APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2011, p. 6918���6925 Vol. 77, No. 19 0099-2240/11/$12.00 doi:10.1128/AEM.05709-11 Copyright �� 2011, American Society for Microbiology. All Rights Reserved. Comparison of Surface Sampling Methods for Virus Recovery from Fomites ��� Timothy R. Julian,��� Francisco J. Tamayo, James O. Leckie, and Alexandria B. Boehm* Department of Civil and Environmental Engineering, Environmental and Water Studies, Stanford University, Stanford, California 94305-4020 Received 2 June 2011/Accepted 15 July 2011 The role of fomites in infectious disease transmission relative to other exposure routes is difficult to discern due, in part, to the lack of information on the level and distribution of virus contamination on surfaces. Comparisons of studies intending to fill this gap are difficult because multiple different sampling methods are employed and authors rarely report their method���s lower limit of detection. In the present study, we compare a subset of sampling methods identified from a literature review to demonstrate that sampling method significantly influences study outcomes. We then compare a subset of methods identified from the review to determine the most efficient methods for recovering virus from surfaces in a laboratory trial using MS2 bacteriophage as a model virus. Recoveries of infective MS2 and MS2 RNA are determined using both a plaque assay and quantitative reverse transcription-PCR, respectively. We conclude that the method that most effectively recovers virus from nonporous fomites uses polyester-tipped swabs prewetted in either one-quarter- strength Ringer���s solution or saline solution. This method recovers a median fraction for infective MS2 of 0.40 and for MS2 RNA of 0.07. Use of the proposed method for virus recovery in future fomite sampling studies would provide opportunities to compare findings across multiple studies. Preclusion of infection is the most effective method to com- bat the respiratory and gastrointestinal diseases that cause over 6 million annual deaths worldwide (12, 59). Successful inter- ventions to reduce disease burden include hand and environ- mental hygiene (9, 76), but the impact of these interventions is difficult to quantify, as the importance of contact with contam- inated surfaces, or fomites, relative to other transmission routes is uncertain (17, 62). Evidence of the importance of fomites comes from both laboratory and field studies. Laboratory studies have demon- strated that the handling of either artificially or naturally contaminated fomites by susceptible hosts indoors results in subsequent infection (41, 43). Additionally, virus can be trans- ferred between hands and fomites on contact and survive on fomites for hours or days (1, 8, 72). As such, it is not surprising that fomites, such as carpets (29, 65), towels, and medication cart items (60) have been implicated as the primary cause of multiple outbreaks. Environmental hygiene intended to miti- gate fomite-mediated transmission has been shown to signifi- cantly reduce illness-related absences in classrooms (18). De- spite this evidence, questions remain regarding both the efficacy of fomite-mediated transmission relative to other ex- posure routes (5, 46) and the likelihood of virus transfer from fomites to hosts (66). To better understand the role of fomites in disease trans- mission, characterization of the level and distribution of virus on surfaces is required. Surface contamination is most often described by the positivity rate, defined as the fraction of total samples collected on which the organism is detectable (19). However, the positivity rate does not provide an indication of infection risk, which depends on exposure magnitude (42). To estimate the risk of infection, information about the quantity of virus on the surface is required. Virus density, expressed as the number of virions or number of virus equivalents per unit surface area, has been measured only in a few studies (10, 68, 73). Both positivity rate and virus density are influenced by the sampling method and detection assay: more sensitive sampling methods and detection assays will yield increases in positivity rates and higher measured concentrations even though the actual level of virus contamination may be unchanged. Use of a sensitive, standard method would limit bias introduced by the use of multiple different sampling methods. Three previous studies have compared or evaluated virus surface sampling methods and suggested that implement type (the tool used to collect the sample, such as a swab) and eluent type (the liquid used to aid in removal, such as saline solution) significantly influence virus recovery. Carducci et al. (20) re- covered a greater fraction of hepatitis C virus from a seeded surface using beef extract than using bovine serum albumin when swabbing with a cotton-tipped applicator. The study demonstrated that eluent type can significantly impact virus recovery from surfaces. Similarly, Taku et al. (79) demon- strated the impact of implement type by comparing calicivirus recovery from food surfaces for four sampling methods. Rins- ing a surface in 0.05 M glycine buffer, rubbing with a cell scraper, and then aspirating the buffer was recommended over (i) rinsing the surface in buffer and then aspirating, (ii) swab- bing the surface with a cotton-tipped applicator, or (iii) swab- * Corresponding author. Mailing address: Department of Civil and Environmental Engineering, Environmental and Water Studies, Stan- ford University, Stanford, CA 94305-4020. Phone: (650) 724-9128. Fax: (650) 725-3164. E-mail: aboehm@stanford.edu. ��� Present address: Department of Environmental Health Sciences, Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205. ��� Supplemental material for this article may be found at http://aem .asm.org/. Published ahead of print on 5 August 2011. 6918

