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1
Moffett PT Laboratory
Year in Review
2019 IFSH Annual Meeting
Ravinder Reddy, Ph.D.
2
PT Program Overview
ISO/IEC 17043
accreditation
renewed in 2019
3
Regulatory and Food Safety
Programs Milk
Shellfish
Vitamins
Liquid and Powdered Infant Formula
Food Microbiology
Workshop on Method Validation and Statistics for Qualitative Microbiological Methods
Inter/Intra-Agency
Programs FDA/USDA FERN
ORA-ORS
• Dietary Supplement Adulteration
• QA Programs
CVM Vet-LIRN
Emergency Preparedness/Capacity
Building
Mission & Collaboration
Total of 24 PTs this FY
4
2019 Microbiology PT - Examples
Analyte Matrix Method(s)
Food pathogens, indicators Liquid infant formula,
powder infant formula
Culture, PCR,
Enumeration
B. anthracis Sweet potato puree Culture, PCR
SET Chicken salad ELISA, VIDAS
Vibrio spp. Oyster puree MPN, PCR
Listeria, Salmonella Raw dog food Culture, PCR
Triage (Ricin, B. anthracis, EHEC) Salsa Culture, PCR,
ELISA
5
2019 Chemistry PT - Examples
Analyte Matrix Method(s)
Glyphosate Cornmeal LC-MS
Tetramine Baby food GC-MS
Thallium Raw breakfast sausage ICP-MS
Aflatoxin B1 Dog food Charm test kit
Somatic cell count, drugs, added water Raw Milk NCIMS-approved
methods
Health Fraud
(Dietary Supplement Adulteration) Dietary supplements IMS, LC-MS
Carbamates Rumen LC-MS
6
Method Validation and Statistics Workshop
• Qualitative Microbiological Methods
Workshop • 2018 at Moffett – 20 participants
• 2019 at JIFSAN – 26 participants
• 2020 Location and Dates – TBD
• Quantitative Microbiological Methods
Workshop – In development
7
Organizing Laboratory for Multi-Laboratory
Method Validations
• Detection method for Cyclospora in agricultural water
• Pilot MLV program
• Successfully validated Cyclospora method in agricultural
water - pending publication in the FDA BAM
8
MLV Projects in the Pipeline for FY20
Detection of STEC in sprout spent irrigation waters.
Detection of Salmonella by qPCR method.
Molecular serogrouping method for L. monocytogenes
Neogen Veratox histamine kit for primary quantitative
analysis of histamines in seafood.
9
Multi-Laboratory Validation Study-
Detection of Cyclospora in
Agricultural Water
2019 IFSH Annual Meeting
Kaiping Deng, Ph.D.
10
Cyclospora cayetanensis
Source: http://www.cdc.gov/dpdx/az.html
• Protozoan, coccidian parasite
• Sphere shape, size 8-10 µm
• Excreted as un-sporulated
oocysts, takes 7-10 days to
sporulate under favorable
conditions
10 µm
11
Food Safety Related to Cyclospora cayetanensis
Source: http://www.cdc.gov/dpdx/az.html
• C. cayetanensis is the only species of
the genus Cyclospora known to infect
humans
• Not multiplying outside of host; very
hardy and chlorine resistant
• There are no in vivo or in vitro methods
available to propagate C. cayetanensis
from clinical or food samples
• Foodborne outbreaks associated with
vegetables and fruits
12
US Cyclosporiasis Outbreaks - 2018
Multiple Laboratories Validation (MLV) –
Cyclospora Detection from Agricultural Water
“Our experts worked with both McDonald’s and Fresh Express to trace the bagged
salad mix ingredients back to where they were grown. We found that the products
came from primarily domestic growers. ……They also stress the need to broaden the
tools, like import alerts, that we have up to this point used to prevent Cyclospora
illnesses in the U.S. to also include actions that are more appropriate for
addressing domestic contamination events.”
13
Previous Method of Detecting C. cayetanensis in Water
2004
EnvirochekTM
14
Detection of Cyclospora cayetanensis in Irrigation Water:
Single Laboratory Validation (SLV) Study Report
April 2018
FDA CFSAN OARSA Parasitology Team
Newly Developed Method
dead-end ultrafiltration (DEUF) filter
15
Flow Diagram of Filter Sample Analysis
Backflush C. caytanensis
from the filter
Backflushing
Filter the 10-L water
through a DEUF Filtration
16
Flow Diagram of Filter Sample Analysis (cont.)
Centrifuge the ~650 ml
backflush effluent at 4,000 x g
for 45 min
Centrifuge the ~120 ml of
concentrated backflush effluent
at 4000 x g for 45 min
Centrifuge the final concentrate
in a single 15ml tube
at 4000 x g for 45 min
17
Flow Diagram of Filter Sample Analysis (cont.)
