Document ID: EPA-HQ-OPP-2016-0467-0031
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2016-08-10T04:00Z

Suitability of the Protocol:  The protocol has been revised to include additional methodology for preparing the test microbe, chemical and physical abrasion, details on materials and supplies, and quality control practices. Refer to Appendix A for an overview of the protocol changes. Please comment on the following issues:   
 Do the revisions summarized in Appendix A provide a substantial improvement and technically sound approach for testing the antimicrobial properties and product durability of solid copper/copper alloy materials?  If not, please provide advice on any additional elements that should be addressed or modified.   
            
 The approach appears to be sound.  However, unlike other disinfectant and sanitizers resistance does occur to some extent and a number of copper resistance genes have been identified (eg., copB, copC, copY, cueO, tcrA, tcrB, tcrZ, etc).  Should a cooper resistant/tolerant strain be included in the efficacy testing?
            
 What do we know about neutralizing capacity of the recovery medium Cu[2][+] so that any potential carry-over of copper into the culture media would not interfere with recovery of the test organisms.  Microbiologic methods from the enclosed studies are relatively lacking sufficient detail to ensure.  The references by Schmidt cite an early study (again lacks detail but looks like lecithin was used as a neutralizer).  Should recovery media include a known metal chelating agent (EDA, DETA, EDTA, thioglycollate or other known chelating agent).  Currently published studies do not appear to use a chelating agent.
            
 Should we incorporate thioglycollate (see:  Landeen LK, Yahya MT, Gerba CP.  Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.  Appl Environ Microbiol 1989;55(12):3045-3050 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC203221/) 
            
            
 Is the protocol of sufficient detail so that it may be conducted by a qualified testing laboratory and is likely to result in reproducible results when conducted in different testing facilities and/or at different times within the same laboratory?  If the protocol is not of sufficient detail, which areas require improvement? 

            So if MB-25 is used with TSB as the growth medium do we know whether some small amount of copper that may end up in solution is not inhibitory to the test organisms leading to false results.  Should thioglycollate be included be incorporated?  Need to include a more detailed section regarding neutralization and give some examples of materials to be used in the neutralization of Cu[2][+].
            
 Is the protocol suitable for evaluating the antimicrobial efficacy of solid copper/copper alloy materials, as well as copper-impregnated or coated surface materials?  
            
            Yes, once neutralization or chelation of residual Cu[2+] is addressed in sufficient detail to allow a standardized approach for different laboratories to use.
            
 If the protocol is not suitable for one or both, please provide advice on how to modify the protocol to cover both materials. 
 Copper tolerance and resistance does occur in nature among strains representing several genera of both gram-negative and positive bacteria (see 3b). Including some strains of MRSA (see below Emergence of copper resistant MRSA in Irish hospitals).  This is especially true among strains among food animals (cows and pigs).  Its theoretically possible that some of these strains could colonize humans.
 Kinnevey PM, Shore AC, Brennan Gi, Sullivan DJ, Ehricht R, Monecke S, Slickers F, Coleman DC.  Emergence of sequence type 779 methicillin resistant Staphylococcus aureus harboring a novel pseudo staphylococcal cassette chromosome mec (SCCmec)-SSC-SCCCRISPR composite element in Irish hospitals.  Antimicrobial Agents Chemother 2013;57(1):524-531 (Available from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535981) 
                  
 There is an interest in using the protocol to evaluate other types of hard non-porous surfaces impregnated with antimicrobial agents other than copper (other solid metals, metal alloys, fabricated materials etc.).  Is the protocol suitable for testing the antimicrobial activity of other types of hard, non-porous surfaces treated or impregnated with antimicrobial agents?  
            
 The general protocol could serve as template for other surfaces.  The carriers for controls however, may not necessarily be stainless steel.  They may reflect the base (non-impregnated material). 
            
 As for nonporous surfaces, until and agreed upon standardized method is available to test the efficacy of disinfectants against nonporous surfaces this is probably not ready to move forward as of yet.
            
            If not, please explain why and offer advice on how to change the protocol so that it would produce reliable, reproducible results when testing these other surface types.
            
