2004 West Nile Bird Testing

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2004 West Nile Bird Testing
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Oral Swab Collection

• Collect specimens on birds from the family
  Corvidae (American Crows, Blue Jays) and birds of
  prey.
• Collect only specimens on birds that are no more
  than 24 hours old with out signs of decay.
• If the carcass is soft and mushy, has an obvious
  odor, skin discoloration, feathers or skin that
  rub off easily, and has insects or maggots DO NOT
  submit for testing. It is too old.

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Oral Swab Collection Cont.

• Submit specimens to OLS in the supplied boxes or
  a clean or unmarked box properly labeled with the
  shipping address and what kind of specimen it
  contains (diagnostic specimen oral swab).
• Fill out the submission form COMPLETELY.

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What To Send OLS

• After you swab in oral cavity of the bird, place
  the swab into a vial of transport media. If
  needed break off the wooden shaft. Tightly cap
  the media tube, and label the tube with the
  unique ID number that matches the submission
  form. Unique ID should be obtained by calling
  IDEP at 1-800-423-1271 or 304-558-5358
• Place the tube of media into a biohazard bag that
  is in the kit. One tube per bag.
• Place the bag (s) into the shipping box and mail
  to OLS. Please mail on the day of collection or
  refrigerate and mail the following day. Do not
  mail on Fridays.

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What You Will Receive In A Kit From OLS

• When you order a kit from OLS you will
• receive in it the following
• A mailing box for up to 3 specimens
• 3 tubes of media
• 6 swabs
• 3 parts of gloves
• 3 bags for collection
• 2 biohazard bags
• 1 mask

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Instructions for Sending Oral Swabs to WVDHHR/OLS
for West Nile Virus Testing

• Test request form
• REFRIGERATE the MEDIA UPON ARRIVAL until ready
  to use
• Please include a test request/submission form for
  each specimen. Fill out the form as completely
  as possible.
• Mailing container
• Please package the specimen as directed below.
  DO NOT CARRY IT ON YOUR PERSON!
• After swabbing the oral cavity place the cotton
  tip of the swab into the provided transport media
  (one swab per tube of media). Break off the
  wooden end if needed. Tightly cap the media.
  Place the tube into the provided biohazard bag.
  Place this bag into the provided bubble wrap
  container and place up to 6 tubes in each box for
  mailing. Mail or return the box to OLS the same
  day as collection. If you can not mail the day
  of collection wait no longer then the next day
  refrigerate the specimen until shipped. Please
  DO NOT mail specimens on Fridays.

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Instruction Forms

• OLS Containers
• Our laboratory will supply containers and forms
  upon request by written requisition. Contact the
  West Nile section to receive supplies.
• Reporting of testing results
• Test results will normally be available 4 days
  to 2 weeks after receipt of specimen. During
  periods of heavy submission, turn-around times
  may be longer. Receipt of a hard copy of the
  results will be mailed within one day after
  completion of testing.
• Shipping address
• West Virginia Department of Health Human
  Resources
• Office of Laboratory Services/Arbovirus Testing
• 167 11th Avenue
• South Charleston, WV 25303
• Please address questions to Wendy Hess at the
  Serology/Arbovirus Section at 304-558-3530 Ext.
  2402.

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Human Sample Submission Form

• WEST VIRGINIA DEPARTMENT OF HEALTH AND HUMAN
  RESOURCES
• Office of Laboratory Services (OLS)
• 167 11th Avenue
• South Charleston, WV 25303
• Phone [phone]
• FAX [phone]
• ARBOVIRUS TEST SUBMISSION FORM
• Patient Name Date of birth______/______/____Addr
  essCity CountyState______ Zip____________ Da
  te symptoms began ______/______/______
• Recent Travel?
• Specimen Information
• Date serum collected ______/______/______Date CSF
  collected ______/______/______
• Indicate the clinical syndrome(s) Was a lumbar
  puncture performed?
• Headache and fever Yes Total WBC count
  __________
• Aseptic meningitis Protein
  __________________
• Encephalitis No
• To whom should the results be forwarded
• Name ___________________________________________
• Address _________________________________________

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Human Specimen Collection Instructions

