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Previous Article | Next Article 
Clinical Microbiology Reviews, October 2004, p. 863-893, Vol. 17, No. 4
0893-8512/04/$08.00+0 DOI: 10.1128/CMR.17.4.863-893.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Bode Chemie GmbH & Co., Scientific Affairs, Hamburg,1 Institut für Hygiene und Umweltmedizin, Ernst-Moritz-Arndt Universität, Greifswald, Germany2
SUMMARY INTRODUCTION TYPES OF SKIN FLORA MICROBIAL AND VIRAL FLORAS OF HANDS AND THEIR EPIDEMIOLOGIC ROLE Gram-Positive Bacteria Role in NIs. Frequency of colonized hands. Role of hand colonization in cross-transmission. Survival on hands and surfaces. Gram-Negative Bacteria Role in NIs. Frequency of colonized hands. Role of hand colonization in cross-transmission. Survival on hands and surfaces. Spore-Forming Bacteria Role in NIs. Frequency of colonized hands. Role of hand colonization in cross-transmission. Survival on hands and surfaces. Fungi Role in NIs. Frequency of colonized hands. Role of hand colonization in cross-transmission. Survival on hands and surfaces. Viruses Role in NIs. Frequency of contaminated hands. Role of hand colonization in cross-transmission. Persistence of infectivity on hands and surfaces. MINIMUM SPECTRUM OF ANTIMICROBIAL ACTIVITY AGENTS FOR REDUCTION OF THE NUMBERS OF PATHOGENS ON HANDS Nonmedicated Soap (Social Hand Wash) Effect on microorganisms and viruses. (i) Spectrum of activity. (ii) Testing under practical conditions. (iii) In-use tests. (iv) Risk of contamination by a simple hand wash. Effect on human skin. Chlorhexidine Effect on microorganisms and viruses. (i) Spectrum of activity. (ii) Testing under practical conditions. (iii) In-use tests. (iv) Resistance. Effect on human skin. Triclosan Effect on microorganisms and viruses. (i) Spectrum of activity. (ii) Testing under practical conditions. (iii) In-use tests. (iv) Resistance. Effect on human skin. Ethanol, Isopropanol, and n-Propanol Effect on microorganisms and viruses. (i) Spectrum of activity. (ii) Testing under practical conditions. (iii) In-use tests. (iv) Resistance. Effect on human skin. EFFECT ON NOSOCOMIAL INFECTIONS Plain Soap (Social Hand Wash) Chlorhexidine and Triclosan (Hygienic Hand Wash) Ethanol, Isopropanol, and n-Propanol EFFECT ON COMPLIANCE WITH HAND HYGIENE PRACTICES CONCLUSION REFERENCES
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| INTRODUCTION |
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In 2002, a new Centers for Disease Control and Prevention (CDC) guideline for hand hygiene in health care settings, entitled Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force, was published (71). It provides health care workers with a review of data on hand washing and hand antisepsis in health care settings and provides specific recommendations to promote improved hand hygiene practices and reduce the transmission of pathogenic microorganisms to patients and personnel in health care settings. As a clinical guideline, its chief aim is to reduce the incidence of NIs by providing detailed recommendations on two main aspects of hand hygiene: (i) choice of the most appropriate agents for hand hygiene in terms of efficacy and dermal tolerance and (ii) different strategies to improve compliance in hand hygiene, including hand hygiene practices among health care workers, behavioral theories, and methods for reducing adverse effects of agents. Our review is intended to support the CDC guideline by presenting specific additional aspects of the various agents, such as a broader evaluation of the in vitro and in vivo efficacy in various test models and their mode of action, resistance potential, and effect on compliance in hand hygiene.
Hand hygiene has been considered to be the most important tool in NI control (403, 462) ever since Semmelweis observed its immense effect on the incidence of childbed fever (473). Health care workers have three opportunities for the postcontamination treatment of hands: (i) the social hand wash, which is the cleaning of hands with plain, nonmedicated bar or liquid soap and water for removal of dirt, soil, and various organic substances; (ii) the hygienic (Europe) or antiseptic (United States) hand wash, which is the cleaning of hands with antimicrobial or medicated soap and water ("scrub"); most antimicrobial soaps contain a single active agent and are usually available as liquid preparations; and (iii) the hygienic hand disinfection (Europe), which normally consists of the application of an alcohol-based hand rub into dry hands without water.
For the preoperative treatment of hands two options are available: (i) the surgical hand wash (Europe) or surgical hand scrub (United States) which is the cleaning of hands with antimicrobial soap and water; and (ii) the surgical hand disinfection (Europe), which is the application of an alcohol-based hand rub into dry hands without water.
Three main types of preparations can be used for the different procedures of hand hygiene. (i) The first is plain, nonmedicated soap (social hand wash). (ii) The second is medicated soap (antiseptic and surgical hand wash). The most commonly used agent is chlorhexidine, usually at a concentration of 4 or 2%. Triclosan can also be found in medicated soaps, usually at a concentration of 1%. Hexachlorophene has now been banned worldwide because of its high rate of dermal absorption and subsequent toxic effects, especially among newborns (84, 98). Levels of 0.1 to 0.6 ppm in blood were found among health care workers who regularly used a 3% hexachlorophene preparation for hand washing (323). These findings speak strongly against the topical use of this active agent. The Food and Drug Administration classifies this agent as not being generally recognized as safe and effective for use as an antiseptic hand wash (21). Hexachlorophene is therefore not included in this review. Other active agents such as povidone iodine have rarely been used for the postcontamination treatment of hands and therefore are also not addressed in this review. (iii) The final type is the alcohol-based hand rub (hygienic and surgical hand disinfection). This is a leave-on preparation and this applied to the skin without the use of water.
