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Comparison of 3 in vivo methods for assessment of alcohol-based hand rubs
American Journal of Infection Control xxx (2015) 1-4
Contents lists available at ScienceDirect
American Journal of Infection Control
American Journal of Infection Control
journal homepage: www.ajicjournal.org
Major article
Comparison of 3 in vivo methods for assessment of alcohol-based hand rubs Sarah Edmonds-Wilson MS a, *, Esther Campbell BS b, Kyle Fox BS a, David Macinga PhD a a b
Research & Development GOJO Industries, Inc, Akron, OH BioScience Laboratories, Bozeman, MT
Key Words: Hand hygiene ASTM Test methods Hand sanitizer
Background: Alcohol-based hand rubs (ABHRs) are the primary method of hand hygiene in health-care settings. ICPs increasingly are assessing ABHR product efficacy data as improved products and test methods are developed. As a result, ICPs need better tools and recommendations for how to assess and compare ABHRs. Methods: Two ABHRs (70% ethanol) were tested according to 3 in vivo methods approved by ASTM International: E1174, E2755, and E2784. Log10 reductions were measured after a single test product use and after 10 consecutive uses at an application volume of 2 mL. Results: The test method used had a significant influence on ABHR efficacy; however, in this study the test product (gel or foam) did not significantly influence efficacy. In addition, for all test methods, log10 reductions obtained after a single application were not predictive of results after 10 applications. Conclusions: Choice of test method can significantly influence efficacy results. Therefore, when assessing antimicrobial efficacy data of hand hygiene products, ICPs should pay close attention to the test method used, and ensure that product comparisons are made head to head in the same study using the same test methodology. Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
Alcohol-based hand rubs (ABHRs) are typically evaluated for efficacy in a clinical laboratory setting using standards set by ASTM International. The most commonly used method in North America, the health care personnel handwash method (ASTM E1174), was originally developed to evaluate efficacy of antimicrobial handwashing products before widespread use of ABHR. ASTM E1174 has characteristics that limit its ability to accurately evaluate ABHRs.1,2 Specifically, increasing hand wetness and a buildup of soil load from repeated application of the bacterial suspension dilutes the alcohol, which reduces alcohol’s antimicrobial activity. Both the Centers for Disease Control and Prevention and the World Health Organization acknowledge weaknesses in the methods and emphasize the need to develop more effective methods.3,4 For this reason new methods have been developed by ASTM in recent years, including ASTM E2755 and ASTM E2784, which aim to make hand contamination and product assessment more realistic.1,5e7
* Address correspondence to Sarah Edmonds-Wilson, MS, GOJO Industries Inc, PO Box 991, Akron, OH 44309. E-mail address: [email protected] (S. Edmonds-Wilson). Conflicts of interest: None to report.
Before sale of new ABHRs into health care settings, manufacturers must evaluate their antimicrobial efficacy performance. Whereas the Food and Drug Administration currently requires evaluation of ABHR by a previous version of E1174,8 recent articles have questioned the use of E1174 for assessment of ABHR9 and have implied that use of the newer methods (eg, E2755) would provide different results that would invalidate conclusions made with the widely accepted E1174 standard. However, there are no published studies to date that directly compare the efficacy of an ABHR when tested by the 3 in vivo ASTM methods. The purpose of our study was to directly compare the antimicrobial efficacy results of ABHRs tested by E1174, E2755, and E2784 standards. MATERIALS AND METHODS Test products were Purell Advanced Instant Hand Sanitizer and Purell Advanced Instant Hand Sanitizer Foam, both containing 70% ethanol as the active ingredient, and manufactured by GOJO Industries, Inc. Participants were recruited from the general population according to standard operating procedures for BioScience
0196-6553/$36.00 - Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajic.2015.01.025
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S. Edmonds-Wilson et al. / American Journal of Infection Control xxx (2015) 1-4
Table 1 Comparison of in vivo test method inoculum application instructions ASTM Method E1174
E2755
E2784
Inoculum application instructions Three aliquots of 1.5 mL (4.5 mL total) containing w 1 109 CFU/mL Serratia marcescens were placed in participant’s cupped hands and distributed evenly over both hands after each application for 20 sec, then allowed to dry for 30 sec between applications and 90 sec after final application A 0.2-mL aliquot containing w1 1010 CFU/mL S marcescens was transferred into the participant’s cupped hands and distributed evenly over both hands for 30 sec Two 30-mL aliquots containing w1 109 CFU/mL S marcescens were distributed and allowed to evenly saturate the surface of 2 sterile paper towels. The participant centered each hand directly above the individual paper towels, and pressed down firmly for 5 sec. The entire palm, fingers, and finger pads were in contact with the saturated towel. Hands were then held motionless and allowed to dry for 90 sec.
