Chemical disinfection in healthcare settings: critical aspects for the development of global strategies.

Chemical disinfection is an indispensable means of preventing infection. This holds true for healthcare settings, but also for all other settings where transmission of pathogens poses a potential health risk to humans and/or animals. Research on how to ensure effectiveness of disinfectants and the process of disinfection, as well as on when, how and where to implement disinfection precautions is an ongoing challenge requiring an interdisciplinary team effort. The valuable resources of active substances used for disinfection must be used wisely and their interaction with the target organisms and the environment should be evaluated and monitored closely, if we are to reliable reap the benefits of disinfection in future generations. In view of the global threat of communicable diseases and emerging and re-emerging pathogens and multidrug-resistant pathogens, the relevance of chemical disinfection is continually increasing. Although this consensus paper pinpoints crucial aspects for strategies of chemical disinfection in terms of the properties of disinfectant agents and disinfection practices in a particularly vulnerable group and setting, i.e., patients in healthcare settings, it takes a comprehensive, holistic approach to do justice to the complexity of the topic of disinfection.

Antibiotic resistance: What is so special about multidrug-resistant Gram-negative bacteria?

In the past years infections caused by multidrug-resistant Gram-negative bacteria have dramatically increased in all parts of the world. This consensus paper is based on presentations, subsequent discussions and an appraisal of current literature by a panel of international experts invited by the Rudolf Schülke Stiftung, Hamburg. It deals with the epidemiology and the inherent properties of Gram-negative bacteria, elucidating the patterns of the spread of antibiotic resistance, highlighting reservoirs as well as transmission pathways and risk factors for infection, mortality, treatment and prevention options as well as the consequences of their prevalence in livestock. Following a global, One Health approach and based on the evaluation of the existing knowledge about these pathogens, this paper gives recommendations for prevention and infection control measures as well as proposals for various target groups to tackle the threats posed by Gram-negative bacteria and prevent the spread and emergence of new antibiotic resistances.

Lesser-known or hidden reservoirs of infection and implications for adequate prevention strategies: Where to look and what to look for.

In developing hygiene strategies, in recent years, the major focus has been on the hands as the key route of infection transmission. However, there is a multitude of lesser-known and underestimated reservoirs for microorganisms which are the triggering sources and vehicles for outbreaks or sporadic cases of infection. Among those are water reservoirs such as sink drains, fixtures, decorative water fountains and waste-water treatment plants, frequently touched textile surfaces such as private curtains in hospitals and laundry, but also transvaginal ultrasound probes, parenteral drug products, and disinfectant wipe dispensers. The review of outbreak reports also reveals Gram-negative and multiple-drug resistant microorganisms to have become an increasingly frequent and severe threat in medical settings. In some instances, the causative organisms are particularly difficult to identify because they are concealed in biofilms or in a state referred to as viable but nonculturable, which eludes conventional culture media-based detection methods. There is an enormous preventative potential in these insights, which has not been fully tapped. New and emerging pathogens, novel pathogen detection methods, and hidden reservoirs of infection should hence be given special consideration when designing the layout of buildings and medical devices, but also when defining the core competencies for medical staff, establishing programmes for patient empowerment and education of the general public, and when implementing protocols for the prevention and control of infections in medical, community and domestic settings.

The role of surface disinfection in infection prevention.

BACKGROUND: The Rudolf Schuelke Foundation addresses topics related to hygiene, infection prevention and public health. In this context a panel of scientists from various European countries discussed "The Role of Surface Disinfection in Infection Prevention". The most important findings and conclusions of this meeting are summarised in the present consensus paper. AIM: Although the relevance of surface disinfection is increasingly being accepted, there are still a number of issues which remain controversial. In particular, the following topics were addressed: Transferral of microbes from surface to patients as a cause of infection, requirements for surface disinfectants, biocidal resistance and toxicity, future challenges. METHODS AND FINDINGS: After discussion and review of current scientific literature the authors agreed that contaminated surfaces contribute to the transmission of pathogens and may thus pose an infection hazard. Targeted surface disinfection based on a risk profile is seen as an indispensable constituent in a multibarrier approach of universal infection control precautions. Resistance and cross-resistance depend on the disinfectant agent as well as on the microbial species. Prudent implementation of surface disinfection regimens tested to be effective can prevent or minimize adverse effects. CONCLUSIONS: Disinfection must be viewed as a holistic process. There is a need for defining standard principles for cleaning and disinfection, for ensuring compliance with these principles by measures such as written standard operating procedures, adequate training and suitable audit systems. Also, test procedures must be set up in order to demonstrate the efficacy of disinfectants including new application methods such as pre-soaked wipes for surface disinfection.

