Lab Matters Fall 2024 | Page 29

FOOD SAFETY

By Rhodel Bradshaw , senior specialist , Food Safety

In 2022 , APHL brought together leaders from bioMérieux , member laboratories and federal partners to address an issue with the Cryptosporidium spp . target on the BioFire ® FilmArray ® Gastrointestinal ( GI ) Diagnostic Panel . The BioFire GI Panel screens for up to 22 gastrointestinal pathogens using stool specimens from patients and is a US Food and Drug Administration ( FDA ) -cleared tool for clinical and public health laboratories to diagnose enteric illness and investigate foodborne disease outbreaks .

CryptoNet National Surveillance Program

The US Centers for Disease Control and Prevention ( CDC ) established CryptoNet in 2015 to increase national reporting of cryptosporidiosis and better understand outbreaks and the transmission of Cryptosporidium spp . The CryptoNet network utilizes molecular subtyping to detect clusters of illness , help identify a source for those clusters and facilitate real-time sharing of Cryptosporidium case data in the United States . Many public health and clinical laboratories conduct testing for Cryptosporidium using multiplex nucleic acid amplification tests ( NAATs ) such as the BioFire FilmArray GI Panel . Positive Cryptosporidium specimens via NAATs , direct florescent antibody ( DFA ), or enzyme immunoassay are forwarded to CryptoNet-certified public health laboratories that partner with CDC to conduct additional molecular subtyping and whole-genome sequencing ( WGS ) on those specimens .

CryptoNet uses the Sanger sequencing protocol and WGS . Sanger utilizes primers for amplification of two gene targets and is performed on all specimens . CryptoNet laboratories then utilize bioinformatics to perform quality control checks to ensure sequencing data aligns with original NAAT results . The Minnesota , Wisconsin and Nebraska public health laboratories are in the process of validating Cryptosporidium WGS protocols , which will eventually be rolled out to all CryptoNet sites . WGS is performed on two species , C . parvum and C . hominis , but CryptoNet ’ s overall goals include expansion to all 50 Cryptosporidium species and other potential undiscovered subspecies . WGS will provide higher resolution including Actin , heat shock protein hsp70 , 18srRNA and GP60 genes .

CryptoNet Detects an Issue

In April 2022 , four CryptoNet sites — Wisconsin , Minnesota , New York and Georgia — noticed inconsistencies between original submitter results and in-house testing . When looking at the method used , the majority of inconsistent results were from the BioFire platform . In Wisconsin ’ s case , the negative specimens forwarded to CDC had originally tested positive for Cryptosporidium on the BioFire platform at the clinical laboratories . Concerns were raised as to why public health testing was not matching the initial results being observed and reported by clinical laboratories . After performing quality checks and investigating potential causes , Wisconsin notified CDC of a suspected false positive issue with the Cryptosporidium target on the BioFire . Wisconsin also observed that there was a shift in the median and mean age of all cases that were negative via 18srRNA and GP60 gene analysis .

Shortly following Wisconsin ’ s report , Minnesota contacted CDC with similar discrepancies between the confirmation rate from the original submitter tests and their in-house PCR test results . Minnesota noted that BioFire specimen confirmation rates went from 90 % to 20 % between October 2021 and April 2022 . In their initial investigation , 31 out of 74 BioFire confirmed specimens were negative . After reanalyzing the data later in the year , all 2022 specimens that tested positive via BioFire came back negative . Like Wisconsin , Minnesota noted an increase in age distribution and immunocompromised cases . New York and Georgia observed similar trends . New York saw a three-fold increase in submission numbers and a considerable number of negatives that had previously tested positive using the BioFire platform ( 38 %). Georgia had 32 of

53 BioFire-positive specimens ( 60 %) that tested negative . The age demographic of reported cases increased by 8.3 years from 2019 – 2022 .

Leveraging APHL Connections and Partnerships

APHL previously collaborated with sustaining member bioMérieux on a separate initiative , and had existing connections to BioFire staff , which proved to be crucial in the investigation . APHL facilitated and ensured timely communication between bioMèrieux , member laboratories and federal partners by setting up calls with affected states , distributing the bioMérieux Client Letter , posting in ColLABorate communities , sending emails and arranging an All-Laboratory Director presentation in January 2023 . Additionally , APHL developed multiple resources to assist members , including a FAQ document drafted in consultation with key members and cleared by CDC , and a tracking sheet intended to capture epidemiologic and demographic data on suspected false positive specimens .

CDC sent 68 suspected false positive specimens and an additional five positive specimens to bioMérieux for BioFire scientists to evaluate onsite . Through their investigative research , bioMérieux scientists determined that the BioFire software was misreading products of the non-specific Cryptosporidium target within the crypt2 assay . As indicated by BioFire scientists , the non-specific product has a melting temperature that lands in a temperature range that the software used for background signal normalization and therefore expects to be “ negative .” When this product is present and generates a melt signature in this temperature range , it creates an artifact or distortion of the signal in the expected temperature range for a positive Cryptosporidium result . This distortion ( an inappropriate signal subtraction ) is sometimes interpreted as “ positive .” Once the issue was clear , bioMérieux was able to develop an

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