Lab Matters Fall 2023 | Page 82

APHL 2023 POSTER ABSTRACTS

laboratories into large-scale public health emergency responses , as well as identifying critical gaps or vulnerabilities that need to be considered for overall public health emergency response and preparedness .

Presenter : Sean Courtney , xcz1 @ cdc . gov

Comparative Data of Clinical Specimens from Southern Nevada Public Health Laboratory on SARS-CoV-2 Variant Detection in Clark County , Southern Nevada with Wastewater Surveillance

S . C . Phung 1 , E . Buttery 1 , M . Picker 1 , H . Zhang 1 , V . Vo 2 , A . Harrington 2 , E . Oh 2 , H . Y . Kan 1 ; 1 Southern Nevada Public Health Laboratory ,

2

University of Nevada , Las Vegas

Shortly after the first reporting of the Omicron variant in the United States , the variant began to dominate Clark County , Southern Nevada by mid-December 2021 . Due to the increased transmissibility of the Omicron variant , the Southern Nevada Public Health Laboratory ( SNPHL ) of the Southern Nevada Health District significantly expanded the implementation of Next Generation Sequencing ( NGS ) for SARS-CoV-2 RNA extracts from clinical specimens . During this time , our team increased its capacity to identify the variants of interest and variants of concern from 48 specimens / week to 192 specimens / week for 2022 . By February 2022 , the government had increased access of free at-home test kits to the community . This impacted the public health sector in identifying variants of interest and concern circulating in Southern Nevada due to the reduction of PCR testing . To consolidate the investigation of the Omicron lineages circulating in the Southern Nevada community , SNPHL collaborated with the University of Nevada , Las Vegas ( UNLV ) on wastewater surveillance for SARS- CoV-2 . When comparing the data with the wastewater surveillance results , all the Omicron variants that were detected from the wastewater surveillance were detected in the clinical specimens at the SNPHL . We compared the timeframe between the clinical specimen with the wastewater specimen for each of the omicron variants . We observed that the timeframe between the clinical specimens and wastewater was comparative and close . One of the major observations when comparing the data was the BF . 7 variant . The UNLV team was able to detect the presence of BF . 7 variant in May but the clinical specimen was not detected until August . This was due to the relatively low number of specimens received by the health district and its community partners , leading to a delay in the detection of the variants . The other observation was the re-introduction of BA . 2 variant where the wastewater specimen detected BA . 2 again in October and the clinical specimen in December . The trend observed from the wastewater was similar to our clinical specimens with BA . 1 dominating by February 2022 and BA . 2 begun circulating and displacing BA . 1 as the dominant strain . We also observed that two of the variants BA . 2.75 and BQ . 1 were first observed from the wastewater and two weeks later , SNPHL observed the same variants from clinical specimens . In summary , six of the variants detected by SNPHL were detected roughly the same time as the wastewater specimens , three of the variants were detected early from the wastewater with two variants detected two weeks after the first discovery through wastewater surveillance . The wastewater surveillance data from Southern Nevada has proven to be a beneficial source of data . Together , wastewater surveillance can complement public health surveillance to alert the community of new SARS-CoV-2 variants in Clark County , despite the low PCR testing by the community .

Presenter : Sui Ching Phung , phung @ snhd . org

Including Peptide Enrichment in a Mass Spectrometrybased Workflow for the Absolute Quantitation of SARS- CoV-2

R . Gibson , Y . Song , S . Samra ; Thermo Fisher Scientific

Introduction : SARS-CoV-2 particles contain proteins that are biomarkers of a COVID-19 infection that may be detected by bottom-up mass spectrometry ( MS ). Proteolytic digestion of proteins generates peptides , which can be separated by liquid chromatography ( LC ). A workflow had previously been developed that achieved low on-column detection limits for spiked nasal fluid samples in viral transport media ( VTM ). The addition of a peptide enrichment step , such as stable isotope standards and capture by anti-peptide antibodies ( SISCAPA ), could increase sample purity . This may reduce background , thereby improving detection limits and reducing LC run-times . Furthermore , SISCAPA could remove any need for sample clean-up and allow the concentration of samples , thereby increasing detection limits . Methods : A mass spectrometrybased SARS-CoV-2 absolute peptide quantification method was developed using a Thermo Scientific™ Vanquish™ MD LC system and a Thermo Scientific TSQ Altis™ MD MS . Recombinant SARS- CoV-2 proteins and stable isotope-labeled standards ( SISs ) were spiked into pooled nasal fluids , before being added to VTM . Samples were then precipitated , centrifuged , and enzymatically digested ( Thermo Scientific SMART Digest™Trypsin kit ). The resulting peptides were enriched with peptide-specific SISCAPA antibodies and separated using a 2-minute LC gradient with a Hypersil GOLD™ C18 column ( 1.9 um , 2.1 x 50 mm ), coupled with a single reaction monitoring method . All assays were performed in triplicate and data was analyzed using Thermo Scientific TraceFinder™ LDT software . Results : Full workflow testing with four peptide-specific antibodies confirmed the antibodies specificity and ability to work in conjunction . Coupling a mass spectrometry-based method with peptide enrichment allowed robust data acquisition with a two minute LC run time . Absolute quantitation of targeted peptides was then performed by including the corresponding SIS for each peptide to mitigate measurement uncertainty , confirm the retention times , and correct for any possible matrix effects . LODs and LOQs were determined for each peptide , with all % RSD / CV 0.99 . Clear chromatographic separation was observed for each nucleocapsid peptide with minimal variance in retention times observed (± 0.01 minutes ). LODs and LOQs were determined to be between 0.25 and 2.5 femtomole on column for the three best performing peptides . Conclusion : SISCAPA allowed 4-fold concentration of samples and eliminated the need for any further sample clean-up . This greatly increased the detection limits when the amount of nucleocapsid protein on each nasopharyngeal swab is considered , meaning that the protein could be detected at lower concentrations or on swabs with less spiked nasal fluid present . The increased sample purity also facilitated the reduction of LC-MS run-times from four to two minutes .

Presenter : Richard Gibson , richard . gibson @ thermofisher . com