Application of a gloved hand model for multiparameter measurements of skin degerming activity

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Application of

a

Gloved-Hand Model for Multiparameter

Measurements of Skin-Degerming Activity

R. N. MICHAUD,* M. B. McGRATH, AND W. A. GOSS'

Departments of Microbiologyand Biometrics, Sterling-Winthrop ResearchInstitutL, Rensselaer, New York12144

Received for publication 17November 1975

The application ofanestablished gloved-hand modeltomultiparameter meas-urements ofskin-degerming activity is described. In particular, appropriate

experimental designs are illustratedwhich allow characterization of the per-formance of topical skin-cleansing preparations in terms of rapid, sustained, cumulative, andpersistent skin-degerming effectsonthe hand. Single-contact

studieswereusedtodefine the degermingactivityprofiles of selected commer-cial surgical scrub preparations, and to establish the optimal post-treatment

sampling interval for individual preparations. Rapid and sustained

skin-de-germingeffectsweremeasured andcontrasted. Rapid skin-degermingactivity, namely, that occurring during actual handcontact, was demonstrated foran

iodophor preparation. Sustained skin-degerming activity, namely, that occur-ringontheglovedhandduringapostcontactinterval,wasshown and character-ized fortwohexachlorophene preparations. Multiple-contact studies witha 3% hexachlorophene preparationwereusedtoillustratecumulative andpersistent

skin-degermingeffects. Cumulativeskin-degermingactivitywasdemonstrated in terms of progressive bacterial reductions after repeated contacts within a

single day.Persistentskin-degermingactivitywasshown intermsoftheprofile

ofdaily pretreatment bacterial counts aftermultiple contactsover successive

days. Uniformity oftreatment response was established for a broad range of pretreatment bacterial countsextendingfromapproximately log4 to log7per

hand.Theimportanceof thepretreatmentbacterialcount measurementand of adequateneutralization of handextractsamplesis stressed. Arandomized-hand

experimental design is discussed relative to its versatility and amenabilityto

statistical analysis.

The fundamental features of a model for measuring skin-degerming activity on the hand have been previously reported from this laboratory (4). The basic elementof the model describedinthe earlier reportwas animproved gloved-hand samplingtechnique for the quanti-tativeandreproducibledetermination of bacte-riallevelsonthehand. Thegloved-hand proce-dure involves theuseofasterile rubber

surgi-cal glove as a direct sampling device for the

controlledextraction of skin bacteria fromthe

hand. This direct

sampling

technique was

shown to be a significantly more efficient

mi-crobiologicalextractionmethod(4) than the in-direct glove-washing technique used by some

investigators in the past (3, 7, 8). Recently, gloved-hand sampling methodshave beenused

in several reported studies related to the

de-germingperformanceof varioussurgicalscrub

' Present address: CalgonConsumerProducts Research Laboratories, Merck &Co.,Inc.,Rahway,N.J.07065.

products (2, 5, 6).

The present report deals with theutility and versatility of the gloved-hand modelin its ap-plication to multiparameter measurements of skin-degerming activity. Appropriate experi-mental designs are illustrated that allow the measurement of (i) rapid and sustained de-germing activity after single-contact scrub-bing, (ii) degerming activity profiles, (iii)

cu-mulative activity aftermultiple-contact

scrub-bingand, (iv) persistent activity after extended

multiple-contact scrubbing. A factor of basic

importance is that data obtained from these

designed experiments are readilyamenableto statistical analysis for the evaluation of

uni-formity oftreatment response and/or specific

treatment comparisons with a high degree of confidence.

(This paperwaspresentedinpartatthe74th Annual Meeting ofthe American Society for Microbiology,Chicago,Illinois,15May 1974.)

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MATERIALS AND METHODS

Skin-cleansingagents.pHisoHex(Winthrop Lab-oratories, NewYork, N.Y.),Betadine Surgical Scrub (The Purdue Frederick Co., Yonkers, N.Y.), Septisol (Vestal Laboratories, St. Louis, Mo.), and Maxine nonmedicated bar soap (Swift Chemical Co., Ham-mon, Ind.) wereobtained fromcommercial sources. pHisoHex is an anionicliquiddetergent containing 3%hexachlorophene (HCP). Betadine Surgical Scrub is aniodophor liquid cleanser containing povidone-iodine at anunspecified concentration. Septisol is a soap-based liquid preparation containing 0.75% HCP (0.375% HCP at usedilution). The undiluted products have pH values of approximately 5.5, 5.0, and 9.0,respectively.

