EVALUATION OF THE PHYSICAL AND DIAGNOSTIC NATURE OF SWABS W. Nugent, M.D. 1, M.J. Russell 1 S. Beck 2, and G.P. Leonardi, Ph.D. 1,3 NASSAU 1 M44 Department of Pathology, Nassau University Medical Center, East Meadow, NY ; UNIVERSITY Department of Biology, Nassau Community College, Garden City, NY2 and Sunrise Medical Laboratories, Hauppauge, NY 3 medicalcenter Abstract: Conclusions: Proper specimen collection is the single most important factor in obtaining a reliable and and reweighed. Following addition of water (1.0ml), tubes were shaken at high speeds ß Fiber confi guration and size of the nylon-fl ocked swab allowed for greater accurate diagnostic result. Swabs are often employed to collect specimens for a variety for 1 minute, followed by centrifugation using a table-top Hermle Z180M centrifuge absorbance than the actual nylon material itself. of microbiologic assays. A novel, nylon-tipped, “fl ocked“ swab (Copan, Inc; Corona, CA) (Universal Labnet Co., Edison, NJ) for 5 minutes at maximum speed. Unabsorbed water FIG. 1 FIG. 2 has recently become commercially available. Unlike conventional swabs in which fi bers ß There are numerous other factors, not presently examined, which can aff ect was removed by decanting and wiping the inner tube surface with bibulous paper, swab performance. are woven on a shaft material, fl ocked swabs have a “one piece” construction with nylon and the tube was reweighed. This exercise was repeated twice. fi bers radiating from the shaft tip. This construction may enhance the amount of specimen ß Nevertheless, our data suggests that using of nylon-fl ocked swabs would available for diagnostic testing. This investigation compared absorption, swab tip ultra- (2) Swab tip area appreciably enhance the recovery of virus from a clinical specimen as structure and viral quantitation properties of commonly used nasopharyngeal swabs with ß A crude estimate of swab tip area was determined by measuring the swab length and compared to the other swabs presently examined. those of fl ocked swabs both the maximum and minimum widths. Values (mm2) were calculated using: The absorbance characteristics of rayon, polyester and nylon-fl ocked nasopharyngeal length x average width. swabs (Copan Diagnostics, Inc; Corona, CA) were evaluated by placing swabs in 1.0ml Bibliography: of water and removing them after 15 seconds. The weight of the remaining liquid was (3) Determination of 50% culture infectivity endpoints 1. Goldschmidt, P. Rostane, H., Saint-Jean, C., Batellier, L., Alouch, C., Zito, E., Bourcier, T., measured using an OHAUS Brainweight B300 electronic scale (OHAUS, Florham Pk., ß A frozen isolate of herpes simplex type 2 virus (NYSDOH Profi ciency test # 851) was Larouche, L and C. Chaumeil. 2006. Eff ects of topical anesthetics and fl euorescein on NJ). Mean water absorbance values (g) of 0.04, 0.02 and 0.08 were obtained for the rayon, thawed and diluted 1:10 with tissue culture refeeding media (Bartels, Inc., Carlsbad, CA). the real-time PCR used for the diagnosis of Herpes viruses and Acanthamoeba keratitis. polyester and nylon swabs, respectively. These were found to be statistically signifi cant by Brit. J. Opthalmol., 90: 1354-1356 ANOVA (p< .005). Similar values were observed when a more viscous solution (22% bovine ß Swabs were placed separate tubes containing viral isolate for 15 seconds. They were albumin; Immucor, Inc., Norcross, GA) was substituted for water. then removed, placed in aliquots of refeeding medium and simultaneously shaken 2. Gibb, A.P and S Wong. 1998. Inhibition of PCR by Agar from Bacteriological Transport Media. using a multi-tube vortex (VWR Scientifi c) at high speeds for 15 seconds. The diagnostic ability of the swabs to collect and elute virus was measured by calculating, J. Clin. Microbiol., 36: 275-276. FIG. 3 FIG. 4 in duplicate, the 50% endpoints (TCID50) of an HSV-2 viral isolate. Under identical ß Serial dilutions of isolate for each swab type were inoculated in duplicate, 3. Lauer, B.A. and H.B. Masters. 1988. Toxic eff ect of calcium alginate swabs on Neisseria experimental conditions, TCID50 values (log10) of –1.73, -2.82 and –3.56 were obtained for into 5 wells each, of human foreskin fi broblast cells (96 well plates, lot 870220; gonorrhoeae. J. Clin. Microbiol., 26: 54-56. the rayon, polyester and nylon-fl ocked swabs, respectively. Diagnostic hybrids, Inc; Athens GA). Plates were incubated at 35o C and examined 4. Mardh, P.A. and B. Zeeberg. 