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Moffett PT Laboratory

Year in Review

2019 IFSH Annual Meeting

Ravinder Reddy, Ph.D.

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PT Program Overview

ISO/IEC 17043

accreditation

renewed in 2019

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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

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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

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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

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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

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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

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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.

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Multi-Laboratory Validation Study-

Detection of Cyclospora in

Agricultural Water

2019 IFSH Annual Meeting

Kaiping Deng, Ph.D.

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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

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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

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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.”

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Previous Method of Detecting C. cayetanensis in Water

2004

EnvirochekTM

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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

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Flow Diagram of Filter Sample Analysis

Backflush C. caytanensis

from the filter

Backflushing

Filter the 10-L water

through a DEUF Filtration

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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

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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

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• 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

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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

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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

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MLV- Method Training

Lecture Hands-on Training

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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.

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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

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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

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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%

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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).

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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.

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• 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

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Pond

River

Well

Water Collection

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Lake

Water Collection

Arrival at IFSH

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Thank You!