Lab Matters Fall 2024 | Page 136

APHL 2024 POSTER ABSTRACTS

and allows users to customize BED files used depending on what primer set is used .

Results : Validation of SPLiNTER was performed using in silico datasets provided by the Centers for Disease Control and Prevention ( CDC ) -led National Wastewater Surveillance System ( NWSS ) team . Nearly identical relative lineage abundance proportions were recovered with minimal deviations from the expected results provided by NWSS . A routine updating and verification process of the version of Freyja used in SPLiNTER was put into place to ensure the most up-to-date data is used to identify and calculate emerging variants in wastewater samples .

Conclusion : SPLiNTER allows DCLS to provide sequence analysis and lineage abundance results to Virginia public health partners quickly and efficiently . Future releases will incorporate customizable R Markdown reports for results sharing with DCLS ’ s public health partners as well as other user feedback-driven updates .

Presenter : Shelby Bennett , shelby . bennett @ dgs . virginia . gov

Targeted Wastewater Surveillance Employing Cultural Methods

M . Davis , D . Waggoner , South Carolina Public Health Laboratory

As part of the US Food and Drug Administration ( FDA ) Laboratory Flexible Funding Model grant ( LFFM ) -COVID-19 wastewater supplement , the South Carolina Department of Health and Environmental Control ( SCDHEC ) Advanced Molecular Laboratory was required to sequence 400 isolates per year of Shiga-toxin Escherichia coli ( STEC ) to meet GenomeTrakr requirements . The Advanced Molecular Laboratory was not able to acquire the isolated needed , so other ways were investigated to see how we could meet this goal . In order to accomplish this , culture dependent wastewater sampling was implemented . The Food Microbiology Laboratory started setting up wastewater samples for STEC in the beginning of 2023 from 24 hr composite influent one liter samples received the SCDHEC Virology Laboratory for COVID-19 wastewater testing . This proved to be ineffective , so permission was obtained from GenomeTrakr to use wastewater for other pathogens of interest . In July of 2023 , the Food Laboratory took a more targeted approach to isolation of organisms of interest . These samples were set up using a combination of methods used in the Food Laboratory for testing samples for routine surveillance and methods used by the National Antimicrobial Resistance Monitoring System ( NARMS ) for testing retail meat and seafood . Bacteria that have been isolated from wastewater include : 30 Listeria spp ., three Cronobacter sakazakii , 38 Enterobacter spp ., 83 Escherichia coli spp ., 17 Pseudomonas spp ., 26 Enterococcus spp ., 29 Salmonella spp . and 135 isolates of other various organisms of interest . Several isolates of interest in ongoing investigations have been isolated including Salmonella javiana , Salmonella braenderup and Proteus mirabilis . Since implementing these methods , the Advanced Molecular Laboratory has been able to meet the requirements for the LFFM grant for Whole Genome Sequencing ( WGS ). In the future , the Food Laboratory will focus on targeted isolation of ESKAPE organisms .

Presenter : Dana Waggoner , waggond2 @ dhec . sc . gov

Tiled Amplicon Primer Development and Optimization for Sequencing of Influenza Hemagglutinin ( HA ) Gene Segment in Wastewater

M . Hetherington-Rauth , V . Nguyen , K . Castro-Vital , A . Rossheim , L . Bankers , S . Matzinger , Colorado Department of Public Health and Environment

Introduction : Although methods exist for sequencing Influenza viruses ( IV ) from clinical specimens , widespread methods for sequencing IV from wastewater samples do not exist . This is in part due to 1 ) the genomic diversity and complexity of IV , including a segmented genome , high mutation rates , the presence of multiple subtypes and 2 ) the complexity of wastewater samples , which represent a pooled population of IV with expected fragmented gene segments . Because gene segments are expected to be fragmented in wastewater samples , the current primers designed to target IV in clinical samples cannot be used as they target a complete , intact gene segment . Therefore , we developed a set of tiled amplicon primers to sequence the hemagglutinin ( HA ) gene segment of IV .

Methods and Results : We designed custom tiled amplicon primer sets for each Influenza virus A ( IVA ) H1 and H3 HA gene segments and Influenza virus B ( IVB ) HA gene segment using the program Olivar , which accounts for nucleotide variant diversity across the length of the target sequence . To determine the nucleotide variant diversity across the HA gene segment , we downloaded sequences from January 2018 - September 2023 of the HA gene segment using NCBI ’ s Influenza Virus Resource database , resulting in 4,660 IVA-H1 , 7,721 IVA H3 and 2,992 IVB HA sequences . Next , we performed alignment and variant calling for each sequence against the vaccine reference strain for the 2023-2024 influenza season . The resulting primer schemes resulted in 12-13 amplicons that captured 90.3 %, 86.4 % and 85.1 % of the IVA-H1 , IVA-H3 and IVB-HA gene segment sequences , respectively , relative to the reference HA gene segment from which the primer scheme was designed . Amplicon sizes averaged 165 , 147 and 146 base pairs in length for IVA-H1 , IVA-H3 and IVB-HA , respectively . We tested the primers on positive controls and mixed positive controls following a modified version of our SARS-CoV-2 sequencing protocol using the Illumina sequencing platform . The mixed positive controls consisted of IVA H1 , IVA H3 and IVB HA sequences in order to simulate wastewater samples , which are expected to have more than one influenza type and subtype within the sample . Next , we analyzed the sequencing data to determine primer specificity , depth of coverage and percent of the gene segment sequenced for each primer set . Each primer set was specific to the targeted subtype , with minimal reads mapping to alternative subtypes . For each primer set , there was coverage across all amplicons , although the average depth of coverage varied across amplicons . Results were similar between positive controls and mixed positive controls , suggesting these primers can be used in wastewater samples .

Future Directions : We are optimizing these primers by testing modified thermocycler conditions , one- and two-step enzyme reactions , modified primer concentrations and revised primer sequences for increased depth of coverage across specific amplicon regions that displayed low sequencing depth . Additionally , we will assess sensitivity of the protocol by performing a limit of detection ( LOD ) study . Next , we will test the primers on real-time wastewater samples . Finally , we will incorporate the use of these primers with a