In addition, non-alcohol-based waterless antiseptic agents are available for use by health care workers. Some of these contain quaternary ammonium-type compounds. They were not discussed in the CDC hand hygiene guideline because there was insufficient evidence at the time to promote their use; therefore, they are not further evaluated here.
This review provides an in-depth comparison of the several options for hand hygiene, with the aim of further supporting the CDC guideline on hand hygiene.
| TYPES OF SKIN FLORA |
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The resident flora consists of permanent inhabitants of the skin. They are found mainly on the surface of the skin and under the superficial cells of the stratum corneum (379). These bacteria are not regarded as pathogens on intact skin but may cause infections in sterile body cavities, in the eyes, or on nonintact skin (292). Resident skin bacteria survive longer on intact skin than do gram-negative transient species (325). The protective function of the resident flora, so-called colonization resistance, has been demonstrated in various in vitro and in vivo studies. Its purpose is twofold: microbial antagonism and the competition for nutrients in the ecosystem (12). Nevertheless, the interactions between bacteria and fungi on the skin are still inadequately understood. Many such interactions have been demonstrated experimentally. Their contribution—which is thought to be a major mechanism of preventing the adherence of pathogens—to the stability of the dermal ecosystem, however, remains unclear (375).
The dominant species is Staphylococcus epidermidis, which is found on almost every hand (311, 454, 522). The incidence of oxacillin resistance among isolates of S. epidermidis is up to 64.3% (311) and is higher among health care workers who have direct contact with patients than in those who do not (522). Other regular residents are Staphylococcus hominis and other coagulase-negative staphylococci, followed by coryneform bacteria such as propionibacteria, corynebacteria, dermabacteria, and micrococci (137, 315, 401). Among fungi, the most important genus of the resident skin flora is Pityrosporum (Malassezia) (201). Viruses are usually not resident on the skin but can proliferate within the living epidermis, where they may induce pathological changes (361).
Total counts of bacteria on the hands of medical staff have ranged from 3.9 x 104 to 4.6 x 106 (294, 309, 338, 447). Their number increases with the duration of clinical activities, on average by 16 cells per min (438). Some clinical situations are associated with a higher bacterial load on the hands of health care workers: direct contact with patients, respiratory tract care, contact with body fluids, and after being interrupted while caring for a patient (438). In general, however, it is difficult to clearly assign a specific risk of hand contamination to certain patient care activities. Nurses can contaminate their hands with 100 to 1,000 CFU of Klebsiella spp. during "clean activities" (81), while 10 to 600 CFU/ml can be found on nurses' hands after touching the groins of patients heavily contaminated with Proteus mirabilis (129). In intensive care units (ICU), the number of direct contacts between the hands of the health care workers and the patients is particularly high, leading to a higher risk of NI (148).
The transient skin flora consists of bacteria, fungi, and viruses that may be found on the skin only at times (447). They usually do not multiply on the skin, but they survive and occasionally multiply and cause disease (15). They may come from patients or inanimate surfaces. Between 4 and 16% of the hand surface is exposed by a single direct contact, and after 12 direct contacts, up to 40% of the hand surface may have been touched (74). The transmissibility of transient bacteria depends on the species, the number of bacteria on the hand, their survival on skin, and the dermal water content (230, 344, 418).
In addition, there is the temporary resident skin flora, which persists and multiplies for a limited period on the skin. The definition is more or less identical to that of transient skin flora, because the duration of residence on human skin is uncertain and variable but never permanent (5). In addition, the temporary resident skin flora often includes nosocomial bacteria and fungi (5, 201, 399, 400).
| MICROBIAL AND VIRAL FLORAS OF HANDS AND THEIR EPIDEMIOLOGIC ROLE |
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Enterococcus spp. are isolated in up to 14.8% of patients with NI (484). The most common species are Enterococcus faecium and E. faecalis (385), which frequently cause urinary tract infections (533). The emergence of vancomycin resistance among enterococci (VRE) has led to an increased recognition of cross-transmission of VRE, including the role of health care workers' hands (29, 347).
Coagulase-negative staphylococci, such as S. epidermidis, mainly cause catheter-associated primary bloodstream infections. In ICUs, approximately one-third of all blood culture isolates from patients with nosocomial bloodstream infections were found to be coagulase-negative staphylococci (463, 533).
Frequency of colonized hands. Colonization of health care workers' hands with S. aureus has been described to range between 10.5 and 78.3% (Table 1). Up to 24,000,000 cells can be found per hand (33). The colonization rate with S. aureus was higher among doctors (36%) than among nurses (18%), as was the bacterial density of S. aureus on the hands (21 and 5%, respectively, with more than 1,000 CFU per hand) (101). The carrier rate may be up to 28% if the health care worker contacts patients with an atopic dermatitis which is colonized by S. aureus (608, 609). MRSA has been isolated from the hands of up to 16.9% of health care workers. VRE can be found on the hands of up to 41% of health care workers (Table 1).
