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. 2023 Jul 11;95(27):10211-10220.
doi: 10.1021/acs.analchem.3c00462. Epub 2023 Jun 26.

At-Home Saliva Sampling in Healthy Adults Using CandyCollect, a Lollipop-Inspired Device

Wan-Chen Tu  1 , Anika M McManamen  1 , Xiaojing Su  1 , Ingrid Jeacopello  1 , Meg G Takezawa  1 , Damielle L Hieber  1 , Grant W Hassan  1 , Ulri N Lee  1 , Eden V Anana  1 , Mason P Locknane  1 , Molly W Stephenson  1 , Victoria A M Shinkawa  1 , Ellen R Wald  2 , Gregory P DeMuri  2 , Karen N Adams  3 , Erwin Berthier  1 , Sanitta Thongpang  1   4 , Ashleigh B Theberge  1   5
Affiliations

At-Home Saliva Sampling in Healthy Adults Using CandyCollect, a Lollipop-Inspired Device

Wan-Chen Tu et al. Anal Chem. .

Abstract

Respiratory infections are common in children, and there is a need for user-friendly collection methods. Here, we performed the first human subjects study using the CandyCollect device, a lollipop-inspired saliva collection device .We showed that the CandyCollect device can be used to collect salivary bacteria from healthy adults using Streptococcus mutans and Staphylococcus aureus as proof-of-concept commensal bacteria. We enrolled healthy adults in a nationwide (USA) remote study in which participants were sent study packages containing CandyCollect devices and traditional commercially available oral swabs and spit tubes. Participants sampled themselves at home, completed usability and user preference surveys, and mailed the samples back to our laboratory for analysis by qPCR. Our results showed that for participants in which a given bacterium (S. mutans or S. aureus) was detected in one or both of the commercially available methods (oral swab and/or spit tubes), CandyCollect devices had a 100% concordance with the positive result (n = 14 participants). Furthermore, the CandyCollect device was ranked the highest preference sampling method among the three sampling methods by 26 participants surveyed (combining survey results across two enrollment groups). We also showed that the CandyCollect device has a shelf life of up to 1 year at room temperature, a storage period that is convenient for clinics or patients to keep the CandyCollect device and use it any time. Taken together, we have demonstrated that the CandyCollect is a user-friendly saliva collection tool that has the potential to be incorporated into diagnostic assays in clinic visits and telemedicine.

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Conflict of interest statement

CONFLICTS OF INTEREST

ABT has ownership in Stacks to the Future, LLC; ST: ownership in Stacks to the Future, LLC, Tasso, Inc., and Salus Discovery, LLC; and EB has ownership in Stacks to the Future, LLC, Tasso, Inc., and Salus Discovery, LLC and employment by Tasso, Inc. However, this research is not related to these companies.

Figures

Figure 1.
Figure 1.
(A) The photo of the CandyCollect device. The CandyCollect device is composed of a polystyrene stick with a microfluidic channel and red isomalt candy. The open-fluidic channel is designed to prevent the tongue from removing the collected bacteria, also accumulating bacteria during the sampling time. The candy flavoring functions as a built-in timer for sampling time (i.e., dissolving time of the candy). (B) This figure is reproduced from Lee et al. (Figure 1B) with permission from the Royal Society of Chemistry.
Figure 2.
Figure 2.
Participant flow diagram. (A) Human subjects samples for biological analysis. (B) User feedback surveys.
Figure 3.
Figure 3.
CandyCollect efficiently captures S. mutans and S. aureus and facilitates quantitative bacterial detection by qPCR. (Ai) Fluorescence microscopy images of S. mutans and S. aureus before elution (left), and after elution with Proteinase K in ESwab™ buffer (ESB) (middle) and with Proteinase K (Pro. K) in PBS (right). (Aii) Quantification of fluorescence microscopy images. Each data point represents data from one CandyCollect device and is the average of the integrated density per area of 12 (10000 μm2) regions of interest (ROI) from 4 images of the CandyCollect device (3 ROIs per image). The bar graph represents the mean ± SEM of n = 3 CandyCollect devices. Data sets were analyzed using one-way ANOVA with Tukey’s multiple comparison test; p-values are indicated for pairwise comparisons: ***p=0.0009, ****p<0.0001. (B) Reported concentration of S. mutans (left) and S. aureus (right) from qPCR analysis of bacteria eluted from CandyCollect devices using proteinase K in PBS as the CandyCollect elution buffer. Bacteria were incubated in-lab at concentrations of 103, 104 and 105 CFU/mL. S. mutans and S. aureus were fluorescently labeled with WGA.
Figure 4.
Figure 4.
S. mutans and S. aureus can be captured on CandyCollect devices from all the participants who had positive results from spit tube and/or ESwab™ samples. CandyCollect, ESwab™, and SpeciMAX Stabilized Saliva Collection Kits (Spit tube) were sent to 14 research participants for a proof-of-concept test. The concentrations of (A) S. mutans and (B) S. aureus from participants’ saliva, collected over two days by three different methods, were analyzed via qPCR and converted to estimated copy number of bacterial DNA/mL in the original saliva sample (see Methods section “Human subject data analysis” for detailed description of calculations). Each dot represents the average of two qPCR technical duplicates from one sample (three samples were collected for the CandyCollect device and ESwab™, and one for the Spit tube each day). Bar graphs represent the mean ± SEM of n = 3 CandyCollect devices or ESwab™. Participants only completed one spit tube per day.
Figure 5.
Figure 5.
Participant feedback shows overall preference for CandyCollect devices. (A) Participants were asked to rank the three sampling methods (CandyCollect, ESwab™, and Spit Tube) in terms of best overall sampling method to worst overall sampling method. (B) Participants were asked to select one sampling method that most accurately fits the above descriptions (best sampling experience, most sanitary, least disgusting/uncomfortable, and least invasive). The CandyCollect device was the most frequently selected sampling method for all of the above user feedback questions.
Figure 6.
Figure 6.
Shelf life tests demonstrate that CandyCollect devices effectively capture S. pyogenes after 1 year of storage. Devices were plasma treated and stored at room temperature for 0 days (control group), 3 months, 4 months, and 1 year. (A) Fluorescence microscopy images indicate capture of S. pyogenes after 1 year of storage is similar to the control, with ~25% decrease in bacteria captured. (B) Quantification of the integrated density per area (pixel/μm2). Data sets were analyzed using one-way ANOVA and Tukey’s multiple comparison tests (*p<0.05). No significant difference between 0 days (control group) and 3 months of storage was found. Note: depicted images are from CandyCollect devices incubated with S. pyogenes at a concentration of 1×109 CFU/mL for 10 minutes. Each data point represents an individual CandyCollect device (4 images were taken per device, and the data point plotted is the average); the bars represent the mean ± SEM of n=3 CandyCollects. S. pyogenes was fluorescently labeled with WGA.
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