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.

Analytical Method for the Detection of Residual Active Ingredients Found in Neutralized Suspensions of Antimicrobial Products.

An analytical method for determining the presence and levels of residual active ingredients found in neutralized suspensions of phenolic and quaternary ammonium salt-based antimicrobial products was developed using solid-phase extraction in combination with LC-tandem MS. A single-laboratory validation of the method was performed at three concentration levels for the quaternary ammonium compounds (also referred to as benzalkonium chlorides or BACs) and the phenols in the presence of letheen broth neutralizer at 2.5 and 2.75 µg/mL, respectively, as well as at dilutions of 1:10 and 1:100 in those concentrations. The method's lowest LODs were 0.005 µg/g for BACs and 0.006 µg/g for phenols. The average recovery of the fortified samples for both active ingredients ranged between 80 and 124%, and RSDs were generally <20%. In a related study, the effectiveness of letheen broth with and without sodium thiosulfate was evaluated as a neutralizer for sodium hypochlorite. The results showed that letheen broth without sodium thiosulfate neutralizes chlorine concentrations up to 60 ppm, and that 200 µg sodium thiosulfate are required to neutralize a 72 ppm concentrated chlorine solution in letheen broth.

A statistical model for assessing performance standards for quantitative and semiquantitative disinfectant test methods.

A performance standard for a disinfectant test method can be evaluated by quantifying the (Type I) pass-error rate for ineffective products and the (Type II) fail-error rate for highly effective products. This paper shows how to calculate these error rates for test methods where the log reduction in a microbial population is used as a measure of antimicrobial efficacy. The calculations can be used to assess performance standards that may require multiple tests of multiple microbes at multiple laboratories. Notably, the error rates account for among-laboratory variance of the log reductions estimated from a multilaboratory data set and the correlation among tests of different microbes conducted in the same laboratory. Performance standards that require that a disinfectant product pass all tests or multiple tests on average, are considered. The proposed statistical methodology is flexible and allows for a different acceptable outcome for each microbe tested, since, for example, variability may be different for different microbes. The approach can also be applied to semiquantitative methods for which product efficacy is reported as the number of positive carriers out of a treated set and the density of the microbes on control carriers is quantified, thereby allowing a log reduction to be calculated. Therefore, using the approach described in this paper, the error rates can also be calculated for semiquantitative method performance standards specified solely in terms of the maximum allowable number of positive carriers per test. The calculations are demonstrated in a case study of the current performance standard for the semiquantitative AOAC Use-Dilution Methods for Pseudomonas aeruginosa (964.02) and Staphylococcus aureus (955.15), which allow up to one positive carrier out of a set of 60 inoculated and treated carriers in each test. A simulation study was also conducted to verify the validity of the model's assumptions and accuracy. Our approach, easily implemented using the computer code provided, offers a quantitative decision-making tool for assessing a performance standard for any disinfectant test method for which log reductions can be calculated.

Use of statistical modeling to reassess the performance standard for the AOAC use-dilution methods (955.15 and 964.02).

The AOAC Use-dilution methods (UDM) 955.15 (Staphylococcus aureus) and 964.02 (Pseudomonas aeruginosa) are laboratory methods used to substantiate antimicrobial efficacy claims for liquid disinfectants on inanimate surfaces. The UDM is accepted by the U.S. Environmental Protection Agency for the purpose of product registration. To attain a hospital-level claim, testing against S. aureus and P. aeruginosa is required, and the product must pass against both microbes. Currently, the UDM's performance standard for a single 60-carrier test is the same for both microbes, and allows up to one positive carrier for the product to be considered as a pass. In this paper, the performance standards for these methods are reassessed using data from a 2009 five-laboratory collaborative study and a recently published statistical model. The reassessment focuses on the pass-error rate for ineffective products and the fail-error rate for highly effective products. The calculations indicate that the pass-error rate is between 9 and 24% and the fail-error rate between 18 and 23% when the current performance standard is used for a single test. For product registration, a smaller pass-error rate (1%) historically has been maintained by requiring that a disinfectant pass three UDM tests for each of the two microbes; however, the calculations also indicate that the fail-error rate is between 42 and 45%. This large fail-error rate is a compelling reason to consider a new performance standard for the two UDM methods, 955.15 (S. aureus) and 964.02 (P. aeruginosa). One alternative performance standard allows no more than six positive carriers out of 60 as a pass when using P. aeruginosa and no more than three positive carriers out of 60 when using S. aureus. In addition, the new performance standard requires that three UDM tests be performed with each of the two microbes, and the disinfectant must pass all six tests to be considered efficacious. The statistical calculations for this alternative performance standard indicate that the pass-error rate is no more than 3%, while the fail-error rate is as small as 5%. Based on these error rate calculations, proposed revisions to the performance standards for AOAC Methods 955.15 and 964.02 are provided.