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Survival on hands and surfaces. S. aureus can survive on hands for at least 150 min; VRE survives on hands or gloves for up to 60 min (Table 1). On inanimate surfaces, S. aureus and MRSA may survive for 7 months, with wild strains surviving longer than laboratory strains (Table 1). VRE may survive on surfaces for 4 months. The long survival on surfaces, together with the relatively short survival on hands, suggests that contaminated surfaces may well be the source of transient colonization despite negative hand cultures.
Frequency of colonized hands. Colonization rates of gram-negative bacteria on the hands of health care workers have been described as ranging from 21 to 86.1% (Table 1), with the highest rate being found in ICUs (271). The number of gram-negative bacteria per hand may be as large as 13,000,000 cells (33). The colonization may be long-lasting (302). Even in nursing homes, a rate of 76% has been described for nurses hands (610). Colonization with gram-negative bacteria is influenced by various factors. For example, it is higher before patient contact than after the work shift (187). Hands with artificial fingernails harbor gram-negative bacteria more often than those without (207). Higher colonization rates with gram-negative bacteria also occur during periods of higher ambient temperature and high air humidity (358).
Different species of gram-negative bacteria exhibit different colonization rates. For instance, the colonization rate is 3 to 15% for Acinetobacter baumannii, 1.3 to 25% for Pseudomonas spp., and 15.4 to 24% for Serratia marcescens (Table 1). Klebsiella spp. were found on the hands of 17% of the ICU staff sampled, with up to 10,000 bacteria per hand (81). Artificial fingernails have been associated with a higher risk for colonization with P. aeruginosa (144).
Role of hand colonization in cross-transmission. Transient hand carriage of various gram-negative bacterial species has quite often been suspected to be responsible for cross-transmission during outbreaks resulting in various types of NI (155, 426, 514, 571). Most reports of cross-transmission of specific gram-negative bacteria come from critical-care areas, such as neonatal ICUs and burn units. Contaminated hands (Table 1), brushes, contaminated plain soap, and contaminated antiseptic soap have been associated with various types of NI, which were quite often caused by S. marcescens (Table 2).
Survival on hands and surfaces. Most gram-negative bacteria survive on the hands for 1 h or more. Survival on inanimate surfaces has been reported to be different for the different gram-negative species, with most of them surviving for many months (Table 1). In general, gram-negative bacteria survive for longer on inanimate surfaces than on human skin (151).
1%) (156). Patients can be contaminated from, for instance, the hands of hospital personnel and from inanimate surfaces (40). Frequency of colonized hands. In one study, the hands of 59% of 35 health care workers were C. difficile positive after direct contact with culture-positive patients. Colonization was found mainly in the subungual area (43%), on the fingertips (37%), on the palm (37%), and under rings (20%) (362). In another study, 14% of 73 health care worker were culture positive for C. difficile on their hands. The presence of C. difficile on the hands correlated with the density of environmental contamination (491). During a third outbreak, caused by Bacillus cereus in a neonatal ICU, 11 (37%) of 30 fingerprints from health care workers were positive for Bacillus spp. (569).
Role of hand colonization in cross-transmission. Transmission of C. difficile in an endemic setting on a general medical ward has been shown to occur in 21% of patients, with 37% of them suffering from diarrhea (362). An outbreak of necrotizing enterocolitis among neonates was associated with clostridial hand carriage in four of seven health care workers (173). Another spore-forming bacterium has been described as well: B. cereus was transmitted to the umbilicus in 49% of newborns on a maternity ward; the hands of 15% of the health care workers were found to be culture positive (62).
Survival on hands and surfaces. Vegetative cells of C. difficile can survive for at least 24 h on inanimate surfaces, and spores survive for up to 5 months (Table 1).
Frequency of colonized hands. In an ICU, 67 (46%) of the hands of 146 health care workers were colonized with a yeast. The most common species were Candida and Rhodotorula spp. Respiratory therapists were found to have the highest colonization rate (69%) (221). In another study of nurses and other hospital staff, 75% of the nurses and 81% of the other hospital staff were colonized with a yeast (541). In a long-term-care facility, 41% of 42 health care workers were found to have Candida spp. on their hands (378). Yeasts quite often also colonize artificial fingernails (207). Acquisition of C. albicans on the hands of health care workers immediately after attending systemically infected patients was reported to occur in 2 of 17 nurses (79).
Role of hand colonization in cross-transmission. Only a few studies are found in the literature which demonstrate the role of hands in cross-transmission (Table 2), sometimes despite negative hand cultures (572). The analysis of an outbreak revealed that caring for a patient who is colonized with Candida parapsilosis can lead to positive hand cultures and finally to severe infections or colonization among patients (501). The transmissibility of yeasts from hand to hand is high (Table 3).