 Is it possible to use a modified version of the AATCC TM 100 Antibacterial textile testing for some of the porous materials (American Association of Textile Chemists and Colorists).  This may require broader discussion.
 Even for EPA registered disinfectants there are no products registered for porous materials.  There are registered carpet and upholstery cleaners and 

 Controls:  Stainless steel was selected as the control carrier material due to the inert nature of the material.  The final log reduction values are calculated by taking the log 10 difference between the stainless steel control carriers and the product test carriers.
 In the protocol, the stainless steel control carriers are not subjected to the mechanical surface abrasion or the chemical treatments (A, B, and C). Please comment on the suitability of this approach.  If this approach is not appropriate, please provide advice on how to address the management of the control carriers.
 The untreated stainless steel coupons provide a baseline control of organisms deposited onto an untreated surface.  
 One should remember that different surface materials have different characteristics that can influence drying, persistence, and survival.  Some surface characteristics also impact bacterial recovery (eg., ability to remove some organisms from the surfaces) when other parameters are equivalent (temp, RH, matrices, etc).  This is true of most persistence studies (see studies on Ebola, Yersinia pestis, and others.
Cook BW, Cutts TA, Nikiforuk AM, Poliquin PG, Court DA, Strong JE, Theriault SS.  Evaluating environmental persistence and disinfection of the Ebola virus Makona variant. Viruses 201514;7(4):1975-86. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411685/) 
Rose LJ, Donlan R, Banerjee SN, Arduino MJ.  Survival of Yersinia pestis on environmental surfaces.  Appl Environ Microbiol  2003;69(4):2166-2171 (http://www.ncbi.nlm.nih.gov/pmc/article/PMC154802)
Sagripanti JL, Rom AM, Holland LE.  Persistence in darkness of virulent alphaviruses, Ebola virus, and Lassa virus deposited on solid surfaces.   Arch Virol. 2010;155(12):2035-9 (http://link.springer.com/article/10.1007%2Fs00705-010-0791-0). 
                  
 Please comment on whether the comparative analyses of log reduction values (i.e., the difference in the level of microbes on exposed carriers vs. unexposed carriers and stainless steel control carriers) is a technically sound approach to the assessment of the antimicrobial activity of the copper and copper alloy products.
            
            One might also consider comparing surface abraded and chemically treated control stainless steel carriers to see how these compare to the exposed carriers.  While the Stainless steel control carriers base ideal (smooth surface, clean surface), a set of abraded, chemically treated carriers might represent an additional test comparable challenge to the exposed carriers.  This assumes that exposed carriers (physical and chemically treated) may provide areas where microbes would be harder to remove, or offer areas of protection when combined with soil.

 Contact Time:  EPA's current guidance requires that a hospital disinfectant kill between five to six logs (100,000 to 1,000,000) of the target microbe in a qualitative test system within the time frame specified on the product labeling.  The use of copper and copper alloy products in medical care facilities is a supplement to (not a replacement for) standard infection control practices and use of EPA registered hospital disinfectants.  
 In a standard chemical disinfectant test, the microbe is applied to the carrier surface, allowed to dry, and then exposed to the disinfectant.  The contact time for the chemical disinfectant begins upon application of the disinfectant.  For copper and copper alloy materials, the surface serves as the antimicrobial agent.  The protocol specifies that the contact time begin upon application of the microbe to the surface, not after the microbe has dried on the surface.  Please comment on whether it is appropriate for the contact time to begin upon inoculation of the surface, and if not, please offer alternative approaches for this step in the protocol.
 The theory behind the copper impregnated surfaces is the the activity begins on contact with the surface.  So the activity begins at inoculation.  The current protocol accounts for this the initiation of contact time (CT) begins upon inoculation of the carrier and then the carrier is allowed to sit for 1 hr (EPA.  Protocol for the evaluation of bactericidal activity of Hard, non-porous copper/copper-alloy surfaces, rev 02/03/15) and is adequate. 
                  
 Similar studies are performed when evaluating the disinfection of pathogens in water, where the measurement of CT starts at the onset of inoculation. 
 Please comment on whether a single inoculation per carrier (4-5 logs bacteria per carrier) for both Staphylococcus aureus and Pseudomonas aeruginosa provides adequate challenge to evaluate the level of antimicrobial activity.  A soil load (three-part) is also added to the inoculum before carrier inoculation.  If a single inoculation is not appropriate, explain why and provide suggestions on how to improve the inoculation procedure.
              
 A single inoculum per carrier should be sufficient as long as data from replicates are consistent.  By using 4-5 logs of test organisms one should be able to note a 3 log reduction.  If wanting to measure more than 3 logs one might use a single inoculum containing > 10[5]-10[6] CFU.  
            
 There are organisms that exhibit heavy metal resistance/tolerance and in some of these are copper resistant or tolerant (eg. Strains of Cronobacter sakazakii, Cupriavidus, Enterococcus faecalis, Escherichia coli, Ralstonia, Salmonella etc.)  I've highlighted a few references:
            
Brown NL, Rouch DA, Lee BT. Copper resistance determinants in bacteria.  Plasmids 1992;27(1):41-51.
            
