Dipstick-based pathogen detection for wastewater surveillance: Variability analysis using gage repeatability and reproducibility

COVID-19 redefined the outlook on pandemic preparedness, accelerating research toward establishing a global consortium for wastewater surveillance. Due to sample heterogeneity and low pathogen loads, microbial concentration remains a key challenge in developing low-cost, point-of-use wastewater monitoring assays. To address this challenge, we have developed a simplified version of dipstick method for RNA capture and isolation from sub-milliliter sample volumes, which simplifies RNA isolation. Given the manual steps involved in executing the dipstick method, variability is a major concern. In this work, we assessed dipstick variability through a multi-operator gage repeatability & reproducibility (gage R&R) study. We focused on detecting pepper mild mottle virus (PMMoV) and bacteriophage Phi6 in wastewater samples collected from a sewage pumping station at IIT Bombay. Our study demonstrated that the repeatability and reproducibility for the dipstick method are less than the acceptability limit of 30%, and could detect changes in PMMoV load associated with change in the population density due to summer break in our campus. Phi6 is a widely accepted surrogate for enveloped viruses such as SARS-CoV-2, and was therefore also chosen to demonstrate utility of this method for monitoring spread of infectious diseases. This work underscores the effectiveness of gage R&R in assessing and understanding the sources of variations in such assays, including operator-induced and part-to-part differences, essential for developing robust, manually-operated assays.