Microbial Diagnostic Sampling Swab

Abstract

A method of using a Defined Swab for genetic identification of microbes, said method comprising contacting said swab with a target to absorb a sample of said target; extracting said sample from said swab; and microbiologically testing said sample to identify said microbe.

Claims

1. A method of using a Defined Swab for genetic identification of microbes, said method comprising: contacting said swab with a target to absorb a sample of said target; extracting said sample from said swab; and microbiologically testing said sample to identify said microbe.

2. The method of using a swab of claim 1, wherein said target is a biological surface.

3. The method of using a swab of claim 2, wherein said target is a biofilm.

4. The method of using a swab of claim 3, wherein said biofilm is on an animal.

5. The method of using a swab of claim 4, wherein said animal is a human.

6. The method of using a swab of claim 5, wherein said biofilm is on a surface of an implant in said human.

7. The method of using a swab of claim 1, wherein said extraction comprises debriding.

8. The method of using a swab of claim 1, wherein said microbiologically testing comprises testing for DNA/RNA of said microbe.

9. The method of using a swab of claim 1, wherein said testing comprises mechanically lysing said sample and extracting using the Qiagen TissueLyser in combination with the Roche High Pure PCR Template Preparation kit.

10. The method of using a swab of claim 1, wherein said swab comprises a polyester knitted fabric.

11. The method of using a swab of claim 10, wherein said swab comprises a swab tip comprising a double layer of woven polyester fabric having standard warp and weft patterns.

12. The method of using a swab of claim 1, wherein said swab comprises a swab tip an area of at least 350 mm2,

13. The method of using a swab of claim 12, wherein said swab tip has two flat sides having a combined area of at least 350 mm2.

14. The method of using a swab of claim 13, wherein said combined area is at least 375 mm2.

15. The method of using a swab of claim 14, wherein said combined area is at least 400 mm2.

16. The method of using a swab of claim 12, wherein said swab tip has two flat sides and approximate dimensions of 5-20 mm W×25-30 mm H×0.5-0.6 mm D.

17. The method of using a swab of claim 1, wherein said swab is sealed in a medical grade sterile package and sterilized.

18. The method of using a swab of claim 17, wherein said swab is sterilized by gamma-irradiation.

Description

BRIEF DESCRIPTION OF FIGURES

[0012] FIG. 1 shows Ct values of various swabs for planktonic S. aureus and E. coli.

[0013] FIG. 2 shows Ct values of various swabs for biofilm S. aureus and E. coli.

[0014] FIG. 3 shows Ct values of prior art swabs following 24-hour hold.

[0015] FIG. 4 shows Ct values of various swabs using buffer solution for S. aureus.

[0016] FIG. 5 shows Ct values of various swabs using buffer solution for E. coli.

[0017] FIG. 6 shows Ct values of various swabs using buffer solution compared to dry 36-hour hold.

[0018] FIG. 7 shows microscopic views of polyester fabric with (A) Sheath Pull Tight over Fixed Member, and (b) Standard Woven Pattern

[0019] FIG. 8 shows a standard swab's deficiency in surface area to collect specimens and its inability to scrap against tissue or surfaces to lift the DNA and RNA compared to the swab of the present invention.

DETAILED DESCRIPTION

[0020] In the following paragraphs, the present invention will be described in detail by way of example with reference to the attached drawings. Throughout this description, the preferred embodiment and examples shown should be considered as exemplars, rather than as limitations on the present invention. As used herein, the “present invention” refers to any one of the embodiments of the invention described herein, and any equivalents. Furthermore, reference to various feature(s) of the “present invention” throughout this document does not mean that all claimed embodiments or methods must include the referenced feature(s).

[0021] The SurfCHECK Cleaning Validation Swab, Foamtec International WCC as disclosed in U.S. Pat. No. 8,745,806 is currently marketed as a tool to validate cleaning processes, specifically within the pharmaceutical industry. As used herein, the term “Defined Swab” refers to the swab disclosed in in U.S. Pat. No. 8,745,806) It is offered as a multi-use tool, including multiple swab heads that can be used to swab a surface, and then be ejected into a container by way of a push button mechanism on the handle. This mechanism reduces the risk of cross contamination between samples or from the swab becoming contaminated by the user. It is used to verify cleaning through the use of several chemical analyses, including total organic carbon (TOC), high performance liquid chromatography (HPLC), ion-mobility spectrometry (IMS), and ultraviolet-visible spectroscopy (UV-Vis).