Laboratories following approval of the protocol by the Gallatin Institutional Review Board. Seventy-two participants were assigned randomly using the Minitab statistical computer package (Minitab Inc., State College, PA) to evaluate 1 of the 2 test products by 1 of the 3 ASTM methods, for a total of 12 participants per test configuration. For all test methods the indicator microorganism, Serratia marcescens (ATCC #14756), was prepared as described previously.2,5,6 Table 1 describes the hand contamination steps for each method. For all test configurations hands were contaminated 11 times, with the first hand contamination followed by a sample for baseline microbial counts and the remaining 10 contaminations followed by product applications. Microbial samples were taken after baseline and product applications 1, 3, 7, and 10. Samples taken at product applications 3 and 7 were not processed for microbial enumeration. For product application, 2 mL gel product and 3 pumps of the foam product (about 2 mL) were dispensed into participants’ cupped hands, then product was rubbed by the participant over the entire surface of the hands and fingers until dry. Samples were taken using the Glove Juice Sampling Procedure, where powder-free sterile latex gloves were placed on participants’ hands, 75 mL sampling solution (previously described2,5,6) was introduced into each of the gloves, and hands were massaged for 60 seconds. Samples were serially diluted in a dilution solution with neutralizer (previously described2,5,6) and plated on tryptic soy agar plates incubated at 35 C until sufficient growth was observed. Only colonies producing a red pigment indicative of S marcescens were counted. Statistical comparisons were made between test products, ASTM methods, and combinations of each to evaluate efficacy. All statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, NC) and R version 3.0.3 (R Foundation for Statistical Computing, Vienna, Austria) with a type I error rate of 5%. Mean log10 reductions were calculated using log10 reduction values for both the left and right hand of each participant. Paired t tests were used to determine the differences between log10 reductions for application 1 and application 10 for each product and method. Two-way analysis of variance (ANOVA) models that combined gel and foam product were created to specifically examine the effects of the ASTM test method at different applications. Two-way ANOVA with interaction at both application 1 and application 10 was used to determine if product and/or test method had the greatest influence on the outcome log10 reduction. StudentNewman-Keuls tests and Tukey’s method tests were performed to evaluate the direction and magnitude of pairwise combinations of product and test. P values < .05 were considered to be significant.
Table 2 Comparison of products by method: Mean baseline log10 recovery summary statistics among each product by ASTM International method with StudentNewman-Keuls test comparison
Product
N
Foam 12 Gel 12
Method E1174 E2755 E2784 E1174 E2755 E2784
Mean log10 recovery
Standard deviation
95% Confidence interval
9.21 9.11 7.44 9.28 9.14 7.28
0.250 0.201 0.368 0.171 0.316 0.445
(9.05-9.37) (8.98-9.24) (7.21-7.67) (9.17-9.38) (8.94-9.34) (7.00-7.56)
P value <.0001*
<.0001*
*Statistically significant at the 95% confidence level.
