Lab Matters Fall 2023 | Page 78

APHL 2023 POSTER ABSTRACTS

that non-B . anthracis organisms within the Bacillus cereus sensu lato group may partially adopt the virulence factors encoded by pXO1 and / or pXO2 . However , it is unclear how this occurs in cases where neither plasmid is detected . To explore this phenomenon , we selected archived isolates previously identified through classic microbiological methods as B . cereus from the Special Bacteriology Reference Laboratory ’ s historical collection at the US Centers for Disease Control and Prevention , which were originally sampled from infections that are atypical for B . cereus . As a preliminary screening , we performed the Laboratory Response Network B . anthracis realtime PCR protocol and whole genome sequencing on 10 isolates . No isolates indicated the presence of either B . anthracis virulence plasmid . However , two of the resulting genomes were found to contain an apparent homolog to the capD gene , a gene normally housed on pXO2 that supports escape from host immune defenses through capsule depolymerization in B . anthracis . Work will continue screening the reference archives for similar genetic phenomena and incorporate long read sequencing on identified isolates to clarify the genomic location of capD .

Presenter : Susanna Sabin , qwg8 @ cdc . gov

Surveillance Whole Genome Sequencing in Denver County , Colorado

A . Smith , L . Bankers , A . Rossheim , N . Pysnack , E . Austin , A . Wheeler , S . Matziner ; Colorado Department of Public Health and Environment

The spread and prevalence of continually evolving SARS-CoV-2 variants is primarily monitored through the testing of symptomatic people and subsequent sequencing of the viral sample . However , test results are not available in real time due to factors such as delayed onset of symptoms , access to testing , and test result reporting . As the pandemic has progressed , the number of clinical samples collected has reduced due in part to the increase in both at-home test use and vaccinations that have helped reduce transmission and symptom severity . Such changes in testing patterns can influence estimates of SARS-CoV-2 variant prevalence . On the other hand , people who are infected with SARS-CoV-2 can shed viral RNA in fecal material , which is detectable in wastewater , regardless of symptoms or testing method , if even performed . Wastewater data obtained from community sources can represent an aggregate sample of the variants currently circulating in the population , whereas submitters to our surveillance efforts are skewed towards samples from symptomatic patients at inpatient healthcare facilities . As such , variant proportions estimated from wastewater whole genome sequencing are expected to be less biased . However , whether this expectation holds , and whether variant proportions estimated from clinical surveillance are congruent with wastewater data , remain open questions . Since March 2021 , CDPHE has partnered with wastewater facilities from across Colorado to collect bi-weekly wastewater samples , which are used for detection , quantification , and genomic characterization of SARS-CoV-2 . In addition , clinical samples are submitted from across the state for whole genome sequencing as part of our sentinel surveillance program . Here , we analyzed wastewater data from June 2022 through October 2022 in Denver County . SARS-CoV-2 weekly lineage abundances were estimated from whole genome sequencing data using Freyja . SARS-CoV-2 viral concentrations ( genome copies per liter ) were quantified using ddPCR . We are using multivariate time series analyses following the Johansen procedure to evaluate whether the time series of lineage frequencies estimated from wastewater and sentinel surveillance sequencing and viral load can form a cointegrating relationship , indicating a correlation between these variables . If so , this would suggest that we are obtaining accurate representations of variant trends and prevalence of the broader community . If not , this shows that variant proportions estimated from our sentinel surveillance system differ from those estimated from our wastewater surveillance data . This may inform whether sentinel surveillance strategies should be adjusted by increasing right-sizing parameters and / or including underrepresented populations . Additionally , this may inform us of ways that wastewater data may supplement our current sentinel surveillance program .

Presenter : Arianna Smith , arianna . smith @ state . co . us

Validation of Illumina ’ s Viral Surveillance Panel at New Jersey Public Health and Environmental Laboratories

C . Barnes , B . Schwem , D . Woell , R . Siderits , T . Kirn ; New Jersey Department of Health Public Health and Environmental Laboratories

Next-generation sequencing has become a cornerstone in public health as it allows us to trace origins of outbreaks , understand evolution of pathogens over time and potentially identify novel variants . In late 2022 , Illumina released the Viral Surveillance Panel ( VSP ), which uses hybrid-capture enrichment technology to sequence 66 DNA and RNA viruses — including viruses identified as significant risk to public health . There are many viruses on the panel that are tested at New Jersey Public Health and Environmental Laboratories ( PHEL ) through other techniques , such as real-time quantitative PCR and whole genome sequencing . The use of the VSP at PHEL would strengthen the sequencing capabilities and viral surveillance program in New Jersey . In order to test the VSP , Influenza and SARS-CoV-2 samples with CT values ranging between 15 and 20 were selected from samples submitted to PHEL . The library preparation process includes the generation of cDNA , tagmentation , capture of hybridized probes and enrichment of the library . The final libraries were run on both the MiSeq and iSeq to determine which instrument would be better for this study . The sequencing results were then analyzed using Genome Detective , DRAGEN Microbial and Kraken . Two library preparations were completed , and it was determined that decreasing the final elution volume from 32 µ L to 16 µ L dramatically increased the concentration of the library . It was also determined that iSeq would be more appropriate instrument to use for sequencing compared to MiSeq . Overall , the sequencing analysis using Genome Detective , DRAGEN Microbial and Kraken were accurately able to assemble and identify the expected virus with coverage over 95 % and confidence fraction over 0.94 . In several samples , there were other viruses also found showing the ability of the VSP to detect all viruses present . After confirming the panel worked for the Influenza and SARS-CoV-2 samples , the panel will be tested further with samples of different Ct values to determine accuracy and precision . The panel will also be tested on other viruses in three virus types : Respiratory viruses , Bloodborne viruses and arborviruses to further show the capability of the panel for surveillance of infectious diseases of public health concern .

Presenter : Cherrelle Barnes , clbarnes @ odu . edu