August 8, 2019 

Donna M. Garren, Ph.D. 

Executive Vice President, Science and Policy

American Frozen Food Institute

2345 Crystal Drive, Suite 801 

Arlington, VA 22202 

Dear Dr. Garren: 

Thank you for your recent correspondence, on behalf of the American Frozen Food Institute 

(AFFI), regarding the U.S. Food and Drug Administration (FDA) microbiological sampling 

assignment for frozen berries. The FDA shares your concerns about the safety of the U.S. food 

supply and the importance of maintaining the public trust. By letter dated July 24, 2019,1 we

addressed the sampling assignment protocols, regulatory actions, and reporting issues raised in 

your letters of June 13, 2019, and July 2, 2019. 

The purpose of this letter is to address your concerns about FDA’s test methods for hepatitis A

virus (HAV) and norovirus (NoV) in berries, raised in your earlier letters and a letter dated July 

10, 2019, which asserts that FDA obtained a false positive test result for sample 1084927 due to 

laboratory contamination. The July 10 letter also expresses concern about information sharing. 

You request that FDA discontinue its frozen berries surveillance sampling program until and 

unless AFFI’s concerns are addressed.

For the reasons explained below and in our earlier response, FDA is continuing with a modified 

frozen berries sampling assignment, consistent with our mission to help ensure the safety of the 

U.S. food supply. 

FDA Sample 1084927 Matches a Clinical Specimen

No Laboratory Contamination 

Your July 10 letter asserts that FDA’s test result on sample 1084927 was a false positive. You 

indicate that an external expert conducted a comprehensive analysis of the HAV sequence FDA 

obtained, and the expert concluded that it is highly likely that FDA’s result is a consequence of 

laboratory cross contamination. 

1 See Letter from Frank Yiannas, Deputy Commissioner, FDA, to Donna Garren, Executive Vice President, Science 

and Policy, AFFI, dated July 24, 2019, enclosed.
Garren – Page 2 

As discussed in FDA’s letter of July 24, FDA’s RT-qPCR detection assay for HAV and NoV 

method has undergone multi-laboratory validation, while viral extraction from soft fruit is a 

matrix extension of the FDA BAM method (Chapter 26B) that had undergone a multi-laboratory 

validation. We have incorporated a series of positive and negative controls in analyzing samples 

associated with the berry assignment. The initial RT-qPCR assay incorporates a negative PCR 

control. If HAV is detected in a sample and the negative PCR control is negative, that sample is 

subjected to the Control Exclusion Assay (CEA). The purpose of the CEA is to ensure that the 

laboratory control strain was not responsible for the positive RT-qPCR result. The CEA was 

performed on FDA sample 1084927 and confirmed the laboratory control strain was not the 

cause of the positive result.

In analyzing the sequence of FDA sample 1084927, FDA found that the sequence obtained was 

consistent with other HAV sequences (M59809; most similar BLAST result) in the National 

Center for Biotechnology Information (NCBI) database.2 This sequence comparison was not 

intended to determine HAV strain relatedness, for exclusionary purposes, nor to establish 

geographic origin. 

FDA also submitted the HAV sequence from FDA sample 1084927 to the Centers for Disease 

Control and Prevention (CDC) for comparison to its non-public, internal HAV database of 

sequenced clinical specimens. After closely reviewing and comparing the sample sequence to its 

database, CDC experts concluded the HAV sequence from FDA sample 1084927 is a 100% 

match (315 of 315 base alignment) to an HAV positive specimen detected during CDC’s clinical 

surveillance sampling in 2002. 

FDA performed additional analysis using the CDC 2002 sequence, along with the best 250 

matches, when BLAST-ing sample 1084927 against the nr/nt NCBI database to determine 

relatedness to other HAV strains. The agency then aligned sequences using muscle (Multiple 

Sequence Comparison by Log-Expectation) and sparsely populated flanking regions were 

trimmed. (NB: The one base difference between FDA1084927 and M59809 found by AFFI 

resided in the trimmed region.) Phylogenetic inference was performed using GARLI (Genetic 

Algorithm for Likelihood Inference) with the GTR (General Time Reversible) model of 

evolution and selecting the best of 10 non-bootstrapped replicates. DNA distances were 

computed using the Ape package in R with pairwise deletion set to true. Additionally, 

comparisons of FDA sample 1084927 to sequences in the public NCBI database showed other 

strains (from sewage and humans in addition to control strains) clustering closely with FDA 

sample 1084927. FDA is providing the results from the above analysis in the dendrogram below 

(Figure 1). This analysis confirmed that the HAV sequence from sample 1084927 is identical 

(315 of 315 bases align) to a CDC clinical HAV specimen from 2002. The laboratory positive 

control is 3 SNPs different from these matching sequences. 

2 NCBI provides public access to biomedical and genomic information, including genome sequences. BLAST is a 

program for sequence similarity searching developed at NCBI and is instrumental in identifying genes and genetic 

features. 
Garren – Page 3 

Figure 1. Dendrogram demonstrating relatedness of the HAV sequence from sample 1084927, 

the laboratory positive control strain, a CDC clinical surveillance specimen (from CDC’s internal 

database), and related strains from the NCBI public database. 

Based on this analysis, FDA scientists have concluded that the HAV sequence FDA obtained 

from sample 1084927 is not the result of laboratory contamination. 
Garren – Page 4 

Results with High Cycle Threshold Values Have Public Health Significance

Recommended Cut-off Value Unnecessary

Your letters recommend that FDA establish a cycle threshold (Ct) cut-off value above which RTqPCR results should be considered false positives and not sequenced. The July 2 letter states: 

“In reviewing the scientific literature and discussing this issue with experts in the field, we 

understand that above Ct values of 37 or 38 the results are suspicious of being false positives due 

to the limits of the assay and potential for cross contamination.” In support, you cite Gao et al. 

in which a sample with a Ct cut-off value of more than 38 was considered NoV negative.3 

FDA disagrees with the assumption that all high Ct values should be considered false positives. 