Test panels. Test panels of 12 to 36 volunteer

subjects were randomly selected from laboratory

and secretarial personnel employedatthis institu-tion. The mixed male and femalesubjects were re-quested to avoid contact of the hands with all medi-cated and antiseptic materials beginning 3 days prior to, and for the duration of, the test period. During this interval, eachsubject was provided with non-medicated barsoap (Maxine) to be used exclusively for all handwashing exposures except those pre-scribed experimentally. No other restrictionswere imposed on the subjects, who were allowed to carry ontheirnormal activities.

Washingandscrubbingprocedures. After remov-ing alljewelry, subjects scrubbed or washed their hands and wrists under supervision for 2 to 6 min using tap waterand the appropriate assigned prod-uct.Eachproductwasusedessentiallyinthe man-nerrecommendedby the manufacturer. Hands and wrists werefirst wet, and each hand wasscrubbed or washedseparately for the specified timeperiod us-ingeither 2 or 3 mlof the 3% HCP preparation, 5 ml of the iodophor preparation, or 2ml of thediluted 0.75% HCP preparation (diluted 1:2 with an equal volume of watertogive 0.375%HCP). In one study, the opposing hand to the one being scrubbed or washed was isolated and protected with a sterile surgical glove,and the procedure was then reversed. A nylon-bristle surgical brush was used for scrub contacts, with special attention given to scrubbing

the fingernails and interdigital surfaces. After

scrubbingorwashingfor therequiredtimeinterval, subjectsrinsed their handsthoroughlyunderrunning tap water, driedthem withasterile cotton towel, and donned sterilerubbersurgical gloves. Pertinent de-tails relative to thescrubbingorwashing procedures for each studyarenoted in thelegendsof the tables andfigures.

Hand-sampling procedures. Microbiological sam-ples were obtained by a gloved-hand sampling tech-niquepreviously described (4). Sterile 0.85% saline (pH 6.8) was used as the sampling fluidineach case except for the post-treatmentsamples after iodophor contacts, where saline containing 0.1% sodium thio-sulfatewasusedas aneutralizer for residual iodine. In all cases, 10-ml samples ofthe resulting hand extracts were immediately added to an equal vol-ume of chilled lecithin-phosphate buffer (LPB) (4), which served asaneutralizer forresidual HCP. For

studies of crossover design, a pooledpretreatment

sample wasprepared using 5-ml aliquotsfrom the right and left hands of each subject. Decimal dilu-tions of the neutralized samples were prepared in chilledlecithin-phosphate buffer and aliquots were plated intriplicate with Letheen agar(BBL) using thepour-plate technique.

Gross et al. (1)reported that 0.5% sodium thiosul-fate incorporated into solid agar media markedly inhibits the growth of cutaneous staphylococci. In the present studies, 0.1% sodium thiosulfate was used, as necessary, in the saline sampling fluid, but was not incorporated directly into the buffer diluent or solid culture media. Under these con-ditions, the maximal concentration of sodium thiosulfate transferred to the culture media was 25

Ag/ml,

alevel that was established in control stud-ies to be noninhibitory to normal skin bacteria. Moreover, Kundsin and Walter (2) have demon-strated that the viabilityofStaphylococcus epider-midis is unaffected after suspension of this

predomi-nantskin inhabitant for upto 1hinsaline contain-ing 0.1%sodium thiosulfate.

Experimental designs and statistical analysis. Depending on thespecificobjective(s)ofaparticular

study, either a crossoverexperimentaldesignora randomized-hand experimentaldesignwasused. In crossover studies, each subject was randomly

as-signedto oneoftwo or moresubgroupsfor thefirst treatmentinterval. After a minimal3-dayrest pe-riod to permit reestablishment of normal levels of skin bacteria, the experimental variable was crossed-over with respect to subjects. In this way, comparativemeasurements were obtained for each

subject, therebyprovidingawithin-subjectcontrast. Incomparative treatment studies, both hands of each subject were sampled for the enumeration of bacteria immediately before each scrub (Pre). Post-treatmentsamples were obtained from one hand of eachsubject immediately after each scrub (To)and from the opposite hand after a glove-wearing period of either 60 min(T60)or 180min(T180).

