Chlorhexidine: A hand sanitizer monograph

Originally developed in England in 1954, chlorhexidine first became available in the United States on September 17, 1976, under the brand name Hibiclens®.^1-3 Chlorhexidine alone is insoluble in water, so it is typically combined with glucuronic acid to form a chlorhexidine gluconate (CHG) salt that is soluble in both water and alcohol.^3,4

A membrane-active agent, Chlorhexidine is primarily used to reduce the number of bacteria found on the skin, and is recommended for both surgical hand and general skin antisepsis due to its persistent presence on the skin.³ Despite these generally accepted roles, actual evidence of benefit is conflicting and continued use of chlorhexidine seems largely unjustified.

Mechanism of action

Chlorhexidine is believed to exert its antimicrobial effects through three mechanisms. The cationic nature of chlorhexidine enables it to specifically target the negatively charged bacterial envelope. At low concentrations, chlorhexidine disrupts the lipid bilayer, inducing leakage of ionic potassium, inorganic phosphates, amino acids, and other cytoplasmic material.^3-7 This effect is primarily bacteriostatic in nature and neutralization or removal of chlorhexidine may allow the cell to recover.⁴ As the concentration of chlorhexidine increases to bactericidal levels, membrane leakage decreases as the biguanide causes nucleic acids and proteins to precipitate and coagulate.^3-7 Additionally, high concentrations of chlorhexidine are thought to inhibit membrane-bound adenosine triphosphatase (ATPase), thereby reducing the cell’s ability to produce useable energy.^4,5 Cytoplasmic coagulation and inhibition of ATPase result in cell death.⁴

Efficacy

The in vitro antimicrobial effects of chlorhexidine exposure have been repeatedly demonstrated; however, concerns regarding insufficient neutralization and conflicting in vivo results have cast serious doubt on the practical value of this agent. Chlorhexidine acts far more slowly than alcohol-based antiseptics and appears to exert an immediate antimicrobial effect similar to non-antimicrobial soap, leaving its clinical value highly debatable. Despite the demonstrated superiority of alcohol-based antiseptics both in vivo and in clinical trials, chlorhexidine has remained in use due to its purportedly persistent antimicrobial activity following application. However, in vivo studies indicate similar long-term reductions in normal flora for both alcohol and chlorhexidine. In light of the available literature, the rationale for chlorhexidine-based antisepsis appears largely unfounded.

In vitro spectrum

Chlorhexidine is effective against most Gram-positive and Gram-negative bacteria, has some activity against enveloped viruses and fungi, but is not sporicidal and lacks substantial activity against mycobacteria and nonenveloped viruses.^3-9 Gram-positive and Gram-negative bacteria are typically susceptible to the bacteriostatic effects of chlorhexidine at minimum inhibitory concentrations (MICs) of 1 µg/mL and 2-2.5 µg/mL, respectively.^5,7 Bactericidal effects are generally observed at concentrations ≥ 20 µg/mL.^5,7 In a test of 1,165 bacterial isolates, the majority of strains were inhibited by 12.5 µg/mL of chlorhexidine and 100 µg/mL was sufficient to inhibit all evaluated strains.⁴ Since typical in-use concentrations range from 0.5-4% (5,000-40,000 µg/mL), clinical levels of exposure should exceed most reported MICs by several orders of magnitude.^3,4

In vivo results

Despite the broad antimicrobial spectrum of chlorhexidine, in-use efficacy is at best marginally superior to plain soap. For example, a 15-second handwash with chlorhexidine showed no advantage over plain soap in reducing normal hand flora. Chlorhexidine did not demonstrate superiority until 15 washes had been performed, and 5 consecutive days of 15 hand washes resulted in a mean log₁₀ reduction factor (RF) of 2.04 (vs. 0.59 for plain soap).¹⁰ In another study, fingertip-contamination with methicillin-resistant Staphylococcus aureus (MRSA) showed equivalent log₁₀ RFs for chlorhexidine 4% and plain soap (1.91 vs. 1.96) following inoculation with 10³ bacteria. Following inoculation with 10⁶ bacteria, chlorhexidine was inferior to plain soap (1.37 vs. 1.77).¹¹  Chlorhexidine was also inferior to plain soap in eliminating 10⁶ Clostridium difficile (47% spores) from gloved hands (1.3 vs. 1.7) and 10⁶ Bacillus anthracis surrogates (1.4 vs. 2.2).^12,13 After a comprehensive literature review, Kampf et al (2004) estimated that chlorhexidine reduces transient flora by 2.1-3 log₁₀ and resident flora by 0.35-1.75 log₁₀ (vs. 0.5-3 and ≤ 0.4, respectively, for plain soap).⁷

