Lab Matters Fall 2024 | Page 101

APHL 2024 POSTER ABSTRACTS determine which variants have been circulating in Colorado over time .

Conclusions : From this work , we established a pathogen genomic surveillance system for RSV capable of differentiating and evaluating the prevalence of subtypes and clades across seasons . This system will provide valuable information for treatment and vaccine development and will establish a framework for public health action in future RSV seasons .

Presenter : Daniel Polanco , daniel . polanco @ state . co . us

Evaluating The Use of Hybridization Capture Metagenomic Sequencing for Public Health Laboratory Surveillance .

M . Ramuta , A . Mooney , C . Jossart , R . Griesser , E . Hanson , K . Florek , A . Bateman , Wisconsin State Laboratory of Hygiene

The COVID-19 pandemic highlighted the benefits of using next generation sequencing ( NGS ) for tracking the spread of disease , monitoring microbial evolution and investigating outbreaks . Following the pandemic , there ’ s been interest in expanding the NGS capacity of laboratories for other pathogens to improve surveillance and help guide public health decision making . Existing surveillance strategies largely focus on using pathogen-specific NGS approaches . However , developing , implementing and validating these assays can be expensive and time consuming . Additionally , relying on these methods requires a priori knowledge of the microorganism present in a sample .

Metagenomics provides a pathogen-agnostic approach for the simultaneous detection of known and novel pathogens from samples . Metagenomics can also be used to provide full genome coverage for further characterization . However , it ’ s not as sensitive as targeted NGS approaches , which can lead to low or uneven coverage across the genome , limiting our ability to perform further analysis . Several techniques have been used to improve the sensitivity of metagenomics , including hybridization capture to enrich nucleic acids of interest . This approach relies on probe-based target capture to retain nucleic acid strands that match the reverse complement of the probes .

In this study , we implemented a hybridization capture metagenomic sequencing approach at the Wisconsin State Laboratory of Hygiene using the Illumina viral surveillance ( VSP ) and respiratory pathogen ID / AMR enrichment ( RPIP ) panels to detect 66 viral and 42 viral , 187 bacterial and 53 fungal pathogens , respectively . We compared the performance of these panels relative to the amplicon-based ARTIC SARS-CoV-2 and shotgun sequencing protocols . We also assessed their ability to detect a variety of microorganisms from clinical samples previously identified by PCR testing .

To compare the sensitivity of the VSP and RPIP panels to ARTIC and shotgun sequencing , we sequenced 7 SARS-CoV-2 positive nasal swabs with Ct values ranging from 23 to 29 . We analyzed the data using the viralrecon pipeline . ARTIC was the most sensitive sequencing protocol for detecting SARS-CoV-2 and calling variants , followed by VSP , RPIP and shotgun sequencing . The VSP and RPIP panels were both able to detect SARS-CoV-2 reads in every sample and identify the correct viral lineage in samples that had Ct values below 29 and 27 , respectively .

To assess the panel ’ s ability to detect various microbes in clinical specimens , we sequenced a total of 22 samples with 10 different viral and bacterial pathogens . The VSP and RPIP panels were able to identify the same pathogens detected by PCR in 21 / 22 samples . The VSP panel provided near-full genome sequences (> 90 %) for multiple viruses including influenza virus types A and B , RSV A and adenovirus .

This study demonstrates the potential for incorporating hybrid capture sequencing approaches in public health surveillance . Next , we plan to use this approach to analyze clinical specimens collected from ORCHARDS , an established surveillance program in a local school district . We will sequence specimens from participants who had influenza-like illness but were negative for 24 pathogens on the Luminex Respiratory Pathogen Panel , to assess if hybrid capture sequencing is useful to identify pathogens not covered in the respiratory panel .

Presenter : Mitchell Ramuta , mitchell . ramuta @ slh . wisc . edu

Expanding Horizons in Public Health : Metagenomic Surveillance of Viral Diversity in Wastewater Using a Hybridcapture Approach

P . Gupta , J . Hergert , K . Oakeson , Utah Public Health Laboratory

Wastewater genomic surveillance has become a powerful public health tool in the last decade . Despite its growing importance , the detection of multiple viruses from wastewater presents significant challenges , often limiting the scope of genomic surveillance to a few specific pathogens such as SARS-CoV-2 , RSV and influenza . While recent efforts have focused on monitoring these well-known viruses , there is a critical need to expand the current genomic surveillance efforts to characterize a broader spectrum of pathogenic viruses and gain a more comprehensive understanding of public health threats . Our study addresses this gap by utilizing a novel hybridcapture approach to obtain whole genome sequencing data of multiple viruses . We examined the diversity of viruses in wastewater using the Illumina viral surveillance panel , which targets 66 highrisk public health viruses . We conducted targeted metagenomic sequencing of over 200 samples collected from 35 wastewater treatment plants across Utah using the Illumina NextSeq 2000 platform . We analyzed the whole genome sequencing data using the DRAGEN microbial enrichment pipeline . We found that the relative abundance of viruses varied across sampling sites . Our analysis revealed a diverse array of enteric and non-enteric viruses , with a predominance of human coronavirus OC43 , polyomaviruses ( including BK Polyomavirus and JC Polyomavirus ) and Astroviruses . Our findings demonstrate the potential of targeted metagenomic sequencing in monitoring and detection of multiple viruses of concern from wastewater . Implementation of broad viral surveillance offers significant benefits over traditional surveillance methods and holds great promise for future outbreak investigations and improving public health preparedness and response .

Presenter : Pooja Gupta , pgupta @ utah . gov