A crossover design was also used for studies in which the degerming activity profile of single prod-ucts wasexamined. After a 30-s prewash with non-medicated bar soap to remove transient bacteria (4), both hands of each subject were sampled for the enumeration of resident skin bacteria immediately before scrubbing (Pre). Each subject then conducted a single-contact scrub with the preparation under

study. Post-treatment sampleswere obtained from onehand of eachsubject immediatelyafterthe scrub

(To)and from theopposite handafter a glove-wear-ingperiod of either 1, 2, 3, or4h.The procedure was repeatedoneach of4separate scrubdays,allowing a 3-day rest period before each scrub day. Followinga crossoverdesign,eachsubjectwassampledoncefor each glove-wearing intervaloverthecourse of the study.

Intherandomized-hand study, the hands of each subject were randomly and separately assigned to one ofsixtreatment regimens involving thesame product. Thedesign involved the isolation and pro-tectionofonehand with a surgicalglove while the opposing hand was subjected to the specified

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ment regimen. For each treatment regimen, four contacts (either wash or scrub)were conducted on the assigned hands withina6-hperiod, allowinga

minimum of1 h between contacts. The study was conductedover3successivedays, with subjects and treatmentregimensbalanced withrespect to days. Extract sampleswereobtainedfrom each hand for theenumeration of resident skin bacteria immedi-ately before (Pre) andat60min after (T60) the first and fourth contacts. Sterile surgical gloves were worn after each of these contacts beforeT60 sam-pling.

Inthe extended multiple-contact study, each sub-ject scrubbed both hands 20 times with a single product over a 5-day period, but only one hand (either rightorleft)wasrandomly assigned for mi-crobiological monitoringoverdays. This designwas similartothe randomized-handdesign,exceptthat asingle-treatment regimenwasused. Microbiologi-cal samples wereobtained from the assigned hand immediately before (Pre) the firstcontactandat60 minafter (T.)the first and fourthcontactsoneach day. A sterile surgical glove was worn after the lattercontactsbeforeT. sampling. Thesamehand ofeach subjectwasalso sampledonce ondays3, 4, and5after discontinuation of the scrubbing regimen (days 8, 9, 10).

The model for studies usingcrossover (or split-hand) designs has been described previously(4).The randomized-hand design is described by the fixed effects covariance model (9):

Yij = A + B(Xij -X) + yj+ eu

i = 1, . ..,n hands j = 1, ...,mtreatments

That is, thepost-treatmentbacterial densityonthe hand (Yu) canbe represented as a function of the populationmean (,u),the influence of the pretreat-ment bacterial count [,B(Xij - X)], the treatment

effect (yj), and the residual error (e1j). The covari-ance component, 8(Xij - X), is derived from the

regression estimate of the influence (,3)of the

indi-vidual pretreatment count (Xij) and the overall

meanpretreatmentcount(X)onthe post-treatment bacterial level on the hand. The inclusion ofthe covariance componentin this modelprovidesa more

preciseestimateof residualerrorthan could be ob-tained withasimplefixed effects model of the form Yij = ,u + yj +eij.

Bacterialcountdatawereroutinelytransformed to logarithms before statistical analysis (4). The primarymeansfortesting hypothesesand for deter-mining statistical significance of associated

con-trasts wasthrough the useofanalysis of variance andanalysisof covariancetechniques.Standard

er-rorsbasedonthe residualerrorterm,and probabil-ity values associated with the calculated Fratios have been includedtosummarize the salientpoints of each analysis. Data from subjects who did not completeaparticular studywerenotincluded in the statisticalanalyses.

RESULTS

Rapidversus sustained degerming activity. One of the parameters that may be used to

evaluateatopical antimicrobial cleansing prep-aration is therate ofskin-degerming activity. To examine thisvariable, apreliminary study was conducted to directly compare the skin-degerming activity of the 3% HCP preparation with that of theiodophor preparation. A treat-mentcrossoverdesignwasutilized with atest panel of 12 subjects. Each subject used each productonceduring the study.

