Swab Sample Transfer for Point-Of-Care Diagnostics: Characterization of Swab Types and Manual Agitation Methods

<div><p>Background</p><p>The global need for disease detection and control has increased effort to engineer point-of-care (POC) tests that are simple, robust, affordable, and non-instrumented. In many POC tests, sample collection involves swabbing the site (e.g., nose, skin), agitating the swab in a fluid to release the sample, and transferring the fluid to a device for analysis. Poor performance in sample transfer can reduce sensitivity and reproducibility.</p><p>Methods</p><p>In this study, we compared bacterial release efficiency of seven swab types using manual-agitation methods typical of POC devices. Transfer efficiency was measured using quantitative PCR (qPCR) for <i>Staphylococcus aureus</i> under conditions representing a range of sampling scenarios: 1) spiking low-volume samples onto the swab, 2) submerging the swab in excess-volume samples, and 3) swabbing dried sample from a surface.</p><p>Results</p><p>Excess-volume samples gave the expected recovery for most swabs (based on tip fluid capacity); a polyurethane swab showed enhanced recovery, suggesting an ability to accumulate organisms during sampling. Dry samples led to recovery of ∼20–30% for all swabs tested, suggesting that swab structure and volume is less important when organisms are applied to the outer swab surface. Low-volume samples led to the widest range of transfer efficiencies between swab types. Rayon swabs (63 µL capacity) performed well for excess-volume samples, but showed poor recovery for low-volume samples. Nylon (100 µL) and polyester swabs (27 µL) showed intermediate recovery for low-volume and excess-volume samples. Polyurethane swabs (16 µL) showed excellent recovery for all sample types. This work demonstrates that swab transfer efficiency can be affected by swab material, structure, and fluid capacity and details of the sample. Results and quantitative analysis methods from this study will assist POC assay developers in selecting appropriate swab types and transfer methods.</p></div

Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations M184V and G190A.

<p>Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in pink and orange bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.</p

Organism recovery for dried samples.

<p>(A) Schematic of the experimental set up. 15 µL of <i>S. aureus</i> solution (∼10⁴ CFU, equivalent to 6×10⁴<i>ldh1</i> copies, as measured by qPCR) was spotted on a 25/64-inch diameter PDMS punch and left to dry. A dry or pre-wet swab was rubbed on the PDMS surface (10 times), agitated in 128 µL lysis buffer using 10 second 1 Hz side twirl, and removed. (B) Comparison of % organism recovery for pre-wet and dry swabs based on a control sample and an assumption of 100% collection efficiency.</p

Organism recovery for low-volume samples.

<p>(A) Schematic of the experimental set up. 15 µL <i>S. aureus</i>/TE (∼100, ∼10⁴, or ∼10⁶ CFU, equivalent to 500, 6×10⁴, or 4×10⁶<i>ldh1</i> copies, respectively, as measured by qPCR) was spiked onto the swab, which was then agitated in 128 µL lysis buffer using 10 second 1 Hz side twirl, and removed. (B) Comparison of the % Organism Recovery in four swabs at three different organism input numbers (mean ± SE, N = 5), which was calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105786#pone.0105786.e016" target="_blank">Equation 5</a> in the text. (C) Comparison of the % Organism Recovery (mean ± SE; N = 5) using ∼10⁴ CFU/swab of <i>S. aureus</i> in the presence and absence of simulated nasal matrix (SNM).</p

Plate and paper formats for Capture, Denaturation and Detection (CDD) in the oligonucleotide ligation assay.

<p>(A) Plate CDD procedure. Products from the ligation step (both ligated and non-ligated products) are captured on a streptavidin-coated plate. Non-ligated probes are released during oligonucleotide denaturation, and ligated MUT and WT probes are then detected in sequential enzyme-based immunoassays (labeling by different detection antibodies, alkaline phosphatase yellow substrate development, optical density reading at 405nm, wash steps, tetramethylbenzidine (TMB) development, stop solution, and optical density reading at 450nm). (B) Paper CDD procedure. Similar to the plate CDD procedure, products from the ligation step (both ligated and non-ligated products) are captured. However, here the products are captured on paper strips by immobilized streptavidin. Non-ligated probes are released during oligonucleotide denaturation. Antibodies targeting the end-labels of the mutant (MUT) or wild-type (WT) probes have conjugated horseradish peroxidase (POD) that converts 3,3’ diazoaminobenzidine substrate (DAB) into brown precipitates. Signals were captured by the scanner (600 DPI). Reported signals represent capture spot intensity minus a background region from the strip.</p

Analysis of clinical specimens and plasmid standards by paper capture, denaturation, and detection (CDD) and plate CDD for mutations K103N and Y181C.

