Comparison of PMMoV RNA, crAssphage DNA and human mitochondrial DNA/RNA as normalization markers in wastewater-based epidemiology

Wastewater-based epidemiology (WBE) may benefit from normalization to account for wastewater dilution and temporal variation in the contributing population. In this study, we compared pepper mild mottle virus (PMMoV) RNA, bacteriophage (crAssphage) DNA targets (CPQ056 and CPPol), and human mitochondrial DNA/RNA (MT-CYB) as normalization biomarkers in a national surveillance setting. A four-target multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed to simultaneously quantify PMMoV RNA, CPQ056 DNA, CPPol DNA, and MT-CYB DNA/RNA and applied to analyse a total of 922 influent wastewater samples from 29 wastewater treatment plants (WWTPs). All markers were highly abundant and demonstrated robust analytical performance. The two crAssphage DNA markers were strongly correlated, with no site-specific or temporal differences in detection, showing that the CPQ056 DNA marker is sufficient to reliably quantify crAssphage DNA in our study. All markers responded similarly to changes in flow, indicating comparable performance for dilution normalization. The variability was primarily driven by WWTP-specific characteristics, with the largest deviations observed during very high flow events. Overall markers performed well and largely similar, however MT-CYB DNA/RNA showed minimal temporal variation and stable person-equivalent estimates across sites and time. PMMoV RNA had the lowest day-to-day variability but showed a seasonal variation. When applied to normalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, all markers reduced variability compared with raw concentrations and per capita normalization, which include flow data. Overall, all three markers performed well, with MT-CYB DNA/RNA a slightly more robust marker for population normalization in our setting.