Revisiting the Potential Role of Urine in Wastewater Surveillance: COVID-19 and Beyond

Clinical microbiology studies and systematic reviews have largely concluded that SARS-CoV-2 RNA is rarely present in the urine of COVID-19 patients. Thus, wastewater surveillance has largely focused on human stool and defecation as the relevant matrix and behavior. But, the over-performance of wastewater testing during near-source surveillance, especially in settings where on-site defecation is presumed to be de minimis such as aircraft and non-residential schools, suggests the possibility of SARS-CoV-2 RNA input from a non-fecal source. To revisit the possibility of urine as an explanation, data was compiled from 45 studies reporting on SARS-CoV-2 RNA in the urine of 1,924 COVID-19 patients. In general, the reporting quality was low with most studies (86.7%) failing to report even basic details such as the volume of urine collected. Only 11 reported quantitative data and of these only a single study reported its experiments to the standards established for publication of quantitative PCR data. Taking the data at its face value, the pooled prevalence of urinary SARS-CoV-2 RNA shedding was 11.3% (95%CI: 8.4 - 14.3), which roughly agrees with previous analyses. However, the percentage of variability in the estimate attributable to heterogeneity between studies (I2 = 93.66%) warrants careful consideration. Notably, for the 8 studies reporting sufficient workflow information, the observed urinary RNA shedding prevalence was normally distributed (r2 = 1.00) as a function of the volume of urine being assayed in a single PCR reaction (i.e., the equivalent sample volume, ESV). The study achieving the highest ESV (83.3 µL of urine) detected SARS-CoV-2 RNA in urine sediments of 95.5% of COVID-19 patients. Conversely, during two studies achieving ESVs of only 2 µL and 11.7 µL of urine, SARS-CoV-2 RNA was not detected in the urine of 72 and 132 patients, respectively. For wastewater surveillance of COVID-19, these observations suggest the rarity of SARS-CoV-2 RNA in urine may be a methodological artifact. Further, the data on which rarity is presumed are of low quality. Beyond COVID-19, the findings demonstrate that clinical studies, by virtue of their design to answer clinically relevant questions, could produce datasets subject to large biases that make them less useful when assessing the feasibility of wastewater surveillance of infectious agents. Wastewater surveillance practitioners must carefully consider the appropriateness and limitations of clinical data considering the important differences between the methods and aims of the two disciplines. Given the great potential of wastewater testing, clinical and environmental microbiologist should collaborate to produce robust quantitative datasets for reliable feasibility assessments for both community and near-source public health surveillance.