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Effect of sampling time on bacterial yield from the hands
Elaine L. Larson, R.N., Ph.D., FAAN Paul I. Eke, M.Sc. Barbara E. Laughon, Ph.D. Baltimore,
Maryland
Despite the Food and Drug Administration’s publication in 1978l of guidelines for methods to assay bacteria from the hands, investigators studying handwashing and scrubbing products continue to use a variety of sampling techniques, stripping solutions, and timing procedures.2”’ The need for standardization in assaying techniques and thorough descriptions of protocols used has been identified.5. ’ In this study we evaluated the effect of stripping (sampling) time on microbial yield from the hands. Forty healthy adult volunteers were assigned by block randomization to one of four handwashing products, 10 subjects per product: a nonmedicated liquid soap, control (Safe ‘n Sure; Calgon Corp., St. Louis); an antiseptic containing 4% (wt/vol) chlorhexidine gluconate, CHG (Hibiclens; Stuart Pharmaceuticals, Wilmington, Del.); and two alcohol-based hand rinses, AlcA and AlcB, (CalStat, Calgon, and Hibistat, Stuart). Within each of these four groups, subjects were assigned by block randomization to a 1.5second or a 3-minute stripping time. After an initial “social” handwash with the nonantiseptic soap, a baseline hand culture was obtained by using the sterile bag technique described below. For this baseline sample, stripping (rubbing the hand through the wall of the bag) was done for 3 minutes for all subjects. All
From ease,
the School of Nursing and the Division of infectious School of Medicine, The Johns Hopkins University.
Funded
in part
Reprints
not available.
272
by the Calgon
Corporation,
St Louis.
Dis-
subjects were then trained to use a standardized 15-second handwashing procedure using 3 ml of their assigned soap for each wash. Hand cultures were obtained after one handwash and again after 15 consecutive handwashes. Rings and nail polish were not worn during this testing. The sterile bag technique’ was used to assay organisms on the hand. The subject inserted the dominant hand into a sterile polyethylene bag containing 50 ml sampling solution (sterile distilled water containing, per liter: lecithin, 20 gm; sodium thiosulfate, 6 gm; sodium oleate, 6 gm; protease peptone, 1 gm; tryptone, 1 gm, and Tween 80, 50 ml, pH 7.2 to 7.4). In preliminary studies we determined that there was no significant increase or decrease in colunyforming units (CFU) in this solution within 2 hours. The entire surface of the hand was rubbed vigorously through the wall of the bag for the specified time (either 15 seconds or 3 minutes). All specimens were plated within 2 hours of sampling. A 0.1 ml volume of serial dilutions up to lo-” was placed on trypticase soy agar containing yeast extract, 5 gm/L, and Tween 80, 1 ml/L, and incubated at 37” C for 48 hours. CFU were counted and reported as total CFU per hand. For each of the subjects, logarithms (base 10) of the differences in microbial coums from baseline sampling to samples collected after one and 15 handwashes were calculated. A Tukey oncway analysis of variance8 was used to compare these differences for each product, between the group whose hands were sampled for 15 sec-
Volume
15 Number
December
6
Effect of sampling
1987
time on bacterial
yield
273
Table 1. Reductions from baseline in mean aerobic CFU (log IO) by handwashing agent and sampling time CHG 15 set sampling Baseline Reduction Reduction No signlflcant
counts after1 after15
6.63 1.13 1.78
handwash handwashes
differences
in counts
between
AlcA 3 min sampling
15 sac sampling
3 min sampling
6.80 1.52 2.60
7.06
6.20 0.71 1.45
the two sampling
AicE
1.50
2.83
times were found
onds and the group whose hands were sampled for 3 minutes. This sample size would detect a 50% difference in log CFU between sampling times with a power of 75% and an cx value of p < 0.05.9 Initial aerobic log CFU from hands of the 40 subjects ranged from 5.9 to 8.5. As expected, reductions in log CFU were much greater among subjects using antiseptics rather than plain soap. For the entire group, there were no significant differences in mean reductions in log CFU among subjects whose hands were sampled for 15 seconds or 3 minutes after one handwash (p = 0.45) or after 15 washes (p = 0.49) (Table 1). We conclude that the difference in bacterial harvest from the skin of the hands with sampling time longer than 1 minute probably adds little information of clinical importance, and therefore a l-minute sampling should be sufficient for most studies.