1981. Toxic eff ect of sampling swabs and transportation test The absorbance of swab-tip material as a function of fi ber weight (water weight absorbed for viral cytopathic eff ect for 10 days. tubes on the formation of Intracytoplasmic inclusions of Chlamydia trachomatis in McCoy cell per .050g fi ber) was evaluated to determine whether the enhanced performance of the ß Calculation of the 50% tissue culture infective dose (TCID ) values (log ) nylon-fl ocked swab was a result of the nylon material itself. Enhanced absorption of 50 10 cultures. Brit. J. Venereal Dis., 57: 268-272. was determined using the method of Reed and Muench5. nylon was not observed as rayon demonstrated the greatest mean absorbance values of 5. Reed, L.J. and H. Muench. 1938. A simple method for estimating fi fty percent endpoints. .45 ± .02g as compared with that obtained for polyester (.29 ± .02g) or nylon-fl ocked (4) Scanning electron Microscopy (SEM) Am. J. Hyg., 27: 493-497. (.35 ± .02g). One reason for increased water absorption by rayon may result from the ability ß Swab tips were placed on SEM stubs and prepared for microscopic examination by of the rayon fi bers to greatly expand following vortexing the mixture. evaporating metallic gold on their surfaces. Specimens were examined using a Hitachi TABLE I. Physical and diagnostic measurements of rayon (RAY), The fi ber structure of each swab type was investigated by scanning electron micros- S-2400 SEM at 20kV accelerating voltages. Images at varying magnifi cations were polyester (POLY) and nylon-fl ocked (NYL) nasopharyngeal swabs. copy. Rayon and polyester swabs were composed of fi bers, wrapped around (sometimes captured and downloaded on a personal computer fi le. tightly) a metallic shaft. Depressions or crevices on the tip surface were often observed. In Measurement RAY POLY NYL p value comparison the nylon-fl ocked swab demonstrated a “brush-like” pattern of smooth fi bers ß Swab tips were placed in a 1.27% aqueous suspension of polystyrene microspheres Absorbed water/swab (g) radiating from the plastic shaft. The fl ocked swab tips were clearly larger than the others. (Polysciences, Inc; Warrington, PA) for 15 seconds, then removed, air dried and prepared ± SEM .04 ± .001 .02 ± .002 .08 ± .003 p < .005 Our fi ndings seem to suggest that the fi ber confi guration and size of the nylon-fl ocked for SEM as above. (n = 10) FIG. 5 FIG. 6 swab allowed for greater absorbance rather than the actual nylon material itself. There are ß In another exercise, swabs were removed from the microsphere suspension, placed in Absorbed fl uid/swab (g) numerous other factors not evaluated (e.g., electrical conductivity, pH) which may have 2 ml of water, vortexed at high speed for 10 seconds and then were air dried and 22% serum albumin .04 ± .004 .03 ± .002 .07 ± .005 p < .001 infl uenced swab performance. Nevertheless, the absorbance and the HSV-2 viral TCID 50 prepared for SEM. ± SEM data in this investigation clearly suggest that the use of nylon-fl ocked swabs would signifi - cantly enhance the recovery of virus from a clinical specimen as compared with the other (5) Statistics Absorbed water/.050g swabs tested. ß Calculation of mean, standard error of the mean (SEM) and ne-way ANOVA were swab tip ± SEM .45 ± .02 .29 ± .02 .35 ± .02 p = .060NS Background: made using a computer-assisted EXCEL program (Microsoft Inc, Redmond, WA). (n = 3) Specimen collection is among the most important processes, necessary for obtaining Results: Swab tip area (mm2)242840 a reliable and accurate diagnostic result. Numerous studies have demonstrated the ß Nylon-fl ocked swabs demonstrated signifi cantly greater ability to absorb water deleterious eff ects that specimen collection variables can have on the diagnosis of infectious agents, even when employing “gold standard” culture or sophisticated, (.08 ± .003g) than that observed for rayon (.04 ± .001g) or polyester (.02 ± .002g) swabs [TABLE I]. TCID (log ) amplifi ed molecular techniques. Sources of variability include the application of topical 50 10 -1.73 -2.82 -3.56 (n = 2) anesthetics prior to specimen collection, the addition of agar in transport media, ß Similar fi ndings occurred when a more viscous solution (22% serum albumin) was specimen collection with wooden-shafted or calcium alginate-tipped swabs and the substituted for water. choice of collection tube.1-4 FIG. 7 FIG. 8 ß Water absorbance as a function of swab tip material demonstrated non-signifi cant Specimen swabs come in a variety of shapes and types depending on the infectious agent fi ndings (p = .060) as absorbed weight (g) values of .45 ± .02, .29 ± .02 and in question, the assay to be used and the body site collected. Swab materials include Figure legend: “natural” fi bers such as cotton, treated natural fi bers such as rayon (formed by treating .35 ± .02 were obtained per .05g of rayon, polyester and nylon, respectively. cellulose with alkali and carbon disulfi de) and synthetic fi bers such as polyester (formed ß Glue material (used to secure rayon and polyester swab tips) and nylon fi ber base FIG. 1: SEM of an untreated polyester swab tip. Note the wound appearance of tip and striation seen on on some fi ber surfaces. 