Interlaboratory evaluations of a standardized quantitative test method for determining the bactericidal and tuberculocidal efficacy of antimicrobial substances on hard non-porous surfaces.

The development, validation, and use of new quantitative methodologies for testing the effectiveness of antimicrobial products are necessary to meet the regulatory challenges associated with an ever-changing marketplace, novel product claims, new infection control practices, and the emergence of new clinical pathogens. A series of four interlaboratory studies were conducted in a standardized manner on an interim quantitative method for testing liquid treatments against bacteria to assess its statistical performance. The Quantitative Method, a derivative of ASTM E2197, is designed to enumerate the number of viable microbes remaining on a test carrier following exposure to a liquid antimicrobial treatment; a log10 reduction (LR) in viable bacteria is calculated based on the difference between the mean log10 density values of the untreated control and treated carriers. The Quantitative Method uses 1 cm diameter disks (carriers) of brushed stainless steel as the material to represent a hard, non-porous surface. The LR value is used as the measure of product effectiveness, where higher LR values are indicative of greater microbial kill. The test microbes were Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium terrae. The liquid antimicrobial treatments used in these studies were highly relevant to those in the marketplace and provided a wide range of mean LR outcomes. The focus of the statistical assessment was on the repeatability of the LRs across experiments within a lab (Sr) and the reproducibility of the LRs across labs (SR). Due to the additional sources of variability, the SR is expected to be higher than the variability within a laboratory (Sr); this was observed in the studies reported here. Across the studies, the Sr values for LR were small (i.e., less than 0.84), most notably for treatments generating high mean LRs (5 or above) where the Sr was as small as 0.12. Overall, the SR values ranged from 0.227 to 1.217. Only three of the twenty-four treatment combinations over the study period resulted in SR values above 1.0 - the associated LRs for the three treatments ranged from 2.22 to 3.26. Antimicrobial treatments with a LR of 4.5 or higher exhibited SR of 0.561 or less. The statistical attributes reported here for the draft Quantitative Method when used to test P. aeruginosa, S. aureus, and M. terrae provide information for decision makers when considering the method as a candidate regulatory procedure. The data and statistical analyses contained in this report are historical in nature and provide useful baseline information for individuals conducting additional technical review of the method. Based on the data, the Quantitative Method displays a statistical profile consistent with other standard methods approved by standard-setting organizations where method performance data are available.

Comparison of 3M Petrifilm Aerobic Count Plates to standard plating methodology for use with AOAC antimicrobial efficacy methods 955.14, 955.15, 964.02, and 966.04 as an alternative enumeration procedure: collaborative study.

A multilaboratory study was conducted to determine the equivalence of the 3M Petrifilm Aerobic Count Plate and standard plating methodology for measuring viable bacteria and spores recovered from hard-surface carriers (stainless steel and porcelain), also known as "control carrier counts," used in AOAC antimicrobial efficacy test methods. Six laboratories participated in the study in which carriers inoculated with Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, and spores of Bacillus subtilis were evaluated using 3M Petrifilm Aerobic Count (AC) plates and standard plating side-by-side. The data were analyzed using a matched-pair t-test to determine the between-method effect with confidence intervals. For all test organisms pooled across all laboratories, the mean difference in log10 concentration between the standard plate count method and 3M Petrifilm AC Plates was -0.012, with a 95% confidence interval of (-0.090, +0.066), which was well within the -0.5, +0.5 interval established as the acceptance criterion. The between-carrier SD averaged 0.139; the between-replicate SD was 0.050. The carrier reproducibility, given that a single replicate per carrier is done, was estimated to be 0.148. Although differences were seen in the final concentrations of the test organisms among laboratories, there were no statistical differences between the enumeration methods. Based on the results from this study, 3M Petrifilm AC Plates are equivalent to standard plating methodology and can be used as an alternative procedure for the enumeration of test organisms used in AOAC Methods 955.14, 955.15, 964.02, and 966.04.

Procedural revision to the use-dilution methods: establishment of maximum log density value for test microbes on inoculated carriers.

(Staphylococcus aureus) and 964.02 (Pseudomonas aeruginosa), were revised in 2009 to include a standardized procedure to measure the log density of the test microbe and to establish a minimum mean log density value of 6.0 (geometric mean of 1.0 x 10(6) CFU/carrier) to qualify the test results. This report proposes setting a maximum mean log density value of 7.0 (geometric mean of 1.0 x 10(7) CFU/carrier) to further standardize the procedure. The minimum value was based on carrier count data collected by four laboratories over an 8-year period (1999-2006). The data have been updated to include an additional 4 years' worth of data (2006-2010) collected by the same laboratories. A total of 512 tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed in each test, for a total of 3072 carriers. Mean log densities for each of the 512 tests were at least 6.0. With the exception of two tests, one for P. aeruginosa without OSL and one for S. aureus with OSL, the mean log densities did not exceed 7.5 (geometric mean of 3.2 x 10(7) CFU/carrier). Across microbes and OSL treatments, the mean log density (+/- SEM) was 6.80 (+/- 0.07) per carrier (a geometric mean of 6.32 x 10(6) CFUlcarrier) and acceptable repeatability (0.28) and reproducibility (0.31) SDs were exhibited. A maximum mean log density per carrier of 7.0 is being proposed here as a validity requirement for S. aureus and P. aeruginosa. A modification to the method to allow for dilution of the final test cultures to achieve carrier counts within 6.0-7.0 logs is also being proposed. Establishing a range of 6.0-7.0 logs will help improve the reliability of the method and should allow for more consistent results within and among laboratories.

Use of alternative carrier materials in AOAC Official Method 2008.05, efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface, quantitative three-step method.