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Frequency of contaminated hands. The risk of direct contact with blood and thereby with blood-borne viruses is variable. In general, it must be assumed that a health care worker wears protective gloves if contact with blood is expected. However, there are still clinical situations in which contamination with blood is unexpected. Health care workers in invasive radiology have blood contact in 3% of clinical activities, surgeons have blood contact in 50%, and midwives have blood contact in 71% (48). Surgical gloves should protect from direct contact with blood, but perforations are found on average in 17% of gloves, which correlates with the detection of blood under surgical gloves in 13% of surgeons (392). Perforations in most gloves (83%) remain undetected by the surgeon (557). Up to 82.5% of protective gloves have invisible perforations (276). In an acute viremic state, HBV may be present in blood at a concentration of 5 x 108 IU per ml of blood (623). A 1-µl volume of blood, which is hardly visible on a hand, may still contain 500 IU of HBV. For HCV, a concentration of 104 to 107 IU was found in blood (105). Virus detection on the hands has been investigated in a few studies. In a dialysis unit, 23.8% of samples obtained from health care workers' hands were positive for HCV RNA after treatment of HCV-positive patients despite the use of standard precautions, whereas the rate was 8% after treatment of HCV-negative patients (11).
Viruses from the respiratory tract are often found on hands, e.g., rhinoviruses in up to 65% from persons with a common cold (191, 457). Adenovirus has been found on the hands of healthcare workers during outbreaks of keratoconjunctivitis (380) and was isolated from the hands of 46% of patients with epidemic keratoconjunctivitis (35), which emphasizes the potential of virus transfer to hospital personnel through casual hand contact. No data were available regarding the detection of severe acute respiratory syndrome (SARS) virus on hands during the outbreaks in Asia and Canada in 2003.
Rotaviruses can be found on the hands in up to 78.6% of individuals sampled (Table 1) and also on surfaces with frequent hand contact, e.g., TV sets, toys, and patient charts (9). At the peak of a bout of rotavirus gastroenteritis, every gram of feces may contain more than 107 to 108 infectious viral particles (590).
Cytomegalovirus has been isolated from the hands of day care workers (224), but exotic viruses such as hemorrhagic fever viruses have to date not been detected on health care workers' hands.
Role of hand colonization in cross-transmission. Hands play a major role especially in the transmission of blood-borne, fecal, and respiratory tract viruses. The transmission of some viruses from the hands of health care workers has been described (Table 2). In addition, transient hand carriage is associated with the transmission of many viruses, such as rhinovirus (99, 191), RSV (194, 488), astrovirus (136), and cytomegalovirus (109). For the SARS virus, a similar correlation has been described, since hand hygiene was found to be the second most effective measure to prevent cross-transmission of the SARS virus in a hospital (510). Most viruses are easily transmitted from hand to hand, food, or surfaces (Table 3).
Persistence of infectivity on hands and surfaces. Persistance of viruses on the hands has been investigated mainly for fecal and respiratory tract viruses. Artificial contamination of hands with HAV led to an immediate-recovery rate of 70.5% (59). HAV persisted for several hours on human hands (354, 355). With poliovirus, the immediate-recovery rate was 22% but the whole inoculum was recovered after 150 min, indicating an almost complete persistence of poliovirus on hands (505). Rotavirus has been described as persisting on hands for up to 260 min, with 57% recovery after 20 min, 42.6% recovery after 60 min, and 7.1% recovery after 260 min (22). It can be transferred from contaminated hands to clean hands, with 6.6% of the viral contamination transferred 20 min after contamination (Table 3), and 2.8% of the viral contamination transferred 60 min after contamination (22). Rotavirus has been described to persist better on hands than rhinovirus or parainfluenzavirus (24).
Many enveloped viruses such as influenza virus, parainfluenza virus (Table 1), and cytomegalovirus (139) may survive on the hands for 10 to 15 min or even up to 2 h (herpes simplex virus type 1 [Table 1]). Adenoviruses have been described to persist on human skin for many hours (499).
Only a few studies of the persistence of viruses on surfaces have been performed. Rotavirus and HAV can persist for up to 60 days (Table 1) depending on the room temperature, air humidity, and type of surface (495). HIV remains infective on surfaces for up to 7 days, depending on the inoculum and the type of preparation (cell-associated virus or cell-free virus). HIV obtained from clinical specimens remains infective for a few days (568). Influenza A virus may persist on steel for up to 48 h; on other materials, such as paper or handkerchiefs, the virus persists for up to 12 h (46). Rhinovirus may persist for up to 7 days (Table 1).
| MINIMUM SPECTRUM OF ANTIMICROBIAL ACTIVITY |
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| AGENTS FOR REDUCTION OF THE NUMBERS OF PATHOGENS ON HANDS |
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(ii) Testing under practical conditions. The use of plain soap and water reduces the numbers of microorganisms and viruses by mechanical removal of loosely adherent microorganisms from the hands. Many studies are available which address the reduction of the transient hand flora. The most common type of artificial contamination of hands for test purposes in the United States is S. marcescens (21), whereas E. coli is the main contaminant used in Europe (115). Regarding the transient flora, a reduction between 0.5 and 2.8 log10 units can be found within 1 min for E. coli (Table 6). Other types of artificial contamination have been used as well, such as VRE, rotavirus, Klebsiella spp., or spores of Bacillus atrophaeus. A simple hand wash still leads to a mean reduction of up to 2.4 log10 units within 1 min (Table 7). There is basically no effect on resident hand flora after a 2-min hand wash; after a 5-min hand wash, a reduction of 0.4 log10 unit was found, and after 3 h of wearing gloves, no reduction at all was observed (Table 8).