15 set sampling
after one handwash
6.81 0.63 2 04
Control 3 min sampling
15 set sampling
6.41 0.84 2.40
6.63 0.45 0.78
(p = 0 45) or after 15 handwashes
3 min sampling 6.18 0.08 0.29 (p = 0 49).
References 1. Proposed rules. Federal Register 1978;43( Jan 6): 1242-3. Rotter ML, Keller W, Wewalka G, Werner HP, Ayliffe GAJ, Babb JR. Evaluation of procedures of hygiene hand-disinfection. J Hyg 1986;96:27-37. 3. Selwyn S. Evaluating skin disinfectants in vivo by excision biopsy and other methods. J Hosp Infect 1985; 6(suppl):37-43. 4. Goldblum SE, Ulrich JA, Goldman RS, Reed WP, Avasthi PS. Comparison of 4% chlorhexidine gluconate in a detergent base (Hibiclens) and povidone-iodine (Betadine) for the skin preparation of hemodialysis patients and personnel. Am J Kidney Dis 1983;2:548-52. 5. Editor. Data lacking on safe, effective antiseptics and disinfectants. Hosp Infect Control 1986;14:29-35. 6. Larson E. Handwashing and skin. Physiologic and bacteriologic aspects. Infect Control 1985;6: 14-23. 7. Larson E, Strom MS, Evans CA. Analysis of three variables in sampling solutions used to assay bacteria of hands. J Clin Microbial 1980;12:355-60. 8. SPSS Inc. SPSS X user’s guide. 2nd ed. New York: McGraw-Hill, 1986:466-70. 9. Lachin JM. Introduction of sample size determination and power analysis for clinical trials. Controlled Clin Trials 1981;2:93-113. 2.
Maryland
Despite the Food and Drug Administration’s publication in 1978l of guidelines for methods to assay bacteria from the hands, investigators studying handwashing and scrubbing products continue to use a variety of sampling techniques, stripping solutions, and timing procedures.2”’ The need for standardization in assaying techniques and thorough descriptions of protocols used has been identified.5. ’ In this study we evaluated the effect of stripping (sampling) time on microbial yield from the hands. Forty healthy adult volunteers were assigned by block randomization to one of four handwashing products, 10 subjects per product: a nonmedicated liquid soap, control (Safe ‘n Sure; Calgon Corp., St. Louis); an antiseptic containing 4% (wt/vol) chlorhexidine gluconate, CHG (Hibiclens; Stuart Pharmaceuticals, Wilmington, Del.); and two alcohol-based hand rinses, AlcA and AlcB, (CalStat, Calgon, and Hibistat, Stuart). Within each of these four groups, subjects were assigned by block randomization to a 1.5second or a 3-minute stripping time. After an initial “social” handwash with the nonantiseptic soap, a baseline hand culture was obtained by using the sterile bag technique described below. For this baseline sample, stripping (rubbing the hand through the wall of the bag) was done for 3 minutes for all subjects. All
From ease,
the School of Nursing and the Division of infectious School of Medicine, The Johns Hopkins University.
Funded
in part
Reprints
not available.
272
by the Calgon
Corporation,
St Louis.
