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Test Organism

A nontoxigenic strain of C. difficile was used for the study (ATCC 700049, American Type Culture Collection). The organism was incubated anaerobically in brain‐heart infusion broth at 37°C for 48 hours to produce a seed culture, which was used to inoculate flasks containing 500 mL of brain‐heart infusion broth. The flasks were incubated anaerobically at 37°C for 48 hours and portioned into 20‐mL aliquots (henceforth referred to as “the inoculum”). The inoculum was stored at −80°C until use. Quantitation of the inoculum was assessed by serial dilution and incubation on cycloserine‐cefoxitin fructose agar plates supplemented with 5% sheep's blood and 0.1% taurocholate (CCFA‐T),¹³ and the percentage of spore forms in the inoculum was measured with the use of an alcohol shock method followed by serial dilution onto CCFA‐T.¹⁴

Subjects

Ten hospital laboratory workers volunteered for this study, and informed consent was obtained prior to participation in the study. No volunteers had any dermatologic diseases affecting their hands at any point during the study. No limitations were set on fingernail length or on the presence of rings or other hand jewelry, nor were any data collected on these variables. No specific training in handwashing techniques was provided to the volunteers at any time during the study. Ethical approval was obtained from the Research Ethics Board of the McGill University Health Centre.

Hand Hygiene Interventions

A crossover format was used so that all volunteers would be exposed to all interventions once for each contamination protocol during the observation period of June–July 2007. The order of interventions for each volunteer was assigned by means of computer‐generated random number lists. The hand hygiene interventions studied were (1) warm (30°C) water with plain soap (Hygenipak Instafoam, Deb Canada), (2) cold (15°C) water with plain soap (Hygenipak Instafoam, Deb Canada), (3) warm (30°C) water with 2% chlorhexidine antibacterial soap (Hygenipak 2% CHG foaming skin cleanser, Deb Canada), (4) antiseptic hand wipes with 40% vol/vol ethanol and 0.5% parachlorometaxylenol (Sani‐dex antimicrobial hand wipes, PDI), (5) alcohol‐based handrub with 70% vol/vol isopropanol (Manorapid, Antiseptica, Pulheim‐Brauwiler), and (6) a no‐wash negative control. Each intervention using soap and water was timed for 10 seconds of lathering, followed by brief rinses with water and drying with paper towels. Use of antiseptic hand wipes and alcohol rubs was each timed for 15 seconds. Hands were allowed to air dry for 1 minute after use of antiseptic handwipe and alcohol‐based handrub. Participants washed their hands with plain soap and water every time they entered or exited the laboratory. A minimum of 24 hours between interventions for each volunteer was mandated to reduce carryover of residual handwashing agents or bacteria.

Whole‐Hand Protocol

The 20 mL of inoculum was poured into a clean, tight‐fitting nitrile glove. Each hand was sequentially placed into the glove, and the inoculum was gently dispersed throughout the glove for 30 seconds. The hands were allowed to air dry for 1–2 minutes, and then one of the hand hygiene interventions was performed. Subsequently, each hand was placed into a new glove containing 20 mL of sterile brain‐heart infusion broth, which was gently dispersed for 30 seconds. A 1‐mL sample of the broth was removed via pipette for quantification by serial 10‐fold dilution on CCFA‐T and incubated anaerobically at 37°C for 48 hours. If a plate yielded 1–100 colony‐forming units (CFUs), the colony counts were recorded.

Surface Contamination Protocol

A ceramic tile measuring cm was painted with 1 mL of the inoculum and dried for at least 30 minutes at room temperature. Both palms were placed on the contaminated surface for 5 seconds, and then one of the hand hygiene interventions was performed. Subsequently, hand impressions were made onto 6 separate CCFA‐T plates for 5 seconds each: 1 plate each for the fingertips, hypothenar surface, and thenar surface of each hand. If a plate yielded 0–50 colonies, the colony counts were recorded; plates with more than 50 colonies per plate or confluent growth were evaluated as “too numerous to count.”