Trends in the testing timeline provide further perspective. The vast majority of studies demonstrating the superiority of chlorhexidine over plain soap occurred around the time the biguanide was approved by the FDA, while most trials conducted in recent years have shown the two to be roughly equivalent. Assuming a lack of research bias, this can be largely attributed to differences in application time. Many early studies assessed the efficacy of chlorhexidine after 2 minutes of application. Since changes in time of exposure have shown to increase the RF of chlorhexidine from 1 log₁₀ at 30 seconds to 1.74 log₁₀ at 2 minutes and handwashing duration is typically 10 seconds or less, the results of these early studies cannot be assumed to apply to normal practice.^3,14 In contrast to their predecessors, more recent studies utilizing relevant exposure times show a general lack of effect. These differences in methodology and results further undermine in vivo efficacy claims, particularly in settings that mimic clinical practice.

The Neutralization Controversy

Further complicating the efficacy debate, recent reevaluation of neutralizing agents used in chlorhexidine testing has cast considerable doubt on the validity of many in vitro and in vivo trials.²⁸ Since residual chlorhexidine persists on surfaces after initial application, incomplete neutralization of chlorhexidine results in continued bacteriostatic or bactericidal effect in the sampling fluid, diluents and/or agar. This reportedly leads to a 2 log₁₀ overestimation of reduction factor in vitro and in vivo.^29-32 The discovery that commonly used neutralizing agent combinations (eg, 3.5% Tween 80, 0.5% lecithin, and 0.5% sodium thiosulfate) failed to inactivate chlorhexidine has generated significant concern regarding the dependability of the majority of chlorhexidine studies.^28,32-34

The relevance of these issues is particularly evident when considering the in vivo chlorhexidine studies referenced by the CDC.³ While all of these studies utilized some sort of neutralizing agent, many failed to validate their neutralization process. The absence of demonstrable chlorhexidine neutralization renders the results of these trials unreliable. Since trials conducted without validation of neutralization can be assumed to err on the side of efficacy overestimation, it is reasonable to presume that trials which failed to show benefit in the absence of proven neutralizing agents would have failed to show benefit in their presence as well. Using this logic, of the dependable in vivo studies referenced by the CDC guideline, there is only a 5:3 superiority-to-inferiority ratio when comparing chlorhexidine with plain soap. Notably, the CDC guideline does not reference two articles with negative efficacy results authored prior to release of the guideline.^12,28 Taking these trials into account, there is a 5:5 superiority-to-inferiority ratio of validated chlorhexidine vs. soap studies conducted prior to compilation of the CDC guidelines.

Clinical studies

Not surprisingly, the clinical benefits of chlorhexidine use are controversial.^7,35 Positive efficacy studies are few and generally limited by poor compliance, inconsistent results, or comparison solely against povidone-iodine. One study showed similar efficacy between 2% chlorhexidine and 61% ethanol in a neonatal intensive care unit (ICU) with poor handwashing compliance over a 22-month period.³⁶ Similarly, another ICU study showed improvement in total nosocomial infection rate with chlorhexidine vs. alcohol use; however, this was not associated with difference in length of stay, mortality, or total number of infected patients.³⁷ Chlorhexidine 4% also demonstrated better general antibacterial activity than triclosan 1% (0.27 vs. 0.07 log₁₀ RF) during a 16-month longitudinal study, though chlorhexidine was ineffective against MRSA.³⁸ Additionally, multiple clinical trials have demonstrated the superiority of chlorhexidine over povidone-iodine; however, both biocides appear to be less effective than alcohol-based antiseptics.^4,39-41

In contrast to these studies, chlorhexidine use has been frequently associated with a lack of clinical benefit. Despite its purported persistent antimicrobial activity, chlorhexidine was unable to maintain resident bacterial counts below baseline values during operations lasting ≥ 3 hours (4.5 preoperatively vs. 5.2 postoperatively).⁴² Frequent handwashing with chlorhexidine in a neonatal ICU was associated with a doubling in baseline bacterial colonization over the course of a month.⁴³ Marena et al reported that chlorhexidine was significantly less effective than plain soap in reducing hand contamination in a 4-month crossover study of two surgical wards.⁴⁴ The results of these and other studies leave the clinical value of chlorhexidine use highly debatable.^45,46