RESULTS Table 2 illustrates a summary of baseline results by product and ASTM method. The Student-Newman-Keuls test value for each individual mean log10 reduction allows for pairwise comparisons among ASTM methods. Statistically significant differences are observed where log10 recoveries obtained with ASTM E1174 and ASTM E2755 were equivalent, and the baseline values for the ASTM E2784 were significantly lower. Table 3 summarizes test product log10 reductions by product, application, and method. Efficacy was found to increase significantly from application 1 to application 10 for the ABHR gel using all 3 test methods. Similarly, for the ABHR foam, log10 reductions were significantly higher at application 10 than at application 1 when tested according to E1174 and E2755. However, no significant difference (P ¼ .0940) in log10 reduction for foam was observed from application 1 to application 10 when E2784 was used. The Student-Newman-Keuls test value for each individual mean log10 reduction allows for pairwise comparisons among ASTM methods at applications 1 and 10. Statistically significant differences were observed according to specific combinations of product, test, and method as shown in Table 3. These results demonstrate that efficacy is influenced by multiple factors, including product formulation, application number (1 or 10), and test method. There was a significant interaction between ASTM method and product (P ¼ .0272), suggesting that it is not possible to understand efficacy by only knowing the test product; one must also consider the method. In addition, when evaluating application 10, main effect results from a 2-way ANOVA, including ASTM method (P ¼ .0010) and product (P ¼ .2589), are indicative of a stronger effect of the test method and a nonsignificant effect of the specific product formulation for this study. DISCUSSION The results presented in Table 3 indicate that in this study the ASTM method used has a significant influence on the observed efficacy of ABHR formulations. These differences are likely attributable to differences in the hand contamination procedures between the 3 methods, as highlighted in Table 1. For example, the baselines obtained with the E2784 method were significantly lower than the baselines for E1174 and E2755, and E2784 was associated with the largest log10 reductions. Starting with lower baseline levels of bacteria, and therefore a lower bioburden, may make killing microorganisms easier, particularly after multiple uses because there would be less influence from microorganism buildup. Furthermore, previous studies10 have shown that rubbing the inoculum into hands makes the bacteria more difficult to kill. The E1174 and E2755 methods both include a rub-in step during inoculum application, whereas E2784 does not. This also likely
S. Edmonds-Wilson et al. / American Journal of Infection Control xxx (2015) 1-4
3
Table 3 Statistical comparison of mean log10 reductions, by ASTM International method Product Foam
Application
N
Method
1
12 12 12 12 12 12 12 12 12 12 12 12
E1174 E2755 E2784 E1174 E2755 E2784 E1174 E2755 E2784 E1174 E2755 E2784
10
Gel
1
10
Mean log10 reduction
Standard deviation
95% Confidence interval
P value
2.69b 3.29b 4.57a 4.35ab 4.06b 5.06a 2.54a 3.33b 4.48c 3.91b 5.06a 5.24a
0.556 0.941 1.132 1.376 0.889 0.465 0.498 1.066 0.821 0.890 0.987 0.518
(2.33-3.04) (2.70-3.89) (3.85-5.29) (3.48-5.23) (3.50-4.63) (4.77-5.36) (2.22-2.85) (2.65-4.00) (3.96-5.00) (3.34-4.47) (4.43-5.69) (4.91-5.57)
<.0001*
.0506
<.0001*
.0006*
NOTE. For each individual test (ie, application 1 and application 10), common superscript letters (a, b) for each mean log10 reduction indicate no statistically significant difference at the 95% confidence level. *Statistically significant at the 95% confidence level.
contributed to the consistently higher log10 reductions observed with E2784. Consistent with Macinga et al,1 log reductions were higher when E2755 was used compared with when E1174 was used, this due to increased wetness and soil load associated with the E1174 hand contamination procedure (4.5 mL) compared with E2755 (0.2 mL). The reduced soil load and overall volume of inoculum (and associated reduced hand wetness) applied to hands is lower and more realistic in both the E2784 and E2755 methods,1,7 which enables ABHR to more effectively reduce bacteria on hands with these methods than in E1174, and likely contributed to the consistently lower log10 reductions observed with E1174. The significant influence of method choice on product efficacy outcome highlights the recommendation that direct comparisons of products be done within the same study using the same test methodology. Minimal differences were observed between the 2 test products evaluated in our study. This observation was not unexpected because the test products each contain 70% ethanol and contain nearly identical excipient ingredient composition.11 A previous study evaluating these formulations using the ASTM E1174 method demonstrated them to be equivalent to each other.11 Additionally, a critical component of efficacy is the amount of time it takes for an ABHR to dry on the skin, and the gel and foam tested in our study were previously shown to have equivalent dry times, which further supports the finding of similar efficacy with the various test methods.12 It is anticipated that if other hand hygiene products based on different formulation technologies or that contain different active ingredients were tested, there would be a significant influence on efficacy. The efficacy of both the gel and foam formulations in this study increased significantly from application 1 to application 10, for all methods except when the foam was evaluated by E2784, which showed a nonsignificant increase of approximately 0.5 log10. This change in efficacy over multiple uses highlights the importance of evaluating efficacy after multiple uses when products are intended for use in health care settings. Efficacy results after a single use, regardless of method used, were not predictive of results after multiple10 uses. As shown previously, product buildup after multiple uses can have significant positive (or negative) influence on efficacy.11 In addition, based on current regulations for health care personnel handwashes, the Food and Drug Administration requires a 3-log reduction after 10 applications using the E1174 method, and based on our study it is not possible to extrapolate data from application 1 to application 10.8 Therefore, infection control professionals (ICPs) should ensure that ABHRs used in their facility are evaluated using in vivo methods after both a single application and multiple applications.
