Novel methods for global water safety monitoring: comparative analysis of low-cost, field-ready *E. coli* assays

Water quality monitoring is crucial for public health, particularly in low- and middle-income countries (LMICs) where resource constraints often limit access to standard microbiological testing. These standard methods require dedicated laboratory space, expensive equipment, specialized consumables, and trained personnel—resources often scarce in areas with poor water safety. The need for simple, scalable, and low-cost microbiological water safety tests is significant, especially in the context of achieving Sustainable Development Goal (SDG) 6, which aims to ensure access to safe water and sanitation for all. This study focuses on evaluating two novel, low-cost assays for *E. coli* detection as potential alternatives to standard membrane filtration assays. The hypothesis was that these assays would demonstrate high sensitivity and specificity (≥90%) compared to the EPA Method 1604 (the reference method), under both standard and ambient incubation conditions. The successful development of such methods would facilitate large-scale global water quality monitoring and contribute to progress towards SDG 6.

The paper reviews existing literature on the limitations of current microbiological water safety testing methods, particularly in LMICs. It highlights the high cost and resource demands associated with standard methods like EPA Method 1604. The authors discuss the need for simpler, low-cost alternatives and mention some previously developed methods, referencing studies that have evaluated such alternatives. The literature review informs the selection of the two candidate assays (CompactDry™ and Aquatest) for this study, based on cost-effectiveness and a lower limit of detection of 1 colony-forming unit (cfu) *E. coli* in 100 ml of water. The review emphasizes the importance of finding assays that can provide reliable *E. coli* detection data with high sensitivity and specificity, even under non-ideal conditions.

The study involved collecting 315 bulk tap water samples from 14 locations in Peenya, Bangalore, India. Each sample was tested in triplicate using three methods: (1) The standard reference method (membrane filtration followed by incubation on MI agar, abbreviated as MF-MI), (2) The Aquatest (AT) presence-absence test (using 10 and 100 ml sample volumes), and (3) A modified CompactDry™ test using membrane filtration and pre-prepared dehydrated culture media. The samples were tested within 5 hours of collection. Both the AT and CompactDry™ tests were conducted under standard (37°C for 24 hours) and ambient (27°C mean, 25-30°C range, for 48 hours) temperature incubation conditions. Negative controls were also run for each method. The MF-MI method involved filtering samples through 0.45 µm pore size filters and incubating the filters on MI agar. The AT test involves adding the sample to pre-measured AT medium; a color change indicates the presence of *E. coli*. The CompactDry™ test uses membrane filtration followed by placement on a pre-sterilized plate with dehydrated culture medium. Data analysis included descriptive statistics, Bland-Altman plots, scatter plots, Spearman's correlation coefficients, and receiver operating characteristic (ROC) curves to assess sensitivity, specificity, and positive predictive values for both candidate tests compared to the MF-MI reference method. The area under the curve (AUC) of the ROC curves was calculated to evaluate the overall performance of the candidate tests.

The study found high sensitivity and specificity for both candidate tests compared to the MF-MI reference method. For the 100 ml AT test, the sensitivity and specificity were 97% and 96% (24h at standard temperature) and 97% and 97% (48h at ambient temperature). The CompactDry™ test showed >99% sensitivity and 97% specificity under both standard and ambient incubation conditions. The CompactDry™ method demonstrated good quantitative agreement with the MF-MI method across a wide range of *E. coli* concentrations. The 10 ml AT test, however, showed poor sensitivity (51.4% and 51.6% at standard and ambient temperatures, respectively), suggesting it is unreliable for detecting *E. coli* counts ≥11 cfu/100ml. The results from both ambient and standard temperature incubations were generally comparable for both candidate tests, indicating that ambient temperature incubation could be feasible in the field. Tables 1 and 2 provide detailed summaries of test performance characteristics for CompactDry™ and AT assays, respectively. Figures 3, 4 and 5 present graphical representations of the *E. coli* count data comparisons between the methods. The AUC estimates for AT exceeded 0.97, and 0.99 for CompactDry™, suggesting near-ideal performance compared to the reference method.

The findings suggest that both the CompactDry™ and the 100 ml AT presence-absence tests are suitable for large-scale *E. coli* monitoring to assess drinking water safety. The CompactDry™ test offers better quantitative data, while the 100 ml AT test provides a simpler and field-ready presence-absence assessment when quantitative data is not required. The importance of using an adequate sample volume (100 ml) for accurate *E. coli* detection was demonstrated, as the 10 ml AT test proved to be unreliable. These findings are consistent with and complement a previous study, although some differences in performance at ambient temperatures were observed. The study highlights the potential of these low-cost, field-ready methods to improve global water quality monitoring and support progress towards SDG 6.1.

This study successfully evaluated two novel, low-cost methods for *E. coli* detection in drinking water, demonstrating high sensitivity and specificity compared to a standard method. The 100 ml AT test is suitable for presence-absence assessments in the field, while CompactDry™ provides more accurate quantitative data. The results support the use of these methods for large-scale water safety monitoring, contributing to global efforts to achieve SDG 6. Future research could explore the application of these methods in diverse settings and further optimize the protocols for even greater field applicability.

The study was conducted in a single location in India and the generalizability of the findings to other regions with different water quality characteristics might be limited. The study only examined two specific low-cost methods and a broader comparison of various low-cost alternatives could provide a more comprehensive understanding. While ambient temperature incubation was found to be comparable to standard incubation in this specific environment, further investigation in different temperature ranges might be needed to assess the robustness of these methods across diverse climates.