In this study, two cleaning robots were selected from the previous experiments described in Lee et al. (2013). Aerosol deposited B. atrophaeus spores were used as a surrogate of B. anthracis spores. Robotic cleaners were evaluated for their performance when challenged with two different contamination scenarios: hot spot and widely dispersed contamination in the test chamber. Test results were compared to currently used surface sampling methods (vacuum sock and sponge wipe).
Two types of commercially available cleaning robots, a vacuum-based robot (R2 from the previous study, XV-11, Neato robotics®, Newark, CA) and a wetted-wipe-based robot (R4 from the previous study, Mint 4200, Evolution Robotics, Inc. Pasadena, CA), were selected from the previous study results and purchased from an internet retail store. R2 and R4 were tested for sampling efficiency on carpet and floor laminate surfaces, respectively, by following the factory manuals procedures for use. The sterilization procedure and sterility checks of test robots are addressed in the previous paper in detail (Lee et al., 2013). In brief, all tested robots were sterilized with vaporized hydrogen peroxide (H2O2; VHP®, 1000ED, Steris, Mentor, OH) before testing and the sterility of robots was confirmed by sampling at least one robot per sterilization batch by swabbing the robot surfaces. Test robots retained their factory settings during testing, and each robot was used only once before being discarded. The R4 wipe material was soaked with sterile phosphate buffered saline with 0.05 weight percent Tween 20 (PBST) before testing.
Robot sampling tests were conducted with two floor surface types: carpet (Model 6666-01-1200-AB, Beaulieu Laredo Sagebrush loop carpet) and floor laminate (PE-191113, Pergo Estate Oak). These materials were purchased from a local retail store (Home Depot, Durham, NC). Coupons were fabricated into 107 cm × 107 cm and 71 cm × 71 cm size pieces for robot sampling tests and 36 cm × 36 cm for vacuum or sponge wipe sampling tests to fit into the Consequence Management and Decontamination Evaluation Room (COMMANDER; Exhibit 1; Wood et al., 2013). Both coupon types were backed with an equal-sized piece of 1.1 cm thick Oriented Strand Board (OSB) plywood. Test coupons were treated before testing to remove the detachable foreign debris and particles. This entailed vacuuming the carpet coupons and the laminate coupons were cleaned with a dry wipe (SIMWyPE tack cloth, Babcock & Wilcox Technical Services Y-12 L.L.C., Oak Ridge, TN). After surface cleaning, all test coupons were sterilized by exposure to 250 ppm of H2O2 for 4 hr inside the COMMANDER. The sterilized coupons were stored in sterilization bags (P/N 63636TW, General Econopak, Inc., Philadelphia, PA) until tested. All fumigated coupons and apparatus were degassed for at least 3 days and their sterility was confirmed by sampling at least one coupon and one deposition apparatus per sterilization batch by swabbing (P/N R12100, Remel Products, Lenexa, KS) their respective surfaces. Sterility check and coupon preparation procedures were described previously (Lee et al., 2013).
The B. anthracis surrogate used for this study was a powdered spore preparation of B. atrophaeus (ATCC 9372, Manassas, VA) and silicon dioxide particles. The preparation procedure is reported in Brown et al. (2007). The powdered preparation was loaded into metered dose inhalers (MDIs). The MDI preparation and characteristics have been reported (Calfee et al., 2013; Carrera et al., 2005; Lee et al., 2011). The sterilized coupons were inoculated using the MDI following the method described in the study by Calfee et al. (2013). Coupons were inoculated with between 10−1 CFUs/cm2 and 104 CFUs/cm2, depending on defined target inoculum levels (Exhibit 2). The consistency and loading levels of inoculums were verified using four stainless steel control coupons during each inoculation event. Test coupons used for robot sampling tests were inoculated in the centermost 30.5 cm × 30.5 cm area of the coupon (36 cm × 36 cm). The same size area was inoculated on the comparative surface sampling method coupons. All test coupons underwent the same inoculation procedures, and were stored (less than 24 hr) together until used in testing. Each coupon was inoculated independently using separate dosing chambers, originally designed for inoculation of the centermost 30.5 cm × 30.5 cm areas of 36 cm × 36 cm coupons. Following a metered dissemination, spores were allowed to settle onto the coupons for a minimum period of 18 hr.
Exhibit 2. Test matrix for two scenarios: Hot spot and widely dispersed contamination
|Scenario Type||Surface||Robot||Target Spore Loading (CFUs/cm2)|
|Widely dispersed contamination||Carpet||R2||101|
|Widely dispersed contamination||Carpet||R2||101|
|Widely dispersed contamination||Laminate||R4||10−1|
|Widely dispersed contamination||Laminate||R4||10−1|
Robot Testing Procedure
All Robot sampling tests were conducted inside COMMANDER and the test environment was controlled for the temperature (22 ± 0.7 °C) and relative humidity (57 ± 5 percent). More detailed information about the COMMANDER can be found in a prior publication (Wood et al., 2013). The test initiated with a blank robot sampling. After the blank sampling, B. atrophaeus spore-inoculated test coupons were sampled with robots. After the completion of sampling, the robots were powered off, removed from the testing chamber one at a time, and disassembled for retrieval of the sample. The robot components were placed in a sterilized plastic bag. Each bag was then secondarily contained in another bag and transported to another laboratory for processing.