Future studies should evaluate the efficacy of additional types of product formulations using these various methods to determine if all are equally adept at differentiating between products. In previous studies E2755 was found to be more appropriate than E1174 for comparing ABHRs, and our study showed E2755 differentiated products effectively, particularly at application 10.1 This is particularly important when screening formulations during product development or when making comparisons between products from different manufacturers, because you must be able to detect real differences in product efficacy that may have an influence on clinical efficacy in hospital settings. CONCLUSIONS ICPs should pay attention to the antimicrobial efficacy data of hand hygiene agents, including ABHRs, and ensure that products are tested by multiple test methods if possible and that product comparisons are made head to head in the same study using the same test methodology. As a result ICPs will be able to make more informed decisions about the hand hygiene products to use in their facilities. References 1. Macinga DR, Beausoleil CM, Campbell E, Mulberry G, Brady A, Edmonds SL, et al. Quest for a realistic in vivo test method for antimicrobial hand-rub agents: Introduction of a low-volume hand contamination procedure. Appl Environ Microbiol 2011;77:8588-94. 2. American Society for Testing Materials International. Standard test method for evaluation of the effectiveness of health care personnel or consumer handwash formulations. Designation: E1174e10. West Conshohocken [PA]: American Society for Testing Materials; 2010. 3. Boyce JM, Pittet D, Healthcare Infection Control Practices Advisory Committee. Society for Healthcare Epidemiology of America. Association for Professionals in Infection Control. Infectious Diseases Society of America. Hand Hygiene Task Force. Guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the HICPAC/ SHEA/APIC/IDSA hand hygiene task force. Infect Control Hosp Epidemiol 2002; 23(12 Suppl):S3-40. 4. WHO guidelines on hand hygiene in health care. Geneva, Switzerland: World Health Organization; 2009. 5. American Society for Testing Materials International. Standard test method for determining the bacteria-eliminating effectiveness of hand sanitizer formulations using hands of adults. Designation: E2755e10. West Conshohocken [PA]: American Society for Testing Materials; 2010. 6. American Society for Testing Materials International. Standard test method for evaluation of the effectiveness of handwash formulations using the paper towel (Palmar) method of hand contamination. Designation: E2784e10. West Conshohocken [PA]: American Society for Testing Materials; 2010. 7. Fuls JL, Rodgers ND, Fischler GE, Howard JM, Patel M, Weidner PL, et al. Alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions. App Environ Microbiol 2008;74:3739-44.
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8. Food and Drug Administration. Tentative final monograph for healthcare antiseptic drug Products; proposed rule. Fed Regist 1994;59:31441-52. 9. Eggerstedt S. Comparative efficacy of commercially available alcohol-based hand rubs and World Health Organization recommended hand rubs. Am J Infect Control 2013;41:472-4. 10. Rotter M, Koller W. Test models for hygienic handrub and hygienic handwash: the effects of two different contamination and sampling techniques. J Hosp Infect 1992;20:163-71.
11. Edmonds SL, Macinga DR, Mays-Suko P, Duley C, Rutter J, Jarvis W, et al. Comparative efficacy of commercially available alcohol-based hand rubs and world health organization-recommended hand rubs: formulation matters. Am J Infect Control 2012;40:521-5. 12. Macinga DR, Shumaker DJ, Werner HP, Edmonds SL, Leslie RA, Parker AE, et al. The relative influences of product volume, delivery format and alcohol concentration on dry-time and efficacy of alcohol-based hand rubs. BMC Infect Dis 2014;14:511.
