CHAPTER 3. Dx-FS for EPOCT/ POCT urinary tract infection diagnosis
3.1 Urinary tract infection diagnosis using Dx-FS
3.1.3 Bacterial cell detection using colorimetric assay in Dx-FS
Bacterial counting via the colorimetric assay in Dx-FS was carried out using a commercial kit (Microbial Viability Assay Kit-WST; Dojindo Molecular Technologies, Inc., Figure 3.2) according to the manufacturer’s recommended protocols. In brief, 100 μL of the detection solution (20 μL WST-8 diluted in 80 μL distilled water) was added to Dx-FS and incubated. Through a gentle spinning, the liquid was transported to the top of the membrane and incubated for 45 minutes at 37 °C. The solution turned orang depending on the bacterial cell count. To retain the same volume of the detection solution on top of the membrane, we removed the liquid from the back chamber before loading the detection solution. For the practical utility of Dx-FS as a POCT platform, we simplified the overall operation to three manual steps: raw sample injection, bacterial enrichment, and final detection (Figure 3.1a). First, 1 mL of a raw urine sample is introduced into the device preloaded with the FAST solution (Supplementary Figure 7) using a plastic pipette through the inlet hole (Figure 3a-1). The bacterial cells in the urine sample are enriched by spinning the device (Figure 3.1a-2). Spinning is repeated until the sample is completely filtered. Finally, the FAST solution is removed to retain the detection solution on top of the membrane, followed by addition of the detection solution (see Methods), which is then spun for short interval until the solution is localized to the membrane chamber (Figure 3.1a-3). The color change is measured after 45 minutes and translated into the bacterial load (Figure 3.1a-4).
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Figure 3.2. WST-8 bacterial cell detection assay in Dx-FS. Schematics of bacterial cell detection assay reaction. WST-8 is reduced by the mediator (e.g. dehydrogenases) in cells with sodium ions and then the orange color is produced (formazan). The reagent, tetrazolium salt, can be stored in a salt form, which is better suited for usage in resource-limited settings.
Figure 3.3. Dx-FS kit used for urinary bacterial detection. The Dx-FS kit used for urinary bacterial detection contains a sample container for urine, Dx-FS, and sample and reagent pipettes for loading 1 mL of urine sample and 100 μL of detection reagent, subsequently. Detection reagent in a vial. The kit can have additional components, namely two more Dx-FS devices and antibiotic containing vials of four different concentrations if intended for Fidget-AST. The liquid form of the detection reagent, WST- 8, was used in this study, which function was retained for 30 days when stored at room temperature. In addition, the reagent can be in salt form, which would be better suited for the POCT usage. The salt form of the formazan dye can be dissolved in water to prepare the detection solution stock following the manufacturer’s protocol. The color chart allows easy interpretation of the results.
Color Chart
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Figure 3.4. Colorimetric calibration of the bacterial cell count and colony-forming unit (CFU). Two standard methods exist for bacterial cell counting, i.e., hemocytometer counting and bacterial cell colony counting after culturing (colony-forming unit, CFU). (a) Different counts of bacterial cells, i.e., 104, 105, 106, 107, and 108 bacterial cell count (#)/mL (upper panel) or 102, 103, 104, 105, and 106 CFU/mL (lower panel) tested using Dx-FS. (b) Color intensities shown in (a) and plotted versus the bacterial cell count (#)/mL (red) and CFU/mL (blue). Linear functions were fitted to the logarithmic concentration measurements (red and blue lines). The slope ratio and offset difference between red and blue lines are 1.04 and 100.5, subsequently.
Figure 3.5. Bacterial cell detection assay with (Dx-FS) and without (Tube) the enrichment step. (a) Samples of 1 mL with 106 bacterial cells/mL were kept in a tube or processed using Dx-FS. A bacterial cell detection assay was performed in the tube (top) or Dx-FS (bottom). (b) Optical density (OD) measurements of the detection assay solution with samples of 102−105 CFU/mL in the tube or Dx-FS.
The OD measurements were carried out using a plate reader (Infinite 200 PRO, Tecan, Switzerland) for comparison.
