1 / 28

The Role of Pathatrix ® in Outbreak Analysis

The Role of Pathatrix ® in Outbreak Analysis. The Role of Pathatrix ® in Outbreak Analysis . Dr Adrian Parton . Matrix MicroScience May 28 th 2008 . Presentation Overview . • Pathatrix ® - how does it work? • Role of Pathatrix ® in outbreak scenarios .

blaze
Download Presentation

The Role of Pathatrix ® in Outbreak Analysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. The Role of Pathatrix® in Outbreak Analysis The Role of Pathatrix® in Outbreak Analysis Dr Adrian Parton Matrix MicroScience May 28th 2008

  2. Presentation Overview • Pathatrix® - how does it work? • Role of Pathatrix® in outbreak scenarios - E.coli O157:H7 - Spinach outbreak (US) - Viruses (HAV) - Frozen Berries (Canada) - Salmonella spp - Produce (US)

  3. PATHATRIX ULTRA : SCHEMATIC PUMP FOOD SAMPLE DIVERTER VALVE CAPTURE AREA MAGNET TO WASH ELUTION VESSEL PIPETTE

  4. Figure 1: Antibody coated beads capturing on surface of capture phase Direction of Flow Magnet Capture phase = Antibody coated beads

  5. Figure 2: Capture of Target in Food Debris Non Specific bacteria Antibodies Magnetic beads Target Bacteria Magnet

  6. Figure 3: Captured target bacteria after wash WASH Antibodies Magnetic beads WASH Captured Target Bacteria WASH Magnet

  7. Figure 4: Captured target bacteria eluted from phase Magnet Removed

  8. DETECTION OPTIONS PCR BACTERIA EIA BEAD POST PATHATRIX Agar Plate COMPLEX = Specific Antibody

  9. TECHNICAL RATIONALE FOR SUPERIOR SENSITIVITY OF PATHATRIX KEY = 225ml +25g food sample - standard Pathatrix = 50ml Composite sub-sample = 0.05 - 0.1 ml PCR/ELISA sub-sample ENTIRE SAMPLE 250,000µl POOLING SAMPLE 50,000µl PCR / ELISA 5µl - 100µl

  10. PATHATRIX - ULTRA

  11. PATHATRIX - ULTRA : Top View Grip lock seal bag Diverter Valves

  12. MATRIX: TEST OPTIONS Viruses + Salmonella + Campylobacter + + + E.coli O157:H7 Listeria PATHATRIX E.sakazakii MAP PATHATRIX CUSTOM POOLING COATING

  13. PATHATRIX POOLING STRATEGY: ULTRA Format 5 x Individual Samples Individual Enrichment of samples Post - Enriched samples Pooling of 50ml 50ml 50ml samples 50ml 50ml 250ml Pooled Sample (1x Pathatrix test) = Positive post enrichment PATHATRIX PCR Agar Plate If Pooled Sample is +ve. Then RE-Test all 5 If Pooled Sample is -ve. Then all 5 individual samples Post-Enrichment individual samples are -ve

  14. Spinach Outbreak EHEC Results • ~12,000 culture plates were streaked • ~1,800 isolates picked for identification • 350 isolates confirmed as E.coli O157:H7 • 94 E.coli non O157 isolated • 14 E.coli non-O157 w/Stx isolated Information kindly provided by California Department of Public Health

  15. Comparison of Methods BAM CDPH • 6+ day procedure • 5+ day method that was shortened to 4+ days during •24 hr enrichment the outbreak (including •Immunomagnetic PFGE) separation using only • 5 hr enrichment 1ml of enrichment broth •Immunomagnetic separation using entire enrichment •Method not successful broth - Pathatrix® in isolation of E. coli •PCR results same day O157:H7 during •Successful isolation of spinach outbreak environmental E. coli O157:H7 strains which matched the outbreak strain by PFGE analysis

  16. Salinas Studies • Participation in the Salinas studies allowed CDPH to optimize the methods for the recovery of E. coli O157:H7 from the environment • Methods were optimized for the E. coli O157:H7 recovery from water, soil, leafy green vegetables and swabs including the modified Moore swabs • Further studies were conducted with a variety of fecal sample types.

  17. Salinas Valley Spinach Field This is one of the Salinas Valley fields growing spinach.

  18. And next door……….. Salinas Valley Cows ! • These cows reside in the Salinas Valley • They graze all over • Frequently, they will stand in a stream of surface water while urinating or defecating

  19. DETECTION OPTIONS PCR BACTERIA PFGE - CDC BEAD POST PATHATRIX Agar Plate COMPLEX = Specific Antibody

  20. Real Time PCR • DNA from the lysed target cells is placed in a smart cycler block for Real Time PCR to determine whether E. coli O157:H7 is present and the toxin type of the strain: • STX 1 and/or 2 • uidAm

  21. Real Time PCR Data output on screen indicates if sample is positive or negative for the presence of E.coli O157:H7

  22. Culturing of Beads 30 ul of washed beads are placed on each plate of TC- SMAC and CHROM agars.

  23. PFGE Analysis It takes approximately 4 days to complete the electrophoresis and compare the gel pattern to those on CDC’s PulseNet.

  24. Frozen Berries: Hepatitis A Outbreak • Several cases of Human disease observed* • Frozen berries were implicated e.g. blueberries, raspberries, strawberries • Samples sent to Canada Health labs for analysis by Pathatrix® (using cationic beads) + RT-PCR *

  25. Frozen Berries: Hepatitis A Outbreak • Used cationic bead approach i.e. using the fact that virus particles have net negative charge and are attracted to the “+ve” cationic beads •30 minute Pathatrix®run time • Entire process is ~4 hrs from point of sample to result !

  26. Frozen Berries: Hepatitis A Outbreak • Hepatitis A (HAV) was successfully isolated using Pathatrix® in multiple berry samples • Secondary laboratory also identified the same strain, in the same product type • The strain of HAV isolated from the berries was matched to the strain that caused the clinical disease

  27. ACKNOWLEDGMENTS I would like to thank the following people for providing data / slides for this talk; • Dr Linda Guthertz (CDPH) • Dr Sunee Himathongkham (CDPH)

More Related