Previous Compartment Bag Test Performance Evaluations

Prior laboratory performance studies were conducted by McMahan et al, to characterize the ability of the CBT to quantify E. coli in drinking water by comparing it to the IDEXX Colilert® Quanti-Tray 2000 (QT), a recognized standard method for microbial water quality testing (McMahan et al, manuscript in preparation). A total of 884 surface water samples were collected over eleven sampling dates from surface water sites in Chapel Hill, North Carolina (United States), supplemented with specific growth medium for E. coli, either Colilert for QT or lauryl tryptose broth (LTB)

supplemented with X-gluc for CBT, and incubated for 24 to 48 hours at three different incubation temperatures, 44.5°C, 37°C, or 27°C.

The comparison between the QT and CBT analytical methods using the Mann-Whitney test in Figure 2.1 and the Bayesian hierarchical analysis in Figure 2.2 both indicate that the new CBT

procedure yields results consistent with those of the Colilert QT procedure. This assessment is based, in part, on the frequency with which the confidence intervals for the difference between the E. coli median values derived from the two testing procedures contain zero. Because these intervals are expected to contain zero roughly 95% of the time, it is expected that of 30 intervals, between 24 to 30 of them contain zero. More specifically, of the 30 confidence intervals constructed (excluding trial 6) for the difference between the QT and CBT results for each statistical procedure, 24 from the

Bayesian analysis include zero, 16 from the conventional analysis excluding right-censored data include zero, and 28 from the conventional analysis including right-censored data include zero. Because two of the three analytical comparison procedures (the Bayesian analysis, and the MPN method including right-censored data) appear to provide an adequate explanation for the observed

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differences, there appears to be sufficient statistical evidence to reject the hypothesis that the procedures are different.

Results recorded at incubation periods of both 24 and 48 hours indicate that the CBT yields results consistent with those from the Colilert QT system for E. coli detection, and that the CBT samples incubated at non-standard temperatures (temperatures above 25°C and below 37°C) gave results equivalent to standard temperatures. Therefore, ambient temperature incubation is possible and the CBT has the potential to serve as a low-cost, accessible solution for practical microbial drinking water quality testing in low resource settings.

Figure 2.1 Water quality sample analysis results for E. coli for each combination of sampling trial, incubation temperature, and incubation period, including Bayesian and conventional (MPN-derived) medians using QT (top panel) and CBT (bottom panel). Bayesian credible intervals which end in an arrow (see trial 6, CBT results) indicate that the interval bound is several orders of magnitude greater than the panel y-axis limits. Note that y-axes in both panels are on a logarithmic scale.

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Figure 2.2 Difference between QT and CBT results, measured as the difference between the assessed E. coli concentration median for each combination of sampling trial and incubation temperature. Bayesian credible intervals which end in an arrow (trial 6) indicate that the interval bound is several orders of magnitude less than the panel y-axis limits.

Results from culture-based confirmatory identification of E. coli bacteria isolated from positive CBT compartments after incubation were further obtained in diagnostic studies to screen for presumptive E. coli in both positive and negative compartments by streaking initially from wells of the QT and compartments of the CBT for E. coli isolation on Bio-Rad’E. coli 2TM _agar medium and observing for characteristically colored E. coli colonies. Table 2.3 lists the calculated identification statistics from these confirmatory analysis results. Based on the appearance of characteristic colonies on streaked plates of the Bio-Rad chromogenic E. coli agar medium, E. coli presence was confirmed in all color positive compartments of plastic bags. Surprisingly, however, E. coli presence was also found in 27% of color negative compartments of plastic bags.

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Table 2.3 Summary statistics for E. coli detection in presumptive positive and negative CBT compartments by streak plate isolation on a chromogenic E. coli agar medium

24 Hours 48 Hours

Sensitivity 73% 82%

Specificity 100% 100%

PPV 100% 100%

NPV 68% 66%

False Positive Rate 0 0

False Negative Rate 27% 18%

Accuracy 71% 79%

Sensitivity refers to how well a test correctly identifies true positives. Therefore, in this study, sensitivity indicates how well the CBT can perform at detecting low concentrations of E. coli (e.g., an E. coli MPN of 1 or several per 100mL). As shown in Table 2.3, the sensitivity of the CBT increased from 73% after 24 hours incubation to 82% after 48 hours incubation, indicating that some E. coli present in the water may not be detected by visual observation for the distinctive color of the hydrolysis product of the ß-D-glucuronide chromogenic substrate after only 24 hours of incubation. Specificity refers to how well the test correctly identifies true negatives, and the positive predictive value (PPV) is a measure of the proportion of positive compartments that are correctly identified, which is also a measure of the precision of the test. This study found very high specificity and PPV for the CBTs as shown in Table 2.3 with values of 100% for both after 24 and 48 hours of incubation. The negative predictive value (NPV) is a measure of the proportion of negative compartments that are correctly identified. At 24 hours, less than a third (32%) of the negative compartments tested were positive for E. coli. At 48 hours, one of the negative compartments had changed color to become visually positive, but 34% of the color negative compartments remained positive for E. coli based on isolation of characteristic E. coli colonies on a differential and selective chromogenic agar medium.

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