USDA Licensed Test Kits and Enhanced Surveillance for BSE

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USDA Licensed Test Kits and Enhanced
Surveillance for BSE
Larry Ludemann1 and Lawrence Elsken2 Policy,
Evaluation, and Licensing1 Inspection and
Compliance2 Center for Veterinary
Biologics Veterinary Services Animal and Plant
Health Inspection Service United States
Department of Agriculture 510 S. 17th Street,
Suite 104 Ames, Iowa 50010 USA
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The Causative Agent of TSEs

• Prions
• Abnormally folded protein
• Not a virus. Not a bacteria. No DNA or RNA
• No known immune response
• Two forms of prion protein
• Normal prion protein (PrPc) on most cells
• Infectious Prion (PrPSc)
• Relatively resistant to disinfectants,
  sterilizations, proteinases
• Accumulates and kills the cells

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BSE Diagnostics

• No effective live animal test for BSE
• Need brain tissue
• Histopathology of brain
• IHC, WB, ELISA PrPres
• Negatives do not guarantee absence of
  infectivity - tests are NOT intended as Food
  Safety Tests

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BSE Diagnostic Tests

• Histology
• ImmunoHistochemistry (IHC)-an ELISA performed on
  a tissue section mounted on a slide
• Rapid tests- ELISA, EIA, Western Blot

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Rapid Tests

• As name implies, generally more rapid than IHC
• Associated with occasional false positive
  initial reactions (especially ELISA)
• Western Blot Test lower throughput, slower, and
  more involved than ELISA
• All approved tests have very good sensitivity and
  specificity
• BUT - only serve as screening tests

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IMMUNOHISTOCHEMISTRY

• Adds immunological confirmation to observation of
  lesions
• Stain observed in specific (definitive) anatomic
  areas associated with BSE
• No false positives
• Generally requires several days to complete test

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• January 8, 2004
• CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 04-02
• Subject Diagnostic Test Kits for Bovine
  Spongiform Encephalopathy
• To Biologics Licensees, Permittees, and
  Applicants
• Veterinary Services Management Team
• Directors, Center for Veterinary Biologics
• Area Veterinarians in Charge, VS
• State Veterinarians
• I. PURPOSE
• The purpose of this notice is to inform
  interested parties that the Center for
  Veterinary Biologics (CVB)
• is accepting veterinary biologics product license
  and permit applications for diagnostic test kits
  
• (including rapid tests) intended as an aid in
  the diagnosis of bovine spongiform encephalopathy
  
• (BSE).

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Authorities, Regulations, Guidance

• Virus-Serum-Toxin Act 21 USC
• Authorizes Secretary of Agriculture to regulate
  manufacture and distribution of biologics
  including diagnostics (test kits) intended for
  use in diagnosis of disease in animals.
• Code of Federal Regulations Title 9
• VS Memoranda
• 800.73 (Immunodiagnostic Test Kits), others

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What is a regulated test kit?

• Diagnostic test kits contain
• All unique reagents required to conduct test
• Complete instructions for the test
• Instructions for interpretation of test results
• Claims, uses, and limitations
• Used to diagnose existence of (usually) or
  susceptibility to disease agents of animals
• Reagents are not regulated

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Prelicense Assessment of Kits

• Accuracy and precision
• Diagnostic sensitivity
• Dynamic range
• Analytical sensitivity
• Diagnostic specificity
• Ruggedness, repeatability, suitability
• Predictive values for specific disease prevalence
  (especially for Program Diseases)

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Prelicense Validation

• Test large number of known positive and known
  negative animal samples covering a range of
  reactions and all sample types
• Determine onset of detection of disease
• Evaluate (in at least 3 laboratory settings)
• Serum or antigen panel (ruggedness)
• Well-characterized patient samples
• Compared to Gold standard assay
• Suitability of test kit
• Adequacy of instructions
• Authorization from USDA per 9 CFR 103.3
  (experimental biological products)