Asanticipated, there was no significant dif-ference between themeannumbers of bacteria recovered from thehands immediately priorto

scrubbing (Pre) for thetwo treatments(Table 1, study 1). This importantcontrastestablisheda reliable reference for subsequent treatment

comparisons. Themean number of skin bacte-ria recoveredimmediately after scrubbing (To)

TABLE 1. Comparison of rapidversussustainedskin-degerming activity Meanlogno. of bacteriarecovered/handa

Measurement Study 1 Study2

3%HCP Iodophor Standarderrorb 3%HCP lodophor Standarderror6 Sampling timer

Pre 6.02 5.76 ±0.21 (P >0.05) 5.29 5.02 ±0.15(P >0.05)

To 5.86 4.75 ±0.20(P = 0.01) 4.95 3.92 ±0.22 (P =0.01)

T60 4.66 4.96 +0.35(P >0.05)

T180 4.26 4.74 ±0.40 (P >0.05)

Mean differences

(Pre -To) 0.16 1.01 ±0.23 (P = 0.01) 0.34 1.10 ±0.20 (P =0.01) (Pre -T60) 1.35 0.79 ±0.32 (P >0.05)

(Pre-T180) 1.03 0.28 ±0.40 (P >0.05)

a Crossoverdesign;12-subjectpanels;3%HCP,4-minscrub with2ml; iodophore,6-minscrub with5ml. bStandarderrorof the difference betweenmeansbasedon awithin-subject comparisonof each treatment, and theprobability associated with this linearcontrastinthecorrespondinganalysisof variance.

cPre, Immediately before scrub; To, immediately after scrub; T60, 60 min postscrub; T180, 180 min

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MEASUREMENTS ACTIVITY 409

was signficantly lower (P = 0.01) for the iodo-phortreatment than that observed for the 3% HCP treatment. Correspondingly, the mean

(Pre-T,)reductioninbacteriallevelswas

signif-icantly greater (P = 0.01) for the iodophor

treatment(90%)versusthe3%HCPtreatment (31%). In contrast, the comparative mean bac-terial levels on the hand at 60 min postscrub

(Tm,,)

were notsignificantly different for thetwo treatments. Likewise, nosignificant difference

wasobserved between the

(Pre-T6o)

reductions

in bacterial levels for the iodophor treatment

(84%) compared to the 3% HCP treatment (96%). It will benoted, however, that themean

post-treatment bacterial levels decreased

con-siderably between

T,,

and T,,) after scrubbing with the 3% HCP preparation, whereas the corresponding bacterial levelswere essentially unchanged in the case of the iodophor

treat-ment.

A second study of similar design was

con-ducted with the 3% HCP preparation and the iodophor preparation to furtherexamine

com-parative differences between thetwo products with respect to rates ofskin-degerming

activ-ity. In thisstudy the post-treatment sampling interval wasextendedto 180 min

(T18o).

As was the case in the previous study, no

significant differences were observed between the mean pretreatmentbacterial levelsonthe hands for thetwo treatments(Table1,study2). The3%HCPtreatmentand theiodophor treat-ment again differed significantly (P = 0.01)

withrespect tothecomparative meanTo bacte-rial levelsonthe hand and also for the observed

(Pre-T,,)

reductionsinbacterial levels. Results for the latter parameter were in good

agree-ment with those noted in the previous study. The mean numbers of bacteriarecovered from the handat

T1,,

were notsignificantly different for thetwotreatments. Comparative

(Pre-T180)

reductionsin meanlevelsof skin bacteriawere

indicative, but not significantly different for the 3% HCP treatment (91%) versus the iodo-phor treatment (48%). It will be noted,

how-ever, thatmeanbacterial levels after the iodo-phortreatment increased appreciably between

T,,

and

Tl,

such thatthe

T1,,8

valueapproached thepretreatment value. In contrast, a further decrease in mean bacterial levels between To

and

T,1,8

wasobserved after the3% HCP

treat-ment.

Degerming activity profiles. It isclear from the above studies that thepretreatment

bacte-rialcount is animportantreference from which skin-degerming activitycanbemeasured. It is

also obvious that a single comparison of

pre-andpost-treatmentbacterial levelscannot

ade-quately characterize the performance of skin-degerming preparations. Therefore, it was of

interest to simultaneously examine a number ofpost-treatment sampling intervals to

deter-mine the pointofmaximaltreatmenteffectand

to gain insight into postscrub trends in the reestablishment ofresident skin bacteria.