Modified AOAC three step method (officialmethod 2008.05): consolidation of fractions B and C.

The AOAC Quantitative Three Step Method (TSM; AOAC Official Method SM 2008.05) is validated for testing the efficacy of liquid sporicides against spores of Bacillus subtilis and Bacillus anthracis on selected hard, nonporous, and porous surfaces. The TSM uses 5x5x1 mm inoculated coupons (carriers), which are placed in 400 microL liquid sporicidal agent contained in a microcentrifuge tube. Following exposure of inoculated carriers to the test chemical and subsequent neutralization, viable spores are recovered in three fractions: A (gentle tapping), B (sonication), and C (gentle agitation). The spores in suspension are serially diluted and plated on a recovery medium for enumeration. The plate counts are summed over the three fractions to provide the number of viable spores per carrier, which is log10-transformed to generate a mean log density (LD) value across carriers. As a measure of product efficacy, a log reduction (LR) value is calculated by subtracting the mean LD for treated carriers from the mean LD for control carriers. This paper reports on the comparative evaluation of the current and modified versions of the TSM in order to support a modification to simplify the procedure. The proposed modified TSM (mTSM) consolidates fractions B and C in the same tube. Thus, the sonication (fraction B) and gentle agitation (fraction C) steps are carried out in the same tube, thereby reducing the number of tubes and associated resources and time necessary to complete the test. Glass, steel, pine wood, and ceramic tile carriers were included in the comparative study. Inoculated carriers were evaluated against two preparations of sodium hypochlorite to generate two presumed levels of efficacy (intermediate and high); the control LD and LR values associated with testing each carrier type for the TSM and the mTSM were compared. For control carriers, the mean log densities per carrier (for each carrier material) were not significantly different based on the TSM compared to the mTSM. Furthermore, the treated carrier data showed comparable LR values for the TSM and mTSM. The data provided in this report demonstrate equivalency between the TSM and mTSM and support the proposed procedural modification to consolidate fractions B and C.

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.

Development and assessment of disinfectant efficacy test methods for regulatory purposes.

The United States Environmental Protection Agency regulates pesticidal products, including products with antimicrobial activity. Test guidelines have been established to inform manufacturers of which methodology is appropriate to support a specific efficacy claim. This paper highlights efforts designed to improve current methods and the development and assessment of new test methods.

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.

Performance of the AOAC use-dilution method with targeted modifications: collaborative study.

The U.S. Environmental Protection Agency (EPA), in collaboration with an industry work group, spearheaded a collaborative study designed to further enhance the AOAC use-dilution method (UDM). Based on feedback from laboratories that routinely conduct the UDM, improvements to the test culture preparation steps were prioritized. A set of modifications, largely based on culturing the test microbes on agar as specified in the AOAC hard surface carrier test method, were evaluated in a five-laboratory trial. The modifications targeted the preparation of the Pseudomonas aeruginosa test culture due to the difficulty in separating the pellicle from the broth in the current UDM. The proposed modifications (i.e., the modified UDM) were compared to the current UDM methodology for P. aeruginosa and Staphylococcus aureus. Salmonella choleraesuis was not included in the study. The goal was to determine if the modifications reduced method variability. Three efficacy response variables were statistically analyzed: the number of positive carriers, the log reduction, and the pass/fail outcome. The scope of the collaborative study was limited to testing one liquid disinfectant (an EPA-registered quaternary ammonium product) at two levels of presumed product efficacies, high and low. Test conditions included use of 400 ppm hard water as the product diluent and a 5% organic soil load (horse serum) added to the inoculum. Unfortunately, the study failed to support the adoption of the major modification (use of an agar-based approach to grow the test cultures) based on an analysis of method's variability. The repeatability and reproducibility standard deviations for the modified method were equal to or greater than those for the current method across the various test variables. However, the authors propose retaining the frozen stock preparation step of the modified method, and based on the statistical equivalency of the control log densities, support its adoption as a procedural change to the current UDM. The current UDM displayed acceptable responsiveness to changes in product efficacy; acceptable repeatability across multiple tests in each laboratory for the control counts and log reductions; and acceptable reproducibility across multiple laboratories for the control log density values and log reductions. Although the data do not support the adoption of all modifications, the UDM collaborative study data are valuable for assessing sources of method variability and a reassessment of the performance standard for the UDM.

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.

AOAC method 966.04: preliminary evaluation of cooked meat medium with manganese sulfate for the cultivation of Clostridium sporogenes: precollaborative study.

AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. For regulatory purposes, Method 966.04 is accepted by the U.S. Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA) for the generation of product performance data for sporicides and sterilants. In this study, we report on findings associated with proposed improvements (modifications) to the Clostridium component of the method. Egg meat medium (EMM), the culture medium for C. sporogenes currently specified in the method, is no longer commercially available and finding a suitable replacement is critical. In addition, the use of a nonstandardized extract of raw soil as an amendment to EMM, as stipulated in the current method, may result in a highly variable spore suspension. The primary focus of this study was to find replacements for EMM and soil extract. A carrier count procedure, the establishment of target carrier counts (spores/carrier), and a neutralization confirmation procedure were also evaluated. The study was limited to liquid products tested against Clostridium on a hard surface carrier (porcelain penicylinder). Spore suspensions of C. sporogenes were generated using: (1) EMM with soil extract (EMM/SE), (2) cooked meat medium with soil extract (CMM/SE), and (3) cooked meat medium with 5 microg/mL manganese sulfate (CMM/MnSO4). The titer of the spore suspension, carrier counts, resistance to hydrochloric acid (HCI), and efficacy against 3 liquid sporicidal agents were used to evaluate the potential of CMM and MnSO4 as replacements. The study was performed by the EPA Office of Pesticide Programs Microbiology Laboratory, Fort Meade, MD. Use of CMM/SE and CMM/MnSO4 resulted in comparable results for titer of spore suspensions (approximately 10(8) spores/mL) and carrier counts (approximately 3 x 10(6) spores/carrier). The carrier counts for the EMM/SE were approximately 1 log lower than CMM-based treatments; however, no attempt was made to dilute the CMM spore suspensions prior to carrier inoculation to reduce the carrier counts for CMM. Resistance of spores to 2.5 M HCI was acceptable across the 3 media types. Treatments for comparative efficacy testing were designed to provide a range of sporicidal activity, i.e., high and low efficacy treatments. Sodium hypochlorite (bleach), hydrogen peroxide/peracetic acid, and glutaraldehyde were used as test chemicals. The number of carriers resulting in growth (positive) for the low treatments for all 3 chemicals ranged from 9 to 59 out of 60 across the 3 media types--EMM exhibited fewer positives overall. The high efficacy treatments for sodium hypochlorite and hydrogen peroxide/peracetic acid yielded a range of 0 to 2 positives out of 60 across the 3 media. However, the high glutaraldehyde treatment generated 3, 20, and 20 positives out of 60 for the EMM/SE, CMM/SE, and CMM/MnSO4, respectively. The lower number of positive carriers for EMM/SE may be due to the reduced carrier counts. CMM, either with SE or MnSO4, appears to be a suitable replacement for EMM/SE. On the basis of the results of this study, the Study Director recommends that CMM/MnSO4 and the spore enumeration target carrier count and neutralization procedures be considered for collaborative study to officially modify the Clostridium x porcelain component of Method 966.04.

Comparative evaluation of two quantitative test methods for determining the efficacy of liquid sporicides and sterilants on a hard surface: a precollaborative study.

Two quantitative carrier-based test methods for determining the efficacy of liquid sporicides and sterilants on a hard surface, the Standard Quantitative Carrier Test Method-ASTM E 2111-00 and an adaptation of a quantitative micro-method as reported by Sagripanti and Bonifacino, were compared in this study. The methods were selected based on their desirable characteristics (e.g., well-developed protocol, previous use with spores, fully quantitative, and use of readily available equipment) for testing liquid sporicides and sterilants on a hard surface. In this paper, the Sagripanti-Bonifacino procedure is referred to as the Three Step Method (TSM). AOAC Official Method 966.04 was included in this study as a reference method. Three laboratories participated in the evaluation. Three chemical treatments were tested: (1) 3000 ppm sodium hypochlorite with pH adjusted to 7.0, (2) a hydrogen peroxide/peroxyacetic acid product, and (3) 3000 ppm sodium hypochlorite with pH unadjusted (pH of approximately 10.0). A fourth treatment, 6000 ppm sodium hypochlorite solution with pH adjusted to 7.0, was included only for Method 966.04 as a positive control (high level of efficacy). The contact time was 10 min for all chemical treatments except the 6000 ppm sodium hypochlorite treatment which was tested at 30 min. Each chemical treatment was tested 3 times using each of the methods. Only 2 of the laboratories performed the AOAC method. Method performance was assessed by the within-laboratory variance, between-laboratory variance, and total variance associated with the log reduction (LR) estimates generated by each quantitative method. The quantitative methods performed similarly, and the LR values generated by each method were not statistically different for the 3 treatments evaluated. Based on feedback from the participating laboratories, compared to the TSM, ASTM E 2111-00 was more resource demanding and required more set-up time. The logistical and resource concerns identified for ASTM E 2111-00 were largely associated with the filtration process and counting bacterial colonies on filters. Thus, the TSM was determined to be the most suitable method.

Modification to the AOAC Sporicidal Activity of Disinfectants Test (Method 966.04): collaborative study.