Statistical Analysis

Previous studies of hand hygiene have shown that effective agents can reduce bacterial loads by approximately 2.0 log₁₀, with a standard deviation of 0.4 log₁₀.⁷ A difference of at least 1 log₁₀ between interventions was considered to be an important difference. With 10 paired assessments for each product, a power of more than 99% to detect a 1.0 log₁₀ difference was calculated. Hierarchical models, adjusted for the effect of multiple observations on each subject, were used to compare the different interventions. For the whole‐hand data, the model was fit by using the base‐10 logarithm of the C. difficile colony count (CFUs per milliliter). For the surface data, a 2‐part model was used that estimated (1) the proportion of observations with scores of “too numerous to count” for each intervention and (2) the median colony count among all plates with countable colonies (0–50 colonies per plate). Both models adjusted for the effect of right versus left hand. The surface model also adjusted for the hand area (hypothenar, thenar, or fingertips) tested. Both models were estimated using a Bayesian approach. A noninformative prior distribution was used for both models, and the posterior median and 95% credible interval (CrI) for the parameters of interest were extracted. The R statistical software package (version 2.3.1; R Foundation for Statistical Computing) was used for descriptive statistics and graphs. The models were fit with WinBUGS (version 1.4.1; MRC Biostatistics Unit).

Inoculum of Test Organism

The concentration of C. difficile test organism used was CFU/mL, consisting of 62% spores. Preliminary testing, including turbidimetry, indicated that this concentration was routinely achievable (data not shown). This concentration was selected to ensure that C. difficile colonies remain detectable following hand hygiene interventions, thus allowing a precise measurement of effect size. Furthermore, this concentration is biologically and clinically relevant, because in a previous study, 1 research group has measured concentrations of C. difficile directly from stool specimens of patients both before and during treatment for CDI that were within 1.5 log₁₀ of this value.¹⁵

Whole‐Hand Protocol

A total of 120 observations (10 participants, 2 hands each, 5 interventions, and 1 negative control) were compared in a pairwise fashion. The results are summarized as mean log₁₀ colony counts after intervention (Table 1) and comparison of log₁₀ colony count reductions between pairs of interventions (Table 2). The largest reductions were observed for plain soap with the use of either warm or cold water versus either no hand hygiene or alcohol‐based handrub. Antibacterial soap had the next largest statistically significant effect, followed by antiseptic hand wipes. Finally, the effect of the alcohol‐based handrub was not significantly different from the effect of not washing.

Table 1.  Mean Clostridium difficile Colony Counts after Different Hand Hygiene Interventions According to the Whole‐Hand Protocol

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Table 2.  Pairwise Comparison of Mean Reduction in Clostridium difficile Colony Count (Intervention 1 vs Intervention 2) for Hand Hygiene Interventions Used According to the Whole‐Hand Protocol

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Palmar Surface Protocol

A total of 318 observations (for interventions: 10 participants, 2 hands each, 3 surfaces per hand, 5 interventions; for negative control: 3 participants, 2 hands each, 3 surfaces per hand) were taken; note that the negative control group included only 3 participants, as colony counts were uniformly too numerous to count. For each intervention, the probability of observing plates evaluated as heavily contaminated (too numerous to count) is displayed in Table 3. The likelihood of heavy contamination was high for alcohol‐based handrub although significantly lower than for no hand hygiene intervention. Thus, alcohol‐based handrub did produce a significant reduction in contamination although of a lesser magnitude than did the other hand hygiene interventions. The comparison of colony count reductions between pairs of interventions is shown in Table 4. The largest reductions in colony counts were observed after any of the interventions involving plain or antibacterial soap versus alcohol‐based handrub; the next largest reductions were after antiseptic handwipe versus alcohol‐based handrub, with soap‐based interventions demonstrating a small advantage over antiseptic hand wipes.

Table 3.  Probability of Heavy Contamination (Colonies Too Numerous to Count) Following Different Hand Hygiene Interventions Used According to the Palmar Surface Protocol

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Table 4.  Pairwise Comparison of Mean Reduction in Clostridium difficile Colony Count (Intervention 1 vs Intervention 2) for Hand Hygiene Interventions Used According to the Palmar Surface Protocol

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Comparing the 3 different anatomical areas of the hand, odds ratios for a score of “too numerous to count” for observations from hypothenar versus fingertips (median odds ratio, 7.98 [95% CrI, 1.96–37.65]) and thenar versus fingertips (median odds ratio, 5.14 [95% CrI, 1.25–23.41]) both suggested that fingertips were less likely than other hand areas to remain heavily contaminated with C. difficile following a hand hygiene intervention.