Modified World Health Organization hand rub formulations comply with European efficacy requirements for preoperative surgical hand preparations.

BACKGROUND: The World Health Organization (WHO) has published "Guidelines on Hand Hygiene in Health Care" recommending 2 hand rub formulations based on 80% vol/vol ethanol or 75% vol/vol isopropanol for local production in healthcare settings where commercial products are not available or are too expensive. Previous investigations have shown that neither formulation meets the efficacy requirements of European norm (EN) 12791, which is the most stringent available norm for surgical hand rub preparations. Even when modified with approximately 5% higher alcohol content, the formulations proved to be inferior to the reference of the norm when measured after 3 hours. OBJECTIVE: Because the high glycerol content of the formulations was suspected to negatively influence their efficacy, additional investigations were performed with varying glycerol content. METHODS: Modified formulations with higher alcohol concentration (mass instead of volume percentage) and lower glycerol concentration (0.725% instead of 1.45%) or without the addition of glycerol were evaluated for their conformity with the efficacy requirements of EN 12791, which demands noninferiority in comparison with a reference hand antisepsis procedure immediately and 3 hours after treatment on volunteers' hands. DESIGN: Randomized Latin-square design. SETTING: Microbiology laboratory of the Medical University of Vienna, Vienna, Austria. PARTICIPANTS: Twenty-five healthy volunteers. RESULTS: Reducing the concentration of glycerol or omitting it completely rendered both WHO formulations noninferior to the reference, both immediately and 3 hours after surgical hand antisepsis. CONCLUSIONS: Both WHO-recommended formulations meet the efficacy requirements of EN 12791 by increasing their alcohol concentrations by 5%, prolonging their application to 5 minutes and reducing the glycerol concentration to 0.725%.

Testing of the World Health Organization recommended formulations in their application as hygienic hand rubs and proposals for increased efficacy.

BACKGROUND: In Central Europe, alcohol-based hand rubs have been the preferred choice for hand hygiene, whereas, in other countries, other preparations have been used that are based on other active agents. Recently, a move towards alcohol-based hand rubs has begun, but they may be costly and unaffordable to some. Therefore, the World Health Organization (WHO) has recommended 2 hand rub formulations (WHO I and WHO II) for local production in health care settings where commercial products are not available or are too expensive. OBJECTIVES: WHO I, based on ethanol 80% (vol/vol), and WHO II, based on isopropanol 75% (vol/vol), were investigated for their bactericidal efficacy in their application as hygienic hand rubs. METHODS: The investigation took place at the Institute for Hygiene and Applied Immunology, Medical University Vienna, Austria, as a prospective, randomized, in vivo laboratory study, comparative in crossover design. Both formulations were tested according to the European Standard EN 1500 in 2 applications (1 × 3 mL/30 seconds or 2 × 3 mL/2 × 30 seconds). Additionally, modifications with increased alcohol concentrations (weight instead of volume percent) were tested in the short application. Bactericidal efficacies were compared with those of the respective reference procedure "R," ie, rubbing 2 × 3 mL 60% vol/vol isopropanol for 2 × 30 seconds onto hands artificially contaminated with Escherichia coli K12. RESULTS: The short application of either WHO formulation resulted in bacterial reductions significantly inferior to the respective ones of R. However, prolonging the contact time to 60 seconds or increasing the alcohol content produced reductions similar to those of R. CONCLUSION: Both WHO-recommended formulations meet the efficacy requirements of EN 1500 within 60 seconds but not within 30 seconds. Increasing the respective alcohol concentrations from 80% vol/vol to 80% wt/wt and 75% vol/vol to 75% wt/wt renders the formulations sufficiently active to conform to the norm also within 30 sections.

Surgical hand antisepsis with alcohol-based hand rub: comparison of effectiveness after 1.5 and 3 minutes of application.