bing the surface with a nylon filter. However, Taku et al.���s (79) recommended method is not easily adapted to the geometry of most fomites. Finally, Scherer et al. (75) evaluated one imple- ment-eluent combination (cotton-tipped swabs wetted in phos- phate-buffered saline [PBS]) for efficacy of rotavirus and noro- virus RNA recovery and reported a mean recovery of 7 to 53%, dependent on surface type and initial seeded titer. However, they did not compare the method to any other methods. Fur- ther research is needed to refine implement and eluent choice for sampling fomites to maximize virus recovery. In the present study, we reviewed the literature on virus sampling of fomites and used a laboratory-based trial to com- pare methods of virus detection on surfaces. We identified, summarized, and analyzed 59 articles that include unique data sets on virus detection on surfaces. A subset of the sampling methods identified from the meta-analysis were compared in a laboratory-based study of the removal of bacteriophage MS2, as measured using both culture-dependent and culture-inde- pendent (quantitative reverse-transcription PCR [qRT-PCR]) methods, from plastic and stainless steel surfaces. Based on both the literature review and experimental results, we identi- fied the implement and eluent combinations that most effec- tively remove infective virus and virus RNA from nonporous fomites. MATERIALS AND METHODS Review of virus surface sampling literature. Literature on virus surface sam- pling was identified by searching PubMed and Embase databases for records added on or before 28 June 2011 and reviewing the references of the identified studies. For further details, see the supplemental material. For analysis, data from the articles were separated into data sets according to the virus, the presence/absence of a clinically infected individual, and the location of sampling. If the authors included clinical or food samples, those samples were removed from data analysis. Further details on the criteria of the separation of articles into data sets are presented in the supplemental material. In summary, 98 data sets from 59 articles were obtained. The positivity rate was determined, as the outcome variable, for each data set. The positivity rate was the only feasible outcome variable, as most of the studies identified report only the presence/absence of virus on surfaces. Only a fraction (9 of 98, or 9%) reported quantitative data. To allow for logit transformation, the positivity rate for studies that detected the virus on none or all of the samples was adjusted to a detection limit of 1/n or (n 1)/n, respectively, where n is the study���s total number of samples collected. The positivity rate is an inherently biased outcome variable because the lower limit of detection (LLOD) varies across studies for reasons described previously. As few studies (30%) reported either the quantitative concentration of the virus or the LLOD of the sampling method, the positivity rate could not be adjusted to account for the bias. We assessed the influence of the implement and eluent used to collect and analyze the samples on the positivity rate. Similar implements and eluents were grouped for data analysis. Polyester and Dacron swabs were both categorized as polyester. The eluent used was categorized into one of four groups: media (defined here as any eluent with a carbon source and includes Amies medium, beef extract, brain heart infusion broth, Letheen broth, minimum essential me- dium, RPMI 1640, and tryptose phosphate broth with 0.5% gelatin), saline (defined as any isotonic eluent without a carbon source and includes phosphate- buffered saline, 0.8% saline, and Ringer���s solution), water, or unreported. Ad- ditives and constituents of eluents, such as antibiotics, were ignored for data analysis to avoid overparameterization. Statistics. All statistical analyses were performed using R (version 2.9.0 R Foundation for Statistical Computing, Vienna, Austria). The normality of trans- formed data was assessed using the Kolmogorov-Smirnoff test. Two bivariate linear models were used to determine the statistical significance of implement and eluent, separately, on the transformed positivity rate the positivity rate was weighted by the total number of samples in each study. Laboratory-based surface sampling method comparison. In a laboratory- based trial, we compared fractions of virus recovered from surfaces for a subset of the implement and eluent choices identified in the literature. Virus and preparation of inoculum. MS2 bacteriophage was obtained from the American Type Culture Collection (ATCC). MS2 (ATCC 15597-B1) is a posi- tive-sense single-stranded RNA (ssRNA) virus with an icosahedral, tailless cap- sid approximately 27 nm in diameter. The