[0022] Applicants have discovered that in addition to cleaning validation and chemical analyses, this swab has a unique ability to absorb and release microbial particles that is superior to many swabs currently used for this application.

[0023] Real-time polymerase chain reaction (RT-qPCR) is a common microbial molecular diagnostic technique. This technique allows quantification of microbes within a sample. This is achieved by amplifying microbial genetic material in cycles. As genetic material is amplified, a fluorescent signal intensifies until it crosses a particular threshold, signifying a positive result. Fewer cycles required to cross threshold corresponds to higher quantities of microbes in the original sample.

[0024] This swab, with a 15×25 mm tip and double-woven polyester fabric, was gamma-irradiated to sterilize and compared to four sampling swabs currently marketed. When adjusting for the difference in surface area between swabs, this swab required approximately 30% fewer cycles in detecting Staphylococcus aureus, a common gram-positive pathogen, to the next highest-performing swab and 40% fewer cycles in detecting Escherichia coli, a common gram-negative pathogen. Although a swab having a 15×25 mm tip and double-woven polyester fabric provided sufficient results, other embodiments exist, for example, a swab with an area of at least 250 mm2, or at least 300 mm2, or at least 350 mm2, or at least 375 mm2, or at least 400 mm2, or at least 450 mm2 or at least 500 mm2.

[0025] The swab, as described in the previous paragraph, was tested against the Clinical & Laboratory Standards Institute (CLSI) M40-A2 Standard for Quality Control of Microbiological Transport Systems. This includes testing the ability to detect several bacterial species representing the entire diverse spectrum, after several time points from collection, including 0, 24, and 48 hours. This swab met all the requirements described in this standard.

[0026] The swab is not only useful for detecting bacteria but can be used for other microbial pathogens. The swab was tested in its ability to detect the novel SARS-CoV-2 virus. Samples collected using the swab were found to exceed the threshold of detection even after a five-fold dilution, even after a 36-hApplicants' hold at room temperature. Oropharyngeal swabbing with this swab demonstrated a detectable lower limit that was 32 times lower samples collected from nasopharyngeal swabs. With the addition of a buffer solution to preserve genetic material, samples collected using this swab were able to detect viral concentrations as low 0.1 viral copies per microliter.

[0027] The preferred form of the swab for the described application is as a single-use device that comes pre-loaded with a single tip encased in two layers of woven polyester fabric. In one embodiment, the woven polyester fabric comprising a double layer of woven polyester fabric utilizing a standard warp and weft patterns, such as a plain weave, twill or leno type of patterns that provide open channels while remaining strong and durable. The swab is sealed in medical grade peel pack packaging and sterilized to reduce the risk of contamination.

Examples

[0028] In these non-limiting examples, Applicants demonstrate that the swab material disclosed herein is highly effective in its absorption and release of S. aureus and E. coli. Specifically, through comparative materials testing, Applicants identify a swab effective in detecting these organisms both as planktonic bacteria and in a biofilm, throughout an extended holding period, and in various buffer solutions. After identification of an optimal swab material, Applicants assessed the final swab using the CLSI M40-AD standard for microbiological culture.

[0029] Methods

[0030] Comparative testing of materials for absorption and extraction of planktonic Gram-positive and Gram-negative organisms on PCR

[0031] The first experiment aimed to compare materials and weave patterns for optimal absorption and extraction of planktonic organisms. A panel of 11 candidate gauze and swab weave patterns were included in the experiment. All materials were spiked with a standardized inoculum of Gram-positive (Staphylococcus aureus), or Gram-negative (Escherichia coli) planktonic organisms. Following a holding period, each candidate sample was extracted and tested via PCR as described below.