OBJECTIVE: Research has shown 1.5 minutes of surgical hand antisepsis with alcohol-based hand rub to be at least as effective under experimental conditions as the 3-minute reference disinfection recommended by European Norm 12791. The aim of the present study was to validate the effectiveness of 1.5 minutes of surgical hand antisepsis in a clinical setting by comparing the effectiveness of 1.5- and 3-minute applications of alcohol-based hand rub (45% vol/vol 2-propanol, 30% vol/vol 1-propanol, and 0.2% mecetronium ethylsulphate). DESIGN: Prospective crossover trial in which each surgeon served as his or her own control, with individual randomization to the 1.5- or the 3-minute group during the first part of the trial. SETTING: Basel University Hospital, Switzerland. PARTICIPANTS: Thirty-two surgeons with different levels of postdoctoral training. METHODS: We measured the bactericidal effectiveness of 1.5 minutes and 3 minutes of surgical hand antisepsis with alcohol-based hand rub by assessing the mean (+/-SD) log10 number of colony-forming units before the application of hand rub (baseline), after the application of hand rub (immediate effect), and after surgery (sustained effect) so as to follow European Norm 12791 as closely as possible. RESULTS: The immediate mean (+/-SD) log10 reduction in colony-forming units (cfu) was 2.26 +/- 1.13 log10 cfu for the 1.5-minute group and 3.01 +/- 1.06 log10 cfu for the 3-minute group (P = .204). Similarly, there was no statistically significant difference between the 2 groups with respect to the sustained effect; the mean (+/-SD) log10 increase in bacterial density during surgery was 1.08 +/- 1.13 log10 cfu for the 1.5-minute group and 0.95 +/- 1.27 log10 cfu for the 3-minute group (P = .708). No adverse effects were recorded. CONCLUSION: In this clinical trial, surgical hand antisepsis with alcohol-based hand rub resulted in a similar bacterial reduction, regardless of whether it was applied for 3 or 1.5 minutes, which confirms experimental data generated with healthy volunteers.

Surgical hand rub: Influence of duration of application on the immediate and 3-hours effects of n-propanol and isopropanol.

BACKGROUND: The recommended duration for surgical hand treatment has been changed from 10 over 5 to 3 minutes and even shorter. OBJECTIVES: Our objective was to study the impact of the length of surgical hand antisepsis with n-propanol 60% (vol/vol) or isopropanol 70% (vol/vol) applied for 1, 3, or 5 minutes on the reduction of resident hand flora in the setting of the microbiologic laboratory for experimental and applied testing of disinfectants and antiseptics at the Medical University Vienna, Austria, using a Latin Square design. METHODS: Our methods were according to the Austrian Guidelines for Testing Products for Surgical Hand Antisepsis. The release of bacterial hand flora of 21 subjects is assessed before and immediately after disinfection from one hand and 3 hours later from the other, meanwhile gloved, hand. Mean reduction factors (RF) are calculated. RESULTS: The immediate mean log(10) RFs with n-propanol or isopropanol were 1.05, 2.03, and 2.30 and 0.74, 1.48, and 2.12, respectively, when applied for 1, 3, or 5 minutes, respectively. After 3 hours, the respective mean log(10) RFs were 0.45, 1.01, and 1.60 and 0.19, 0.79, and 1.03. Thus, with increasing length of application, a highly significant trend (P < .001) toward higher log(10) reductions was demonstrated. At both sampling times, n-propanol was more effective than isopropanol at the corresponding treatments. Furthermore, a highly significant (P < .001) association was found between the individual volunteers and the effect of the antiseptics on their hands. CONCLUSION: The efficacy of surgical antisepsis is significantly associated with the length of application.

A novel noninvasive genotyping method of Helicobacter pylori using stool specimens.

BACKGROUND & AIMS: The source(s) of the infection and the route(s) of transmission of Helicobacter pylori have not yet been clarified. This is to introduce a noninvasive protocol allowing molecular typing of H pylori using stool specimens. METHODS: The genotyping method is based on 2 H pylori-specific biprobe real-time polymerase chain reaction assays using fragments of the glmM and the recA genes as target sequences. Discrimination between strains results from differences in the melting temperature during melting curve analysis. In case of identical melting temperatures in both assays, sequence analysis of the glmM amplicon was performed to confirm strain identity. The method was validated using gastric biopsy specimens and stool specimens of 97 unrelated individuals suffering from abdominal pain and stool specimens of members of 10 families in Austria (infected index child and family members) and 8 African households. RESULTS: Of the 97 patients, 27 were infected as shown by culture, histology, and rapid urease test. The sensitivity of each of the assays was 100% in gastric biopsy specimens and 92.2% in stool specimens; the specificity was 100%. The discriminatory capacity of the method was 100%. Clonal identities were found in 9 of 10 (90%) European and 7 of 8 (87.5%) African households. In 2 African households, 2 different clonal lineages each were found. CONCLUSIONS: The genotyping protocol introduced allows for both accurate detection and discrimination of H pylori strains in stool samples. Large-scale studies using this protocol may contribute to the clarification of the transmission pathways of infection with H pylori.