isoelectric point (pI) of MS2 is 3.9. MS2 was chosen because of its prior use as a surrogate for human viruses, such as norovirus (26), and the availability of plaque assay and qRT-PCR methods to enumerate both infective phage and copies of nucleic acids (63, 80). Escherichia coli HS(pFamp)R (ATCC 700891) was used to propagate and enumerate viable MS2, measured as the number of PFU. The inoculum was prepared using the method described by Pecson et al. (67). In brief, the log-phase E. coli host was inoculated with MS2 at a multiplicity of infection of 0.1 and incubated at 37��C for 4 to 6 h. Chloroform was added to complete cell lysis, and the sample was clarified by centrifugation for 15 min at 4,000 g. The supernatant was stored at 4��C overnight in 10% polyethylene glycol (PEG 6000 Sigma-Aldrich, St. Louis, MO) and 0.5 M NaCl. The solution was centrifuged at 7,000 g for 45 min, and the pellet was resuspended in dilution buffer (5 mM NaH2PO4, 10 mM NaCl, pH 7.4). A chloroform-only extraction was used to remove the remaining polyethylene glycol (PEG), and the sample was concentrated in an Amicon Ultra centrifugal filter device (100,000 nominal molecular-weight limit Millipore, Billerica, MA), triple washed with dilution buffer, and then filtered through a 0.1- m-pore-size polyvinylidene di- fluoride filter (Millipore, Billerica, MA). The propagated virus was then enu- merated using the double-agar-layer method and diluted in the dilution buffer to a virus stock of 1 106 PFU/ml. Immediately before being seeded on the surface, the virus stock was mixed with tryptic soy broth (TSB) to form a 50% solution. Implements and eluents tested. The implements tested were cotton-tipped (Thermo Fisher Scientific, Waltham, MA) and polyester-tipped (Thermo Fisher Scientific, Waltham, MA) swabs as well as antistatic cloth (Bel-Art Products, Pequannock, NJ). The antistatic cloth used in the study is a microfiber cloth wet by the manufacturer with 1,1-difluoroethane. Prior to sampling, the cloth was cut into single-ply square swatches of approximately 9 cm2 and stored in sterile, sealed 5-ml containers for 5 to 10 days. The eluents tested include 0.85% saline, one-quarter-strength Ringer���s solu- tion (EMD Chemicals, Inc., Gibbstown, NJ), and viral transport media (Copan Diagnostics, Murietta, CA). A fourth eluent, termed acid/base, was added to assess an eluent adapted from a method to concentrate virus from environmental water samples (48). The acid/base eluent relies on knowledge of the virus surface charge to improve recovery from fomites. Briefly, a weakly acidic (0.5 mM dihydrogen sulfate, H2SO4) eluent is used to wet the implement prior to sam- pling. Viruses with low isoelectric points adsorb to negatively charged surfaces (like cotton) under acidic conditions (48). After sampling, the implement is placed into a weakly basic (1 mM sodium hydroxide, pH 10.5 to 10.8) eluent which reverses the surface charge of the virus to elute the virus from the imple- ment. The implements/eluents were chosen based on a subjective analysis of the literature. Specifically, we included implements/eluents that were either com- monly used (e.g., cotton and saline) or had the potential to improve virus recovery (e.g., antistatic cloth and acid/base). Surfaces tested. To determine the method most effective in removing virus from surfaces, we compared recovery from both high-temperature polyvinyl chloride (PVC) plastic (part no. 8748K21) and type 304 stainless steel with a mirror-like finish (part no. 9785K11), both obtained from McMaster-Carr (Santa Fe Springs, CA). Many of the surfaces identified in the literature review that were frequently contaminated (e.g., door knobs, faucet handles, drains, medical instruments, toys, playmats, computer parts, and telephones) were composed of plastic or metal. PVC plastic and stainless steel were chosen as representative samples, as it was infeasible to test every potential type of surface material. Between 8 and 10 replicates for each eluent and implement combination were tested on both surfaces. In total, 230 samples were collected (3 implements, 4 eluents, 2 surfaces, and 8 to 10 replicates). All 230 samples were tested using the double-agar-layer plaque assay method and a subset (213 samples) using qRT-PCR. Study design. For both plastic and stainless steel surfaces, a 5- l inoculum of MS2 stock was seeded in the center of 120 5-cm by 5-cm surface swatches, resulting in a seeded surface density of 3.7 log10 PFU per swatch with a standard deviation of 0.13 log10 PFU per swatch, equivalent to 5.4 log10 RNA target copies per swatch with a standard deviation of 0.16 log10 RNA target copies per swatch. An additional 8 surface swatches were seeded with bacteriophage-free TSB to act as negative controls to confirm that there was no cross-contamination of samples (e.g., due to aerosolization and deposition of MS2). The seeded aliquot was dried for 45 1 min under ambient conditions (temperature of 20 to 22��C and relative humidity of 45 to 60%, determined by a thermometer and hygrometer Spring- field Precision Instruments, Wood Ridge, NJ), outside a laminar flow hood. The order of implement and eluent combinations used to recover MS2 from the surfaces was randomized prior to the start of the study. VOL. 77, 2011 SAMPLING METHODS FOR VIRUS RECOVERY FROM FOMITES 6919