Cervantes C, Gutierrez-Corona F. Copper resistance mechanisms in bacteria and fungi.  FEMS Microbiol Rev 1994;14(2):121-137
Dupont CL, Grass G, Rensing C.  Copper toxicity and the origin of bacterial resistance -- new insights and applications.  Metallomics 2011;3(11):1109-18.
Silveira E, Freitas AR, Antunes P, Barros M, Campos J, Coque TM, Peixe L, Novais C.  Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages. J Antimicrob Chemother 2014;69(4):899-906. (available from http://jac.oxfordjournals.org/content/69/4/899.long) 
            
 Based on the Agency's experience in utilizing hard non-porous carriers in standard efficacy test methods, microbial populations on environmental surfaces decline naturally over time mainly due to desiccation.  This natural decline presents challenges in determining whether the decline in a microbial population is due to desiccation or antimicrobial activity. An antimicrobial surface such as copper should be capable of accelerating the decrease in the number of surface-associated bacteria.  The Agency expects that an antimicrobial effect due to the product should be measureable within a one hour timeframe. The original protocol specified a 99.9% reduction of viable bacteria within two hours of inoculation while the new protocol specifies a one hour timeframe.   Please comment on the suitability of reducing the timeframe from two hours to one hour, or if the specified timeframe is not reasonable, provide advice on a suitable timeframe.  
            
 Including the use of the untreated controls may assist (stainless steel) in determining the loss upon drying and there actually may be no way to actually account for this loss. 
                  
 However, one must realize that survival on surfaces is a complex and involves suspending media (matrices), surface characteristics, organism factors, temperature and relative humidity.   Depending on the test organisms being used loss with in an hour may be minimal (in many of our ongoing studies of healthcare associated pathogens) > 3 log reduction has been seen after 24 hrs with the exception of Acinetobacter baumannii and the gram positive organisms (eg enterococci, staphylococci, and bacterial endospores).  We do know that Y. pestis has a more rapid decline.
            See:  
Esteves DC, et al.  Influence of biological fluids in bacterial viability on different hospital surfaces and fomites.  Am J Infect Control 2015 Nov 12 (ahead of print)

Kramer A, Schwebke I, Kampf G.  How long do nosocomial pathogens persist on inanimate surfaces?  A systematice review.  BMC Infect Dis 2006;6:130 (available from http://www.ncbi.nlm.nih.gov/pmc/article/PMC1564025) 
            
Rose LJ, Donlan R, Banerjee SN, Arduino MJ.  Survival of Yersinia pestis on environmental surfaces.  Appl Environ Microbiol  2003;69(4):2166-2171 (http://www.ncbi.nlm.nih.gov/pmc/article/PMC154802) 

Shams AM, Rose LJ, Hodges L, Arduino MJ.  Survival of Burkholderia pseudomallei on environmental surfaces.  Appl Environ Microbiol 2007;73(24): 8001-4 
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168135) 

Zarpellon MN, et al. Survival of vancomycin-intermediate Staphylococcus aureus on hospital surfaces.  J Hosp Infect 2015;90(4):347-5.  
      
 Please comment on whether copper and copper alloy products that kill 99.9% of target microbes within 1 hour would provide a significant benefit in reducing levels of target microbes in medical care facilities.  If you think that killing 99.9% of target microbes within 1 hour would not provide a significant benefit in reducing levels of target microbes in medical care facilities, please offer advice on the level of antimicrobial activity that would provide such benefits.  Please explain the basis for your conclusions
            
 Health outcomes data regarding the use of antimicrobial impregnated surfaces that products is still lacking.  Though there are published studies that claim that the use of these copper impregnated surfaces yield reductions in healthcare-associated infections have been published.  However, a recent review (Muller et al, 2016) finds the data quality of these studies to be quite low.  
                  
 More evidence is needed to show a link with log reduction on surfaces and prevention of HAIs.  So unknown whether a log reduction would have an effect, though it may predict the ability to recover certain HAI pathogens, eg., multiply drug resistant ornganisms from the patient care environment.

 From some of our studies of these "noncritical" surfaces" in patient rooms after routine and terminal cleaning is that contamination is quite broad (2014 IDSA Poster attached).