• Instructions for Sending Specimens to WVDHHR/OLS
  for West Nile Virus Testing Test request form
• Please include a test request form for each
  specimen. Fill out the form as completely as
  possible. Testing will not be initiated without
  inclusion of the date of onset of symptoms and
  date each specimen was collected. Name of
  patient is required also.
• Mailing container
• Please put your specimen in a substantial
  container. Wrap your specimen in absorbent
  material and pack it in 2 different plastic
  containers to insure that any leakage is
  contained. If you are requesting serology, there
  is no need to keep it frozen. If you are hand
  carrying specimens, please observe the packing
  instructions above to secure the specimens in
  transit. DO NOT CARRY IT ON YOUR PERSON!
• OLS Containers
• Our laboratory will supply containers and forms
  upon request by written requisition. Contact the
  Container Room to receive supplies.
• Specimen types and amounts
• Acute and convalescent specimens should be sent
  together whenever available. If one of these
  specimens is unavailable, we will accept single
  specimens for testing. Ideal timing of specimens
  is as follows acute specimens should be taken
  10 days after onset of symptoms. Convalescent
  specimens should be taken 2-3 weeks after the
  acute sample. Keep in mind if the acute sample
  is taken prior to 10 days post-onset of symptoms,
  antibodies may not have had time to develop and a
  false negative test may result.
• Testing for serology is done on serum and
  cerebrospinal fluid (CSF) specimens. Please send
  at least 0.5 ml of serum and at least 1 ml of CSF
  for proper testing to proceed. The larger amount
  of CSF specimen is required because these samples
  are routinely tested undiluted. Do not send
  whole blood.
• Reporting of testing results
• Test results will normally be available 4 days to
  2 weeks after receipt of specimen. During
  periods of heavy submission, turn-around times
  may be longer. Receipt of a hard copy of the
  results will be mailed within one day after
  completion of testing. Initial serologic testing
  will be performed using IgM capture ELISA and IgG
  ELISA. If the initial results are positive,
  further confirmatory testing may delay the
  reporting of final results.
• Shipping address West Virginia Department of
  Health Human Resources
• Office of Laboratory Services/Arbovirus
  Testing
• 167 11th Avenue
• South Charleston, WV 25303
• Contacts Wendy Hess or Kirsten Snowden at the
  Serology/Arbovirus Section at 304-558-3530

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LABORATORY TESTING
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Laboratory TestingWest Nile Virus

• Serological based diagnosis
• Sample Type Human clinical samples
• Detection target Antibody / Antigen
• Molecular based diagnosis
• Sample Type Mosquitoes and Birds (Oral Swabs)
• Detection Target Nucleic Acid (RNA)

The most sensitive WNV detection assay for
field-collected specimens is RNA detection
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Molecular Diagnostics

• Making copies of a specific portion of the
  targets (ex. WNV) genome i.e. DNA or RNA
• Different Methods
• PCR (Usually DNA)
• NASBA (Usually RNA)
• NASBA
• Nucleic Acid Sequence Based Amplification
• Method (to be) used at OLS to diagnose WNV in
  birds and mosquitoes

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How NASBA Works

• Sample
• Grind mosquitoes into a mosquito milkshake
• Transport media containing oral swab (birds)
• Add sample to lysis buffer
• Inactivates the virus
• Releases nucleic acids (RNA)
• Denatures nucleases that destroy RNA
• Stabilizes the RNA for processing or storage

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How NASBA Works

• Extraction and Isolation of the RNA
• The NucliSens Extractor makes this an automated
  procedure
• Some sample prep required
• Addition of silica to bind RNA
• Extractor performs a series of washes to clean
  and isolate the RNA
• 45 minutes for 10 samples

NucliSens Extractor
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How NASBA Works

• Amplification of the RNA
• Make millions of copies of the RNA for detection
• The more you have the easier it is to see
  (detect)
• Requires primers to initiate the amplification
• Short pieces of commercially made RNA that bind
  specifically to your target RNA (ex. WNV)
• Just like priming a pump with a small amount of
  water

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How NASBA Works

• Detection of the RNA
• Is it there?
• NucliSens EasyQ Reader
• Incubator and Analyzer
• Automated
• Real Time
• Simultaneous amplification and detection
• Molecular beacons
• Similar to primers
• Fluorescent attachment
• 90 minutes for 96 WNV samples

NucliSens EasyQ reader
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Questions?