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 28th 2008

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)

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

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

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

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

Figure 4: Captured target bacteria eluted from phase Magnet Removed

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

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

PATHATRIX - ULTRA

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

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

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

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

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

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.

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

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

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

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

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

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

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

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 *

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 !

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

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

The Role of Pathatrix ® in Outbreak Analysis