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Current norms for testing hand disinfectants

Manfred L Rotter
29 May, 2014  
Approved guidelines for the valid evaluation of the antimicrobial efficacy of hand hygiene agents are discussed
 
Manfred L Rotter MD Dip Bact
Professor emeritus for Hygiene and Microbiology;
former director of the Hygiene-Institute,
Medical University Vienna, Austria
 
The purpose of norms for testing the antimicrobial efficacy of products for hand hygiene is, of course, to evaluate this property in a well standardised fashion in order to ensure a highly  reproducible and comparable estimate of their performance for the sake of safe patient care. Hand hygiene, that is, hand washing and, more efficiently, hand disinfection, is namely the single most important measure to prevent infection-generating microbial transfer in health care.(1) It is of note that every new formulation for hand antisepsis should be tested for its antimicrobial efficacy together with all accompanying ingredients added for better skin tolerance even if the active agent(s) is/are known to satisfy an agreed minimum efficacy requirement, because such additives can compromise the antimicrobial activity. 
 
Several methods are available to assess this activity. For example, the reduction of bioburden can be measured in vitro by assessing the minimal microbicidal concentration or perform quantitative suspension tests or establish kill-time curves. But results of such tests do not reflect the inactivating effect on live skin; neither do ex vivo tests on explants of animal or human skin. By contrast, in vivo laboratory tests simulating practical conditions on the hands of volunteers offer this possibility, as do field tests. The latter are, however, difficult to control for extraneous influences. The best evidence for the efficiency of hand hygiene measures in clinical settings to prevent hand-carried transmission of pathogens can probably be provided by clinical trials. But unfortunately, they require large expenditures in money, time and workforce. This is the reason why standardised laboratory-based in vivo tests seem to be the most affordable compromise for evaluating the microbicidal efficacy of products for hand disinfection. To the best of our knowledge, test methods such as these, and which are included in officially approved norms, are presently only available in North America with the ASTM International Standards, and in Europe with the Centre Européen de Normalisation (CEN) norms.
 
The worldwide first, officially approved guidelines for evaluating chemical disinfectants – including hand disinfectants – were published in 1958 by the German Society of Hygiene and  Microbiology (DGHM).(2) They distinguished between tests for products used by healthcare workers (HCWs) for ‘hygienic hand disinfection’ (HHD), which is directed solely against the transient microbial flora of hands, without regard to the autochthonous, resident skin flora, and products for ‘surgical hand disinfection’ (SHD) used for presurgical hand preparation. Products designed for the latter purpose are directed against both transient and resident flora, whereby eliminating the latter is much more difficult. For testing HHD, hands were, in these guidelines, experimentally contaminated with Escherichia coli (E. coli) as a test organism. Products for surgical antisepsis were tested on clean, but not artificially contaminated, hands. 
 
Because, in our eyes, these tests had several shortcomings (only post-treatment values assessed, unsatisfactory pass criteria, no reference procedure included, no statistical proceeding proposed), we developed new test models that were then adopted by the Austrian, and also the German, Societies for Hygiene and Microbiology. In 1974, a model for the hygienic handrub (HHR) with volatile agents, such as alcohols was proposed.(3)
 
A test for the hygienic handwash (HHW) with medicated soaps followed in 1991,(4) and in 1981 the model for SHD was published.(5) The main principle in these models is that, together with the test product, a concomitant reference procedure is tested concurrently by the same subjects in a cross-over fashion (or when testing more than one product in a Latin Square pattern). Briefly: one half of the subjects use the test product according to the manufacturer’s instructions, the other half performs the standardised reference treatment. After a first run, the volunteers reverse roles – in tests for HHR and HHW immediately after the first run, in those for surgical antisepsis one week later to allow regrowth of the skin flora. To establish the microbicidal efficacy of the treatments, pre- and post-treatment samplings are performed by the ‘fingertip method’ in broth.
 