In an effort to improve AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, selected modifications to the procedure were evaluated in a collaborative study. Method 966.04 is used to generate efficacy data to support the product registration of sporicides and sterilants. The method is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. The use of garden soil extract and the lack of standard procedures for the enumeration of spores and neutralization of the test chemicals have been considered problematic for many years. The proposed modifications were limited to the B. subtilis and hard surface carrier (porcelain penicylinder) components of the method. The study included the evaluation of a replacement for soil extract nutrient broth and an establishment of a minimum spore titer per carrier, both considered crucial for the improvement and utilization of the method. Additionally, an alternative hard surface material and a neutralization confirmation procedure were evaluated. To determine the equivalence of the proposed alternatives to the standard method, 3 medium/carrier combinations, (1) soil extract nutrient broth/porcelain carrier (current method), (2) nutrient agar amended with 5 microg/mL manganese sulfate/porcelain carrier, and (3) nutrient agar amended with 5 microg/mL manganese sulfate/stainless steel carrier were analyzed for carrier counts, HCI resistance, efficacy, quantitative efficacy, and spore wash-off. The test chemicals used in the study represent 3 chemical classes and are commercially available antimicrobial liquid products: sodium hypochlorite (bleach), glutaraldehyde, and a combination of peracetic acid and hydrogen peroxide. Four laboratories participated in the study. The results of the spore titer per carrier, HCI resistance, efficacy, and wash-off studies demonstrate that amended nutrient agar in conjunction with the porcelain is comparable to the current method, soil extract nutrient broth/porcelain. The nutrient agar method is simple, inexpensive, reproducible, and provides an ample supply of high quality spores. Due to the current use of porcelain carriers for testing C. sporogenes, it is advisable to retain the use of porcelain carriers until stainless steel can be evaluated as a replacement carrier material for Clostridium. The evaluation of stainless steel for Clostridium has been initiated by the Study Director. Study Director recommendations for First Action revisions are provided in a modified method.

Enumeration procedure for monitoring test microbe populations on inoculated carriers in AOAC use-dilution methods.

The AOAC Use-Dilution methods do not provide procedures to enumerate the test microbe on stainless steel carriers (penicylinders) or guidance on the expected target populations of the test microbe (i.e., a performance standard). This report describes the procedures used by the U.S. Environmental Protection Agency to enumerate the test microbe (carrier counts) associated with conducting the Use-Dilution method with Staphylococcus aureus (Method 955.15) and Pseudomonas aeruginosa (Method 964.02) and the examination of historical data. The carrier count procedure involves the random selection of carriers, shearing bacterial cells from the carrier surface through sonication, and plating of serially diluted inoculum on trypticase soy agar. For each Use-Dilution test conducted, the official AOAC method was strictly followed for carrier preparation, culture initiation, test culture preparation, and carrier inoculation steps. Carrier count data from 78 Use-Dilution tests conducted over a 6-year period were compiled and analyzed. A mean carrier count of 6.6 logs (approximately 4.0 x 10(6) colony-forming units/carrier) was calculated for both S. aureus and P. aeruginosa. Of the mean values, 95% fell within +/- 2 repeatability standard deviations. The enumeration procedure and target carrier counts are desirable for standardizing the Use-Dilution methods, increasing their reproducibility, and ensuring the quality of the data.

Efficacy testing of disinfectants.

The U.S. Environmental Protection Agency's (EPA) Office of Pesticide Programs (OPP) has the responsibility for regulating antimicrobial products, including sporicides, used to treat and decontaminate inanimate surfaces. In response to the anthrax (Bacillus anthracis) attacks of 2001 and the associated need for verifying the performance of chemicals for building decontamination, the EPA initiated research in late 2003 to evaluate and improve efficacy test methods for sporicides. The OPP Microbiology Laboratory located at the Environmental Science Center, Ft. Meade, MD is the lead laboratory. Through funding provided by EPA's Office of Research and Development (Safe Buildings Program), a collaborative research plan has been established to address several key issues. Research is currently being conducted on 2 fronts: (1) the evaluation of quantitative methodology for assessing the efficacy of sporicides, and (2) the development and comparative testing of selected modifications to improve the AOAC Sporicidal Activity Test (AOAC Method 966.04). Future studies will include the evaluation of candidate surrogates of B. anthracis using a quantitative method, and a multilaboratory validation study of a quantitative method-surrogate combination. The General Referee is serving as the Principal Investigator for all research described in this report, and has the overall responsibility for the technical conduct of the projects. In cases where the General Referee has oversight of projects that involve official collaborative studies and validation support from AOAC INTERNATIONAL, AOAC officials and the Committee Chair will determine the appropriate mechanism for formal study review. The 2003 General Referee report provides the background on the development and direction of the research projects. The preliminary data, general conclusions, next steps, and recommendations are provided in this report.