Assessment of crAssphage as a biological variable for SARS-CoV-2 data normalization in wastewater surveillance

This study aimed to assess the use of cross-assembled phage (crAssphage) as an endogenous control employing a multivariate normalization analysis and its application as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data normalizer.

A total of 188 twelve-hour composite raw sewage samples were obtained from eight wastewater treatment plants (WWTP) during a 1-year monitoring period. Employing the N1 and N2 target regions, SARS-CoV-2 RNA was detected in 94% (177) and 90% (170) of the samples, respectively, with a global median of 5 log10 genomic copies per liter (GC l-1). CrAssphage was detected in 100% of the samples, ranging from 8.29 to 10.43 log10 GC l-1, with a median of 9.46 ± 0.40 log10 GC l-1, presenting both spatial and temporal variabilities.

Although SARS-CoV-2 data normalization employing crAssphage revealed a correlation with clinical cases occurring during the study period, crAssphage normalization by the flow per capita per day of each WWTP increased this correlation, corroborating the importance of normalizing wastewater surveillance data in disease trend monitoring.