Test manufacture
The DNA plasmid encoding the fluoride biosensor used in this study was assembled using Gibson assembly (New England Biolabs, Cat#E2611S) and purified using a Qiagen QIAfilter Midiprep Kit (QIAGEN, Cat#12143). Its coding sequence consists of the crcB fluoride riboswitch from Bacillus cereus regulating the production of the enzyme catechol 2,3-dioxygenase, all expressed under the constitutive E. coli sigma 70 consensus promoter J2311939. A complete sequence of the plasmid used is available on Addgene with accession number 128810 (pJBL7025) [https://www.addgene.org/128810/].
Cell-free biosensing reactions used in the tests were set up according to previously established protocols20,40. Briefly, reactions consist of cleared cellular extract, a reagent mix containing amino acids, buffering salts, crowding agents, enzymatic substrate, and an energy source, and a reaction-specific mix of template DNA and sodium fluoride in an approximately 30/30/40 ratio (Supplementary Table 3). Test reactions contained no sodium fluoride, while positive control reactions were supplemented with 1 mM sodium fluoride to induce gene expression. Template DNA concentration for both sets of reactions was 5 nM, determined by the maximal template concentration at which no color change was observed in the absence of fluoride.
During reaction setup, master mixes of cellular extract, reagent mix, and template mix were prepared for both test and positive control reactions in 1.7 mL microcentrifuge tubes. Individual reactions were then aliquoted into 20 µL volumes in PCR tube strips for lyophilization. After aliquoting on ice, PCR tube caps were pierced with a pin, strips were wrapped in aluminum foil, then the wrapped strips were immersed in liquid nitrogen for freeze-drying for approximately 3 min. Reactions were immediately transferred to a Labconco FreeZone 2.5 Liter −84 °C Benchtop Freeze-Dryer (Cat# 710201000) with a condenser temperature of −84 °C and pressure of 0.04 mbar and freeze-dried overnight (≥16 h).
After freeze-drying, tests were vacuum sealed (KOIOS Vacuum Sealer Machine, Amazon, Amazon Standard Identification Number (ASIN) B07FM3J6JF) in a food saver bag (KOIS Vacuum Sealer Bag, Amazon, ASIN B075KKWFYN), along with a desiccant (Dri-Card Desiccants, Uline, Cat# S-19582) (Supplementary Fig. 3). Vacuum sealed reactions were then paced in a light-protective outer bag (Mylar open-ended food bags, Uline, Cat# S-11661) and impulse heat-sealed (Metronic 8-inch Impulse Bag Sealer, Amazon, ASIN B06XC76JVZ) before shipping. Tests were also shipped with single-use 20 µL micropipettes (MICROSAFE® 20 µL, Safe-Tec LLC, Cat# 1020) for field operation.
Test-kit shipment to Nakuru County, Kenya
A first shipment of biosensor tests was used to assess 33 water samples from the first 16 households surveyed. All of these tests resulted in a faint yellow color, regardless of water source or fluoride concentration established via fluorimeter. This was likely caused by thermal degradation of the tests during shipment with the commercial shipping agency. While previous studies report shelf stability for up to a year20,41, these figures were derived from storage in temperature-controlled laboratory conditions. Commercial shipment routes from Illinois, USA to Nairobi, Kenya pass through extremely hot regions, e.g., Dubai for this particular shipment. These conditions were much different from those in the previous study usability study in Costa Rica in which tests were transported by commercial air, with gentler shipping and storage conditions20. A laboratory investigation of test temperature stability indicated that elevated storage temperatures can indeed cause test components to degrade, resulting in a faint yellow color upon rehydration consistent with field observations (Supplementary Fig. 2).
The next batch of tests was therefore shipped refrigerated on January 25th, 2022, which we hypothesized would extend the tests’ shelf stability to align with earlier findings. After the tests were made and packaged, they were placed in a polystyrene foam-lined container before being covered with a NanoCool refrigeration system (Peli BioThermal). The container was then sealed shut and shipped using a standard commercial shipping service. This batch of tests was held in customs, refrigerated, until release on February 28th, 2022. These tests were used in the field from March 5th to March 14th, 2022 to generate the data on test accuracy reported in this manuscript.
As discoloration due to thermal degradation could confound the intended yellow hue in the presence of fluoride (i.e., false positives), we assessed test accuracy using only tests that had been refrigerated during shipping and transport to participants’ houses. The 33 water samples from the first 16 households were therefore excluded from analysis of test accuracy.
Participant recruitment
Participants were recruited from six sublocations (Kelelwet, Kipsimbol, Kigonor, Parkview, Lalwet, and Mwariki) in Barut Ward within Nakuru County (Supplementary Fig. 4, geographic information adapted from OpenStreetMap42). This location was chosen because of high fluoride levels and familiarity with the communities by the study team.
Before any data were collected, community meetings were held in each sub-location to discuss study goals and objectives. After obtaining permission from the community and village assistant chiefs to conduct research, local community mobilizers were engaged to assist with identifying households eligible for participation. Individuals who were 18 years or older, had lived in Nakuru country for more than three months, relied on local water sources for drinking, had a child in the household, were willing to discuss their household water situation, and provide a sample of each source of water in the household for fluoride testing were eligible. We sought to recruit 10–12 participants from each of the five sublocations to ensure a range of sociodemographic characteristics and drinking water sources. Having a child resident was a criterion in order to elucidate community understandings about fluorosis in children.
