Salmonella - bioMerieux

A national reference laboratory’s practical
Salmonella workflow experience
Ben Howard
Microbiology Laboratory Manager,
Certified Laboratories

Overview
• How do we evaluate platform/method performance?
• What can you do to improve your detection protocol?
• What are the options for result confirmation?
• What is the future of Salmonella testing?

Evaluating Performance
• Time to Result
• Cost
• Materials
• Labor
• Ease of Use
• Number transfer steps
• Number of variations between matrices and between targets
• Reliability
• False positives
• False negatives
• No result results

Protocol Overview
• Brief Comparison of Common Salmonella Methods
Developer Platform Assay Method Reference
Minimum Time to
Negative Result* (hours) Transfer Steps*
FDA/USDA Cultural Cultural FDA BAM Chpt 5 67-86 4
Difco Cultural BBL CHROMagar AOAC RI# 020502 60 4
DuPont BAX Salmonella 2 AOAC 2003.09 30.5 3
DuPont BAX RT Salmonella Pending Validation 24.5-28 2-3
3M
MDS
MD Salmonella
Assay AOAC RI# 031208 20 2
bioMerieux VIDAS Salmonella (SLM) AOAC 2004.03 49 3
bioMerieux VIDAS Salmonella (SPT) AOAC RI# 071101 20-24 1
Roka Bioscience Atlas Atlas Salmonella AOAC RI# 031208 ~21 1
*Estimates based on reference methods and existing literature, consult manufacturer for more complete
information

Time to Result
• Time from first enrichment to final result
• Includes:
• Minimum enrichment time
• Any transfers (estimated at 30min)
• Post enrichment steps (i.e. lysis or heating block)
• Instrument run time
• Time to Confirmation (if presumptive)

Time to Result cont.
• Example:
• BAX Salmonella 2 assay =
• 22 hour enrichment +
• 0.5 hour transfer +
• 3 hour BHI regrowth +
• 0.5 hour transfer +
• 0.5 hour lysis +
• 3.5 hour run time =
• ~30 hour total time to negative result
• If presumptive, add standard cultural confirmation:
• Confirmation: 22 + 44 + 18 + 8 = 92h
• Total time to confirmed result = 122h

Cost (Labor)
• (Time required in man hours to run a single test) x (average
rate of pay for technicians required) = Labor Cost
• Includes:
• Sample login
• Media preparation*
• Sample preparation*
• Transfer steps*
• Instrumentation*
• Confirmation*
• Result entry
• Waste handling
*Areas where labor cost vary widely between methods

Cost (Supplies)
• Supply cost = Manufacturers kit (if applicable) +
media + consumables (pipettes, bags, loops, PPE’s,
waste disposal, sanitizers, etc)
• For rapid detection methods, kit cost is usually the
biggest driver
• Can account for up to 80% of supply cost
• Cost per test = cost per kit/number of tests per kit

Ease of Use
• Ease of use = the overall level of complexity and the
amount of effort required to run/train/maintain a
method.
• Other Ease of Use considerations
• Ability to interface instrument software with LIMS
system
• Consideration may also be made based on the size of the
instrument relative to available lab space

Ease of Use cont.
• Factors contributing to ease of use:
• Number of steps between primary enrichment and
result
• Number of protocols across different matrices
• Frequency of calibration
• Making reagents/media
• Time required to train a new employee
• Time required for a new employee to achieve proficiency

Reliability
• Frequency of Correctness
•1 - (False positives + False Negative + Indeterminate / number
of tests performed) = Frequency of Correctness
• False Positives
• Implication: Cost Time, Money, Client Confidence, Hair Loss
• False Negative
• Implication: Depending on the sample type the impact ranges
from nominal to enormous
• Indeterminate
• Implication: Costs time and money, can delay action

False Negatives
• Salmonella present but not detected
• Causes:
• Matrix inhibition in enrichment (i.e. spices)
• High-background microflora masks/inhibits target organism
• Matrix interference with replication process (i.e. PCR reaction)
• Instrumentation error or failure
• Lab error (i.e. expired media)
• Prevention:
• Validate new matrices and formulations to ensure compatibility
• Run a positive and negative control for each variation of a methods
protocol

