Lab Matters Fall 2023 | Page 75

APHL 2023 POSTER ABSTRACTS and multiplex MinION sequencing runs were successfully completed on-site by LRN laboratory scientists after minimal remote training . For 100 % of the runs in this study , scientists sequenced and assembled the bacterial chromosome and associated plasmids from B . anthracis strains , and each yielded the expected predictive genomic AMR profile with no evidence of bioengineering detected in as few as five hours . Data analyses conducted by the LRN scientists were completed in < 2 hours and matched CDC findings with no differences observed in the quality of assemblies generated or in the detection of AMR features . Following analysis , chromosomal single nucleotide variants ( SNVs ) related to fluoroquinolone resistance were interrogated , and correctly identified . However , an SNV located in a homopolymeric region that confers B . anthracis penicillin resistance was not reliably resolved by MinION sequencing alone . In this study , laboratory scientists from all four states successfully profiled plasmids , mutations and genetic signatures associated with AMR in under five hours , demonstrating that trained laboratory scientists could rapidly sequence and analyze B . anthracis in a BSL-3 laboratory using a portable sequencer , strengthening public health preparedness for an emergency anthrax response .

Presenter : Blake Cherney ( lii0 @ cdc . gov ); Luz Medina Cordoba ( gfc8 @ cdc . gov )

Next Generation Sequencing Strategies for Public Health Surveillance and Outbreak Investigation of SARS-CoV-2 and Mpox Infections

F . Kabir , E . Plaisance , J . Stringer , L . Short ; Dallas County Health and Human Services Public Health Laboratory

Introduction : The COVID-19 pandemic has greatly impacted public health preparedness and brought to light the crucial role of advanced molecular investigation and surveillance of genetic mutations in effectively responding to infectious outbreaks . While COVID-19 , caused by the SARS-CoV-2 virus , remains the leading cause of death by a single infectious agent , the emergence of mpox as a threat in the US and worldwide further emphasizes the need for accurate detection of pathogen variants for public health initiatives and outbreak management . The objective of the Dallas County Health and Human Services ( DCHHS ) next generation sequencing ( NGS ) program is to establish and provide sequencing-based pathogen clade analysis and genomic strategies that could effectively assist in outbreak investigation of respiratory pathogens , including influenza and SARS-CoV-2 and the emerging mpox infections . Methods : The workflows for both SARS-CoV-2 and mpox have been designed and developed from Illumina COVIDSeq protocol by differing the genome-specific primer pools . The most updated ARTIC community V4 / V4.1 primer pools ( for SARS-CoV-2 ) and a custom design pool from Yale School of Public Health ( for mpox ) have been employed for highly accurate detection of mutations in the respective viral genomes . The workflow includes steps for viral RNA / DNA extraction , library prep , sequencing in the MiSeq , analysis and report generation . The sequence analysis steps include raw read assembly and consensus sequence mapping to reference genomes and variant identification using Bioinformatics platform , including Illumina BaseSpace , NextClade , Pangolin web tools and CLC Genomics Workbench . Results : COVIDSeq protocol has been validated through cross-analysis verification of 40 deidentified samples provided by another public health lab with 100 % agreement on positive Alpha / Gamma / Delta / Omicron variants and

QC precision samples . About 100 COVID patient samples have been successfully sequenced and detected for ~ 20 Omicron sub-lineages in which BA . 5 was the most prevalent in July – November 2022 , and BQ . 1 spiked in December 2022 . These reports are also in direct agreement with other agency labs , including CDC . Using the parallel workflow , we have sequenced over 170 mpox samples and identified B . 1 lineage of West African Clade IIb as the most dominant variant during the peak surge of mpox in July through November 2022 . The viral genome sequences have been uploaded to global databases ( GISAID , NCBI ). Conclusion : The DCHHS NGS program has successfully created streamlined and modified protocols for the efficient detection of genetic mutations in both SARS-CoV-2 and mpox . These strategies not only aid in real-time surveillance and outbreak investigation of these infectious agents , but also provide valuable educational and technical resources for promoting public health interventions .

Presenter : Farruk Kabir , farruk . kabir @ dallascounty . org

Pathogen and Antimicrobial Resistance Detection in Wastewater using Hybrid Capture Enrichment Provides a Method for Tracking Emerging Infectious Diseases and Antimicrobial Resistance Genes

K . Janssen 1 , M . Roos 2 , J . Huang 3 , K . Broadbent 2 , J . Bierstedt 2 , A . Roguet 1 , 4 , E . Doolittle 1 , G . Knuth 1 , D . Cooper 1 , E . Olsen 1 , R . Schlaberg 2 , C . Wilusz 3 , S . Kuersten 2 ; 1 Wisconsin State Laboratory of Hygiene , 2 Illumina , Inc ., 3 Colorado State University , 4 University of Wisconsin

Monitoring wastewater for pathogens and antimicrobial resistance has emerged as an important epidemiological approach to assist public health professionals in tracking and potentially predicting trends in community disease prevalence . Increased pathogen levels in wastewater often precedes increases in clinical case metrics , providing an opportunity for non-invasive early warning of outbreaks . The use of quantitative PCR methods to detect and quantify pathogens in wastewater has become routine and provides a sensitive and targeted means to track viral dynamics and infection burdens in communities . This approach has distinct advantages for both sensitivity and throughput , however , it is limited to a single ( or if multiplexed , at best a small number of ) pathogens for any one assay . Next-generation sequencing ( NGS ) may provide a powerful alternative approach for monitoring large numbers of specific pathogens . Successful implementation of NGS in wastewater depends on applying robust methods for capture and concentrating the microbes and extracting the nucleic acids from concentrated samples . There is currently limited knowledge on how modern concentration and extraction techniques affects the recovered number and types of microorganisms in wastewater . Here we provide an evaluation of two concentration methods and multiple extraction methods using representative samples from Colorado and Wisconsin . The performance of two concentration approaches ( 1 ) a Concentrating Pipette ( InnovaPrep ) and ( 2 ) Nanotrap Microbiome Particles ( Ceres Nanosciences ) were evaluated . Furthermore , four different extraction techniques were assessed using wastewater samples obtained from college dorms and wastewater treatment facilities in Colorado and Wisconsin . The extracted nucleic acid was sequenced with Shotgun metatranscriptomics-which depleted ribosomal RNA ( rRNA ) using RiboZero PlusTM Microbiome ( Illumina ) coupled with total RNAseq library preps to profile the entire microbial content in the samples and the Urinary Pathogen