APHL 2022 POSTER ABSTRACTS training series . In the future , we look forward to providing more PulseNet training using our Learning Management Portal .
Presenter : Adom Yusuf , Association of Public Health Laboratories , adom . yusuf @ aphl . org
Evaluation of a Novel One-tube Nested Real-time Quantitative Polymerase Chain Reaction Assay to Detect Cyclospora cayetanensis
T Richins 1 , K Houghton 2 , J Barratt 2 , S Sapp 2 , Y Qvarnstrom 2 ; 1 Oak Ridge Associated Universities , Oak Ridge Institute for Science and Education , 2 Centers for Disease Control and Prevention
Cyclospora cayetanensis is a coccidian parasite that causes outbreaks of cyclosporiasis , a food-borne gastrointestinal ( GI ) disease , in North America and other regions . In 2021 , domestic diagnosis of C . cayetanensis was made 38 % of the time by microscopy , 50 % by syndromic tests and 3 % by PCR assays . Microscopy is cheap and time efficient but requires a highly skilled technician . Syndromic tests are powerful diagnostic tools but often require specialized equipment and are expensive . It is imperative to develop sensitive , reliable and inexpensive assays for the diagnosis of C . cayetanensis to protect the health of individuals and keep fresh produce safe for consumers .
In this study , we compare a one-tube nested qPCR targeting 89bp of the mitochondrial cytochrome B ( CytB ) gene to the 18S rRNA ( 18S ) diagnostic qPCR currently used by the US Centers for Disease Control and Prevention . The one-tube nested qPCR approach was evaluated due to the increased sensitivity and specificity usually afforded by nested assays , without the risk of amplicon contamination and lengthy hands-on time usually associated with nested PCR formats . A panel of 151 specimens was used to assess the performance characteristics of this assay , including 101 clinical specimens positive by the BioFire ® gastrointestinal panel , which has a reported 100 % sensitivity and 100 % specificity for Cyclospora and 50 specimens negative by microscopy and PCR for Cyclospora . These negative samples were positive for other related human pathogens , such as Cryptosporidium and Giardia . Difference in sensitivity was assessed with a chi squared ( 2 ) test to determine significance . The novel CytB qPCR test detected 81 / 101 ( 80 %) C . cayetanensis positive specimens while the 18S qPCR detected 58 / 101 ( 57 %) of the positive specimens . Neither test reacted to the negative specimens . The increase in positive specimens detected by the CytB qPCR compared to the 18S qPCR was statistically significant ( p = 0.0007 ). While BioFire ® is more sensitive , the novel CytB assay could be useful in settings where proprietary machines and supplies are not available or accessible due to cost and is a marked improvement over the 18S assay . It is also useful as the one-tube nested qPCR method requires no additional training other than what is already required to run traditional qPCR assays . The CytB qPCR test should make detection of C . cayetanensis more accessible . Further evaluation of this assay will be performed using sorted oocysts for a limit of detection study .
Presenter : Travis Richins , Centers for Disease Control and Prevention , richinstravis @ gmail . com
Foodborne Outbreak Detection and Control using an Automated Whole Genome Sequencing Platform
M Balamotis , T Cheung , O Valencia , H Kang , A Lin , K Hseih , P Thwar and R Khaksar , Clear Labs
Foodborne illnesses impact over 600 million people worldwide each year , leading to more than 420,000 deaths and the loss of 33 million healthy life years . Detection and serovar identification of foodborne pathogens such as Salmonella and Listeria can help limit the impacts of foodborne illness , but these methods lack the resolution needed for public health surveillance . Whole genome sequencing of bacterial isolates ( iWGS ) provides the highest level of strain resolution , determines relatedness to other strains , and can fully characterize the virulence and antimicrobial resistance profiles of a pathogen . CDC ’ s PulseNet and NCBI / FDAs collaboration , GenomeTrakr , use iWGS data for pathogen identification , which facilitates tracing outbreaks to their origins and sharing that information in real time with the FDA , USDA , CDC and public health departments to protect consumers . Over 700,000 food pathogen isolates have been sequenced and added to public databases through these programs . Revealing pathogen relatedness at the DNA level enables greater resolution of strain and lineage , thereby increasing more appropriate responses and interventions to contain food safety outbreaks establishing iWGS as the gold standard for foodborne disease surveillance and outbreak investigation . However , universal adoption of iWGS is challenging due to the specialized expertise and hands-on time that is required with preparing libraries for sequencing and bioinformatics data analysis . Clear Labs fully automated iWGS platform can reduce manual hands-on time from multiple hours to less than 30 minutes and narrow the number of human touch points from multiple steps down to just one . This is achieved through automation of DNA extraction , normalization , library preparation , sequencing and bioinformatics analysis ; helping to democratize genomics so that it can be more widely adopted across any laboratory to support foodborne disease surveillance . To evaluate the performance of the Clear Labs automated iWGS platform , Salmonella enterica Typhimurium isolates from the same FDA SAFE outbreak cluster collection and similar isolates not implicated in the outbreak were analyzed . Genome assemblies were generated with these strains using the Clear Labs end-to-end automated workflow alongside a manual sequencing workflow . The sequences generated were then used to build phylogenetic trees that equally and correctly clustered the Salmonella outbreak strains . Thus , automated iWGS platforms , through generation of near real time genomic data will play a key role in the rapid generation of actionable data for food safety surveillance and investigations .
Presenter : Michael Balamotis , Clear Labs , michael . balamotis @ clearlabs . com
Food Safety