APHL 2024 POSTER ABSTRACTS with the KingFisher Apex System , offering a reliable and high throughput method for WBE surveillance of parasites and fungi in wastewater .
Presenter : Daniel Goldfarb , dgoldfarb @ Ceresnano . com
Comparison of SARS-CoV-2 dPCR Targets in Wastewater- Based Epidemiology
Z . Scott 1 , J . Zimmer 2 , S . Siomko 3 , L . Beversdorf 2 , D . Payne 1 , Milwaukee Health Department Laboratory 1 , City of Milwaukee Health Department Laboratory 2 , University of Bristol 3
In the past , wastewater-based epidemiology ( WBE ) has been a niche branch of microbiology . Due to the emergence of SARS-CoV-2 ( SC2 ) and the ensuing pandemic , WBE is now at the forefront of detecting and combating the spread SC2 , as well as dozens of other pathogens and microbial targets . The City of Milwaukee Health Department Laboratory ( MHDL ) has been using WBE to detect SC2 for nearly two years . Recently , MHDL has been working to expand the wastewater program to include Flu-A / B and respiratory syncytial virus ( RSV ) to better serve and protect the Milwaukee community particularly during respiratory pathogen season . However , as new assays are developed and new variants emerge , assay targets can change . For example , early assays may have targeted S-gene , N1 gene , or N2 gene , with new assays altering their primer design . The aim of this study was to compare to assays with differing SC2 targets . For our preliminary investigation we compared 20 samples over five months , all of which have positive SC2 detects . In both assays , SC2 trends were similar . However , the copy numbers varied greatly . Therefore , as laboratories develop WBE assays and undergo their validation process , it will be important to consider various assay benefits such as absolute gene copy number , sensitivity and variability in targets , or perhaps prioritizing continuing trends will be more relevant for the community that is being served .
Presenter : Zachary Scott , zscott @ milwaukee . gov
Comprehensive Viral Pathogens Detection in Wastewater Using Hybrid Capture Enrichment and Whole Genome Sequencing
L . Immaraj 1 , S . Bhavanam 1 , Q . Jiang 1 , L . Brand 1 , J . Qiu 2 , M . Parkins 3 , C . Hubert 3 , T . Gao 1 , B . Lee 1 , X . Pang 1 , 2 , University of Alberta 1 , Alberta Precision Laboratories ( APL ) 2 , University of Calgary 3
Wastewater surveillance is a powerful tool for monitoring high-risk pathogens that are present and threaten the community . During the COVID-19 pandemic , the major technological platforms for detecting SARS-CoV-2 in wastewater ( WW ) samples have been RT-PCR-based amplification and amplicon sequencing . Although these methods are highly sensitive in detecting targeted viruses , they provide incomplete information regarding the various virome dynamics present in a heterogeneous WW sample , especially those posing a potential threat to human health .
Recent studies utilizing hybrid probe-capture-based genome sequencing to enrich viral targets in WW have been inconsistent . Our study aims to identify aggregate levels of viral pathogens in WW across Alberta using hybrid capture enrichment with viral surveillance probe panels ( VSP ), followed by comprehensive genome sequencing and computational analysis .
The library workflow was prepped using RNA Prep with Enrichment combined with hybridization with VSP probes , which can identify 66 viruses . Following double capture , the enriched and cleaned library was sequenced on an Illumina ® MiniSeq and the DRAGEN™ Targeted Microbial app was employed to identify viral targets and the percentage of confident callable bases in the consensus sequences .
To validate and optimize the VSP method , positive controls were prepared by mixing clinical samples with confirmed viruses and viral loads , including SARS-CoV-2 , Influenza A / B , enteroviruses and others assessed by qPCR analysis ( Ct = 27-30 ), to mimic typical viral loads shed in the WW matrix . We observed that detectable proportions varied , ranging from 45-100 % for SARS-CoV-2 , hCoV- 229E and enterovirus C in confident callable bases , indicating that VSP enrichment panel facilitates the simultaneous capture of multiple viruses at high genome coverages effectively for most viral targets , although adenovirus and rhinovirus were undetectable .
Subsequently , we applied this methodology to WW samples . RNA extracts from 26 WW collected from eleven WW treatment plants across Alberta and tested positive for SARS-CoV-2 , Influenza A / B and enteroviruses using RT-qPCR with Cts ranging from 27-35 were selected randomly . We detected higher levels of astrovirus , polyomaviruses and norovirus GII strains in WW samples with average % confident callable bases at 92.5 %, 70.5 % and 62.9 %, respectively . Smaller proportions of other viral pathogens , such as hCoV-229E at 13.2 % and sapovirus at 7.5 %, were also observed . Interestingly , major pathogens like SARS-CoV-2 and Influenza were positive by qPCR but showed no increase in detection levels . This is likely due to the complex nature of WW , which contains highly fragmented virome material that can interfere with the detection . These observations support that NGS sequencing thresholds or other parameters need to be altered for virome libraries in WW samples to enhance detection sensitivity and assay reproducibility .
Our preliminary data provides a comprehensive view of the viral landscape within the WW matrix using the hybrid capture sequencing approach and refining the methodology can potentially improve the detection of distinct pathogens . In the post-pandemic era , robust surveillance techniques are crucial for identifying highrisk pathogens in WW , serving as an early red-siren signal to guide public health measures .
Presenter : Linnet Immaraj , immaraj @ ualberta . ca
Confirming Subtype Specificity in Influenza-positive Wastewater Samples Through Subtyping PCR and Sequencing
V . Nguyen , M . Hetherington-Rauth , F . Cassidy , K . Castro-Vital , A . Rossheim , S . Matzinger , Colorado Department of Public Health and Environment
This study explores an innovative approach for influenza surveillance in wastewater , leveraging a multiplexed process to deconvolute the complex matrix in wastewater and allowing the confirmation of qPCR or dPCR results for influenza targets . Following the detection of influenza A or B virus targets in sewershed level wastewater samples using digital PCR ( dPCR ), RNA was reextracted from influenza A or B target positive samples and subsequently subtyped using the CDC Human Influenza Virus Subtyping A and B Real-Time RT-PCR Diagnostic Panels . The targeted subtypes include Hemagglutinin
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Fall 2024 LAB MATTERS 127 |