Browsing by Author "Farnleitner, Andreas"
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- Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater SurveillancePublication . Ahmed, Warish; Simpson, Stuart; Bertsch, Paul; Bibby, Kyle; Bivins, Aaron; Blackall, Linda; Bofill-Mas, Silvia; Bosch, Albert; Brandao, Joao; Choi, Phil; Ciesielski, Mark; Donner, Erica; D'Souza, Nishita; Farnleitner, Andreas; Gerrity, Daniel; Gonzalez, Raul; Griffith, John; Gyawali, Pradip; Haas, Charles; Hamilton, Kerry; Hapuarachchi, Chanditha; Harwood, Valerie; Haque, Rehnuma; Jackson, Greg; Khan, Stuart; Khan, Wesaal; Kitajima, Masaaki; Korajkic, Asja; La Rosa, Giuseppina; Layton, Blythe; Lipp, Erin; McLellan, Sandra; McMinn, Brian; Medema, Gertjan; Metcalfe, Suzanne; Meijer, Wim; Mueller, Jochen; Murphy, Heather; Naughton, Colleen; Noble, Rachel; Payyappat, Sudhi; Petterson, Susan; Pitkanen, Tarja; Rajal, Veronica; Reyneke, Brandon; Roman, Fernando; Rose, Joan; Rusinol, Marta; Sadowsky, Michael; Sala-Comorera, Laura; Setoh, Yin Xiang; Sherchan, Samendra; Sirikanchana, Kwanrawee; Smith, Wendy; Steele, Joshua; Sabburg, Rosalie; Symonds, Erin; Thai, Phong; Thomas, Kevin; Tynan, Josh; Toze, Simon; Thompson, Janelle; Whiteley, Andy; Wong, Judith; Sano, Daisuke; Wuertz, Stefan; Xagoraraki, Irene; Zhang, Qian; Zimmer-Faust, Amity; Shanks, OrinWastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.
- Performance of Human Fecal Anaerobe-Associated PCR-Based Assays in a Multi-Laboratory Method Evaluation StudyPublication . Layton, Blythe; Yiping, Cao; Ebentier, Darcy; Kaitlyn, Hanley; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara; Converse, Reagan; Farnleitner, Andreas; Gentry-Shields, Jennifer; Gidley, Maribeth; Gourmelon, Michèle; Soo Lee, Chang; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim; Noble, Rachel; Peed, Lindsay; Reischer, Georg; Rodrigues, Raquel; Rose, Joan; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; Van De Werfhorst, Laurie; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia; Boehm, Alexandria; Shanks, Orin; Griffith, JohnA number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, B. thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman, HumM2, and M. smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman was found to be the most effective marker of human fecal contamination in this California-based study.