[0032] Comparative testing of materials for biofilm collection, absorption and extraction of Gram-positive and Gram-negative organisms on PCR

[0033] Next, Applicants assessed a high-performing subset of the first experiment against the same organisms in biofilm form. To this end, biofilms consisting of monocultures of either S. aureus (ATCC 25923) or E. coli (ATCC 11303) were grown on the surface of 12 mm Polycarbonate transwell inserts. Once the biofilms were grown, each insert was removed from its original growth well and transferred to a new well containing a single gauze testing material (approximately 1 cm.sup.2; each sample was previously weighed for subsequent normalization). Each biofilm was removed via debridement (vortex with uniform force applied). The samples were then transferred for testing of absorptive, biofilm removal, and extraction efficiencies.

[0034] Comparative Testing of Materials for Absorption and Extraction of Gram-Positive and Gram-Negative Organisms on PCR, Following 24 Hours of Simulated Transport Specimen Hold

[0035] Monocultures consisting of either S. aureus (ATCC 25923) or E. coli (ATCC 11303) were grown in 12-well plates. After 24 hours each gauze sample was briefly submerged in culture. The samples were then transferred into tubes for 24 hours at 25° C. to mimic the time of transport. Samples were then assayed to test for absorptive and nucleic acid extraction efficiencies.

[0036] Comparative Buffer Testing

[0037] To assess the buffer transport utility, biofilms consisting of monocultures of either S. aureus (ATCC 25923) or E. coli (ATCC 11303) were grown on the surface of 12 mm Polycarbonate transwell inserts. After biofilms were grown, each insert was removed from its original growth well and transferred to a new well containing a single gauze testing material (approximately 1 cm.sup.2; each sample was previously weighed for subsequent normalization). Each biofilm was removed via debridement (vortex with uniform force applied). The samples were then transferred into different buffers for testing of absorptive, biofilm removal and nucleic acid extraction efficiencies.

[0038] Assessment of Candidate Swab to the CLSI M40 A2 Standard for Microbiological Culture

[0039] The test procedures employed for determining bacterial viability performance were based upon the quality control methods described in Clinical Laboratory Standards Institute (CLSI) M40-A2. Test organisms utilized in this study were those specifically listed in M40-A2 for establishing performance claims and quality control of swab transport systems. These include a representative panel of aerobes, anaerobes and fastidious bacteria. Bacterial viability studies were performed on the candidate swab, as well as another widely used commercially available swab, at room temperature. Swabs accompanying each transport system were inoculated in triplicate with 100 μl of specific concentrations of organism suspension. Swabs were then placed in their respective transport medium tubes and were held for 0 hrs, 24 hrs or 48 hrs. At the appropriate time intervals, each swab was processed according to the Roll-Plate or Swab Elution Method.

[0040] PCR Testing

[0041] In each of the preceding experiments, PCR testing was performed per standardized protocols at CLIA-certified MicroGenDx Laboratories (Lubbock, Tex.). Samples were mechanically lysed and extracted using a Qiagen TissueLyser in combination with the Roche High Pure PCR Template Preparation kit. Each sample was tested for 16S abundance to verify species, and to test for possible contaminates obtained during the procedure. All experimental conditions were repeated in triplicate.

[0042] Results

[0043] Comparative Testing of Materials for Absorption and Extraction of Planktonic Gram-Positive and Gram-Negative Organisms on PCR

[0044] Ct thresholds were calculated for 11 candidate materials and two controls (FIG. 1). The average Ct across all materials for S. aureus was 17.05, while the average Ct across all materials for E. coli was 21.38. Six candidates (highlighted in Table 1) demonstrated consistent absorption and extraction results for planktonic forms of both S. aureus and E. coli (Table 1).

[0045] Comparative testing of materials for biofilm collection, absorption and extraction of Gram-positive and Gram-negative organisms on PCR

[0046] The six remaining candidates that demonstrated optimal results for planktonic bacteria were further analyzed for absorption and extraction of organisms in a biofilm. Ct thresholds were calculated after adjusting for surface area (FIG. 2). No significant differences between candidate for S. aureus biofilm, but significant differences were noted between candidates for E. coli biofilm (Table 3) as well as between S. aureus and E. coli for three of the candidates (Table 4).

