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<p>Wastewater-based epidemiology (WBE) has emerged as a valuable tool for monitoring community-level infections, capturing both symptomatic and asymptomatic cases, as demonstrated during the <i>SARS-CoV-2</i> pandemic. However, WBE implementation is challenged by the complex nature of wastewater, which contains PCR inhibitors and varies significantly between wastewater treatment plants (WWTPs) due to differences in influent sources, industrial discharge, and environmental conditions. Furthermore, the lack of standardized protocols for virus concentration limits the practical and reliable use of WBE. Timely collection, concentration, and analysis of wastewater are crucial for detecting pathogens and informing public health decisions, yet most currently used protocols have been empirically established without a detailed comparison of methods under realistic surveillance conditions. This study compared two virus concentration approaches, filtration and pelleting, to determine which method yields the highest relative detection of <i>SARS-CoV-2</i> RNA. Wastewater samples from six WWTPs were processed using different concentration methods, with and without an inhibitor removal step. WWTPs were selected based on historical <i>SARS-CoV-2</i> RNA detection data, with three characterized by consistently high viral levels during peak infection periods and three with low or undetectable levels. In this context, method comparisons are extended to more accurately reflect real-world monitoring programs. In sites with high RNA concentrations, pellets and 100&#xa0;kDa and 30&#xa0;kDa ultrafiltration methods yielded significantly higher <i>SARS-CoV-2</i> RNA concentrations than the volume-intensive method routinely used in national surveillance. PCR inhibitor removal further improved detection, particularly in pellets, which offer a practical alternative for low-resource settings. In contrast, low-RNA concentration sites showed no significant differences between methods. However, PCR inhibitor removal enhanced detection across all methods. These findings demonstrate that viral RNA detection varies between concentration approaches and that removing PCR inhibitors substantially improves detection. Overall, standardized and optimized protocols for wastewater concentration and PCR inhibitor removal are essential for generating reliable WBE data.</p> Graphical Abstract <p></p>

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Improving SARS-CoV-2 RNA Detection in Wastewater: Comparison of Concentration Methods and the Effect of PCR Inhibitor Removal on Detection Sensitivity

  • 期刊论文

  • 发表日期：

    2026年1月31日

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  • Noor Ul Hudda,
  • Marie De Smedt,
  • Naomi de Roeck,
  • Veronik Hutse,
  • Lotte Jacobs,
  • Sara Van Looy,
  • Martina Ceconi,
  • Sofie Schaerlaekens,
  • Francisco I. Serrano-Cano,
  • Axelle Fransen,
  • Peter L. Delputte

摘要

Wastewater-based epidemiology (WBE) has emerged as a valuable tool for monitoring community-level infections, capturing both symptomatic and asymptomatic cases, as demonstrated during the SARS-CoV-2 pandemic. However, WBE implementation is challenged by the complex nature of wastewater, which contains PCR inhibitors and varies significantly between wastewater treatment plants (WWTPs) due to differences in influent sources, industrial discharge, and environmental conditions. Furthermore, the lack of standardized protocols for virus concentration limits the practical and reliable use of WBE. Timely collection, concentration, and analysis of wastewater are crucial for detecting pathogens and informing public health decisions, yet most currently used protocols have been empirically established without a detailed comparison of methods under realistic surveillance conditions. This study compared two virus concentration approaches, filtration and pelleting, to determine which method yields the highest relative detection of SARS-CoV-2 RNA. Wastewater samples from six WWTPs were processed using different concentration methods, with and without an inhibitor removal step. WWTPs were selected based on historical SARS-CoV-2 RNA detection data, with three characterized by consistently high viral levels during peak infection periods and three with low or undetectable levels. In this context, method comparisons are extended to more accurately reflect real-world monitoring programs. In sites with high RNA concentrations, pellets and 100 kDa and 30 kDa ultrafiltration methods yielded significantly higher SARS-CoV-2 RNA concentrations than the volume-intensive method routinely used in national surveillance. PCR inhibitor removal further improved detection, particularly in pellets, which offer a practical alternative for low-resource settings. In contrast, low-RNA concentration sites showed no significant differences between methods. However, PCR inhibitor removal enhanced detection across all methods. These findings demonstrate that viral RNA detection varies between concentration approaches and that removing PCR inhibitors substantially improves detection. Overall, standardized and optimized protocols for wastewater concentration and PCR inhibitor removal are essential for generating reliable WBE data.

Graphical Abstract