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Manufacturing controls

• Aim is to minimize within-serial and
  serial-to-serial variation by ensuring
  consistency of production Outline of Production
• Master Seed/Master Cell concept
• Validation of new lots of antibody/antisera
• Serial release testing of serum or antigen panel
  with range of reactivities and cross-reactivities
• Serial release panel is critical to minimize
  variation in test results
• Panel includes range of reactivity

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Other Requirements

• Inspection of manufacturing facilities and other
  licensed sites (e.g., Permittee quarantine sites)
• Prelicense serial testing
• Label approval

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USDA Approved Bovine Spongiform Encephalopathy
Antigen Test Kits(Permits, as of September 30,
2004)
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USDA Approved Bovine Spongiform Encephalopathy
Antigen Test KitsLicenses, as of (September 30,
2004)
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Licensed BSE Test KitsFormat or Technique

• All use obex tissue
• Purify normal and abnormal PrP
• Treatment to remove normal PrP
• Western Blot separate protein by Mol Wt
• Antibody binds abnormal PrP
• Indicator enzyme (on antibody) produces visible
  reaction
• ELISA, EIA, chemiluminescence, solid precipitate
  (Western Blot, IHC)

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Diagnostics Under Development

• Cowside or on-site tests
• Capillary electrophoresis blood
• Blood other methodologies - Capture
• Urine UPrPsc
• Increased sensitivity cyclic amplification
• Non-PrP markers Upregulated Markers

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What does licensure provide?

• USDA controlled manufacturing of all components
  and kits
• Minimal variation within and between serials
• USDA evaluation of test kit before and after
  licensure (serial release)
• Monitoring of field performance and
  investigations
• CVB-Inspection and Compliance 800-752-6255
• Ongoing facilities inspections
• Questions? www.aphis.usda.gov/vs/cvb/

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BSE Surveillance Program

• Expanded or Enhanced surveillance began June 1,
  2004. Purpose
• Determine if BSE is present in U.S. cattle
  population
• Determine if risk-management policies adequate
• For animal health NOT food safety

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EU testing Jan-Dec 2001

• Samples Positive positive
• Risk 776,266 756 .097
• Em slaug 96,774 324 .335
• CNS 27,991 34 .121
• Fallen 651,501 397 .061
• Healthy 7,677,576 279 .0036
• Suspect 3,267 1103 33.76

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BSE Surveillance Program

• Experience in Europe has demonstrated that
  targeting surveillance efforts at certain high
  risk populations is the most effective way to
  identify BSE, if it is present.

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BSE Surveillance Program

• US high-risk population estimate about 446,000
• 246,000 on-farm deaths with unexplained causes or
  causes consistent with BSE
• 200,000 ante-mortem condemnations by FSIS
• 129 (2004 data) Foreign Animal Disease
  investigations for CNS disease

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BSE Surveillance Program

• The majority of samples from the following
  categories
• Nonambulatory cattle
• Cattle exhibiting signs of a central nervous
  system disorder
• Cattle exhibiting other signs that may be
  associated with BSE, such as emaciation or
  injury and
• Dead cattle.
• USDA personnel will also sample all cattle
  condemned on ante-mortem inspection by USDAs
  Food Safety and Inspection Service.)

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BSE Surveillance Program

• If 268,500 samples are obtained from a target
  population of 446,000, and if no positives were
  found after completion of the testing plan, then
  we can state that we are 99 percent confident
  that there were less than 5 positive animals in
  the target population

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BSE Surveillance Program

• Enhanced surveillance plan background,
  statistical inferences
• http//cofcs66.aphis.usda.gov/lpa/issues/bse/bse.h
  tml

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BSE Surveillance Program

• As of October 10, 2004, no positive BSE test
  results in Enhanced Surveillance Program
• Cumulative rapid tests from June 1, 2004
  October 10, 2004 79,766
• Also 1,583 IHN negative tests
• Currently More than 5,000/week
• http//www.aphis.usda.gov/lpa/issues/bse_testing/t
  est_results.html