Effects of alcohols, povidone-iodine and hydrogen peroxide on biofilms of Staphylococcus epidermidis.

OBJECTIVES: To test the effects of several biocides [N-propanol, a commercially available propanol/ethanol/chlorhexidine mixture, polyvinylpyrolidone (povidone-iodine) and hydrogen peroxide] on established biofilms of Staphylococcus epidermidis isolated from patients with cardiac implant infections and catheter-related bacteraemia. METHODS: Biofilms were grown in microtitre plates for 24 h, dyed and stained with Crystal Violet. The mean optical density (OD) and the OD ratio (ODr=OD of the treated biofilm/OD of the untreated biofilm) were used for quantification. Biofilms were incubated with 60% (v/v) N-propanol, the mixture of propanol/ethanol/chlorhexidine, hydrogen peroxide at three concentrations (0.5%, 3% and 5%, v/v) and povidone-iodine for 1, 5, 15, 30 and 60 min. Unstained biofilms were sonicated and plated on Columbia agar for time-kill curves. S. epidermidis skin isolates from healthy volunteers were used as controls. RESULTS: Biofilm ODs of the clinical S. epidermidis isolates and the isolates from the healthy volunteers were significantly different (1.17+/-0.512 versus 0.559+/-0.095, respectively; mean+/-SD) (P<0.01). No viable S. epidermidis was detected in biofilms treated with the alcohols, N-propanol or the propanol/ethanol/chlorhexidine mixture. Incubation with povidone-iodine and hydrogen peroxide 3% and 5% led to a log reduction of the viable cells of >5 after incubation for 5 min, however, up to 10(3) viable cells were detected in four isolates after 30 min of incubation with povidone-iodine. CONCLUSIONS: S. epidermidis obtained from infected implants forms thicker biofilms than that of healthy volunteers. Hydrogen peroxide, at a concentration of 3% and 5%, and alcohols rapidly eradicate S. epidermidis biofilms, whereas povidone-iodine is less effective.

Extended double disc synergy testing reveals a low prevalence of extended-spectrum beta-lactamases in Enterobacter spp. in Vienna, Austria.

OBJECTIVES: The aims of this study were to determine the prevalence of extended-spectrum beta-lactamases (ESBLs) in AmpC-carrying Enterobacter spp. in a tertiary care university hospital in Vienna, Austria, and to implement a cost-effective strategy to detect ESBLs in this particular genus on a routine basis. METHODS: Clinical Enterobacter isolates (n=208) were investigated by means of (i) an inhibitor-potentiated diffusion test using cefpodoxime, (ii) an expanded double disc diffusion synergy test (discs of cefotaxime, ceftazidime, cefpodoxime and cefepime placed around amoxicillin/clavulanic acid), (iii) the Etest ESBL screening method and (iv) the cefoxitin-cefotaxime antagonist test. Cefepime MICs were determined by separate Etests. RESULTS: Of 208 isolates, 76 (37%), 18 (9%) and 92 (44%) were derepressed, partially derepressed and inducible AmpC producers, respectively. Eight (4%) ESBL-producing Enterobacter strains could be detected, all of which would have been detected using disc-based tests. Six out of eight strains were genetically not related, as assessed by random amplification of polymorphic DNA. Typing results were confirmed by means of enterobacterial repetitive intergenic consensus PCR. The MIC(90) of cefepime was not different in ESBL carriers (range 2-4 mg/L), and was especially low in inducible AmpC producers (0.125 mg/L). More than half of all Enterobacter isolates (n=110; 53%) were partly derepressed or fully inducible AmpC producers. In the absence of cefoxitin, they appeared susceptible or intermediately susceptible to cefazolin (n=8; 9%), cefuroxime (n=75; 81.5%), ceftazidime (n=91; 99%), cefotaxime (n=92; 100%), cefpodoxime (n=75; 81.5%) and cefepime (n=91; 99%). CONCLUSIONS: Susceptibility to third-generation cephalosporins would have been falsely assumed in more than half of all Enterobacter isolates, but ESBL in Enterobacter is currently rare in our institution. Integration of multiple double disc tests into the routine antibiogram seems a reliable approach to screen for emerging resistance mechanisms. Etests did not provide additional information in this study.

Population kinetics of the skin flora on gloved hands following surgical hand disinfection with 3 propanol-based hand rubs: a prospective, randomized, double-blind trial.

