Lab Matters Fall 2024 | Page 128

APHL 2024 POSTER ABSTRACTS

An Integrated Approach for Pathogen Detection , AMR Monitoring and Functional Analysis in Wastewater

K . Locken 1 , J . Wilkinson 2 , K . Ngo 2 , P . Baybayan 2 , Y . Kim 2 , P . Pham 2 , E . Carrasco 1 , S . Tang 1 , J . Shen 1 , X . Cheng 1 , Zymo Research Corporation 1 , PacBio 2

Wastewater surveillance has emerged as a valuable epidemiological instrument in public health . In this study , we introduce an innovative , integrated methodology for the concurrent detection of pathogens , monitoring of antimicrobial resistance ( AR ) and functional analysis in wastewater . Leveraging the advanced sample preparation solutions provided by Zymo Research and PacBio Onso short-read sequencing , we aimed to enhance our understanding of microbial dynamics in wastewater and provide a report for actionable insights for public health and water treatment facilities . Real wastewater samples from local treatment facilities were processed using Zymo Research nucleic acid purification technologies . The PacBio Onso short-read sequencing system , which yields Q40 + accuracy , captured a comprehensive microbial profile . Metagenomic sequence data was downsampled to eight million reads per sample to be comparable to data obtained using the Illumina NextSeq 2000 system at the same depth , then analyzed using the Zymo wastewater analysis pipeline . By comparing the number of taxa , functional groups and ARGs , this study shows the potential of the high accuracy short-read PacBio Onso system for wastewater surveillance . The outcomes of this study offer multifaceted benefits for public health departments and water treatment facilities . Accurate pathogen detection enables the prediction of potential disease outbreaks , empowering public health authorities to implement proactive measures . AR monitoring provides crucial insights into resistance gene prevalence , informing strategies against the spread of antibiotic resistance . Functional analysis delves deeper into the intricacies of microbial communities within wastewater , specifically highlighting Nitrogen removal species , Phosphorus-accumulating organisms ( PAOs ), methylotrophs , filamentous bacteria and pathogens . This nuanced understanding empowers water treatment facilities to customize effective strategies , optimizing processes for contaminant removal and water safety . This research signifies a significant stride in proactive public health management and water treatment process optimization for enhanced environmental and public well-being .

Presenter : Xiaoxiao Cheng , xcheng @ zymoresearch . com

Automating Genomic Wastewater Surveillance for Efficient Pathogen Detection and Monitoring in the Population

J . Ng 1 , M . Grose 2 , A . Lin 1 , D . Careter-House 1 , S . Shokralla 1 , A . Allred 1 , V . Anderson 2 , I . Powell 2 , C . Bush 2 , R . Khaksar 1 , Clear Labs 1 , Kansas Department of Health and Environment-Kansas Health and Environmental Laboratories 2

Wastewater ( sewage ) can be tested to detect traces of infectious diseases circulating in a community , even if people don ’ t have symptoms . Wastewater monitoring is a robust tool that can reveal changes in disease trends before trends are seen in clinical cases , thereby guiding public health decision making across the nation . Whole genome sequencing ( WGS ) of wastewater samples is a reflex test that provides the added benefits of detecting the emergence of new variants , as well as tracking the dynamics of currently circulating variants .

Here , we automate end-to-end a SARS-CoV-2 wastewater whole genome sequencing ( WGS ) workflow , from extracted RNA as sample input to sequencing data as output , using the ARTIC v . 5.3.2 nCoV- 2019 Amplicon Panel . This fully automated , turnkey solution , with a single human touch point , generated equal or better sequencing results than traditional manual NGS methods . More uniform and consistent results were also obtained using the fully automated workflow as evident both in internal validation studies , as well as field generated data . We have also optimized wastewater extraction methods to guide users in obtaining the best quality RNA as starting material for this fully automated wastewater surveillance WGS application . This turnkey end-to-end fully automated genomic solution strives to provide the wastewater monitoring community a fast , efficient , user-friendly and no frills alternative to monitor changes in COVID-19 cases in communities across the United States and beyond .

Presenter : Justin Ng , justin . ng @ clearlabs . com

Capture and Concentrate Parasites and Yeast Using Nanotrap Particles

D . Goldfarb , L . Saunders , P . Andersen , C . Svezia , B . Lepene , Ceres Nanosciences

Wastewater-based epidemiology ( WBE ) to monitor SARS-CoV-2 has rapidly expanded across the globe as data has shown that WBE trends correlate with clinical case trends . WBE collects aggregate community numbers for a sewer shed , providing an overall picture of disease load that is independent of individual health care behavior or clinical symptom presentation . The success of WBE with SARS- CoV-2 has led to interest in monitoring additional microorganisms , including parasitic species such as Cryptosporidium parvum and fungi such as Candida auris . Waterborne parasites can be present in public water sources and can infect humans with minimal contact , then propagate to high quantities within the epithelium of the human digestive system . Fungal pathogens like Candida can often be spread through physical contact with an infected host or surfaces . Candida and other similar microbes have also been observed to contain genes which confer antimicrobial resistance ( AR ), leading to further need for broad surveillance of infectivity rates , as treatment methods can be more limited . These larger microbes can also present a significant challenge in sample testing , as they contain stronger cell structures and are therefore more difficult to lyse using common nucleic acid extraction workflows . Alternative methods must be implemented before their presence can be quantified .

This study presents a wastewater testing method that utilizes Nanotrap ® Microbiome B Particles to capture and concentrate Cryptosporidium parvum , Giardia Lambdia and Candida auris . Nanotrap Microbiome B Particles were used for concentration of microbes from 40 mL wastewater samples , followed by extraction using the MACHEREY-NAGEL NucleoMag DNA / RNA Water kit . This method employs the use of bead-beating techniques for efficient lysis of the robust cell walls of these microbes . A thirdparty column-based concentration and extraction kit was used with 40 mL samples as a comparison . The Nanotrap Microbiome B Particle method concentrated and recovered all three pathogens successfully and showed improved recovery over the column-based method in 7 / 9 samples tested . This scalable workflow is compatible