Contents lists available at ScienceDirect
American Journal of Infection Control
American Journal of Infection Control
journal homepage: www.ajicjournal.org
Major article
Comparison of 3 in vivo methods for assessment of alcohol-based hand rubs Sarah Edmonds-Wilson MS a, *, Esther Campbell BS b, Kyle Fox BS a, David Macinga PhD a a b
Research & Development GOJO Industries, Inc, Akron, OH BioScience Laboratories, Bozeman, MT
Key Words: Hand hygiene ASTM Test methods Hand sanitizer
Background: Alcohol-based hand rubs (ABHRs) are the primary method of hand hygiene in health-care settings. ICPs increasingly are assessing ABHR product efficacy data as improved products and test methods are developed. As a result, ICPs need better tools and recommendations for how to assess and compare ABHRs. Methods: Two ABHRs (70% ethanol) were tested according to 3 in vivo methods approved by ASTM International: E1174, E2755, and E2784. Log10 reductions were measured after a single test product use and after 10 consecutive uses at an application volume of 2 mL. Results: The test method used had a significant influence on ABHR efficacy; however, in this study the test product (gel or foam) did not significantly influence efficacy. In addition, for all test methods, log10 reductions obtained after a single application were not predictive of results after 10 applications. Conclusions: Choice of test method can significantly influence efficacy results. Therefore, when assessing antimicrobial efficacy data of hand hygiene products, ICPs should pay close attention to the test method used, and ensure that product comparisons are made head to head in the same study using the same test methodology. Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
Alcohol-based hand rubs (ABHRs) are typically evaluated for efficacy in a clinical laboratory setting using standards set by ASTM International. The most commonly used method in North America, the health care personnel handwash method (ASTM E1174), was originally developed to evaluate efficacy of antimicrobial handwashing products before widespread use of ABHR. ASTM E1174 has characteristics that limit its ability to accurately evaluate ABHRs.1,2 Specifically, increasing hand wetness and a buildup of soil load from repeated application of the bacterial suspension dilutes the alcohol, which reduces alcohol’s antimicrobial activity. Both the Centers for Disease Control and Prevention and the World Health Organization acknowledge weaknesses in the methods and emphasize the need to develop more effective methods.3,4 For this reason new methods have been developed by ASTM in recent years, including ASTM E2755 and ASTM E2784, which aim to make hand contamination and product assessment more realistic.1,5e7
* Address correspondence to Sarah Edmonds-Wilson, MS, GOJO Industries Inc, PO Box 991, Akron, OH 44309. E-mail address: [email protected] (S. Edmonds-Wilson). Conflicts of interest: None to report.
Before sale of new ABHRs into health care settings, manufacturers must evaluate their antimicrobial efficacy performance. Whereas the Food and Drug Administration currently requires evaluation of ABHR by a previous version of E1174,8 recent articles have questioned the use of E1174 for assessment of ABHR9 and have implied that use of the newer methods (eg, E2755) would provide different results that would invalidate conclusions made with the widely accepted E1174 standard. However, there are no published studies to date that directly compare the efficacy of an ABHR when tested by the 3 in vivo ASTM methods. The purpose of our study was to directly compare the antimicrobial efficacy results of ABHRs tested by E1174, E2755, and E2784 standards. MATERIALS AND METHODS Test products were Purell Advanced Instant Hand Sanitizer and Purell Advanced Instant Hand Sanitizer Foam, both containing 70% ethanol as the active ingredient, and manufactured by GOJO Industries, Inc. Participants were recruited from the general population according to standard operating procedures for BioScience
0196-6553/$36.00 - Copyright Ó 2015 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajic.2015.01.025
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S. Edmonds-Wilson et al. / American Journal of Infection Control xxx (2015) 1-4
Table 1 Comparison of in vivo test method inoculum application instructions ASTM Method E1174
E2755
E2784
Inoculum application instructions Three aliquots of 1.5 mL (4.5 mL total) containing w 1 109 CFU/mL Serratia marcescens were placed in participant’s cupped hands and distributed evenly over both hands after each application for 20 sec, then allowed to dry for 30 sec between applications and 90 sec after final application A 0.2-mL aliquot containing w1 1010 CFU/mL S marcescens was transferred into the participant’s cupped hands and distributed evenly over both hands for 30 sec Two 30-mL aliquots containing w1 109 CFU/mL S marcescens were distributed and allowed to evenly saturate the surface of 2 sterile paper towels. The participant centered each hand directly above the individual paper towels, and pressed down firmly for 5 sec. The entire palm, fingers, and finger pads were in contact with the saturated towel. Hands were then held motionless and allowed to dry for 90 sec.