Separate studies wereconductedtoexamine thedegermingactivityprofiles of three antimi-crobial cleansingpreparationsafter

single-con-tact use. The preparations studied contained, respectively, 3% HCP, aniodophor,and0.375%

HCP.Foreachstudy,asamplingcrossover de-sign was utilized with a test panel of 16 sub-jects.

Foreach of the three studies, there was no

significant difference in mean pretreatment

levels of skin bacteria over the four separate

scrubdays (Table2). The absence oftreatment carryover effects between scrub days allowed the calculation of overall mean Pre values.

Likewise, ineachstudy, themeanTobacterial levels did not differ significantly over the 4 separate scrub days. Thus, the calculation of overall meanTovalues andoverallmean (Pre-To) reductionswas alsojustified sinceeach sep-arate scrub day could be considered as a

repli-cate experiment. Although the experimental designwas notintended for directcomparisons amongthe threeproductsstudied,itis

notewor-thythat overall

(Pre-TO)

reductionsinbacterial levels were different for the 3% HCP

prepara-tion(60%),theiodophor preparation(84%),and the0.375% HCPpreparation (13%).

The overall mean Pre and overall mean

To

values,aswell asthe postscrubmeanbacterial counts for eachstudy, are summarized in Fig. 1.Referringtostudy1with the3%HCP prepa-ration, itwillbe noted thatthe postscrub

bacte-riallevelonthehand continuedtodecline from the

T0,

level, reaching alow value at 1h

post-scrub,correspondingto an overall reduction of

97%. Thereafter, reestablishment of resident skin bacteria ensued, but the mean bacterial level was significantly depressed (P = 0.01),

relative to the pretreatment level, even at 4 h

postscrub. In study 2, after a single contact

with the iodophor preparation, the maximal

decreaseinbacterial levelswasobservedatTo, and corresponded to a reduction of 84%. This decrease was immediately followed by

reestab-lishment ofresident bacteria, which returned

to the pretreatment levelby 3 h postscrub. In

study 3, after a single scrub with the 0.375% HCPpreparation,skin-degermingactivity

pro-gressedvery slowlyto a maximal effect at 2 h

postscrub, corresponding to an overall reduc-tionof72%. Rapid reestablishment of skin

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TABLE 2. Measurements of rapid degerming activity for three skin-cleansing preparations Meanlog no. of bacteriarecovered/handb

Study Product Sampling Week1 Week 2 Overall Standard error"

timea _Overall__

Scrub 1 Scrub 2 Scrub 3 Scrub 4 mean

1 3%HCP Pre 5.25 5.10 5.20 5.14 5.17 ±0.10 (P > 0.05)

T(, 4.88 4.66 4.68 4.85 4.77 ±0.11 (P > 0.05) (Pre-T,)d 0.37 0.44 0.52 0.29 0.40 ±0.09 (P > 0.05)

2 Iodophor Pre 5.37 5.42 5.38 5.23 5.35 ±0.06(P >0.05)

T,) 4.44 4.59 4.49 4.62 4.54 ±0.13 (P >0.05)

(Pre-TO)' 0.93 0.83 0.89 0.61 0.81 ±0.11 (P >0.05)

3 0.375%HCP Pre 5.20 5.42 5.32 5.48 5.35 ±0.10 (P > 0.05)

T(, 5.23 5.38 5.17 5.36 5.29 ±0.13 (P > 0.05) (Pre-T0)d -0.03 0.04 0.15 0.12 0.06 ±0.06(P > 0.05) Pre, Immediately prior to scrub; T(,, immediately after scrub.

b Crossover design; 16-subject panels;3% HCP,4-min scrub with 2 ml; iodophor, 6-min scrub with 5 ml;

0.375% HCP,4-min scrub with 2 ml; scrubs preceded by a 30-s prewash withnonmedicatedbarsoap. ' Standard error of anindividual mean based on a within-subject comparison among the four scrubs, and the probability associated with this linear contrast in thecorresponding analysis of variance.

dMeandifference.