 
Shams A, Rose LJ, Edwards JR McDonald LC, Arduino MJ, Noble-Wang J.  Assessment of the overall and multi-drug resistant organism bioburden on environmental surfaces in healthcare facilities.  Open Forum Infect Dis 2014; 1(Suppl 1):S358,  (http://ofid.oxfordjournals.org.content/1/suppl_1) 
McDonald LC, Arduino MJ.  Editorial commentary: climbing the evidentiary hierarchy for environmental infection control.  Clin Infect Dis 2013;56(1):36-9 (http://cid.oxfordjournals.org/content/56/1/36.long) 
Muller MP, MacDougall C, Lim C, Ontario Agency for Health Protection, PIDAC-IPC. Antimicrobial surfaces to prevent healthcare-associated infections:  a systematic review.  J Hosp Infect 2016;92:7-13. 
Environmental Hygiene in Healthcare Research (September 2015) http://www.cdc.gov/hai/research/eic-meeting.html

 Abrasion/Chemical Treatment: The proposed protocol includes a requirement that the carriers made from the copper or copper alloy undergo both an abrasion step and a chemical treatment step in order to simulate actual conditions of use and to evaluate how abrasion and/or chemical treatment might affect the level of antimicrobial activity.  Note that some disinfectant and sanitizer products, as well as some cleaning agents, contain chelating agents (e.g., EDTA) intended to bind free metal ions.  
 Please comment on whether abrasion and/or chemical treatment is likely to affect the level of antimicrobial activity displayed by a product.  If not, please explain why.  If so, please comment on how well the proposed abrasion step and chemical treatment step reflect the likely range of actual use conditions.  To the extent that the simulated conditions do not reflect the likely range of actual use conditions, please comment on whether the additional requested information (quantitative and qualitative) about the durability of the product is sufficient to assess the potential for physical disruption of the product surface after long term use.
 The abrasion and chemical treatment steps would represent aging and potential changes to the surfaces with use over time.  Since these surfaces will be routinely cleaned on a daily basis followed by a more thorough cleaning after patient discharge at least cleaned daily and the a more thorough cleaning (terminal cleaning) after patient discharge.
                     
 In this same vane it might be useful to know if there are changes in the amount of loss of active agent over time or improvement by releasing more copper ions.

 Does wiping surfaces (eg., commercially available disinfectant wipes; microfiber cleaning cloths with a hospital grade disinfectant, etc) and scratching the surface increase the likelihood of copper coming off in the leachate.  What is the average life span of these surfaces (especially of this is a coating). So abrasion and chemical treating would simulate the potential use of the product.  It may he helpful to look at the market and to pick the most common disinfectant  cleaners, disinfectants in use at healthcare facilities. (I would suspect that Sodium hypochlorite solutions, quats, and peroxide containing products would be the most widely used)

 If you think that abrasion and/or chemical treatment may affect antimicrobial efficacy, but that the proposed protocol does not adequately evaluate the potential for such effects, please offer advice on how to change the protocol (e.g., what process and/or chemical solutions should be used to treat a carrier) so that the protocol will adequately evaluate the level of antimicrobial activity of a product.  Please comment specifically on whether the use of products containing chelating agents is likely to affect the level of antimicrobial activity of solid metal and metal alloy products. Also, please comment specifically on whether the cleaning step (thoroughly rinse with DI water) between exposure cycles is sufficient to remove residual chemical solutions (solutions A, B and C).   
            
 Have you considered measuring copper concentration in solutions (leachate) used to remove cells from the carriers.  Abrasion and scratching of the surfaces might release copper from the impregnated surfaces.  
            
 I also believe that the protocol should employ a chelator (eg., thioglycollate, EDTA, etc) to remove any potential carry over of copper ions into the culture media thus preventing growth and yielding lower colony counts (so potentially greater log reduction). 

 I think before going to far that a comparison of the method with and with out chelation would be necessary.  If the results of that comparison study are equivalent, then the use of chelators may not be necessary.  
            
            
 Residual/Continuous Activity:  Residual/continuous activity over time is claimed to be one attribute inherent to copper and copper alloy products.  
 Some technology developers would like to claim that solid copper and copper alloy products provide "residual/continuous activity."  Please comment on whether the proposed protocol is capable of determining if copper and copper alloy products provide such activity, and if not, what changes to the proposed protocol (e.g., instituting repeated inoculations of the carrier) would provide data to evaluate such activity
            
 Current protocol addresses the log reduction in within an hour however, these products claim to have continuous activity.  It would seem appropriate to re-challenge the coupons (re-inoculate) at some time period after their culture and determine whether the log reductions are similar log reductions over time. 
               
 For antimicrobial containing dressings FDA recommend that manufacturers follow ASTM E2315-03 Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure or an equivalent method. Test results should demonstrate at least a 4 log reduction.  So this type of testing is performed on items such as antimicrobial impregnated respirators (eg., copper-silver, copper, iodine).  Efficacy for testing must show 4 log reduction within 1 hr (impregnated drapes, bandages, respirators, surgical masks, etc).

 The other testing that FDA accepts is performance testing (bench and animal) so for silver impregnated catheters they look at controls (not coated or impregnated) vs impregnated and evaluate colonization over time (difference here is that they are fluid based assessments, eg central venous or urinary catheters).
 So continuous activity might require a sequential re-inoculation of carriers to see whether the same log reduction occurs.