40x of hydrocarbon backbones containing long-chain ester linkages). These fi bers are wound material contained in the fi bers collected for the weight/absorbance exercise may be a around a shaft substance (wood, metal, plastic) forming a collection surface. source of experimental error, aff ecting fi ber weight and the fl uid absorbance values. FIG. 2: SEM of untreated rayon swab tip. 40x Recently, a nylon-tipped (formed by repeating polymers linked by peptide bonds) swab ß When vortexed, rayon material demonstrated a greater ability to expand than polyester. FIG. 3: SEM of untreated nylon-fl ocked swab tip. 40x has been introduced (Copan Inc., Corona CA) for specimen collection. Instead of being ß Crude measurement of swab-tip area clearly demonstrated that nylon swab area FIG. 4: SEM of a rayon-tipped swab surface placed in a 1.27% suspension of 1μ polystyrene wound around a shaft, these “fl ocked swabs have a one piece construction with the fi bers beads. Beads, too numerous to count are demonstrated on fi ber surfaces. 3000x radiating from the shaft tip. (mm2) was clearly greater than rayon or polyester (40, 24 and 28, respectively). FIG. 5: SEM of a polyester-tipped swab placed in a 1.27% suspension of 1μ polystyrene To demonstrate potential benefi ts of fl ocked swabs in the enhancement of specimen ß The viral HSV-2 isolate collected with nylon swabs required greater dilution to beads. Beads are adhered to fi ber surfaces. 3000x collection, this study investigated physical (absorption of fl uid, size, microscopic structure) reach the 50 percent infectivity endpoint (TCID ) than the same isolate collected 50 FIG. 6: SEM of a nylon fl ocked swab surface placed in a 1.27% suspension of 1μ polystyrene and diagnostic (viral infectivity-TCID50 assay) properties of the new fl ocked swab to that of with rayon and polyester swabs (log values of -3.56, -1.73 and –2.82, respectively). rayon and polyester nasopharyngeal swabs. 10 beads. Beads were clearly visualized on the smooth nylon fi bers. 3000x ß Rayon and polyester swabs looked similar under SEM examination. Rope-like fi bers FIG. 7: SEM of nylon fl ocked swab surface which was vortexed in water follwing placement of FIG. 9 Materials and Methods: were wrapped around a shaft forming the swab-tip (fi g. 1, fi g. 2). At higher the swab in a 1.27% suspension of 1μ polystyrene beads. Less beads were observed on magnifi cations, these fi bers appeared to have ridges or striations on their surface the fi bers after vortexing the swab. 3000x (1) Absorbance studies (fi g. 8). Nylon swabs appeared as smooth-surfaced bristles radiating from the FIG. 8: SEM of a rayon-tipped swab surface which was vortexed in water following placement ß Ten each of rayon, polyester and nylon-fl ocked nasopharyngeal swabs (Copan, Inc., shaft (fi g. 3). in a 1.27% suspension of 1μ polystyrene beads. There was a dramatic reduction in Corona, CA) were immersed in a tube containing 1ml of water for 15 seconds. ß All swabs immersed in a 1.27% suspension of polystyrene beads demonstrated the beads on rayon fi bers and in the crevices between fi bers. 3000x Following swab removal, absorbance values were determined by weighing the presence of numerous, uniform 1 μ structures on their fi bers (fi g. 4, fi g. 5, fi g. 6). FIG. 9: SEM of a polyester-tipped swab which was vortexed in water following placement in a remaining liquid using an OHAUS Brainweight 300 scale (OHAUS, Florham Pk., NJ). A reduction in the number of beads adhering on the fi ber surfaces was clearly 1.27% suspension of 1μ polystyrene beads. Beads were observed to have been eluted ß A more viscous solution (22% serum albumin; Immucor, Inc, Norcross, CT) was used demonstrated for the nylon and rayon swabs (fi g. 7, fi g. 8) following vortexing. form fi ber surfaces but were sometimes demonstrated in great abundance in the crevices formed between adjacent fi bers. 1000x to assess absorbance using the same method as above. However, pools of adhered beads, particularly in crevices where adjacent ß Swab tip material was removed from swab-shafts using tweezers or scalpel blades. fi bers abutted, were often seen in the polyester swab (fi g. 9). Polyester swabs A weight of 0.50g was collected in plastic centrifuge tubes (Sarstedt, Inc.; Newton, CT) failed to appreciably expand following vortexing.