Q uY d bUUY

X 6 3%HCP IODOPHOR 0375% HCP

~50

400

3.Oi

PREO 2 34 RO 0 2 34

MLt P - fR 1 vR)

FIG. 1. Degerming activityprofiles ofthree skin-cleansing preparations. Sixteen-subject panels;

crossoverdesign with respect topostscrub samples;

3%HCP,4-min scrub with 2ml; iodophore, 6-min scrub with 5 ml; 0.375%HCP, 4-min scrub with 2 ml.Scrubspreceded bya30-sprewashwith

nonmed-icated barsoap. The PreandTo valuesrepresentthe overallmeanoffourseparatemeasurementsper sub-ject.Asinglemeasurementpersubject wasobtained foreachoftheotherpostscrub intervals.

teria followedimmediately,suchthat themean

bacterial level had returned to the

pretreat-ment level by 3 h postscrub and was even higher (P = 0.05) at4 h postscrub.

Uniformity oftreatment response. Because of the rather broad distributionofpretreatment

bacterial countsnormallyobservedamong

sub-jects, it was important to ascertain that each

subjectpanel, asagroup, responded uniformly to treatment. This was confirmed for each of thethreeprecedingstudiesbylinearregrdssion analysis of individual subject responses. To

il-lustrate this point, individual

T0,

counts have

beenplottedas afunction ofthe corresponding

pretreatment counts for each study (Fig. 2). It can be seen that, in each case, response to treatment was uniform over a range of pre-treatmentbacterial counts extending from

ap-proximatelylog 4 to log 6.5. That is, the line of

best fit for the data closely approximated the

theoretical line ofuniformresponse.

Cumulative activity after multiple con-tacts.The foregoing studiesallowed

characteri-zation of theskin-degerming activity of topical antimicrobial cleansing preparations after a singlecontact onthehands. Anotherimportant

parameterrelevanttotheskin-degerming

per-formance of these preparations is the effect of

multiplecontacts onthe residentskin bacteria.

Although the basiccrossover experimental de-sign may be adapted to multiple-contact

stud-ies, a randomized-hand experimental designof greaterutilitywasdeveloped forthis purpose,

where the hand, rather than the subject, was usedasthe testobject.

Utilizing the randomized-hand design, the multiple-contact skin-degerming performance

of the 3% HCPpreparation wasevaluated

un-der several use conditions. Specifically, six treatment regimens were examined, corre-spondingto contacttimesof either2, 4,or6min

withorwithout theuseofa surgical brush.

At each of the four sampling intervals, no

significantdifference wasobservedamong the

mean bacterial levels on the hand for the six

treatment regimens (Table 3). Similarly, the

mean(Pre-T,0)reductionsinskinbacteriaafter asinglecontact werenotsignificantlydifferent

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STUDY 1:3% HCP ! STUDY 2:IODOPHOR STUDY 3:0.375%HCP

:5

/,. I

slopeO091 ±010 slope-087 ±015 slope=

n-64 n-60

4 J 6 2 3 6 5

PRETREATMENT (PRE) LOG BACTERIAL RECOVERY/ HAND

082±009

n=60

D 6 7

-HEORETICAL LINEOF UNIFORMRESPONSE(SLOE/O,

---CALCUL4TED LEAST SOUARE FIT

FIG. 2. Uniformityoftreatmentresponsefor subjectsparticipatingin three separatestudies. Individual subject datafor themeanPreand T.values summarized inTable2.

TABLE 3. Cumulativeskin-degermingactivityofa3%HCPpreparation Meanlogno.of bacteriarecovered/hand'

Measurement Wash Scrub Standard errorb

2min 4min 6min 2min 4min 6min

Samplingtime!

Contact1 Pre 5.73 5.50 5.68 5.71 5.98 5.86 ±0.17 (P > 0.05) Contact1T6,0d 4.80 4.69 4.63 4.61 4.85 4.71 ±0.15(P > 0.05) Contact4 Pred 4.49 4.53 4.50 4.58 4.55 4.17 +0.16(P > 0.05) Contact4T60, 3.64 3.20 3.78 3.77 3.97 3.98 +0.21 (P> 0.05) Meandifferences

(C,Pre-CIT60) 0.93 0.90 1.08 1.11 1.03 1.11 ±0.18(P >0.05)

(C1Pre-C4T60) 2.09 2.33 1.90 1.94 1.98 1.86 ±0.25 (P >0.05)

aRandomized-handdesign; 36-subject panel (n = 12); fourwashorscrubcontactswith3 mlas indicated withina 6-hperiod;contacts oneand fourpreceded by a 30-s prewashwithnonmedicated barsoap.