“False” Positives
• What is a false positive?
• Example 1:
• Rapid Screen: presumptive
• Culture Confirmation: negative
• Example 2:
• Rapid Screen: presumptive
• Cultural Confirmation: positive
• Strain typing reveals exact match with positive control strain
or another positive from a different sample set on the same
run

Confirmations - Overview
• Why do you confirm?
• Rule out lab/instrument error
• Obtain an isolate for further analysis (i.e. strain typing)
• Good old fashioned CYA
• What are your options?
• No confirmation at all
• Cultural confirmation
• Cross-platform confirmation
• Hybrid of the two

Confirmation – Schmonfirmation
• If:
• Your response protocol does not change depending on
the outcome of the confirmation OR…
• The corrective action will be complete by the time the
confirmation result is available AND…
• You do not perform any further analysis on recovered
isolates AND…
• You do not have reason to suspect lab error was the
cause of the result…
You may opt not go through with confirmation.

Confirmation Options (Cultural)
• Cultural Confirmations
• Full FDA cultural confirmation
• Time to result from primary enrichment (86h)
• Plus serological (O and H antigens)
• USDA/FSIS
• Time to result from primary enrichment (80h)
• Plus serological (O and H antigens)
• Difco confirmation with BBL CHROMagar
• Time to result (40h)
• Plus serological (O and H antigens)

Confirmation Options (Cultural)
• Advantages
• Allows for the recovery of an isolate
that can be used for further analysis
• Strain typing to rule out lab error
• Strain typing to track contamination spread
• Helpful with determination of appropriate
organisms for challenge studies
• Warm and fuzzy satisfaction in seeing
your typical isolate

Confirmation Options (Cultural)
• Disadvantages:
• Not as sensitive as rapid methodology
• Background microflora frequently masks typical
colonies on selective agar
• Many strains are atypical on selective agar
• Time to confirmed positive result is considerably longer
than time to negative
• Negative cultural confirmation does not erase the initial
presumptive

Alternative Confirmation Options:
• Cross-platform Verification
• Two platforms with different detection targets
• DNA
• Protein
• Different “weaknesses” in one may not be present in the
other
• Cross-reactivity with PCR
• Cross-reactivity with Immunoassay
• Cross-reactivity with Phage

Alternative Confirmation Options:
• Advantages
• Much faster time to confirm
• Similar high level of sensitivity
• Odds of two false presumptive results from platforms
with different targeting mechanisms is very low
• Can still take on to selective broths and agars to recover
an isolate

Alternative Confirmation Options:
• Disadvantages
• No isolate unless taken through at least partial cultural
method
• Will not allow for determination of cross-contamination
events
• As with cultural confirmation, a negative result on the
cross-check platform does not erase the original
presumptive

Confirmation – Hybrid
• Combining cultural and crossplatform
confirmation
• Developing your own protocol
• Example 1:
• If presumptive on ELFA, run PCR
• If presumptive on PCR begin
presumptive response SOP
• If negative on PCR wait for cultural
confirmation
• Either way, initiate cultural
confirmation
• If negative on PCR and via cultural …
refer to false positive SOP

Validation
• When is it worth it?
• Significant improvement to cost, time, reliability
• When do you need to do it?
• When you lack the data supporting the viability of
method with a particular matrix
• When you make adjustments to the reference method
• How do you do it?
• Internally
• Externally: Send it to Certified Laboratories (or another
accredited reference laboratory)

Salmonella in the Crystal Ball
• 2015 – Rapid detection platforms finally realize the potential of
multiplex technology and include a marker for internal control
strains to detect positive control contamination and wild-types
simultaneously
• 2022 – Smart phone app allows users to test for pathogens in
food and food by-products and upload results to the popular
online social networking site “MyFace”, in a matter of seconds
• 2023 – Routine testing labs take on a reduced role in Salmonella
testing (continues to survive on study work, consulting, and free
lunches provided by the remaining rapid detection platform
vendors)

Predictions Continued
• 2033 – FDA approves a Salmonella destroying
phage for use on food products and ingredients
• 2034 - New mutated Salmonella strain discovered
that turns affected into catnip craving zombies. A
terrible, if not playful, zombie apocalypse ensues.

Questions?