Persistent activity

In spite of mediocre efficacy and dubious clinical benefit, chlorhexidine is still used in a variety of roles thanks to its non-volatility and ability to adsorb to surfaces. Five hours after application, 93% of radio-labeled chlorhexidine remains present on uncovered skin, which correlates well with claims of persistent antimicrobial effect for 5-6 hours. Twenty-four hours after application of a 5% tincture, concentrations of 12.2 µg/mL (~0.25%) remain on the skin.⁴ Chlorhexidine appears to adsorb less readily to inorganic surfaces—approximately 1% binds to glass.⁴⁷ Overall, these results indicate the presence of residual chlorhexidine greatly outlasts the original application time, though the clinical relevance of this non-volatility has never been demonstrated.⁷

The CDC defines persistent activity as “prolonged or extended antimicrobial activity that prevents or inhibits the proliferation or survival of microorganisms after application of the product.”³  Multiple trials conducted in an effort to demonstrate the persistent activity of chlorhexidine have reported mixed results. These results are complicated by the synergistic effects of alcohol on residual chlorhexidine activity as well as a lack of neutralization validation. Combination with alcohol appears to enhance the antimicrobial effects of residual chlorhexidine, making it difficult to quantify the independent activity of either agent.⁴⁸ Additionally, while the need for proper, verifiable neutralization appears to add weight to residual antimicrobial activity arguments, insufficient neutralization makes it difficult to tell whether changes in microbial count are due to the residual presence of chlorhexidine on the skin or the presence of chlorhexidine in the growth medium after sampling. This concept is corroborated by the results of an “ex-vivo” study, where the presence of chlorhexidine in solution resulted in a 3-4 log₁₀ greater reduction of pathogenic bacteria than observed when chlorhexidine was applied to skin samples.⁴⁹

Suppression of Normal Flora

The CDC handwashing guideline cites eight studies as proof that chlorhexidine exhibits persistent antimicrobial activity.³ On close inspection, however, the majority of these articles provide little evidence to support this claim.²⁸ One “study” is actually one of the first review articles to point out the need for proper chlorhexidine neutralization, arguing that the reported persistence of chlorhexidine may be due to its action in sampling fluids.²⁹ A second article reports only the immediate effects of chlorhexidine.¹⁸ A third claims to demonstrate the superiority of 0.5% chlorhexidine over 70% isopropyl alcohol in reducing bacterial counts on the hands. However, despite multiple daily washings, chlorhexidine use was associated with increased baseline colonization increased (5.76 to 5.92 log₁₀ CFUs) and 6-hour regrowth that was far more rapid than that observed with alcohol (see below).⁵⁰

Other articles cited by the CDC are less contrary, but still inconclusive. In one, both chlorhexidine and alcohol—alone and in combination—were associated with an approximately 1 log₁₀ larger reduction in normal flora after 3 hours of glove use than after initial administration, but there was no significant difference in RF between agents at any point.¹⁶ Another study reported slower normal flora recovery with 4% chlorhexidine than with a 70% ethyl alcohol and 0.5% chlorhexidine combination, however, overall bacterial reduction was greater with the combination product regardless of number of washings or the length of time after antisepsis.²² Lastly, Pereira et al compared various strengths of chlorhexidine and combinations with alcohol. Though all tested groups showed a greater reduction in CFUs 2 hours after initial administration than at administration, post-exposure CFU counts were higher following a second washing, suggesting swift bacterial recovery between treatments.⁵¹

Many other trials have been conducted in an effort to quantify the persistent activity of chlorhexidine. The majority of these have found chlorhexidine alone or in combination with alcohol to be approximately equal to or slightly inferior to alcohol-based hand-rubs in suppressing the normal flora.^16,23,53-58 Faoagali et al reported a mild persistent effect on the first day of administration, though this disappeared after 5 days of twice-daily antisepsis.²⁵ Results from another study indicated that delay of 72-hour regrowth of the normal flora on the upper arms and back was greater for the combination of chlorhexidine and n-propanol than for n-propanol alone by approximately 1 log₁₀. Baseline CFU counts, however, were far lower than those typically observed on hands (~2.5 vs. ~ 6 log₁₀), and delay of regrowth may have been due in part to virtual elimination of the natural flora during initial antisepsis.⁵⁹ These results, along with those previously discussed, indicate that while chlorhexidine may mildly suppress the normal flora over time, the effects are controversial and of limited clinical relevance.