[0047] Comparative Testing of Materials for Absorption and Extraction of Gram-Positive and Gram-Negative Organisms on PCR, Following 24 Hours of Simulated Transport Specimen Hold

[0048] Three candidates were selected to assess performance following 24-hApplicants' hold, compared to four commercially available competitors. FIG. 3 displays Ct values for S. aureus and E. coli following 24-hApplicants' hold. Candidates 2 and 3 demonstrated significantly lower Ct values compared to competitor for S. aureus, while all three candidates demonstrated lower Ct values for E. coli.

[0049] Comparative Buffer Testing

[0050] Normalized surface-area adjusted Ct values for the six candidates above were calculated in four buffer solutions for performance with S. aureus (FIG. 4) and E. coli (FIG. 5). Each was also compared with the same material in a “dry” control condition (FIG. 6). Minimal, if any, differences were noted between the buffers tested vs. dry 36-hApplicants' hold conditions.

[0051] Assessment of Candidate Swab to the CLSI M40 A2 Standard for Microbiological Culture

[0052] Two candidate swabs were compared to a single competitor swab according the CLSI M40-A2 Standard. Results are reported in Table 5. Both candidates met all requirements per the standard.

[0053] Discussion

[0054] Through a series of in vitro experiments, Applicants have found a specific polyester weave pattern that performs the best among all tested fabrics in absorbing, storing, and releasing DNA of a given pathogen. The material performed well in the collection of S. aureus and E. coli in both planktonic form and in biofilm, which is a crucial aspect of detecting an organism in implant-related infections. The improvements in quality sample collection by use of this swab may aid in reducing the rate of culture-negative PJI. This may have further effect in improving outcomes associated with PJI as cases of culture-negative PJI only have a 70% treatment success rate.

[0055] Furthermore, the swab performed very well even after an extended 24 hours of simulated transport. Molecular testing often occurs in centralized laboratories, rather than individual hospitals. It is therefore necessary for a swab system to maintain organism viability while the specimen is in route to the facility for analysis. Additionally, after a 36-hApplicants' dry hold, the swab demonstrated similar results to those preserved in a series of buffer solutions. While the addition of a buffer solution can stabilize a sample for a prolonged period, availability of molecular testing supplies has become limited in the current epidemic, including supplies used for nucleic acid storage and stabilization.′ The swab developed demonstrates the ability to provide reliable results following transport without the dependence on such buffer solutions.

[0056] While S. aureus and E. coli were chosen to test candidate materials with both Gram-positive and Gram-negative organisms, it is possible that another material may have performed better with other organisms chosen from the same categories. Although several candidates were chosen with potential for high performance, it is possible that a material not tested in this study would demonstrate superior results. From the results of this study, it is not known how the surface being swabbed affects performance or if certain swabs perform better on certain sampling surfaces. However, the swab demonstrated its efficacy against the array of organisms shown in the results of CLSI M40-A2 testing.

[0057] While molecular testing techniques offer the potential for significant improvement in the identification of causative organisms, the performance of these techniques hinge on the quality of samples provided. Use of a superior sampling swab, as demonstrated in Applicants' results, may even further improve the success of these novel techniques.

[0058] Having thus described a few particular embodiments of the invention, various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements as are made obvious by this disclosure are intended to be part of this description though not expressly stated herein, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description is by way of example only, and not limiting. The invention is limited only as defined in the following claims and equivalents thereto.

[0059] Tables

TABLE-US-00001 TABLE 1 CP threshold (Ct) Values Material S. aureus E. coli Candidate 1 19.68 19.25 Candidate 2 17.81 18.2  Candidate 3 17.17 18.04 Candidate 4 16.02 23.23 Candidate 5 16.77 23.75 Candidate 6 16.25 23.11 Candidate 7 15.93 22.93 Ca.ndidate 8 16.22 21.49 Candidate 9 15.98 23.07 Candidate 10 16.08 22.48 Candidate 11 20.21 16.62 Control 1 18.85 18.14 Control 2 16.18 23.13