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Some studies have examined only microorganisms that are left on the hands after a hand wash. Washing hands with soap and water has been described to be ineffective in eliminating adenovirus from the culture-positive hands of a physician and patients, indicating that mechanical removal was incomplete (235). Transient gram-negative bacteria remained on the hands of health care workers in 10 of 10 cases despite five successive hand washes with soap and water (187). Furthermore, transmission of gram-negative bacteria from hands has been shown to occur 11 of 12 cases when a simple hand wash is carried out (129).
(iv) Risk of contamination by a simple hand wash. One risk of using soap and water is the contamination of hands by the washing process per se. This has been reported for P. aeruginosa (143). A possible source is the sink itself, when splashes of contaminated water come in contact with the hand of the health care worker (119). The reason is that the microorganisms are not killed during the hand wash but only removed and distributed in the immediate surroundings of the person, including the clothes. Nonmedicated soaps may also become contaminated and lead to colonization of the hands of personnel and to NIs, e.g., with S. marcescens (492) or Serratia liquefaciens (183).
Although the data involving nonmedicated soap suggest that a simple hand wash has some effect on the transient hand flora, it must be borne in mind that, in reality, a simple hand wash often does not last longer than 10 s (121, 145, 176, 177, 180, 300, 334, 450, 552).
Effect on human skin. Each hand wash detrimentally alters the water-lipid layer of the superficial skin, resulting in a loss of various protective agents such as amino acids and antimicrobial protective factors. Regeneration of the protective film may be insufficient if many hand washes are carried out in a row. This may lead to damage of the barrier function of the stratum corneum by inhingement of intercellular putty substances. The transepidermal water loss (TEWL) increases, and the skin becomes more permeable for toxic agents. At the same time, the superficial skin cells dry out, resulting in dehiscence of the stratum corneum, initially on the microscopic level and in due course on the macroscopic level (280).
The incidence with which simple soaps and detergents affect the condition of the skin of health care workers' hands varies considerably (407). For years, natural soaps that have high pH values were thought to be more irritating to the skin than synthetic detergents with neutral or acidic pHs. However, subsequent studies have found that pH is less important than other product characteristics as a cause of skin irritation (200). In some studies, plain soaps have caused less skin irritation than synthetic detergents, while in others, plain bar soap caused greater skin irritation than did a synthetic antimicrobial-containing detergent (299, 565). Synthetic detergents also vary in their propensity to cause skin irritation (200, 407). The incidence of detergent-related-irritant contact dermatitis is affected by various factors: the concentration of the compound, the type of detergent (anionic, cationic, amphoteric, or nonionic) and its quantity, the refattening, the vehicle, the time of exposure, and area exposed (50, 133, 283, 565). For example, it has been shown in vivo that higher concentrations of sodium lauryl sulfate (a detergent) caused greater skin irritation than lower concentrations did (133). In addition, anionic detergents are known to cause greater skin irritation than amphoteric or nonionic detergents (565).
Another factor is the temperature of the water that is used for the hand wash. Hot water leads to greater skin irritation, as reflected by in vivo measurements of TEWL and in vitro measurements of the penetration of detergent through the skin (50, 133, 405). This is explained by an increased penetration of detergents into the epidermis (405). In addition, scaling of the skin is greater when hands are washed with hot water (50). Only skin hydration does not appear to be affected by higher water temperatures (50, 405).
Frequent hand washing induces irritative contact dermatitis (ICD) and dry skin (70, 275, 525, 611), which may become colonized with nosocomial pathogens. ICD can be found in 18.3% of nursing staff in hospitals and is a major occupational health concern (523). A single hand wash already significantly reduces the dermal sebum content; the reduction lasts for 1 h. Skin hydration drops at the same time (280). If hands are washed four times within 1 h, the skin does not recover to its normal state within this period (337). In a study with 52 volunteers who washed their hands 24 times per day for a total of 5 days, a significant increase of the TEWL was observed, indicating that the skin barrier function is impaired (299). The prevalence of ICD caused by hand washing with antimicrobial soaps (detergents) is related to the factors listed above (540). The hardness of water may also affect the incidence of ICD due to frequent hand washing (591).
In summary, plain soap has basically no antimicrobial activity. A simple hand wash can reduce transient bacteria by 0.5 to 3 log10 units but has no real effect on the resident hand flora. The dermal tolerance is rather poor (Table 9).
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The main target is the bacterial cytoplasmic membrane (360, 464). After chlorhexidine has caused extensive damage to the cytoplasmic inner membrane, precipitation or coagulation of protein and nucleic acids occurs (487). Damage also occurs to the outer membrane in gram-negative bacteria and the cell wall in gram-positive cells (131, 142, 227, 228, 236). Chlorhexidine also damages the cytoplasmic membrane of yeasts (588) and prevents the outgrowth, but not the germination, of bacterial spores (511). If chlorhexidine is hydrolyzed, small amounts of carcinogenic para-chloraniline may develop (87); this chemical has been found even in manufactured chlorhexidine solutions (274). At temperatures above 70°C, chlorhexidine is not stable and may degrade to para-chloraniline (171). An upper limit for para-chloraniline has been set in the British Pharmacopoeia at 0.25 mg per 100 mg of chlorhexidine (17).