Contamination prevention

Bacterial contamination after antisepsis can also be used to assess the persistent activity of a biocide. Studies evaluating the ability of chlorhexidine to prevent new bacterial adherence and survival are limited, though their results are favorable. In 3 separate studies, Lowbury et al inoculated hands immediately following antisepsis with 20-50 µL suspensions of Staphylococcus aureus or Escherichia coli. The skin was allowed to dry for 2 minutes, rinsed and dried on a sterile towel, and then sampled 1 hour later.^15,16,60 The quantity of bacteria administered was not reported in two of these trials, which reported only the number of viable bacteria per mL after sampling for chlorhexidine and plain soap. Both trials showed a substantially greater decrease in surviving bacteria following chlorhexidine use.^15,60 Initial inoculum counts were included in the third trial, which compared 0.5% chlorhexidine in 95% ethyl alcohol with 70% ethyl alcohol.  Alcoholic chlorhexidine demonstrated a much greater ability to prevent artificial S. aureus contamination than alcohol alone (3.47 vs -0.01) after inoculation with 4.7 log₁₀ of bacteria. Intriguingly, while Lowbury also tested both biocides against E. coli, they did not report these results due to an unexpected residual effect in the alcohol group.¹⁶ Unfortunately, prolonged application time and immediate administration of a bacterial suspension make the results difficult to extrapolate to clinical practice.

Resistance

Given the already low efficacy profile of chlorhexidine, increasing reports of bacterial resistance are particularly concerning. Most Gram-positive and many Gram-negative bacteria lack significant resistance to chlorhexidine, though increases in MIC have been noted in some strains of MRSA, Escherichia coli O157, Salmonella enterica and Acinetobacter baumannii.^62-68 In contrast, effective resistance to 0.5% chlorhexidine is often observed in clinical strains of Providencia stuartii (83.3%), Pseudomonas spp. (45.7%), Proteus spp. (38.7%) and Klebsiella spp. (1.8%).⁶² While the link between biocide resistance and antibiotic resistance has not yet been completely elucidated, many strains with significant chlorhexidine resistance also exhibit broad antibiotic resistance.^62-64,69-71 Fortunately, exposure to residual chlorhexidine does not seem to engender meaningful resistance.^47,72,73  However, regardless of reported planktonic MICs—most of which are irrelevant at in-use concentrations—chlorhexidine resistance and subsequent pathogenicity appear to be primarily due to the formation of biofilms.

Biofilms

Bacterial biofilms have high chlorhexidine resistance that allows constituent members to survive in concentrations 100s-1000s of times greater than the MICs reported for their planktonic counterparts.^74-78 Biofilms form readily on almost any surface and are associated with an estimated 65% of nosocomial infections.^77-79 Bacteria exhibit varying propensities for biofilm formation, which correspondingly affects their resistance to chlorhexidine. For example, in vitro biofilm studies demonstrated that 1% chlorhexidine eliminated 1.3 log₁₀ of S. aureus and 1.6 log₁₀ of Salmonella spp. after 5 minutes of exposure and only 0.22 log₁₀ of P. aeruginosa after 30 minutes of exposure time.^80,81 Clearly, biofilm-conferred resistance permits microorganisms to survive for prolonged periods in the presence of chlorhexidine.

Clinically speaking, biofilm formation not only enables biocide survival on fomites and environmental surfaces but may also directly facilitate infection outbreaks through contaminated chlorhexidine solutions. ⁸² Serratia marcescens biofilms are able to survive in chlorhexidine for years and can be cultured from solutions containing 2% chlorhexidine.⁸³ Contamination of chlorhexidine solutions by Serratia marcescens and Pseudomonas aeruginosa has been repeatedly reported and linked to outbreaks of infection.^68,82,83 Ironically, biofilm contamination is such an omnipresent infection risk that it is recommended that stock solutions of chlorhexidine be periodically disinfected with alcohol.^68,82

Hindering factors

Putting clinical efficacy aside, a variety of substances undermine the killing ability of chlorhexidine. As a cationic molecule, chlorhexidine is inactivated natural soaps or other anionic compounds, non-ionic surfactants, cork, and hand creams containing anionic emulsifying agents.^2-8,84 The activity of chlorhexidine is reduced in the presence of organic material (eg, blood, sputum), though it remains more far more effective than other cationic compounds.^2,3,8,85 At concentrations ≥ 0.05%, chlorhexidine precipitates from solution as an insoluble salt when combined with borates, bicarbonates, carbonates, citrates, nitrates, phosphates, sulfates, and most dyes.³ Additionally, chlorhexidine may also be neutralized by hard water.³

Toxicity

Chlorhexidine use is typically well-tolerated and has a good safety record, though reports of skin irritation with chlorhexidine use range as high as 90% and can lead to poor use rates.^{3,4,8,9,55,86} Since chlorhexidine is not absorbed through the skin, systemic exposure following topical administration is extremely unlikely and has not been reported.^4,87,88 There are 5 documented cases of acute toxicity following ingestion of high doses of chlorhexidine that manifested as gastrointestinal erosion, gastritis, and/or acute liver toxicity.⁴ Ocular exposure to preparations containing > 1% chlorhexidine can cause conjunctivitis and/or severe corneal damage and care should be taken to avoid touching the eyes after application.^3,4,89 Sensorineural deafness has been reported in patients who received 0.05% chlorhexidine in 70% alcohol for perioperative disinfection of the ear. Additionally, allergic reactions can occur, particularly when chlorhexidine is used in the genital area or on neonates, and this hypersensitivity has lead to non-fatal anaphylaxis in several cases.⁴ However, considering the widespread, decades-long use of chlorhexidine, the incidence of toxic effects is remarkably low and chlorhexidine appears to be safe for general use.