TABLE-US-00002 TABLE 2 S. aureus E. coli SA-adjusted SA-adjusted Material CP threshold Ct value CP threshold Ct value Candidate A 19.85 0.1985 20.92 0.2092 Candidate B 19.18 0.1918 21.37 0.2137 Candidate C 19.22 0.1922 20.37 0.2037 Candidate D 19.23 0.1923 20.27 0.2027 Candidate E 18.91 0.1891 21.24 0.2124 Candidate F 18.42 0.1842 19.76 0.1976 SA—surface area

TABLE-US-00003 TABLE 4 P-values of comparison between S. aureus and E. coli for each candidate P-value (one- Material P-value tailed) Candidate A 0.37878 0.18939 Candidate B 0.009283 0.004642 Candidate C 0.006465 0.003232 Candidate D 0.052233 0.026116 Candidate E 0.146111 0.073055 Candidate F 0.034463 0.017231

TABLE-US-00004 TABLE 5 CLSI M40A-2 Standard Testing Results Quantitative (Swab Elution) CFU/ml Qualitative (Roll Plate) CFU M40-A2 M40-A2 Organism Swab Temperature 0 hr 48 hr Compliance 0 hr 24 hr 48 hr Compliance Cutibacterium acnes Candidate 1 Room Temp 4.80E+06 1.49E+07 ✓ 312 35 ✓ ATCC 6919 Candidate 2 5.30E+06 1.61E+07 ✓ 174 14 ✓ Competitor 6.37E+06 1.52E+07 ✓ 242 59 ✓ Neisseria gonorrhoeae Candidate 1 RoomTemp 5.60E+06 1.62E+07 ✓ 149 76 ✓ ATCC 43069 Candidate 2 6.18E+06 1.58E+07 ✓ 248 97 ✓ Competitor 6.38E+06 1.48E+07 ✓ 197 96 ✓ Fusobacterium nucleatum Candidate 1 Room Temp 2.20E+07 1.95E+07 ✓ 142 47 ✓ ATCC 25586 Candidate 2 2.18E+07 1.99E+07 ✓ 241 21 ✓ Competitor 2.20E+07 2.03E+07 ✓ 230 96 ✓ Haemophilus influenzae Candidate 1 Room Temp 1.49E+07 1.60E+07 ✓ 269 70 ✓ ATCC 10211 Candidate 2 7.21E+05 3.00E+07 ✓ 320 82 ✓ Competitor 7.68E+06 2.41E+07 ✓ 264 75 ✓ Streptococcus pneumoniae Candidate 1 Room Temp 1.76E+07 2.85E+07 ✓ 170 13 ✓ ATCC 6305 Candidate 2 1.37E+07 1.90E+07 ✓ 182 28 ✓ Competitor 3.75E+06 5.44E+06 ✓ 134 11 ✓ Streptococcus pyogenes Candidate 1 Room Temp 1.71E+07 1.42E+07 ✓ 284 76 ✓ ATCC 19615 Candidate 2 6.35E+06 1.83E+07 ✓ 178 44 ✓ Competitor 1.88E+07 1.09E+07 ✓ 282 92 ✓ Prevotella melaninogenica Candidate 1 Room Temp 1.58E+06 2.31E+07 ✓ 264 64 ✓ ATCC 25845 Candidate 2 1.85E+07 8.50E+06 ✓ 134 61 ✓ Competitor 1.56E+07 1.35E+07 ✓ 178 31 ✓ Bacteroides fragilis Candidate 1 Room Temp 8.95E+06 2.02E+07 ✓ 210 38 ✓ ATCC 25285 Candidate 2 9.72E+06 4.80E+06 ✓ 196 97 ✓ Competitor 2.46E+06 1.81E+07 ✓ 160 83 ✓ Peptostreptococcus Candidate 1 Room Temp 2.08E+07 1.51E+07 ✓ 226 44 ✓ anaerobius Candidate 2 5.15E+06 9.14E+06 ✓ 140 37 ✓ ATCC 27337 Competitor 9.46E+06 1.75E+07 ✓ 280 63 ✓ Pseudomonas aeruginosa Candidate 1 Room Temp ✓ ✓ ATCC Candidate 2 ✓ ✓ Competitor ✓ ✓