratory 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
when to collect serum specimens
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)

nasopharyngeal swab
Nasopharyngeal Swab

nasopharyngeal aspirate nasal wash collection process
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
labeling specimens
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
review question 4
Review Question 4

True or False: Are nasopharyngeal swabs best for seasonal or avian influenza?

Answer: Seasonal

how to store specimens
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 notuse standard freezer; keep on ice or in refrigerator
    • Avoid freeze-thaw cycles
how to store specimens47
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
packing specimens for transportation
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
transporting specimens
Transporting Specimens
  • WHO guidelines for the safe transport

  • Follow local regulations
  • Coordinate with the laboratory
review question 5
Review Question 5

Which of the following is true about storing clinical specimens (sera or specimens in VTM)?

  • They can be stored at 4°C 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 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

items requiring disposal
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

managing contamination or accidents
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
specimen tracking system
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
data management rules
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
  • 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
  • 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
  • If needed, ship specimens to national or regional laboratory
  • Share findings with local health officials and the WHO Global Influenza Program


A portion of a total amount of a solution


A machine that uses high-speed rotation to separate materials with different densities.


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.



A glass or plastic tube used to measure or transfer small amounts of liquid.


A liquid solution made of salt and water.

Viral Transport Medium (VTM)

The preservative liquid in which specimens are stored until they are tested.

references and resources
References and Resources
  • Recommended laboratory tests to identify avian influenza A virus in specimens from humans. World Health Organization, June, 2005.
  • WHO guidelines for the collection of human specimens for laboratory diagnosis of avian influenza infection, 12 Jan 2005.
  • WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus. 12 Jan 2005.
  • Infection control for viral haemorrhagic fevers in the African health care setting. WHO/EMC/ESR/98.2 Section 6: Dispose of Waste Safely