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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
slide9

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

Anti-H5

Anti-H3

Anti-A/NP

Anti-B

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

RAPID TESTS NOT RECOMMENDED

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

management

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

http://www.cdc.gov/OD/ohs/symp5/

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

Remember!

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

http://www.nlm.nih.gov/medlineplus/ency/imagepages/9687.htm

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

http://www.who.int/csr/emc97_3.pdf

  • 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
summary
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
summary60
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
summary61
Summary
  • If needed, ship specimens to national or regional laboratory
  • Share findings with local health officials and the WHO Global Influenza Program
glossary
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.

glossary63
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.

references and resources
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/guidelines/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/guidelines/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/disease/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/publications/ebola/WHO_EMC_ESR_98_2_EN/en/index.html