The influence of a swab type on the results of point-of-care tests

Model samples

In this study we used two types of model samples: (i) diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and (ii) three bacterial strains at different concentrations, i.e., Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 isolated from blood, for investigating the recovery of nucleic acids.

Swabs

We investigated four different commercially available swabs: FLOQSwabs (Copan Italia S.p.A, Italy), which are flocked swabs made of nylon; rayon swabs (Copan Italia S.p.A, Italy); dacron swabs (Copan Italia S.p.A, Italy); and BBL Culture Swabs (Becton, Dickinson and Company, USA), which are swabs composed of polyurethane foam (Fig. 1).

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Measurement of the absorbed volume

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We transferred 500 µl of water to an Eppendorf tube, and then the tube was weighted using a balance Discovery (Ohaus, Germany). The swab was immersed in water for 5-10 s. After removing the swab, the tube was weighted. The weight of absorbed liquid was calculated as a difference between the weight of the tube before swab immersion and after removing the immersed swab. Furthermore, the weight of water absorbed by a swab (m) was converted into volume (v) using the following calculation: v = m/d, where d is density of water (1 kg/m3). The experiment was repeated five times for each type of swab.

Measurement of DNA recovery

In the preliminary study, DNA was extracted from suspensions of the three above mentioned bacterial strains using Wizard Genomic DNA Purification Kit (Promega, Germany) according to the manufacturer’s protocol. A 24-h culture of bacterial strains on Columbia agar with 5% sheep blood (BioMerieux, France) was suspended in saline solution at appropriate densities. We used ten different densities (from 0.5 McF to 9 McF) of the bacterial suspension, and the amount of extracted DNA was measured using a BioPhotometer® model 6131 (Eppendorf, Germany). The extraction was triplicated for each suspension and for each bacterial strain. Based on the results of the pilot study, we selected both the bacterial strains and density of the suspension most suitable for further experiments.

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We used nine types of buffers to wash out bacterial cells and DNA from tested swabs: phosphate-buffered saline (PBS), tris-EDTA buffer (TE), molecular grade water, AL buffer (Qiagen), ATL buffer (Qiagen), lysis buffer (Qiagen), saline, 0.5% Tween 20 and Nucleic Lysis Solution (Promega). We immersed the swabs in a bacterial suspension and manually agitated for ~ 10 s. Then, the swabs were transferred into 200 µl of each different buffer and manually agitated for ~ 10 s to release bacterial cells and DNA. Next, the swab was removed and the obtained suspension was used for DNA extraction using the Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer’s instruction. All the experiments were performed six times for each combination swab/bacterial strain/type of buffer, by two different laboratory workers. We used the bacterial suspension in a volume equal to average volume absorbed by particular type of swabs as a positive control, and the amount of extracted DNA was measured using a BioPhotometer® model 6131 (Eppendorf, Germany).

Measurement of protein recovery

We used the concentration of 2 µg/ml of diphtheria toxoid for the test. We transferred 500 µl of diphtheria toxoid suspension to an Eppendorf tube. Every swab was immersed into the suspension for 5-10 s, and then transferred to an Eppendorf tube containing 500 µl of PBS and manually agitated for 5-10 s and removed. The amount of protein released from the swabs was examined using the Lowry assay according to European Pharmacopoeia (Ph. Eur. 2.5.33; 01/2008:20533). We used eight reference solutions of diphtheria toxoid to prepare the standard curve. For calculating the standard curve, we plotted the absorbance of the reference solutions against the protein concentrations along with linear regression. We determined the concentrations of the diphtheria toxoid in test solutions using a standard curve. The experiment was performed three times for each type of swabs and each concentration of diphtheria toxoid.

Statistical analysis

The arithmetic mean and standard deviations were calculated using Excel. Statistical analysis was performed using the Kruskal-Wallis test, which is suitable for comparing two or more independent samples of the same or different size. The results were regarded as significant at a p value of < 0.05.

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