Laboratories following approval of the protocol by the Gallatin Institutional Review Board. Seventy-two participants were assigned randomly using the Minitab statistical computer package (Minitab Inc., State College, PA) to evaluate 1 of the 2 test products by 1 of the 3 ASTM methods, for a total of 12 participants per test configuration. For all test methods the indicator microorganism, Serratia marcescens (ATCC #14756), was prepared as described previously.2,5,6 Table 1 describes the hand contamination steps for each method. For all test configurations hands were contaminated 11 times, with the first hand contamination followed by a sample for baseline microbial counts and the remaining 10 contaminations followed by product applications. Microbial samples were taken after baseline and product applications 1, 3, 7, and 10. Samples taken at product applications 3 and 7 were not processed for microbial enumeration. For product application, 2 mL gel product and 3 pumps of the foam product (about 2 mL) were dispensed into participants’ cupped hands, then product was rubbed by the participant over the entire surface of the hands and fingers until dry. Samples were taken using the Glove Juice Sampling Procedure, where powder-free sterile latex gloves were placed on participants’ hands, 75 mL sampling solution (previously described2,5,6) was introduced into each of the gloves, and hands were massaged for 60 seconds. Samples were serially diluted in a dilution solution with neutralizer (previously described2,5,6) and plated on tryptic soy agar plates incubated at 35 C until sufficient growth was observed. Only colonies producing a red pigment indicative of S marcescens were counted. Statistical comparisons were made between test products, ASTM methods, and combinations of each to evaluate efficacy. All statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, NC) and R version 3.0.3 (R Foundation for Statistical Computing, Vienna, Austria) with a type I error rate of 5%. Mean log10 reductions were calculated using log10 reduction values for both the left and right hand of each participant. Paired t tests were used to determine the differences between log10 reductions for application 1 and application 10 for each product and method. Two-way analysis of variance (ANOVA) models that combined gel and foam product were created to specifically examine the effects of the ASTM test method at different applications. Two-way ANOVA with interaction at both application 1 and application 10 was used to determine if product and/or test method had the greatest influence on the outcome log10 reduction. StudentNewman-Keuls tests and Tukey’s method tests were performed to evaluate the direction and magnitude of pairwise combinations of product and test. P values < .05 were considered to be significant.
Table 2 Comparison of products by method: Mean baseline log10 recovery summary statistics among each product by ASTM International method with StudentNewman-Keuls test comparison
Product
N
Foam 12 Gel 12
Method E1174 E2755 E2784 E1174 E2755 E2784
Mean log10 recovery
Standard deviation
95% Confidence interval
9.21 9.11 7.44 9.28 9.14 7.28
0.250 0.201 0.368 0.171 0.316 0.445
(9.05-9.37) (8.98-9.24) (7.21-7.67) (9.17-9.38) (8.94-9.34) (7.00-7.56)
P value <.0001*
<.0001*
*Statistically significant at the 95% confidence level.