Effect on microorganisms and viruses. (i) Spectrum of activity. The antimicrobial activity of chlorhexidine is dependent on its concentration. At lower concentrations, chlorhexidine has a bacteriostatic effect against most gram-positive bacteria (e.g., at 1 µg/ml), many gram-negative bacteria (e.g., at 2 to 2.5 µg/ml) (100, 195), and bacterial spores (513). At chlorhexidine concentrations of 20 µg/ml or more, a bactericidal effect can be expected as well as activity against yeasts (487). The actual effective concentration against Burkholderia cepacia and S. aureus varies with different supplements from 0.004 to 0.4% (factor 100), and the actual killing time also varies with different supplements (phenylethanol or edetate disodium) from <15 min to >360 min (465). In most studies, concentrations for rapid inactivation are well in excess of MICs, e.g., for S. aureus (103), E. coli. Vibrio cholerae (237), and yeasts (214). When used in a liquid soap, chlorhexidine usually has a concentration of 4% and exhibits a bactericidal activity against various gram-negative (130) and gram-positive (249) bacteria. In some comparative studies using suspension tests chlorhexidine (4%) was found to be less effective against MRSA than against methicillin-susceptible S. aureus, which has raised concerns about the suitability of the active agent in the prevention of transmission of MRSA (93, 192, 249). This concern has been confirmed with enterococci. Against Enterococcus species and VRE, chlorhexidine (4%) was found to be essentially ineffective in suspension tests if neutralization of residual activity is excluded (247). In a comparison with a nonmedicated hand wash product, a chlorhexidine-based scrub yielded a lower reduction of different antibiotic-resistant test bacteria such as MRSA, VRE, or high-level gentamicin-resistant enterococci (175). Chlorhexidine has no sporicidal activity (513). The data on mycobactericidal activity are not unambiguous but do indicate the relevance of a threshold concentration of chlorhexidine. In one report, 4% chlorhexidine was described as having very good activity against Mycobacterium smegmatis (reduction of >6 log10 units within 1 min) (54), whereas another study with Mycobacterium tuberculosis suggested a low activity of 4% chlorhexidine (reduction of <3 log10 units within 1 min) (55). Chlorhexidine at 1.5% did not reveal sufficient activity against Mycobacterium bovis (56), and chlorhexidine at 0.5% had no activity against Mycobacterium avium. Mycobacterium kansasii, or M. tuberculosis within 120 min (466).
Against dermatophytes such as Trichophyton mentagrophytes, chlorhexidine (1.5%) has been described as having no activity (56).
Antiviral activity has been described as good against most enveloped viruses, such as HIV, cytomegalovirus, influenza virus, RSV, and herpes simplex virus (284, 441), but the virucidal activity of chlorhexidine against naked viruses such as rotavirus, adenovirus, or enteroviruses is low (391, 498).
In comparison to other active agents, chlorhexidine has been described to be less effective in vitro against various nosocomial pathogens than is benzalkonium chloride or povidone iodine (517).
Overall, chlorhexidine seems to have good residual activity (13, 34, 305, 328, 423, 468, 476), but the residual activity must be assessed with caution. It may be false positive due to insufficient neutralization of chlorhexidine in the test design, leading to bacteriostatic concentrations beyond the actual exposure time. Significant difficulties in effective neutralization in in vitro tests have been described, and may yield false-positive activity data for this active agent (246, 516, 517, 600). In addition, the clinical benefit of such a residual effect has never been shown.
(ii) Testing under practical conditions. A 1-min hand wash with soap containing 4% chlorhexidine has been reported to lead to a mean reduction of E. coli of 3.08 log10 units on artificially contaminated hands (478). In a study with 52 volunteers who washed their hands 24 times per day for a total of 5 days, a significant decrease in the number of resident skin bacteria was observed with a 4% chlorhexidine liquid soap (mean reduction of 0.76 log10 unit) compared to nonmedicated bar soap (mean increase of 0.21 log10 unit) and a povidone-iodine soap (mean reduction of 0.32 log10 unit) (299). Under practical conditions with hands artificially contaminated by MRSA, chlorhexidine-based liquid soap was equally effective as simple soap (188, 220). A similar result was reported after contamination of hands with S. aureus (577). A reduction of 2.1 to 3 log10 units was found on hands contaminated with Klebsiella spp. after a 20-s hand wash with a soap based on 4% chlorhexidine (81). If hands were contaminated with rotavirus and treated with chlorhexidine soap for 10 s, the number of test viruses was reduced by 86.9%, which was significantly lower than the reductions achieved with 70% ethanol (99.8%) and 70% isopropanol (99.8%) (23). Treatment with 4% chlorhexidine soap for 30 s on hands contaminated with rotavirus leads to a similar effect of only 0.27 to 0.5 log10 unit (47). Under practical conditions and in terms of removal rate from hands, the efficacy against bacterial spores (e.g., B. atrophaeus) of an antiseptic liquid soap based on chlorhexidine was similar to that of nonmedicated soap, indicating that within 10 s or 60 s, chlorhexidine does not exhibit a significant sporicidal activity (57, 594). The effect of 4% chlorhexidine on the resident hand flora was found to be a reduction of between 0.35 and 2.29 log10 units, depending on the application time (Table 8).