b Standard error ofan individual mean based oneither the pretreatment analysisof variance or the post-treatmentanalysisof covarianceerrorestimate,and theprobabilityassociated with thecorresponding

linearcontrastamongmeans.

cPre,Immediately priortocontact;

T60,

60 minpostcontact;Cl, firstcontact;C4,fourthcontact. dMean values adjusted through covariate analysis based on the highly significant correlation with pretreatmentvalues.

for the varioustreatmentregimens (range87to 92%). Overallmeanreductions in bacterial lev-els from contact 1 Pre through contact 4

T.,

likewise, didnotdiffer significantlyfor the six

treatment groups of hands (range 98.6 to

99.5%). It is clear,however,that each

multiple-contactregimen resulted inprogressive reduc-tions in levelsof skin bacteria. The degerming

efficacy of this product, however, was not en-hancedby washing regimens longer that2min, norbytheuseofasurgical brush regardless of

contacttime.

Persistent activity afterextended multiple

contacts.Tofurther define theskin-degerming

effects of multiple-contact scrubbing, a study wasundertaken thatsimulated extendeduseof the 3% HCPpreparation. Anadaptation of the randomized-handexperimental designwas

uti-lized, wherein the preparation was used four timesdailyfor 5 consecutivedays.

On each of the first 2treatmentdays,

cumu-lative skin-degerming activity was apparent during multiple scrubbing with the 3% HCP

preparation (Fig.3). Themeanbacterial

reduc-tionsonday1were94% atscrub 1

Tw

and 99%

at scrub 4

Tw.

By treatment day3, the mean number ofskin bacteriaonthe handatscrub 1

Tw

wasreducedtoaminimal level andshowed

no further decrease with additional scrub

con-tacts on this day or on subsequent days. The overall mean bacterial reduction achieved by

treatmentday3was99.9%.

Mean levels of bacteria immediately before

scrubbingoneachtreatment day(scrub 1Pre)

showed a progressive and significant decrease (P = 0.01) over the 5days (Fig. 3). It willbe

C.3

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412 MICHAUD, McGRATH, AND GOSS