OBJECTIVE: To study the bacterial population kinetics on gloved hands following hand treatment with 3 optically indistinguishable, alcohol-based surgical hand rubs, with and without supplements to delay bacterial regrowth. DESIGN: Prospective, randomized, double-blind, balanced quasi-Greco-Latin square design. SETTING: Microbiology laboratory of the Medical University Vienna, Austria. PARTICIPANTS: Twenty-four healthy adult volunteers without skin lesions. Surgical hand rubs. The following hand rubs, all stained blue, were applied to the hands for 3 minutes: 1-propanol 60% vol/vol (A); 2-propanol 70% m/m plus chlorhexidine gluconate 0.5% wt/wt (B); 2-propanol 45% wt/wt plus 1-propanol 30% wt/wt plus mecetronium etilsulfate 0.2% wt/wt (C). As a reference formulation (R), 1-propanol 60% vol/vol, unstained, was applied for the same amount of time. METHOD: In 8 once-weekly tests, 24 subjects randomly assigned to use the 4 hand rubs in groups of 6 persons each performed hand hygiene according to the method described in European Norm 12791. Every subject used one preparation at a time, the antimicrobial effect of which was evaluated at 2 sampling times. After week 8, each volunteer had tested every preparation at every preset sampling time. All preparations were tested in parallel. RESULTS: The mean pretreatment counts of viable bacteria (in colony-forming units per milliliter) in fluid samples were not significantly different between week 1 and week 8, nor between the right and left hands (analysis of variance [ANOVA], P>.1). Immediately after applying the formulation (t(0)), bactericidal effects of the blinded formulations A and C were equivalent to that of the reference formulation R, whereas the effect of B was questionable. The population kinetics of the flora on the hands proceeded from large and fast initial reductions of the skin flora by 2.7 log units (A), 3.1 log units (B), 3.3 log units (reference formulation), and 3.5 log units (C), to slow regrowth. However, even after 6 hours wearing gloves viable bacterial counts remained significantly (P<.01) below the baseline values (by 0.9 log [reference formulation], 1.1 log [A and B], and 1.5 log [C]). The slowest regrowth 1 and 3 hours after application (Delta from t(0), 0.1 log and 0.7 log respectively) was seen with formulation C, and the slowest regrowth after 6 hours was seen with formulation B (Delta from t(0), 1.6 log). These differences did, however, not reach statistical significance. CONCLUSIONS: With respect to the rapid and dramatic antibacterial action of suitable alcohols at high concentrations and with appropriate neutralizers, the contribution of supplements to the delay of bacterial regrowth on gloved hands appears rather minor, if a product only exerts an immediate effect equivalent to that of the reference disinfection procedure described in EN 12791.

Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens.

In the present study, novel real-time PCR assays targeting the fungal ITS2 region were developed for the detection and differentiation of medically important Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) and Candida species (Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis) using a LightCycler instrument. The combination of a group-specific and a universal primer with five Aspergillus or six Candida species-specific biprobes in one reaction mixture facilitated rapid screening and species differentiation by the characteristic peak melting temperatures of the biprobes. Both assays can be performed either as single assays or simultaneously in the same LightCycler run. The analytical sensitivity using pure cultures and EDTA-anticoagulated blood, cerebrospinal fluid (CSF), and tissue samples spiked with A. fumigatus and C. albicans cell suspensions was shown to be at least 1 CFU per PCR, corresponding to 5 to 10 CFU/ml blood and 10 CFU/200 microl CSF or 0.02 g tissue. To assess the clinical applicability, 26 respiratory samples, 4 tissue samples from the maxillary sinus, and 1 blood sample were retrospectively tested and real-time PCR results were compared with results from culture, histology, or a galactomannan enzyme-linked immunosorbent assay (ELISA). Twenty samples (64.5%) were both culture positive and positive by real-time PCR. Six samples (19.4%) showed no growth of fungi but were positive by real-time PCR. However, all of the tissue samples were positive by both PCR and histology. The blood sample showed no growth of Aspergillus, but aspergillosis was confirmed by positive galactomannan ELISA, histology, and PCR results. The remaining samples (16.1%) were culture and PCR negative; also, no other signs indicating fungal infection were observed. Our data suggest that the Aspergillus and Candida assays may be appropriate for use in clinical laboratories as simple and rapid screening tests for the most frequently encountered Aspergillus and Candida species and might become an important tool in the early diagnosis of fungal infections in the future.