(iii) In-use tests. The in-use studies yield a heterogeneous picture of the efficacy of chlorhexidine. One of the first studies with chlorhexidine was performed in 1955. A hand cream containing 1% chlorhexidine was rubbed into dry hands and led to a substantial reduction is the number of resident skin bacteria after 30 min (386). In another clinical study, 74 health care workers evaluated plain soap and a liquid soap based on 4% chlorhexidine over 4 months in a neurosurgical unit and a vascular surgery ward. Overall hand contamination was found to be significantly lower after the use of plain soap (mean number of CFU, 125) than after the use of chlorhexidine (mean number of CFU, 150) (343). A hand wash with 4% chlorhexidine was reported to be more effective on the total bacterial count under clinical conditions than was a 1% triclosan hand wash (140). In a prospective crossover study over 4 months with plain soap and a 4% chlorhexidine soap among health care workers in two surgical units, plain soap was found to be significantly more effective than chlorhexidine in reducing bacterial counts from the hands of health care workers (343). After contamination of hands with Klebsiella spp., a 98% reduction was described in 19 of 23 experiments in which a soap based on 4% chlorhexidine was used (81); this is an almost 2 log10 unit reduction. Chlorhexidine failed to eliminate MRSA from the hands (140). In contrast, gram-negative bacteria were more likely to be eliminated after the use of chlorhexidine (140, 357, 573, 580). The mean resident flora of the hands of surgeons was reduced by a 3-min application of 4% chlorhexidine from 3.5 log10 units (preoperatively) to 3.15 log10 units (postoperatively) in operations lasting less than 2 h. It has been shown that for operations lasting more than 3 h, 4% chlorhexidine was unable to keep the resident skin bacteria below the baseline value (4.5 preoperatively and 5.2 postoperatively) (76).
(iv) Resistance.
The definition of chlorhexidine resistance is often based on a report from 1982 in which the MICs of chlorhexidine for 317 clinical isolates of P. aeruginosa were analyzed, leading to the suggestion that resistance to chlorhexidine should be reported if the MIC is 
50 mg/liter (390).
Resistance to chlorhexidine among gram-positive bacterial species is rather uncommon. Among Streptococcus and Enterococcus species, no chlorhexidine resistance has been demonstrated (42, 231). However, gram-negative bacteria, such as E. coli (389), Proteus mirabilis (100, 536), Providencia stuartii (227, 228, 554), P. aeruginosa (390, 556), P. cepacia (348), and S. marcescens (291), have frequently been reported to be resistant to chlorhexidine. The frequency of resistance for the different species is variable. A total of 84.6% of clinical isolates of P. mirabilis must be considered resistant to chlorhexidine (536). Among other gram-negative bacteria, the rate is lower (42, 195). C. albicans was found to have a resistance rate of 10.5% (42, 231).
Acquired resistance to chlorhexidine has been reported to occur in S. aureus (249) and among many gram-negative bacteria (37, 38, 434) which were isolated after recurrent bladder washouts using 600 mg of chlorhexidine per liter (537, 538) or after addition of chlorhexidine to catheter bags for paraplegic patients (584). Some of the isolates were highly resistant, with chlorhexidine MICs of 500 mg/liter (538). The chlorhexidine resistance is quite clearly linked to hospital isolates only. A selection of 196 environmental gram-negative isolates did not reveal a resistance to chlorhexidine (147).
High chlorhexidine MICs correlate with poor reduction in the number of test bacteria in suspension tests, which highlights the potential hazard (555). The MIC may be as high as 1600 µg/ml and correlates well with a slow and insufficient bacterial reduction in suspension tests, as shown with strains of Providencia (539). The resistance may be single (83), but cross-resistance to other anti-infective agents can also occur. Among isolates of P. aeruginosa from industry and hospitals, an association between resistance to antibiotics and chlorhexidine has been described (290). The potential for cross-resistance between antiseptic agents and antibiotics must be given careful consideration (443). Various nonfermenting gram-negative bacteria which were isolated from blood cultures of oncology patients were inactivated only with >500 mg of chlorhexidine per liter (210).
Different mechanisms of resistance have been found. The acquired resistance is probably linked to the inner (227) or the outer (551) membrane of bacterial cells, the cell surface (131), or the cell wall (549). It may also be explained by the presence of plasmids which code for chlorhexidine resistance (269) and may therefore be transfered to other bacterial species (486, 619). A change in lipid content or a reduced adsorption of the antiseptic can be excluded as the main mechanism of resistance, as shown with isolates from urinary tract infections caused by P. mirabilis (554) and S. marcescens (410).
Recurrent exposure of bacteria to chlorhexidine may lead to adaptation and may enhance their resistance. This phenomenen was shown with S. marcescens. One example involves repeated exposure to various contact lens solutions containing between 0.001 and 0.006% chlorhexidine, which enabled S. marcescens to multiply in the disinfectant solution (154). Repeated exposure of P. aeruginosa to 5 mg of chlorhexidine per liter was shown to increase the MIC from <10 to 70 mg/liter within 6 days (556). A similar result was reported with Pseudomonas stutzeri, which became resistant (MIC, 50 mg/liter) after 12 days of exposure to chlorhexidine (550). Even with Streptococcus sanguis, a clear increase of the chlorhexidine MIC during permanent chlorhexidine exposure was observed (601). In general, higher exposures to chlorhexidine in hospitals were reported to be associated with higher rates of resistance (67). Recently, some isolates of P. aeruginosa. K. pneumoniae, and A. baumannii isolated from soap dispensers were reported to multiply in a 1:2 dilution of a 2% chlorhexidine liquid soap; ATCC strains of K. pneumoniae and A. baumannii multiplied only at higher dilutions (73). The latter report highlights the potential danger for the hospital.