<p>Panels A and C show mutant (MUT) detection, and Panels B and D show wild-type (WT) detection. Sample optical density (OD) minus negative control OD (left y axis) for each specimen is shown in white/gray by rank along the x axis, from the lowest MUT OD, followed by the plasmid standards (0%, 5%, 50% MUT) performed in duplicate. Spot intensity minus background intensity (right y axis) for each specimen is shown in blue and green bars followed by the plasmid standards (0%, 5%, 50% MUT) performed in triplicate. Scanned images of the paper CDD detection strip are shown below each specimen’s signal data.</p

Comparison of manual agitation methods for swab transfer.

<p>(A) Schematic of action performed over a period of 1 second for different manual twirling methods. (B) Comparison of % organism recovery of PUR swabs using different twirling methods, which was calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105786#pone.0105786.e016" target="_blank">Equation 5</a> in the text. (C) Schematic of the new forced flow syringe method. (D) Comparison of % organism recovery for PES and rayon swabs, using different twirling methods and the forced flow syringe method. * indicates statistically significant differences (Tukey-Kramer, α = 0.05).</p

Organism recovery for high-volume samples.

<p>(A) Schematic of the experimental set up. Either a dry or pre-wet swab was dipped into 1 mL ∼10⁶ CFU/mL <i>S. aureus</i> solution (equivalent to 6×10⁶<i>ldh1</i> copies/mL, as measured by qPCR) and agitated by 10 second 1 Hz side twirl. The swab was then inserted into 128 µL lysis buffer, agitated by 10 second 1 Hz side twirl, and removed. (B) Comparison of the absolute number of organisms recovered for dry and pre-wet swabs. Absolute organism recovery was reported (rather than %) since the uptake of sample volume was different for each swab; absolute recovery was calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105786#pone.0105786.e015" target="_blank">Equation 4</a> in the text. In all cases, recovery was larger than would be expected based on swab volume and sample concentration by colony counts due to presence of multiple target copies per CFU. (C) The number of organisms recovered from each swab from panel (B) normalized by the number of organisms expected based solely on the sample concentration and volume capacity of the swab (estimated number of organisms collected by the swab  =  swab volume capacity (µL) x bacterial stock concentration (copies/µL from qPCR)).</p

Volume recovery testing.

<p>(A) Schematic of the experimental setup. The tube containing 128 µL TE was weighed (W1), and 15 µL TE was pipetted onto the swab, which was then transferred into the tube using 10 second 1 Hz side twirl, and removed. The tube containing the leftover buffer (eluate) was weighed (W2). The % volume available for analysis (% Volume Recovery) was calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105786#pone.0105786.e003" target="_blank">Equation 3</a> in the text. (B) Mean TE volume (µL) absorbed by each type of swab (N = 5). (C) Comparison of the % Volume Recovery (mean ± SE; N = 5) from each swab. Calcium alginate swabs were resuspended in 1% w/v sodium citrate buffer to dissolve fibrous tip materials, the % Volume Recovery was not reported here due to density change of the buffer during (A). * indicates significant differences (Tukey-Kramer, α = 0.05).</p

Schematic of amplification and ligation in the existing oligonucleotide ligation assay (OLA).

<p>The <i>pol</i> gene of HIV DNA is amplified by nested PCR. Note that the HIV DNA gene map positions are not to scale. A portion of the amplicon is mixed with three oligonucleotide probes: a 5’ fluorescein (F) -conjugated mutant (MUT)-specific HIV probe; a 5’ digoxigenin (D) -conjugated wild-type (WT)-specific probe; and a 5’ phosphorylated, 3’ biotin (B) -conjugated common probe. When specific probes are complementary at the mutation site, they are ligated to the common probe to create a DNA strand with labels at both ends. Only ligated products are detected during the CDD procedure (surface <u><b>c</b></u>apture, <u><b>d</b></u>enaturation of oligonucleotide from target DNA, and enzyme-based detection).</p