If the test formulation contains chemicals exerting sustained microbicidal or microbistatic activity, the post-treatment broth is supplemented with neutraliser. Viable counts are then assessed from these sampling broths by quantitative culture. The differences of the individual log10 pre-treatment and post-treatment counts form the log10 reduction factors. These are the measures for the microbial reduction achieved representing the microbicidal activity of both product and reference treatment. In tests for SHD, post-treatment sampling is carried out immediately after disinfection on one hand to assess the immediate effect and three hours later on the other, meanwhile gloved, hand, with the option to demonstrate sustained activity if such a claim has to be substantiated. 
 
The reference treatment for HHR consists of rubbing 3ml 2-propanol 60% v/v onto hands in a standardised manner for 30 seconds. This operation is repeated with the total application time not exceeding 60 seconds.(3) The requirement is that the efficacy of a product for HHR shall not be inferior to that of the reference product.
 
The reference treatment for HHW is a 60 second standardised handwash with dilute (20%) soft soap.(4) A product for HHW must be significantly more efficacious than the soap.
 
Performing the standardised reference treatment for SHD, volunteers rub their hands with as many 3-ml portions of 1-propanol 60% v/v as are necessary to keep them wet for three minutes.(5) The immediate and three-hour effects of a product must not be inferior to those of the reference treatment. Sustained efficacy is accepted if the three-hour effect of the product is significantly stronger than that of the reference.
 
It has been shown for both hygienic and surgical hand antisepsis that relating the results from concomitant testing of product and reference disinfection to each other by standardising them by intra-individual subtraction of the log10 RF of the product from that of the reference renders results from different laboratories comparable. This is not the case when only the reductions assessed in different laboratories with the test product are being compared with each other.(6,7)
 
Through the successful work of the Technical Committee 216 of the CEN, the above mentioned principles developed into the European Norms EN 1499 (HHW), EN 1500 (HHR) and EN 12791 (SHD). But before this, many details had to be clarified at the Vienna University Hygiene Institute. 
 
There was, for example, the question of how hands, experimentally contaminated or not,  should be sampled for microorganisms. Should this be carried out by washing hands in a sequence of basins containing sampling fluid from which subsequently viable counts are assessed, or in plastic bags with broth, or by kneading and rubbing fingertips at the bottom of Petri-dishes containing broth or by the cylinder scrub method, where a glass cylinder is tightly pressed onto a hand surface – palm or back–filled with stripping fluid, and the skin being rubbed through the cylinder with a glass rod? It was shown that the relative sampling efficacy of the fingertip method was as good as that of the basin and plastic bag method, but because of the small sampling volume of only 10ml, it was the most sensitive and also the most economical method.(7) An additional advantage is that, of the hand surfaces, the fingertips have the most frequent and intense contact with the environment and are therefore the most relevant area.
 
For testing HHR and HHW products, the question arose of how the experimental contamination of hands with a test strain should be handled: by distributing a small volume of microbial inoculum from fingertips onto the rest of the hand surface by rubbing or by immersion of hands into a liquid culture followed by drying on the air or by immersion and rubbing hands against each other until dry? In order to avoid mechanical ‘dilution’ of the inoculum by the first method and for a more even distribution, immersion followed by air-drying was chosen, although the third method results in smaller bacterial reductions making the test stricter and thereby more difficult to demonstrate a product’s efficacy.(9) This argument was, however, disregarded as the same happens with the reference disinfection, the efficacy of which is compared with that of the test product. 
 
The test organism was a strain of E. coli because this bacterium is found among the most frequent microorganisms causing hospital-acquired infections (HAIs) and, because its colony form is easily distinguished on the counting plates from colonies of the Staphylococcal resident skin flora which, at the same time, is also washed into the stripping fluid.(9) In EN 1499 and EN 1500, an apathogenic strain of E. coli K12 is used. With regard to the reality in clinical settings, Staphylococcus aureus (S. aureus) may perhaps be more relevant, because most HAIs are caused by this organism. But the infectious risk for subjects participating in tests for product evaluation is regarded as unacceptable.
 
