Lab Matters Fall 2024 | Page 109

APHL 2024 POSTER ABSTRACTS spp . Sequence data analysis was performed in BioNumerics v 7.6 .

Results : Accuracy , precision , sensitivity and specificity of the ANI test for species identification were calculated using the known results of each bacterial isolate included in the validation test panels as the gold standard . Precision of the ANI test was estimated to be 100 % and accuracy , sensitivity and specificity to be 99 %.

Conclusions : The ANI test reliably identifies enteric bacteria to the species level in defined taxonomic groups by WGS and provides results that are comparable to those obtained by gold standard methods . The ANI test is a reliable and sensitive genomic tool that overcomes challenges posed by traditional laboratory methods for identification of bacterial species .

Presenter : Karim Morey , karim . e . morey @ oha . oregon . gov

Rapid Bioinformatic Analyses and Data Visualization for Enhanced Genomic Surveillance of Candida auris in Massachusetts

M . Doucette , E . Fortes , S . Bhattacharyya , N . Epie , Massachusetts Department of Public Health

Background : Candida auris is an emerging fungal pathogen that is often resistant to antifungal treatments and is a growing public health threat . In January 2024 , the MA State Public Health Laboratory ( SPHL ) received a request from the Healthcareassociated Infections and Antimicrobial Resistance Epidemiology Program of MA Department of Public Health ( DPH ) to reproduce a sequencing analysis and data visualization workflow for C . auris sequencing data generated by the Centers for Disease Control and Prevention ( CDC ) and the New York State Department of Health Wadsworth Center , with the aim of evaluating linkages between the two data sources .

Methods : Raw reads generated by the CDC and Wadsworth that were previously uploaded to National Center for Biotechnology Information ’ s ( NCBI ) Sequence Read Archive ( SRA ) were imported into Terra ( Terra . bio ) using the SRA _ Fetch _ PHB ( v1.3.0 ) workflow . Raw reads were processed , assembled and characterized using the TheiaEuk _ Illumina _ PE _ PHB ( v1.3.0 ) and MycoSNP _ Variants ( v1.5 ) workflows . Single nucleotide polymorphism ( SNP ) distances and a phylogenetic tree were generated using the MycoSNP _ Tree ( v1.5 ) workflow . A tree file , SNP distance matrix and metadata file were uploaded to Microreact for data visualization and interpretation .

Results : Of the 18 C . auris sequences analyzed , 17 resulted as Clade I and one resulted as Clade III . These results were concordant with genotyping data provided by both the CDC and Wadsworth . Several sequences within Clade I clustered together with 1-16 SNP differences . Two sequences within Clade I were more distant than the others ( 27-36 SNPs ) but appeared to be closely related to each other with 0 SNP differences . Four additional sequences within Clade I appeared to be unrelated with over 60 SNP differences .

Conclusion : The Division of the Sequencing and Bioinformatics Core at the MA SPHL was able to rapidly perform bioinformatic analyses and data visualization for enhancing the genomic surveillance of C . auris using publicly available workflows and tools in response to a high priority epidemiologic need within 48 hours from the initial request . This work also emphasizes the importance of sharing genomic data using public repositories such as NCBI ’ s SRA for the rapid development and implementation of analysis workflows to respond to emerging public health threats .

Presenter : Matthew Doucette , Matthew . Doucette @ mass . gov

Sequencing West Nile Virus in Wyoming Using Illumina Microbial Amplicon Prep Kit

C . Rowley , T . Fearing , R . Petit , J . Mildenberger , R . Christensen , Wyoming Public Health Laboratory

West Nile virus ( WNV ) is the leading cause of mosquito-borne disease in the United States and in rare cases can lead to severe , sometimes fatal , illnesses . There currently is no treatment or vaccine to prevent WNV infection . We have detected WNV in most counties of Wyoming , making it a good candidate for ongoing pathogen surveillance for Wyoming . At the Wyoming Public Health Laboratory ( WPHL ), it is important that we continue to expand surveillance of public health pathogens in Wyoming . More importantly , we expand in such a way that utilizes our existing capacity and expertise .

At WPHL we have well established SARS-CoV-2 amplicon sequencing using the COVIDSeq chemistry . We identified the Illumina Microbial Amplicon Prep ( iMAP ) as a potential kit for sequencing as it also utilizes the same chemistry as COVIDSeq . iMAP can be applied to viral pathogens , antimicrobial resistance markers , wastewater and identification of bacterial and fungal organisms . We decided to test iMAP because it has the potential to expand our surveillance capacity without additional training or instrumentation . For this test , we selected WNV which we had previously not sequenced .

We first validated iMAP on SARS-CoV-2 , once validated we applied iMAP for WNV amplicon sequencing . To do so we acquired mosquito pool samples that were previously PCR tested to be positive for WNV from our in-state academic collaborators . A total of 16 different mosquito pools were ground up and underwent sequencing using iMAP protocols . For the amplicon sequencing , primers were ordered from IDT using a WNV primer scheme available from the Grubaugh Lab . We then used the Illumina MiSeq for sequencing . To analyze the sequencing data , we first removed host mosquito and human reads from each sample using Kraken . Then we were able to apply existing SARS-CoV-2 consensus assembly methods to our WNV amplicon sequencing . Many of the consensus assemblies were high enough quality to confirm their types based on previous PCR testing .

Here we demonstrate the application of iMAP for amplicon sequencing of WNV , a public health pathogen of concern in Wyoming . iMAP has the potential to allow the WPHL , as well as other PHLs , to rapidly shift to other pathogens of interest with no need to undergo extensive training of personnel or purchasing new instruments . We plan to continue to explore the application of iMAP for surveillance of other viral pathogens .

Presenter : Robert Petit , robert . petit @ wyo . gov