To evaluate R2 and R4 in the two scenarios, hot spot and widely dispersed contamination, subsections of the floor were inoculated using the method described in the previous section. COMMANDER was fitted with pre-sterilized coupons as shown in Exhibit 1. The set-up consisted of two 107 cm × 107 cm coupons, four 71 cm × 71 cm coupons, and a single 36 cm × 36 cm coupon in the center. The spore inoculation was performed at the center of the room for the hot spot. Approximately 1.7 m2 from two 107 cm × 107 cm coupons and the center coupon (36 cm × 36 cm) were inoculated for the widely dispersed scenario (Exhibit 1).
Surface Sampling Procedure
The number of spores CFUs sampled using test robots was compared to the CFUs obtained by currently used surface sampling methods. Control coupons of carpet and laminate were sampled using currently used surface sampling methods (Brown et al., 2007; GAO, 2005b; Krauter et al., 2012; Rose et al., 2011; Valiante et al., 2003). Floor laminate surfaces were sampled with a sponge wipe sampling method and carpet surfaces with a vacuum sock method. An area of 34 cm × 34 cm, delineated with a sterile stainless steel template placed over the target area was sampled with the sponge wipe. Sponge wipe samples were collected using the following five steps: (1) the surface was sampled using horizontal S-strokes using one flat side of the sponge wipe, covering the entire template area; (2) the sponge wipe was then flipped over to the opposite flat side to sample the surface in a vertical S-stroke pattern, covering the entire template area; (3) using the narrow edges of the sponge wipe, the surface was sampled using the same S-strokes but applied diagonally across the template, (4) rotating the sponge to use the opposite narrow edge at the midway point of the coupon; and (5) the tip of the sponge wipe was then used to sample the perimeter of the sampling area. During vacuum sampling, a 34 cm × 34 cm sterile stainless steel template and a sterile sock/nozzle attachment to a vacuum were used to collect the sample. Holding the nozzle at a 45 degree angle on the sample area, samples were taken using horizontal and vertical S-strokes. The sponge wipe sampling method is described in detail in the study by Rose et al. (2011), and the vacuum sock method is a modified version of the method detailed in the study by Brown et al. (2007).
Sampling Extraction and Spore Recovery
Sponge wipe (PN SSL10NB, 3M Inc., St. Paul, MN) samples were extracted by stomaching (1 min, 260 rpm) in 90 mL of PBST using a Seward® Model 400 circulator (Seward® Laboratory Systems, Inc, Port Saint Lucie, FL). Vacuum sock samples were extracted by first wetting the collection (white) portion of the filter in PBST, then cutting it with sterile scissors (vertically and horizontally) into small pieces (approximately 1 cm × 4 cm). As the filter was fractioned, the resulting pieces were allowed to fall into a 120 mL sterile specimen cup (Starplex Scientific LeakBuster Specimen Containers, Fisher Scientific, Pittsburgh, PA) containing 20 mL sterile PBST. The cups were then agitated (30 min, 300 rpm, ambient temperature) using an orbital platform shaker incubator (Model 3625, Lab-Line Instruments, Inc., Melrose Park, IL). Spores collected by R4 were recovered from the mopping cloth by stomaching the cloth (2 min, 230 rpm) in 133 mL PBST using a Seward® Model 400 circulator (Seward Laboratory Systems, Inc., Bohemia, NY).
Two extraction procedures were required for R2 as collected spores could have partitioned to either the collection bin or the filter. Recovery from the filters proceeded by placing each filter into two 14 cm × 23 cm sterile sample bags (Fisher Scientific, Pittsburgh, PA), one inside the other for double containment. A total of 180 mL of sterile PBST was then added to the innermost bag, and the samples were agitated (30 min, 300 rpm) on an orbital platform shaker incubator (Model 3625, Lab-Line Instruments, Inc., Melrose Park, IL). Spore recovery from the particle bins was accomplished by placing the bins into double layer of 25 cm × 38 cm sterile sample bags, aseptically adding 180 mL of PBST to each bag containing the bin, and then agitating (30 minutes, 300 rpm, ambient temperature) on an orbital platform shaker incubator (Model 3625, Lab-Line Instruments, Inc., Melrose Park, Illinois).
The resulting liquid extracts from the robots and all fractions were individually concentrated by centrifugation where, briefly, each sample was retrieved from its respective extraction bag or cup, and dispensed equally into four 50 mL conical tubes (approximately 45 mL for each tube). The samples were then centrifuged (3,500 × g, 15 min, 4 °C) to sediment the collected spores. All but 5 mL of the supernatant was carefully removed via 50 mL sterile serological pipette. Each spore pellet was resuspended in the remaining 5 mL by three cycles of alternating vortex mixing (30 sec) and sonication (30 sec, 40 kHz, Model 8510, Branson, Rochester, NY).
Following resuspension, the four fractions per sample were recombined into one approximately 20 mL sample extract. All sample extracts (robot, vacuum sock, and sponge wipe) were then subjected to a series of ten-fold dilutions, as necessary, by adding 0.1 mL of the sample to 0.9 mL of PBST using a micropipette. Appropriate dilutions were spread in triplicate (0.1 mL each) onto trypticase soy agar (BD™; Becton, Dickinson, and Company, Franklin Lakes, NJ) plates and incubated at 35 ± 2 °C. Resulting CFUs were counted manually after approximately 18 hr. The recovery for each sample was determined by averaging the observed CFUs from triplicate plates (subsamples), multiplying by the inverse of the dilution factor, dividing by the volume plated (typically 0.1 mL), and multiplying by the total volume of the sample extract. Total recoveries from each of the replicate samples were then averaged to determine mean recovery for each device and material type.