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.     

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.     

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.     

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.     

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.     

Evaluation of Different Interstimulus Rinse Protocols on Smoke Attribute Perception in Wildfire-Affected Wines

Wildfires produce smoke that can carry organic compounds to a vineyard, which are then absorbed by the grape berry and result in wines with elevated levels of smoke-related phenols. These phenols have been found to have a large impact on the flavor of wines, being the cause of a smokey flavor with a lasting ashy aftertaste. When evaluating the sensory profile of these wines, there is an observed problem due to the lasting nature of these undesirable attributes and potential flavor carryover between samples. Through the use of standard and temporal attribute check-all-that-apply, this research desires to better understand the impact of smoke on the sensorial profiles of wines with various levels of smoke phenols (high, moderate, and low). Additionally, through the employment of different interstimulus protocols, the effectiveness of rinses on diminishing the smoke flavor in wines and optimal time separation were investigated. It was determined that a 1 g/L pectin rinse in between samples with a 120 s separation is optimal to ensure the removal of smoke attribute perception. This work also indicated the need to look deeper at the effects of the in-mouth hydrolysis of glyconjugate phenols that impact overall smoke flavor.     

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.