A certain disadvantage of using E. coli, a gram-negative bacterium, may be presumed in its sensitivity to drying. It was, indeed, shown that by air-drying freshly contaminated hands for three minutes that approximately 1 log10 is lost from the fingertips.(10) But with regard to the large inoculum size of 108 –109 colony-forming units (CFU), this is not great. And, again, the same happens to the inoculum on the fingertips of subjects performing the reference disinfection.
 
Sometimes, it was argued that sampling for pre-treatment values would wash most of the  inoculum from the fingertips into the stripping fluid before disinfection. We have disproved this argument by demonstrating that the actual loss does not even amount to one power of ten. This loss wanes asymptotically the more often the fingertips are sampled.(10) Thus, the contaminated fingertips behave similar to a stamp-pad, releasing ink many times, and the same happens during the reference treatment.
 
A question of utmost importance is the choice of a reference disinfection because this not only serves as an intrinsic control – each subject acts as his/her own control – but  also as the standard for efficacy performance. (It must be emphasised here that the necessary potency of the latter to prevent HAI in the clinical setting is still unknown.)  As outlined above, among the aliphatic, short-chained alcohols ethanol, 1-propanol and 2-propanol, the latter was – partly arbitrarily – chosen as a reference antiseptic for testing products for HHR.(11) Ethanol, the least efficacious, would have also been a possibility. But for the strong potency desired for a performance standard, high concentrations of well above 80% V/V would be necessary. This, however, lowers the flash point of the alcohol to an unacceptably low temperature whereby the ignition hazard rises. Finally, the reason, why not the most efficacious of these alcohols, 1-propanol, was taken, is that its inactivating potency is too strong for the purpose of this test insofar as, with concentrations of 50–60%, the post-treatment counts assessed on most subjects would be zero, preventing the exact enumeration of the bacterial reduction to be assessed.(11)
 
Products for the HHW must, of course, reduce the microbial release from hands much more effectively than unmedicated soap. As we could not find any unmedicated soap without association to a certain manufacturer – a prerequisite in each norm – we have chosen sapo kalinus  (soft soap) as the reference agent in EN 1499.(4)
 
The choice of 1-propanol as a reference alcohol for testing products designed for the surgical area is based on the idea that, if for that matter surgical instruments have to be sterile, surgeons´ hands, which cannot be sterilised, should be treated with the strongest agent available, although in an acceptable concentration for the skin.(5)
   
Another, often criticised fact is the long duration of hand treatments (30–60 seconds) required in EN 1499 and EN 1500 for the reference disinfection procedures, although the average duration observed in practice is often less than 15 seconds. The rationale for this requirement, which obviously does not reflect the actual usage among HCWs, is the fact that very short contact times such as 15 seconds are not suitable to demonstrate differences in the efficacy of a test product as compared with the reference because, under valid statistical settings, this would require very large sample sizes, that is, the  number of subjects tested. Therefore, the non-inferiority of a test product can easier and much more economically be demonstrated by using a reference treatment with longer skin contact than in real practice. 
 
In EN 12791, the duration of antisepsis in the respective reference treatment is three minutes. This duration is usually accepted, although nowadays products for SHR exist that achieve the same effect within 90 seconds.(12)
 
The application volume of a hand antiseptic is important as the performance of hand hygiene agents increases significantly with volumes up to 3ml. Unfortunately, many dispensers are reported to deliver much less with one actuation.(13) However, with volumes of 1.0–2.0ml, it is hardly possible to cover all hand surfaces and, thereby, a product often fails to meet the efficacy requirements of the norms.(14) This is the reason why we have proposed application volumes of 3ml in the reference treatments.
 