57 3.1.3.1 Colorimetric detection of UTI Dx-FS
We first explore the possibility of using colorimetric WST-8 assay for detecting bacteria isolated on the memorable. In POCT and EPOCT settings use of such detection method will make adaptation practical. Since it also facilitates easy interpretation for users, we integrated a proven rapid colorimetric WST-8 assay in which the electron mediator in the kit receives electrons from viable bacterial cells and transfers them to the WST, causing changes to orange color of the formazan dye, which can be visually identified by the naked eye (Figures 3.2). The entire activity is based on the metabolic activity of the bacteria isolated and needs time. From our early experiments we understood that 45 minutes for the reaction is enough and can be kept constant for the detection of bacterial load. All the components required to perform a single test based on colorimetric reaction using Dx-FS is put together as a kit. The kit comprises of 6 components as shown in Figure 3.3. A semi-quantitative estimate of the bacterial load can be easily obtained by naked-eye detection by the color chart comparison, while keeping the whole kit to simple (Figures 3.1d, Figures 3.3 and Figure 3.4a).
Two standard methods exist for bacterial cell counting, i.e., hemocytometer counting and bacterial cell colony counting after culturing (colony-forming unit, CFU). For ease of identification across the standards the effective range of detection, samples with bacterial concentrations of 102–106 CFU/mL were tested as shown in Figure 3.4a. The Figure 3.4b shows the Color intensities plotted, versus the bacterial cell count (#)/mL (red) and CFU/mL (blue). Linear functions were fitted to the logarithmic concentration measurements (red and blue lines). The slope ratio and offset difference between red and blue lines are 1.04 and 100.5, subsequently.
The reaction kinetics in the small volume above the membrane in Dx-FS vs the bulk Eppendorf tube is very different resulting in a sensitive visual readout that allows faster detection than the manufacturers recommendation. To confirmed that the enrichment effect of bacteria has affected the sensitivity of the detection in colorimetric detection by comparing the results of different concentration of bacteria with Dx-FS and using Eppendorf tube. Our earlier estimate on enrichment of bacteria shows that ~128-time enrichment of bacteria on the membrane. Without the enrichment step, the signal was too low to detect the bacterial concentration as high as 105 CFU/mL (Figure 3.5a). To quantify the same optical density measuring of both Dx-FS and the tube were measured and plotted in Figure 3.5b. Both visual and quantitative measurement clearly proves that the enrichment effect has significant difference in colorimetric readout.
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Figure 3.6. Effect of mammalian cell contamination. (a and b) To demonstrate robust UTI detection using Dx-FS, samples with mammalian cells (a; 0−105 MCF-10A epithelial cells/mL) and bacteria solutions (b; 0−108 bacterial cells/mL) spiked with mammalian cell contamination (104 MCF-10A cells/mL) were tested, and the optical densities (ODs) were measured. (c) Bacteria solutions (106 bacterial cells/mL) spiked with mammalian cell contamination (0−105 MCF-10A cells/mL) were tested with Dx-FS. (d), The color intensities were not significantly influenced by the presence of contaminated mammalian cells.
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3.1.3.2 Evaluating mammalian cells in urine interference in colorimetric assay.
Since the detection principle of the dye is based on APT and there is presence of puss cells in patient urine when infected, it’s important to verify the influence of mammalian cells in detection.89 To verify this we back calculated the cell number presented from highest dose of pus cells found in microscopic examination of UTI patient. We confirmed that the used microbial viability assay kit is highly favorable for bacterial cells with the use of a specific electron mediator by spiking mammalian cells and performing the assay.90 The breast cell line, MCF-10A, was obtained from the ATCC and maintained by following ATCC’s protocol. The mammalian cell spike test was carried out. In brief, MCF-10A cells were washed with 1×PBS twice by centrifugations. The cells were counted using an automated cell counter (Countess™, ThermoFisher, USA), and prepared with the desired cell concentration. The above cells were spiked along with bacterial cells and the assay was performed (Figure 3.6) The optical density (OD) of the solution was measured using a plate reader, NanoQuant Plate™, for nanodrop measurements (Infinite 200 PRO, Tecan, Switzerland). There was no significant signal from mammalian cells (<105 cells/mL) (Figure 3.6).
As shown in Figure 3.6a, first only the mammalian cells of different concentration were incubating, and the OD were measured and plotted. The results confirm that there is no significant signal in these concentrations of the cells. Similar test was repeated by keeping the mammalian cell number constant (104 cells/mL) and changing bacteria cell number from 105 to 108 (Figure 3.6b). The result clearly demonstrates that mammalian cells from urine don’t interfere in the results. A further validation of the same using colorimetric method was performed with fixed bacteria count and results were quantified. (Figure 3.6c and d)
3.1.3.3 Quantitative analysis of Dx-FS images.
In addition to naked-eye detection of the colorimetric assay in Dx-FS, images were acquired using photographic devices (a cell phone camera, Apple, USA and digital single-lens reflex (DSLR) camera, Canon, Japan) for more quantitative analysis. Automatic photograph analysis was carried out as follows (Figure 3.7): We first performed color calibration of the acquired images with an Imatest®
eSFR chart, which is the ISO 12233:2014 standard for photograph quality.91 To measure the color intensity from the membrane area in Dx-FS, regions of interest (ROIs) were found using a template- based cross-correlation analysis from the image. From each ROI, a circular membrane area was located using a circular Hough transform, and the inner-circle pixels were taken (circular ROI). Color measurements were carried out by converting the image into the profile connection space (PCS) L*a*b*
(L*: lightness, a*: green-red, and b*: blue-yellow) format.92 All computations were performed using MATLAB (MathWorks, MA, USA).