Isolate DNA from the water
pellets using a FAST DNA
FastPrep-24 Kit
Purify the DNA using a
QIAquick PCR Purification Kit
Detect C. cayetanensis in the
DNA sample using
real-time PCR
18
• Objective:
Multi-lab validation (MLV) of Cyclospora in water detection
method
• Project Outline:
- Independent Laboratory Validation (ILV) - Moffett Team
- Method Training and mini-PT – Moffett Team, OARSA Team and 11
labs
- Multiple Laboratories Validation (MLV) - MLV Team, OARSA Team
and 8 labs
Project Objective and Outline
19
Independent Laboratory Validation Results
Inoculation
level
(oocysts per
10 L)
Number of
samples
analyzed
Number of
positive
results
Percentage of
positives (%)
0 10 0 0
6 74 55 74.3
12 12 11 91.7
100 4 4 100.0
200 7 7 100.0
20
MLV- Lab Identification
9. IEH Laboratories
10. NC Department of Agriculture
11. MN Department of Agriculture
12. CDC
1. FDA- ORA-ARKL
2. FDA-ORA-DENL
3. FDA-ORA-NFFL
4. FDA-ORA-PNL
5. FDA-ORA-PSFFL
6. FDA-ORA-SANFL
7. FDA-ORA-SFFL
8. FDA-CFSAN-DI
21
MLV- Method Training
Lecture Hands-on Training
22
MLV- Mini Proficiency Test (Mini-PT)
Lab # Inoculation level (oocysts/10 L)
0 6 12 100
1 - + + +
2 - + + +
3 - + + +
4 - + + +
5 - + + +
6 - + + +
7 - + + +
8 - + + +
9 - + + +
10 - + + +
11 - + + +
Originating lab - + + +
Results of PT samples:
+: C. cayetanensis was detected in the PT sample.
_: C. cayetenensis was not detected in the PT sample.
23
MLV Study Workflow
Day 1-2 Water (900-L) collected and tested for background
C. cayetanensis
C. cayetanensis-free water subdivided into 10-L
carboys, inoculated and filters prepared; Pre-
shipment testing performed
Pre-shipment testing completed and shipment
documents cleared
One set of eight filters shipped to each MLV
participant
The MLV samples received by the participants and
stored until testing
The MLV samples tested by the participants and
results submitted to the MLV organizers
Day 2-4
Day 5
Day 6
Day 7-8
Day 11-15
Water collection
Honda pump
24
MLV – Inoculation Scheme
MLV round Total number of samples
Blank
(0 oocyst/10 L)
Fractional level
(6 oocysts/10 L)
Positive control
(100 oocysts/10 L)
1 1 6 1
2 3 2 3
3 4 0 4
4 1 6 1
25
MLV – Results from Participating Labs
0 oocysts/10 L 6 oocysts/10 L 100 oocysts/10 L
Lab ID # positive #/sample # Specificity positive #/sample # Sensitivity positive #/sample
#
Sensitivity
1 0 / 9 100% 12 / 14 85.71% 9 / 9 100%
2 0 / 9 100% 14 / 14 100% 9 / 9 100%
3 0 / 9 100% 10 / 14 71.43% 9 / 9 100%
4 0 / 9 100% 11 / 14 78.57% 9 / 9 100%
5 0 / 9 100% 14 / 14 100% 9 / 9 100%
6 0 / 9 100% 14 / 14 100% 9 / 9 100%
7* 3 / 9 33.33% 14 / 14 100% 9 / 9 100%
8 0 / 9 100% 11 / 14 78.57% 9 / 9 100%
Number of
positive
Results
0 86 63
Number of
samples
63 98 63
Specificity 100% N/A N/A
Sensitivity N/A 87.8% 100%
26
MLV – Statistical Analysis
POD Curve Across Laboratories:
The LOD95% of a laboratory with median performance is 7.7 oocysts and
the LOD50% is 1.8 oocysts per sample (10 L water).
27
Conclusions
• The method is sufficiently sensitive and specific at large
sample volumes for use as a reference method.
• The FDA Microbiological Methods Validation Subcommittee
(MMVS) approved this method for regulatory uses.
28
• Moffett Team :
- Ravinder Reddy - FDA
- Matthew Kmet - FDA
- Samantha Lindemann - FDA
- Robert Newkirk – FDA
- Vishnu Patel – FDA
- Karl Reineke – FDA
- Jodie Ulaszek – IIT
• Other PT members • Josh Warren - IIT
Acknowledgement
• FDA OARSA Parasitology Team:
- Mauricio Durigan
- Helen Murphy
- Alexandre da Silva
• MLV Participanting Laboratories
• FDA Microbiological Methods
Validation Subcommittee (MMVS):
- Thomas Hammack
• QuoData Quality & Statistics GmbH
29
Pond
River
Well
Water Collection
30
Lake
Water Collection
Arrival at IFSH
31
Thank You!