Dis-
subjects were then trained to use a standardized 15-second handwashing procedure using 3 ml of their assigned soap for each wash. Hand cultures were obtained after one handwash and again after 15 consecutive handwashes. Rings and nail polish were not worn during this testing. The sterile bag technique’ was used to assay organisms on the hand. The subject inserted the dominant hand into a sterile polyethylene bag containing 50 ml sampling solution (sterile distilled water containing, per liter: lecithin, 20 gm; sodium thiosulfate, 6 gm; sodium oleate, 6 gm; protease peptone, 1 gm; tryptone, 1 gm, and Tween 80, 50 ml, pH 7.2 to 7.4). In preliminary studies we determined that there was no significant increase or decrease in colunyforming units (CFU) in this solution within 2 hours. The entire surface of the hand was rubbed vigorously through the wall of the bag for the specified time (either 15 seconds or 3 minutes). All specimens were plated within 2 hours of sampling. A 0.1 ml volume of serial dilutions up to lo-” was placed on trypticase soy agar containing yeast extract, 5 gm/L, and Tween 80, 1 ml/L, and incubated at 37” C for 48 hours. CFU were counted and reported as total CFU per hand. For each of the subjects, logarithms (base 10) of the differences in microbial coums from baseline sampling to samples collected after one and 15 handwashes were calculated. A Tukey oncway analysis of variance8 was used to compare these differences for each product, between the group whose hands were sampled for 15 sec-
Volume
15 Number
December
6
Effect of sampling
1987
time on bacterial
yield
273
Table 1. Reductions from baseline in mean aerobic CFU (log IO) by handwashing agent and sampling time CHG 15 set sampling Baseline Reduction Reduction No signlflcant
counts after1 after15
6.63 1.13 1.78
handwash handwashes
differences
in counts
between
AlcA 3 min sampling
15 sac sampling
3 min sampling
6.80 1.52 2.60
7.06
6.20 0.71 1.45
the two sampling
AicE
1.50
2.83
times were found
onds and the group whose hands were sampled for 3 minutes. This sample size would detect a 50% difference in log CFU between sampling times with a power of 75% and an cx value of p < 0.05.9 Initial aerobic log CFU from hands of the 40 subjects ranged from 5.9 to 8.5. As expected, reductions in log CFU were much greater among subjects using antiseptics rather than plain soap. For the entire group, there were no significant differences in mean reductions in log CFU among subjects whose hands were sampled for 15 seconds or 3 minutes after one handwash (p = 0.45) or after 15 washes (p = 0.49) (Table 1). We conclude that the difference in bacterial harvest from the skin of the hands with sampling time longer than 1 minute probably adds little information of clinical importance, and therefore a l-minute sampling should be sufficient for most studies.
15 set sampling
after one handwash
6.81 0.63 2 04
Control 3 min sampling
15 set sampling
6.41 0.84 2.40
6.63 0.45 0.78
(p = 0 45) or after 15 handwashes
3 min sampling 6.18 0.08 0.29 (p = 0 49).
References 1. Proposed rules. Federal Register 1978;43( Jan 6): 1242-3. Rotter ML, Keller W, Wewalka G, Werner HP, Ayliffe GAJ, Babb JR. Evaluation of procedures of hygiene hand-disinfection. J Hyg 1986;96:27-37. 3. Selwyn S. Evaluating skin disinfectants in vivo by excision biopsy and other methods. J Hosp Infect 1985; 6(suppl):37-43. 4. Goldblum SE, Ulrich JA, Goldman RS, Reed WP, Avasthi PS. Comparison of 4% chlorhexidine gluconate in a detergent base (Hibiclens) and povidone-iodine (Betadine) for the skin preparation of hemodialysis patients and personnel. Am J Kidney Dis 1983;2:548-52. 5. Editor. Data lacking on safe, effective antiseptics and disinfectants. Hosp Infect Control 1986;14:29-35. 6. Larson E. Handwashing and skin. Physiologic and bacteriologic aspects. Infect Control 1985;6: 14-23. 7. Larson E, Strom MS, Evans CA. Analysis of three variables in sampling solutions used to assay bacteria of hands. J Clin Microbial 1980;12:355-60. 8. SPSS Inc. SPSS X user’s guide. 2nd ed. New York: McGraw-Hill, 1986:466-70. 9. Lachin JM. Introduction of sample size determination and power analysis for clinical trials. Controlled Clin Trials 1981;2:93-113. 2.