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Figure 3.7. Photographic analysis of Dx-FS. (a) Photographs of Dx-FS acquired with an Imatest® eSFR chart for color calibration. (b) An image of the Imatest® eSFR chart after calibration. (c) Regions of interest (ROIs) for color measurement detected using a template matching algorithm. (d) Membrane area for color measurement defined from the ROIs using a circular Hough transform (1), orange color intensity within the circle from (1) was measured using a custom L*a*b* color space definition (2), and examples of the color intensity measurements using (2) with the color channel (3) and background channel (4).
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3.1.3.4 Evaluating the effect of temperature and color stability.
In many POCT and EPOCT settings lack of regulated room temperature and availability of incubator or cold storage can cause undesirable results that can be miss interpreted. Due to the simple nature of the colorimetric test we adapted it for this study, however during the experimental validation we observed that temperature below 30 °C causes delay in reaction and can be miss interpreted.
Alternate solution is to use some exothermic reaction that can sustain itself for an hour at a favorable temperature that is suitable for the reaction. Many previous reports have already demonstrated use of hand warmer.93 As a potential alternative for such cases, a Dx-FS bacterial cell detection assay was performed using a commercial hand warmer (Figure 3.8d). Heat map images of Dx-FS were acquired using thermal imaging camera (Ti300 PRO, Fluke, USA). The plot in Figure 3.8a denoted that the hand warmer was purchased from a nearby convenience store for 30 cents per pouch, temperature raises to temperature of about 35-40 °C in 10 minutes’ time. When measure for longer time we found it could maintain 50–60 °C for 6–8 h according to the specification. From Figure 3.8 b and c we can conclude that the devices can be effectively to overcome the temperature problem and the colorimetric assay works efficiently in this case.
We also measured the effect of time on the stability of the color produced during the reaction.
For the study we compared the result of control and 104 CFU/ mL bacteria at 45 minutes which is a standard time for our test as reported vs 24 hours later. In Figure 3.9 the plot clearly shows there is no change. It’s also important to note that we observed evaporation of liquid over time in the 24-hour case which resulted in decrease in intensity when quantified but it remained well within the standard deviation levels.
In short urine samples were accrued and subjected it for colorimetric detection in the device as shown in Figures 3.1d. The acquired images of Dx-FS for bacterial loads in the range of 103–106 CFU/mL were analyzed and plotted (Figures 3.1e). Since CFU being a standard unit of measuring bacteria to relate it to clinical condition, we calibrated CFU to our bacterial count method (Figure 3.4).
The assay works at temperatures above 30C; however, a hand warmer can be helpful in colder environments (Figure 3.7 and Figure 3.8). No significant color difference was observed after 24 hours after the detection was performed (Figure 3.9). Based on all these observations, we conclude that Dx- FS is a versatile platform that meets the World Health Organization (WHO)-ASSURED standards (Supplementary Table 2); therefore, Dx-FS can be an effective diagnostic tool for POCT in resource- limited settings for practical utility we then spiked different bacterial loads in the range of 103–106 CFU/mL in waster
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Figure 3.8. Dx-FS bacterial cell detection assay using a hand warmer. Dx-FS bacterial cell detection assays were performed in an incubator or using a commercial hand warmer. (a) Temperature of the Dx- FS membrane area measured using a thermal imaging camera. Dx-FS was taken from the incubator or placed on a hand warmer at the beginning (0 minutes) or 5 minutes later. (b and c) Dx-FS microbial detection assay using the incubator or hand warmer (b: photographs, c: color intensity measurements).
No significant color difference was identified (p = 0.7501, N =3). (d) Heat signatures of Dx-FS during incubation in incubator (left) or hand warmer (right).
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Figure 3.9. Color stability for Dx-FS bacterial cell detection assay. Dx-FS assay was carried out with samples having no bacterial cells (Neg.) or those with 104 CFU/mL. Color intensity was measured right after bacterial cell detection assay in Dx-FS (45 minutes) or after 24 hours (24h) at room temperature and no significant difference was observed (N=3).