RESULTS Table 2 illustrates a summary of baseline results by product and ASTM method. The Student-Newman-Keuls test value for each individual mean log10 reduction allows for pairwise comparisons among ASTM methods. Statistically significant differences are observed where log10 recoveries obtained with ASTM E1174 and ASTM E2755 were equivalent, and the baseline values for the ASTM E2784 were significantly lower. Table 3 summarizes test product log10 reductions by product, application, and method. Efficacy was found to increase significantly from application 1 to application 10 for the ABHR gel using all 3 test methods. Similarly, for the ABHR foam, log10 reductions were significantly higher at application 10 than at application 1 when tested according to E1174 and E2755. However, no significant difference (P ¼ .0940) in log10 reduction for foam was observed from application 1 to application 10 when E2784 was used. The Student-Newman-Keuls test value for each individual mean log10 reduction allows for pairwise comparisons among ASTM methods at applications 1 and 10. Statistically significant differences were observed according to specific combinations of product, test, and method as shown in Table 3. These results demonstrate that efficacy is influenced by multiple factors, including product formulation, application number (1 or 10), and test method. There was a significant interaction between ASTM method and product (P ¼ .0272), suggesting that it is not possible to understand efficacy by only knowing the test product; one must also consider the method. In addition, when evaluating application 10, main effect results from a 2-way ANOVA, including ASTM method (P ¼ .0010) and product (P ¼ .2589), are indicative of a stronger effect of the test method and a nonsignificant effect of the specific product formulation for this study. DISCUSSION The results presented in Table 3 indicate that in this study the ASTM method used has a significant influence on the observed efficacy of ABHR formulations. These differences are likely attributable to differences in the hand contamination procedures between the 3 methods, as highlighted in Table 1. For example, the baselines obtained with the E2784 method were significantly lower than the baselines for E1174 and E2755, and E2784 was associated with the largest log10 reductions. Starting with lower baseline levels of bacteria, and therefore a lower bioburden, may make killing microorganisms easier, particularly after multiple uses because there would be less influence from microorganism buildup. Furthermore, previous studies10 have shown that rubbing the inoculum into hands makes the bacteria more difficult to kill. The E1174 and E2755 methods both include a rub-in step during inoculum application, whereas E2784 does not. This also likely
S. Edmonds-Wilson et al. / American Journal of Infection Control xxx (2015) 1-4
3
Table 3 Statistical comparison of mean log10 reductions, by ASTM International method Product Foam
Application
N
Method
1
12 12 12 12 12 12 12 12 12 12 12 12
E1174 E2755 E2784 E1174 E2755 E2784 E1174 E2755 E2784 E1174 E2755 E2784
10
Gel
1
10
Mean log10 reduction
Standard deviation
95% Confidence interval
P value
2.69b 3.29b 4.57a 4.35ab 4.06b 5.06a 2.54a 3.33b 4.48c 3.91b 5.06a 5.24a
0.556 0.941 1.132 1.376 0.889 0.465 0.498 1.066 0.821 0.890 0.987 0.518
(2.33-3.04) (2.70-3.89) (3.85-5.29) (3.48-5.23) (3.50-4.63) (4.77-5.36) (2.22-2.85) (2.65-4.00) (3.96-5.00) (3.34-4.47) (4.43-5.69) (4.91-5.57)
<.0001*
.0506
<.0001*
.0006*
NOTE. For each individual test (ie, application 1 and application 10), common superscript letters (a, b) for each mean log10 reduction indicate no statistically significant difference at the 95% confidence level. *Statistically significant at the 95% confidence level.
contributed to the consistently higher log10 reductions observed with E2784. Consistent with Macinga et al,1 log reductions were higher when E2755 was used compared with when E1174 was used, this due to increased wetness and soil load associated with the E1174 hand contamination procedure (4.5 mL) compared with E2755 (0.2 mL). The reduced soil load and overall volume of inoculum (and associated reduced hand wetness) applied to hands is lower and more realistic in both the E2784 and E2755 methods,1,7 which enables ABHR to more effectively reduce bacteria on hands with these methods than in E1174, and likely contributed to the consistently lower log10 reductions observed with E1174. The significant influence of method choice on product efficacy outcome highlights the recommendation that direct comparisons of products be done within the same study using the same test methodology. Minimal differences were observed between the 2 test products evaluated in our study. This observation was not unexpected because the test products each contain 70% ethanol and contain nearly identical excipient ingredient composition.11 A previous study evaluating these formulations using the ASTM E1174 method demonstrated them to be equivalent to each other.11 Additionally, a critical component of efficacy is the amount of time it takes for an ABHR to dry on the skin, and the gel and foam tested in our study were previously shown to have equivalent dry times, which further supports the finding of similar efficacy with the various test methods.12 It is anticipated that if other hand hygiene products based on different formulation technologies or that contain different active ingredients were tested, there would be a significant influence on efficacy. The efficacy of both the gel and foam formulations in this study increased significantly from application 1 to application 10, for all methods except when the foam was evaluated by E2784, which showed a nonsignificant increase of approximately 0.5 log10. This change in efficacy over multiple uses highlights the importance of evaluating efficacy after multiple uses when products are intended for use in health care settings. Efficacy results after a single use, regardless of method used, were not predictive of results after multiple10 uses. As shown previously, product buildup after multiple uses can have significant positive (or negative) influence on efficacy.11 In addition, based on current regulations for health care personnel handwashes, the Food and Drug Administration requires a 3-log reduction after 10 applications using the E1174 method, and based on our study it is not possible to extrapolate data from application 1 to application 10.8 Therefore, infection control professionals (ICPs) should ensure that ABHRs used in their facility are evaluated using in vivo methods after both a single application and multiple applications.