Environmental effects

While chlorhexidine has an excellent topical safety profile, widespread use of the biguanide has raised concerns regarding the role of chlorhexidine in the environment.⁹⁰ Chlorhexidine accumulates in the environment and may reach concentrations as high as 10.3 µg/mL (0.001%) in domestic wastewater.^90,91 Concentrations of 10 µg/mL have been shown to have effects on algal and cyanobacterial biofilm, with bacterial biofilms beginning to change at concentrations of 100 µg/mL. The ultimate effects of this perturbation are unknown, but since biofilms form the foundation of the food chain, changes at this level may have broad consequences. Additionally, microbes appear to lack the ability to render chlorhexidine inert, which may result in progressive environmental accumulation.⁹⁰ Since the EPA has determined that chlorhexidine is not only toxic to microbes but also slightly toxic to birds, moderately to highly toxic to fish and very highly toxic to invertebrates, continued use at current rates may lead to rising levels of environmental exposure with far-reaching ecological effects.^89-91

Uses

Orally, chlorhexidine gluconate is used to reduce flora in the mouth for a variety of therapeutic and prophylactic purposes. Chlorhexidine binds strongly to negatively charged oral surfaces (eg, hydroxyapatite on tooth enamel, pellicle on the tooth surface), and is reported to exert an inhibitory effect on oral flora anywhere from 2 days to 12 weeks.^92,93 The true residual oral activity of chlorhexidine is somewhat debatable; however, since biofilms of resident oral bacteria such as Streptococcus mutans demonstrate marked resistance to 0.2% chlorhexidine and may not be accounted for during the spit tests used to determine persistent activity.⁹⁴ Despite these conflicting reports, chlorhexidine is used clinically to treat gingivitis (0.12%) and periodontitis (biodegradable subgingival pellets). Additionally, chlorhexidine mouthwash is used to prevent dental carries and plaque when normal oral hygiene is impossible, to reduce the incidence of mucositis and other oral complications in immunocompromised patients, and to decrease the risk of nosocomial respiratory tract infections in critically ill patients. Chlorhexidine appears to be highly effective in preventing respiratory tract infections, but efficacy data for other prophylactic oral uses is poor.⁹²

Alternative formulations may offer ways to circumvent some of the limitations of chlorhexidine. Catheters impregnated with chlorhexidine and silver sulfadiazine have been shown to reduce catheter-related bloodstream infections by approximately 60%, though efficacy has been somewhat inconsistent.^95-97 Additionally, while still approved in the United States, these catheters have been discontinued in Japan due to severe anaphylactic reactions associated with use.^97-99 Another formulation currently under development utilizes chlorhexidine-loaded nanocapsules to achieve a slow-release, sustained antimicrobial effect. Nanochlorex® appears to have a similar immediate effect to 62% propanol on resident bacteria and a dramatically superior effect (5.5 log₁₀ vs 1 log₁₀) on the survival of transient Staphylococcus epidermidis. However, this persistent activity disappears after 4 hourly inoculations with 1 mL of 10⁷ bacteria.^100,101 These preparations may provide more clinical benefit than currently available forms.

Conclusion

Chlorhexidine’s persistent presence in the market appears to be as unwarranted as claims of chlorhexidine’s persistent antimicrobial activity. While the membrane permeabilizing abilities of chlorhexidine give it a good in vitro profile, in vivo tests of immediate effect demonstrate a mild, equivocal superiority to plain soap, particularly at in-use application times. The vaunted residual presence of chlorhexidine also appears to be largely irrelevant, since numerous studies have shown an overall equivalence or inferiority to alcohol in long-term reduction of normal flora. Chlorhexidine may have some role in preventing exogenous recontamination, but this is undermined by poor clinical efficacy data, microbial resistance, limited effect against biofilms, skin dehydration and environmental accumulation. Despite this dearth of proven benefit, since the biguanide appears to provide the best persistent activity of currently available agents, chlorhexidine will likely remain in general use until the advent of a practical alternative.

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