We believe Gao et al. has been misinterpreted; it states: 

“In this study, the CT cut-off value was used as a criterion for the determination of NoV 

contamination in berry samples. Although the method has been widely used (Jiang et al., 

2018; Liu et al., 2016), the use of the CT cut-off leads to underestimation of positive 

samples in practice. Therefore, the positive rate of NoV contamination in berries 

collected in this work may be higher.” (emphasis added)

As discussed above, FDA successfully sequenced sample 1084927, in which HAV was detected 

through RT-qPCR with a Ct value of 42, and matched (315 of 315 bases) to a CDC clinical 

specimen. Based on that analysis, FDA scientists have concluded that the qPCR result on 

sample 1084927 is not a false positive, despite the high Ct value. 

FDA achieves successful analytical results with: 

1) An efficient extraction method to reduce the presence of potentially inhibitory

material coextracted with viral RNA;

2) High amplification efficiency in detection and sequencing reactions; and

3) Sufficient quantity and quality of RNA for sequencing.4

Your July 2 letter also suggests that samples yielding Ct values of 42 or higher do not represent a 

demonstrated public health risk, and you recommend that FDA consider high-value Ct results to 

be negative for purposes of enforcement or other regulatory action. In the July 10 letter, you 

request that FDA issue a statement that any prior recalls or market withdrawals based on Ct 

values greater than 37 were unwarranted. 

FDA has data that do not support this assumption. For the past decade, we have successfully 

sequenced samples of foods epidemiologically linked with HAV and NoV illnesses—with Ct 

values as high as 50. These data are reported in Table 1 (below). 

3

 Gao, et al., 2019. Surveillance of norovirus contamination in commercial fresh/frozen berries from Heilongjiang 

province, China, using a TaqMan real-time RT-PCR assay. Food Microbiol. 82, 5812-5817. 4 An inability to successfully sequence the targeted genome after an initial positive RT-qPCR should not necessarily 

be considered a false positive due to the expected low level of the viral RNA. Nonetheless, for this sampling 

assignment FDA will not request initiation of a voluntary recall unless further characterization (i.e., Sanger 

sequencing) is achieved.
Garren – Page 5 

Table 1. Analytical Results of Seafood and Berries Associated with Illness (2009-2018). 

Source Commodity Analysis 

Year 

RTqPCR 

Result

Ct 

value 

(rounded)

Characterized 

Strains/

Genotypes

*Outbreak Related Oysters 2009 NoV GII 39, 42 NoV GII.7

*Outbreak Related Oysters 2009 NoV GII 49, 40 NoV GII.7

*Outbreak Related Oysters 2010 NoV GII 42 Nov GII.4 

Minerva/Den Haag

*Outbreak Related Oysters 2011 NoV GI 

NoV GII

42 

39

NoV GI.8, 

GII.3

Outbreak Related Oysters 2012 NoV GII 37, 41 NoV GII

Outbreak Related Oysters 2012 NoV GII 39, 40 NoV GII

*Outbreak Related Oysters 2013 NoV GII 42 NoV GII

*Outbreak Related Oysters 2013 NoV GI 41 NoV GI.4

*Outbreak Related Oysters 2014 NoV GII 40 NoV GII.12

*Outbreak Related Oysters 2014 NoV GII 43 NoV GII.21

#

Outbreak Related Scallops 2016 HAV 36 to 49 HAV IA

Outbreak Related Strawberries 2016 HAV 39 to 50 HAV IB

Outbreak Related Oysters 2017 NoV GII 41 NoV GII.2 and 

NoV GIIP/GII.10

Outbreak Related Oysters 2017 NoV GII 43 NoV GII.2

Outbreak Related Raspberries 2017 NoV GII 42 NoV GII.2

Outbreak Related Raspberries 2017 NoV GII 49 GII.17B

Outbreak 

Associateda

Crabmeat 2018 HAV 47 HAV IA

a Not meal remnant but associated lot

* Woods, J. W., K. R. Calci, J. G. Marchant, W. Burkhardt III, 2016. Detection and molecular characterization of

norovirus from oysters implicated in outbreaks in the U.S. Food Microbiol. 59, 76-84.

# Viray, M.A. et al., 2018. Public health investigation and response to a hepatitis A outbreak from imported scallops

consumed raw—Hawaii, 2016. Epidemiology and Infection 147, E28: 1-8.

FDA’s experience and the published literature demonstrate that foods yielding high Ct values 

have been identified as the vectors for HAV and NoV outbreaks. 

For the reasons explained above and in our earlier response, FDA intends to continue the frozen 

berries surveillance sampling program as modified and clarified. 

FDA Procedures for Information Sharing

Your July 10 letter references blinded information that was shared in response to an inquiry from 

a state-chartered berry commission. FDA is committed to ensuring that sample results are shared 

in accordance with agency procedures, which we have reviewed and reinforced with agency 

staff. 
Garren – Page 6 

In closing, we value the continuing collaboration with AFFI and its members and share the 

commitment to strengthening the safety of the U.S. food supply to protect American consumers. 

Sincerely,

Frank Yiannas

Deputy Commissioner 

Food Policy and Response 

Enclosure

Letter from Deputy Commissioner Frank Yiannas 

to the American Frozen Food Institute from July 24, 2019

https://www.fda.gov/media/129655/download