Data collection
After obtaining informed written consent, participants participated in a 30-min survey (cf. Supplementary Fig. 1 for a graphical overview of data collection). Topics included household sociodemographic information, knowledge, attitudes, and behaviors about fluoride and fluorosis, and household water insecurity using the validated Household Water Insecurity Experiences (HWISE) scale43. The 12 HWISE items query the frequency of experiences with water insecurity in the prior month; “never” is scored 0, “often/always” scored 3, for a range of 0–36. These data were collected to be able to investigate if user experiences or attitudes about testing varied by experiences with fluorosis or water insecurity. Participants were also asked about the number of sources of their water and willingness to provide and test water samples. Survey responses were recorded on tablets using Open Data Kit (ODK)44.
After completion of the survey, participants provided 1–3 samples of water from different household sources. They then received a brief (~5 min) explanation of the testing process, and then tested their own household samples using the fluoride biosensor tests. Each test consisted of a microtube that was a positive control, and a second microtube in which the sample of interest was tested. To test their samples, participants first removed the tests from the light-protective foil pouch and vacuum sealed pouch containing desiccant, both of which were then discarded (Supplementary Fig. 3). A micropipette was then filled with 20 µL water by slowly immersing it to the fill line. To dispense the water, the thumb and index finger were used to cover the holes in the micropipette while the bulb was squeezed with the other hand. The reactions were then incubated at ambient temperature for up to six hours, shorter if there was a visible color change. During this incubation time, participants were asked to check hourly for yellow color change and note the time taken for it to occur. Tests were expected to turn yellow if fluoride levels were ≥1.5 ppm, with no color change for tests of water below this level. All positive controls were expected to turn yellow. Color change was read after placing reactions against a white background for visual contrast.
The study team returned to conduct a second survey on user experiences with the testing process and to test the water samples using the gold-standard photometer within 6 h. Participants were asked about their experiences with the testing procedure as well as their interpretation of the color of the results of the sample and control tests. Photographs of the completed reactions were also taken at this time. Finally, quantitative fluoride measurements were taken by the field team with a Hanna Instruments Fluoride High Range Photometer Kit (Cat# HI97739C), a gold-standard method used to assess the accuracy of the bioengineered tests. Photometry results on actual measured fluoride concentrations of water samples were shared with and explained to participants. At the conclusion of the second survey, each participant was given KES 500 (USD 4.30) as remuneration for the time and effort spent participating in the research. Each participating household was also given a ceramic drinking water filter.
Data were collected from November 16th to November 23rd, 2021 and March 5th to March 14th, 2022. During surveying and water testing, participants and research assistants maintained COVID-19 protocols as per the local area guidelines. Study staff were vaccinated, maintained appropriate social distancing, sanitized hands, and cleaned field tools after each household visit.
Data analysis
Data were exported from ODK into Microsoft Excel for analysis. Basic descriptive statistics were performed to describe participant socio-demographics and experiences with usability, including if participants’ interpretation of color change matched that of study staff. Open-ended items about fluoride and fluorosis knowledge, attitudes, and behavior were grouped thematically and coded independently by two authors. Knowledge-related responses were characterized as “correct” if consistent with conventional biomedical understanding, “incorrect”, or unfamiliar.
Tests were classified as ‘ON’ by the Kenya-based field team if they were visibly yellow after six hours, and ‘OFF’ if there was no observable color change by eye. These assessments were independently validated by the US-based team from photographs of the completed tests. Tests classified as ‘ON’ were marked true positive if they corresponded to a photometer measured fluoride concentration ≥1.5 ppm, and false positive if they corresponded to a photometer measured fluoride concentration <1.5 ppm. Tests classified as ‘OFF’ were marked as true negative if they corresponded to a photometer measured fluoride concentrations <1.5 ppm, and false positive if they corresponded to a photometer measured fluoride concentrations ≥1.5 ppm. Sensitivity was determined by the ratio of true-positive results to total-positive measurements (combined true and false positives), while specificity was determined by the ratio of true-negative results to total-negative measurements (combined true and false negatives), and calculated in Stata45. Confidence intervals for sensitivity and specificity were calculated using the diagt module in Stata using counts of true positives, true negatives, false positives, and false negatives.
Our target sample size for establishing test accuracy (Aim 1) was 65, based on the observed sensitivity of 0.93, and observed prevalence of 0.7846. Although we obtained 90 water samples, only 57 were suitable for this analysis (see “Test Kit Shipment to Nakuru County, Kenya”); robust estimates were still generated with this sample size. For usability testing (Aim 2), data on experiences with rehydration and interpretation from 36 individuals is well above the number recommended for usability studies47,48.
Human subjects approval
We obtained ethical approval for this study from Northwestern University’s (IRB STU00215306) and Amref Health’s (AMREF-ESRC P1003/2021) Institutional Review Boards. We also received authorization from the Ministry of Planning and Development, Nakuru County, which is responsible for coordinating research activities in the county and relevant Ministries. All participants provided written consent to participate in the study activities, including consent to take pictures of the at-home testing. The authors affirm that human research participants provided informed consent for publication of the images in Fig. 3.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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