"I wash my hands of it!?" - Trends in hand hygiene over the past decades.

Hand hygiene is the most important measure to protect against the spread of nosocomial infections. With the development of in vitro und in vivo test methods for evaluation of the effect of hand hygiene, there has been a sharp increase over the past 50 years in the body of knowledge relating to effective methods for removal from the hands or killing and inactivation of pathogens. In 1958 the German Society of Hygiene and Microbiology (DGHM) published a first "Guidelines for Testing Chemical Disinfectants" and included only those hand disinfection products on its "List of Tested Chemical Disinfectants Found To Be Effective" that had been tested as per the methods cited in the guidelines. The American Society of Testing and Materials (today: ASTM International) was next, with the first test protocols for hand disinfection products, which in 1974 were adopted by the US Food and Drug Agency as "Guidelines" in a "Tentative Final Monograph" (TFM) and in 1994, having revised it to incorporate new insights, it was published once again. Where the user is concerned, guidelines for hand disinfection containing information on indication and implementation are of course more important than methods dealing with efficacy testing of products. Such guidelines are compiled within the hospitals by the infection control teams set up during the 1970s. Written guidelines were also published by several healthcare institutions, scientific societies and associations. The guidelines formulated by the World Health Organisation (WHO), in an expert committee under the direction of Didier Pittet, proved to be the most successful of the attempts undertaken at global level to enhance hand hygiene. The most remarkable changes appear to be the efforts aimed at improving compliance among medical personnel and the increasing international acceptance of hand disinfection by using alcohols in the form of rubs; whether this will be with lotions or gels remains to be seen.

Reproducibility and workability of the European test standard EN 12791 regarding the effectiveness of surgical hand antiseptics: a randomized, multicenter trial.

OBJECTIVES: To evaluate the reproducibility and workability of the in vivo test model of the European test standard EN 12791 regarding the effectiveness of surgical hand antiseptics and, as a secondary objective, to evaluate the power of the model to discriminate between the effectiveness of various formulations of surgical hand antiseptics. DESIGN: Prospective, randomized, multicenter study with a Latin square design. SETTING: Five laboratories at 2 universities, 2 disinfectant manufacturers, and 1 private testing institution. PARTICIPANTS: Twenty healthy adults in each laboratory. INTERVENTION: Surgical hand antisepsis was performed by scrubbing with chlorhexidine gluconate 4% detergent (CHG) or by rubbing the hands with propan-2-OL (70% by volume; Iso 70) or ethanol 85% (E 85); rubbing the hands and forearms for 3 minutes with propan-1-OL (N 60) was used as the reference disinfection procedure. We deliberately chose to use these antiseptics at the given concentrations because they were intended to cover the range of typical antiseptics submitted for approval according to EN 12791. METHODS: In once-weekly tests, the immediate effects of the 4 antiseptics were established according to the method laid down in EN 12791 by assessing the release of skin flora from the fingertips as viable bacteria counts per milliliter of sampling fluids before treatment and viable bacteria counts immediately after treatment, separately for both hands, such that after 4 weeks each volunteer had used every formulation once. RESULTS: The mean log reduction factor (RF) for the release of bacterial skin flora (the log RF was calculated as the log count before treatment minus the log count after treatment) and corresponding standard deviations for the 4 hand antisepsis formulations were as follows: for CHG, 1.1+/-0.3 colony-forming units (cfu) per milliliter of sampled fluid; for Iso 70, 1.7+/-0.3 cfu/mL; for E 85, 2.1+/-0.3 cfu/mL; and for N 60, 2.4+/-0.4 cfu/mL. The differences between these values proved significant (P<.001) by analysis of variance and in Tukey's "honestly significantly different" (HSD) post hoc test. Although, with regard to their immediate antibacterial activity, the same ranking of these antiseptics was found at all laboratories, the levels of efficacy were significantly different across laboratories (P<.001); no statistical difference was found between left and right hands (P>.01). Relating the log RF values of the other 3 formulations to those of the reference formulation (N 60) abolished differences between laboratories (P=.16); in addition, the interclass correlation coefficient decreased from 9.1% to 4.5%. With 20 volunteers, a minimum difference of 0.47 log between the mean log RFs of the reference formulation and an inferior test formulation will be detected as significant at an alpha of .05 (1-sided) and a 1- beta value of .8. CONCLUSION: The test method described in EN 12791 yielded the same conclusion on the effectiveness of the tested formulations in every laboratory and proved, therefore, reproducible and workable.