Resistance to chlorhexidine may even result in nosocomial infections. Occasional outbreaks of NIs have been traced to contaminated solutions of chlorhexidine (345). There is one report that a 0.5% chlorhexidine solution which was used to disinfect plastic clamps for Hickman lines and was handled by health care workers who transmitted the adapted bacteria to intravenous lines led to 12 cases of bacteremia with three fatalities (357). In another outbreak, contamination of a disinfectant solution with Burkholderia multivorans led to nine cases of surgical site infection (45). Especially when chlorhexidine resistance is endemic in gram-negative bacteria, the use of chlorhexidine-based hand antiseptics may lead to an increase of NIs by the chlorhexidine-resistant species (100).
Effect on human skin. Chlorhexidine gluconate is among the most common antiseptics causing ICD (540). However, the frequency of hand dermatitis associated with chlorhexidine-containing detergents is concentration dependent; products containing 4% chlorhexidine cause dermatitis much more frequently than do those containing lower concentrations (540). However, even preparations with the same concentration of chlorhexidine (4%) may cause skin irritation at different frequencies (398, 508). The differences are presumably due to other components of the various formulations. The relatively large number of reports of dermatitis related to chlorhexidine gluconate was partly explained by the fact that it was one of the most widely used antiseptics. In a survey of over 400 nurses working in several hospitals, detergents containing chlorhexidine were reported to cause skin damage less frequently than was nonantimicrobial soap or other detergents containing antimicrobial agents (298). In one 5-day prospective clinical trial, a detergent containing 4% chlorhexidine gluconate caused less irritation than did plain bar soap (300). Nonetheless, dry skin may occur with repeated exposure to preparations containing 4% chlorhexidine gluconate (339, 398).
The potential for contact allergy has been studied as well. Among eczema patients, 5.4% were found to have a positive skin reaction after a single patch test with 1% chlorhexidine, indicating the presence of an allergic contact dermatitis. Repeated exposure resulted in a sensitization rate of ca. 50% (310). In another study, 15 (2.5%) of 551 patients showed a strong and obviously relevant skin reaction in a single patch test with 1% chlorhexidine (415). Although these studies were carried out with patients and not with health care workers, the results nevertheless indicate the potential for sensitization and allergic contact dermatitis during frequent use. Allergic reactions to the use of detergents containing chlorhexidine gluconate on intact skin have been reported and can be severe, including dyspnea and anaphylactic shock (30, 92, 124, 138, 158, 270, 409, 425, 430, 468, 526, 563). Some cases of contact urticaria have also occurred as a result of chlorhexidine use (141, 617).
In summary, chlorhexidine (2 to 4%) has good activity against most vegetative bacteria, yeasts, and enveloped viruses but limited activity against mycobacteria, dermatophytes, and naked viruses. It has a moderate potential for acquired bacterial resistance. A hand wash with a chlorhexidine-based soap can reduce the number of transient bacteria by 2.1 to 3 log10 units; the effect on the resident hand flora is smaller, with a mean reduction between 0.35 and 2.29 log10 units. The dermal tolerance is rather poor, and anaphylactic reactions have been reported (Table 9).
The mode of action of triclosan was identified some years ago. For decades, it has been assumed that triclosan attacks the bacterial cytoplasmic membrane (372, 458). Since 1998, we have known that it blocks lipid synthesis by inhibition of the enzyme enoyl-acyl carrier protein reductase, which plays an essential role in lipid synthesis (367). Mutation and overexpression of the fabI gene—which encodes the enoyl-acyl carrier protein reductase—are able to abolish the blockage of lipid synthesis caused by triclosan (205, 312). The fabI gene was first found in E. coli (366) and was subsequently also found in various other bacterial species such as P. aeruginosa (215), S. aureus (203, 520), and M. smegmatis (365). Some other bacteria, such as Bacillus subtilis, contain orthologous enoyl-acyl carrier protein reductases, namely those encoded by fabI and fabK, which are not inhibited by triclosan (204, 206). A genetic sequence coding for broad-spectrum resistance to triclosan has been identified (239).
The identification of the specific mode of action has raised concerns about the development of resistance to triclosan (313, 366, 506). A recent study has shown that this concern is valid. Strains of P. aeruginosa were exposed to triclosan and subsequently developed multiresistance to various antibiotics, including ciprofloxacin (86). Particular care should be taken in the use of triclosan in ICUs, where P. aeruginosa is the most common nosocomial pathogen, causing lower respiratory tract infection (260).
Effect on microorganisms and viruses. (i) Spectrum of activity. In vitro, triclosan exhibits a bacteriostatic effect at lower concentrations (575); at higher concentrations, it has bactericidal activity (560<