A great disadvantage of the official test norms in North America and in Europe is that their results cannot be compared with each other. Products evaluated for approval by the US Food and Drug Administration according to the Tentative Final Monograph (TFM),(15) which refers to the ASTM E-1174 standard, may pass the test, whereas the same product fails when tested according to the respective CEN norm, and sometimes vice versa. This is probably partly due to the fact that the TFM methods use fixed bacterial log10 reductions as pass criteria, whereas in the CEN methods they are flexible as they are defined in each single test by the performance of the respective concomitant reference treatment. 
 
Conclusions
Fortunately, there exist officially approved standards, today, for valid evaluation of the antimicrobial efficacy of hand hygiene agents. However, intense research and great efforts are still required to ensure that not only effective, but also skin tolerable formulations are being provided, the acceptability of which helps motivating HCWs to comply with the rules of hand hygiene – both being an indispensable prerequisite for safe patient care.
 
References
  1. World Health Organization. WHO Guidelines on Hand Hygiene in Health Care. Geneva: WHO;2009.
  2. Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM). Richtlinien für die Prüfung chemischer Desinfektionsmittel. Zbl Bakt Hyg I. Abt  Orig 1958; 173:307-317.
  3. Rotter M, Mittermeyer H, Kundi M. Investigations on the model of the artificially contaminated hand – Proposal for a test method. (In German). Zbl Bakt Hyg I.Abt Orig B  1974;159:560–81.
  4. Rotter ML, Koller W. A European test for the evaluation of the efficacy of procedures for the antiseptic handwash. Hyg Med 1991;16:4–12.
  5. Anonymous. Guideline of the Austrian Society for Hygiene, Microbiology and Preventive Medicine (ÖGHMP) as of November 4, 1980, for testing the efficacy of procedures for surgical hand antisepsis. (In German). Österreichische Krankenhauszeitung 1981; reprint, pp 3–11.
  6. Rotter ML et al. Evaluation of procedures for hygienic hand disinfection: controlled parallel experiments on the Vienna test model. J Hyg Cambridge 1986;96:27–37.
  7. Rotter M et al. Reproducibility and workability of the European test standard EN 12791 on the effectiveness of surgical hand antiseptics – a randomized, multicenter trial. Infect Cont Hosp Epidemiol 2006;27:935–9.
  8. Männer F, Rotter M, Mittermeyer H. Vergleichende Untersuchungen zur Keimgewinnung von der künstlich kontaminierten Hand. Zbl Bakt Hyg I.Abt Orig B 1975;160:412–31.
  9. Koller W et al. Test method for the evaluation of procedures for the hygienic disinfection of hands. 2nd part: Argumentation and comments illustrated by an example. (In German). Zbl Bakt Hyg I.Abt Orig B 1977;164:507–20.
  10. Koller W et al. Kinetics of the release of testbacteria from the artificially contaminated hand. (In German). Zbl Bakt Hyg I.Abt Orig.B 1976;163:509–23.
  11. Rotter M, Koller W, Kundi M. Usability of three alcohols for a standard [= reference] disinfection method to be employed for the evaluation of procedures for the hygienic disinfection of hands. (In German). Zbl Bakt Hyg I Abt Orig B 1977;164:428–38. 
  12. Suchomel M et al. Surgical hand disinfection using alcohol: The effects of alcohol type, mode and duration of application. J Hosp Infect 2009; 71:228–33. 
  13. Macinga DR et al. Efficacy of novel alcohol-based hand rub products at typical in-use volumes. Infect Cont Hosp Epidemiol 2013;34:299–300.
  14. Kampf G et al. Lesser and lesser – The impact of small volumes in hand disinfection on quality of hand coverage and antimicrobial efficacy. ICPIC congress 2013, Geneva; Abstract # O007.
  15. US Food and Drug Administration. Tentative final monograph for healthcare antiseptic drug products; proposed rule. Federal Register 1994:31441–52.