Future studies should evaluate the efficacy of additional types of product formulations using these various methods to determine if all are equally adept at differentiating between products. In previous studies E2755 was found to be more appropriate than E1174 for comparing ABHRs, and our study showed E2755 differentiated products effectively, particularly at application 10.1 This is particularly important when screening formulations during product development or when making comparisons between products from different manufacturers, because you must be able to detect real differences in product efficacy that may have an influence on clinical efficacy in hospital settings. CONCLUSIONS ICPs should pay attention to the antimicrobial efficacy data of hand hygiene agents, including ABHRs, and ensure that products are tested by multiple test methods if possible and that product comparisons are made head to head in the same study using the same test methodology. As a result ICPs will be able to make more informed decisions about the hand hygiene products to use in their facilities. References 1. Macinga DR, Beausoleil CM, Campbell E, Mulberry G, Brady A, Edmonds SL, et al. Quest for a realistic in vivo test method for antimicrobial hand-rub agents: Introduction of a low-volume hand contamination procedure. Appl Environ Microbiol 2011;77:8588-94. 2. American Society for Testing Materials International. Standard test method for evaluation of the effectiveness of health care personnel or consumer handwash formulations. Designation: E1174e10. West Conshohocken [PA]: American Society for Testing Materials; 2010. 3. Boyce JM, Pittet D, Healthcare Infection Control Practices Advisory Committee. Society for Healthcare Epidemiology of America. Association for Professionals in Infection Control. Infectious Diseases Society of America. Hand Hygiene Task Force. Guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the HICPAC/ SHEA/APIC/IDSA hand hygiene task force. Infect Control Hosp Epidemiol 2002; 23(12 Suppl):S3-40. 4. WHO guidelines on hand hygiene in health care. Geneva, Switzerland: World Health Organization; 2009. 5. American Society for Testing Materials International. Standard test method for determining the bacteria-eliminating effectiveness of hand sanitizer formulations using hands of adults. Designation: E2755e10. West Conshohocken [PA]: American Society for Testing Materials; 2010. 6. American Society for Testing Materials International. Standard test method for evaluation of the effectiveness of handwash formulations using the paper towel (Palmar) method of hand contamination. Designation: E2784e10. West Conshohocken [PA]: American Society for Testing Materials; 2010. 7. Fuls JL, Rodgers ND, Fischler GE, Howard JM, Patel M, Weidner PL, et al. Alternative hand contamination technique to compare the activities of antimicrobial and nonantimicrobial soaps under different test conditions. App Environ Microbiol 2008;74:3739-44.
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8. Food and Drug Administration. Tentative final monograph for healthcare antiseptic drug Products; proposed rule. Fed Regist 1994;59:31441-52. 9. Eggerstedt S. Comparative efficacy of commercially available alcohol-based hand rubs and World Health Organization recommended hand rubs. Am J Infect Control 2013;41:472-4. 10. Rotter M, Koller W. Test models for hygienic handrub and hygienic handwash: the effects of two different contamination and sampling techniques. J Hosp Infect 1992;20:163-71.
11. Edmonds SL, Macinga DR, Mays-Suko P, Duley C, Rutter J, Jarvis W, et al. Comparative efficacy of commercially available alcohol-based hand rubs and world health organization-recommended hand rubs: formulation matters. Am J Infect Control 2012;40:521-5. 12. Macinga DR, Shumaker DJ, Werner HP, Edmonds SL, Leslie RA, Parker AE, et al. The relative influences of product volume, delivery format and alcohol concentration on dry-time and efficacy of alcohol-based hand rubs. BMC Infect Dis 2014;14:511.