The quantitative Three-Step Method (TSM) for testing the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard, nonporous surface (glass) was adopted as AOAC Official Method 2008.05 in May 2008. The TSM uses 5 x 5 x 1 mm coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers and neutralization, spores are removed from carriers in three fractions (gentle washing, fraction A; sonication, fraction B; and gentle agitation, fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. The counts are summed over the three fractions to provide the density (viable spores per carrier), which is log10-transformed to arrive at the log density. The log reduction is calculated by subtracting the mean log density for treated carriers from the mean log density for control carriers. This paper presents a single-laboratory investigation conducted to evaluate the applicability of using two porous carrier materials (ceramic tile and untreated pine wood) and one alternative nonporous material (stainless steel). Glass carriers were included in the study as the reference material. Inoculated carriers were evaluated against three commercially available liquid sporicides (sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde), each at two levels of presumed efficacy (medium and high) to provide data for assessing the responsiveness of the TSM. Three coupons of each material were evaluated across three replications at each level; three replications of a control were required. Even though all carriers were inoculated with approximately the same number of spores, the observed counts of recovered spores were consistently higher for the nonporous carriers. For control carriers, the mean log densities for the four materials ranged from 6.63 for wood to 7.14 for steel. The pairwise differences between mean log densities, except for glass minus steel, were statistically significant (P < 0.001). The repeatability standard deviations (Sr) for the mean control log density per test were similar for the four materials, ranging from 0.08 for wood to 0.13 for tile. Spore recovery from the carrier materials ranged from approximately 20 to 70%: 20% (pine wood), 40% (ceramic tile), 55% (glass), and 70% (steel). Although the percent spore recovery from pine wood was significantly lower than that from other materials, the performance data indicate that the TSM provides a repeatable and responsive test for determining the efficacy of liquid sporicides on both porous and nonporous materials.

Improving the AOAC use-dilution method by establishing a minimum log density value for test microbes on inoculated carriers.

The AOAC Use-Dilution methods, 955.14 (Salmonella enterica), 955.15 (Staphylococcus aureus), and 964.02 (Pseudomonas aeruginosa), are used to measure the efficacy of disinfectants on hard inanimate surfaces. The methods do not provide procedures to assess log density of the test microbe on inoculated penicylinders (carrier counts). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this method deficiency. Based on carrier count data collected by four laboratories over an 8 year period, a minimum log density value is proposed to qualify the test results. Carrier count data were collected concurrently with 242 Use-Dilution tests. The tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 1452 carriers. All 242 mean log densities were at least 6.0 (geometric mean of 1.0 x 10(6) CFU/carrier). The mean log densities did not exceed 7.5 (geometric mean of 3.2 x 10(7) CFU/carrier). For all microbes and OSL treatments, the mean log density (+/- SEM) was 6.7 (+/- 0.07) per carrier (a geometric mean of 5.39 x 10(6) CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.29) and reproducibility (0.32). A minimum mean log density of 6.0 is proposed as a validity requirement for S. aureus and P. aeruginosa. The minimum level provides for the potential inherent variability that may be experienced by a wide range of laboratories and the slight effect due to the addition of an OSL. A follow-up report is planned to present data to support the carrier count procedure and carrier counts for S. enterica.

Determining the efficacy of liquid sporicides against spores of Bacillus subtilis on a hard nonporous surface using the quantitative three step method: collaborative study.

A collaborative study was conducted to validate the quantitative Three Step Method (TSM), a method designed to measure the performance of liquid sporicides on a hard nonporous surface. Ten laboratories agreed to participate in the collaborative study; data from 8 of 10 participating laboratories were used in the final statistical analysis. The TSM uses 5 x 5 x 1 mm glass coupons (carriers) upon which spores have been inoculated and which are introduced into liquid sporicidal agent contained in a microcentrifuge tube. Following exposure to a test chemical and a neutralization agent, spores are removed from carriers in 3 fractions: passive removal (Fraction A), sonication (Fraction B), and gentle agitation (Fraction C). Liquid from each fraction is serially diluted and plated on a recovery medium for spore enumeration. Control counts are compared to the treated counts, and the level of efficacy is determined by calculating the log10 reduction (LR) of spores. The main statistical goals were to evaluate the repeatability and reproducibility of the LR values, to estimate the components of variance for LR, and to assess method responsiveness. AOAC Method 966.04-Method II was used as a reference method. The scope of the validation was limited to testing liquid formulations against spores of Bacillus subtilis, a surrogate for virulent strains of B. anthracis, on a hard nonporous surface (glass). The test chemicals used in the study were sodium hypochlorite, a combination of peracetic acid and hydrogen peroxide, and glutaraldehyde. Each test chemical was evaluated at 3 levels of presumed efficacy: high, medium, and low. Three replications were required. The TSM was validated as it successfully met the statistical parameters for quantitative test methods. Satisfactory validation parameters, such as the repeatability standard deviation (Sr) and reproducibility standard deviation (SR), were obtained for control carrier counts and LR values. Both the TSM and the reference method were responsive to the efficacy levels of the test chemicals. For the 72 total TSM tests conducted, the mean (+/- standard error of the mean) log density of spores per control carrier was 6.86 (+/- 0.08); the Sr and SR were low at 0.15 and 0.27, respectively. Across the range of test chemicals, the Sr and SR estimates associated with LR were also acceptably low. The Sr ranged from 0.17 to 0.72 and the SR ranged from 0.34 to 1.43. Overall, the Sr and SR estimates associated with the efficacy data were within the ranges published for other quantitative methods and meet the performance characteristics necessary for validation.