6.0 PERIOD

PERIOD

M4.00 SCRUB IPRE

~~~ S~~~CRUB T60

3.0-K ~~~~~SCRUB04T60

2.0

123 4 5 6 7

8'

9 10

DAYS

FIG. 3. Cumulative and persistent skin-degerm-ingactivityafterextended multiple-contact scrubbing with a 3%HCP preparation. Twelve-subject panel; onehand of each subject wasrandomly assigned for microbiologicalsampling; four 2-min scrub contacts with 2 ml of the preparation were conducted daily withina6-h interval on days1 through 5; scrub 1 Pre measurementson all days were preceded by a 30-s prewash with nomedicated bar soap.

noted that reduced bacterial levels persisted overnight on successive treatment days, and didnot return to pretreatmentlevelsuntilday

5postscrub (day 10).

DISCUSSION

The application of the gloved-hand modelto

multiparametermeasurementsof

skin-degerm-ing activity allowssystematic characterization

oftheskin-degerming effects exerted by topical antimicrobial cleansing preparations. The key

parameterswhichyield useful information are

measurements ofrapid, sustained, cumulative,

and persistent skin-degerming effects. A sche-maticrepresentation ofthese different parame-ters ofactivity is shownin Fig. 4.

Collectively, the studies reported herein show that asinglecomparison of pre- and post-treatment bacterial levels cannot adequately

characterize theperformanceof a

skin-degerm-ing preparation. Ideally, a systematic evalua-tion should first focus on examination of the

single-contact activityof thepreparationunder

study.

Single-contactstudies are usefultodefinethe

skin-degerming activity profileofapreparation

over anumber ofpost-treatmentsampling inter-vals. The activity profile establishes the point

ofmaximal bacterial reductionandallows

dis-crimination betweenrapid andsustained skin-degerming activity. The (Pre-T0)measurement provides anindex ofrapid skin-degerming

ac-tivity, namely, thatwhichoccursduringactual

handcontact. Conversely, adecrease in

bacte-rial levels subsequenttoTo, denotes sustained skin-degerming activity, namely, that which

occurs ontheglovedhandduringapostcontact

interval. It is clear from the studies reported here that the iodophor preparation exerted rapid activity, but no detectable sustained

ac-tivity after a single exposure. Incontrast, the twoHCP preparations producedaslowerinitial activity, but exhibitedcharacteristic sustained activity afterasingle contact. With the

appro-priate selection ofposttreatment sampling in-tervals, the activity profile defined in single-contactstudies alsoprovidesusefulinsightinto postscrub trendsin the reestablishment of

resi-dent bacteria on skin. Single-contact studies mayalsobe usedtodetermine optimal doseand contacttime foraparticularpreparation.

Multiple-contact studies allow the

measure-HAND

DEGERMING

PARAMETERS

12:

Cr)

singlecontact POST- TREATMENT

PRE TO TX T)

SAMPLING TlIME

multiplecontoct

treatment POST-TREATMENT

12 3 4 x 12 3 x

y

DAYS

FIG. 4. Schematic representation of rapid, sus-tained, cumulative, andpresistent skin-degerming activity parameters. Pre, Immediately before

treat-ment;T,andT.,xandyminutes post-treatment.C1,

Contact1;C4,contact4;x,xthtreatmentdayor

post-treatmentday.

J. CLIN. MICROBIOL.

SCRUBBING POSTSCRUB

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MEASUREMENTS OF SKIN-DEGERMING ACTIVIrY 413

ment of cumulativeskin-degerming activity on the hand afterrepeatedcontacts withinasingle day. This activity parameter is characterized by progressive bacterial reductions as a func-tion of multiple contacts. Whenextendedovera number of successive days, multiple-contact studies also provide ameasurementof persist-entskin-degerming activity over days. The lat-ter paramelat-ter, which is characterized by the profile of daily pretreatment levelsof skin bac-teria, reflects thenetdifference between maxi-maldaily bacterial reductions and subsequent regrowth of skin bacteriaduring overnight non-contact intervals. The studies presented here demonstrate that the 3%HCP preparation

ex-erted both cumulative and persistent skin-de-germing activity after multiple-contact expo-sure.

For both single-contact andmultiple-contact studies,the pretreatmentbacterial countis the criticalreference from which activity

measure-ments aremade. It is important,therefore,that pretreatmentsamples be obtainedimmediately before treatment on each day to accurately measure the microbiological state of the hand atthat specific pointintime.

Itis alsoessential that post-treatment extract samples obtained from the hand be appropri-ately neutralized with a suitable nontoxic agentand/or adequately diluted before cultur-ing to avoid possible interference due to

car-ryover ofresidual amounts of the active antimi-crobial agent. In the case ofmultiple-contact studies, however,itisobvious that neutralizers should not be incorporated directly into the hand-sampling fluid.

Inview of the rather broad range of pretreat-ment bacterial counts normally observed within thegeneral population,itisequally

im-portant thatsubjectsunder study respond

uni-formlyto treatment. Based onthe resultsand analyses presented herein, candidate subjects withpretreatment bacterial countsinthe range fromapproximately log4tolog7per hand may beexpectedtorespond uniformlytotreatment, and, therefore, would be generally acceptable for these types of studies.Appropriateanalyses ofpre-andpost-treatment data may be usedto

verify the satisfaction of this requirement for eachstudy.

The selection of appropriate experimental

de-signsaffordsconsiderableflexibilityforstudies ofthe types described. The majorobjectives of

anappropriate experimentaldesignare to

min-imize the risk ofbias, maximize precision, and provide for corresonding hypothesis testing.

These requirements were an integral feature of the skin-degerming studies reported here, and all data were readily amenable to statistical analysis.

The randomized-hand experimental design described herein is especially useful in that it pernits the simultaneous examination of a

number of experimental variables, or topical cleansing products, with the use of a minimal number ofsubjects. The increased versatility of this design, over traditional crossover and split-hand designs, is achieved at a relatively small and acceptable sacrifice in statistical discrimi-natory power. It has been the experience of this laboratory that a minimum of 12 independent measurements for each variable examined in hand-degerming studies will provide an ade-quatebasis for direct comparisons. This num-ber of observations will usuallypermit the sta-tistical discrimination of differences in bacte-rial reductions as small as 0.5-log unit. Signifi-cantdifferences of this magnitude provide prac-tical and meaningful information related to product comparisons and/or comparisons of var-iables such as dose and contact time.

ACKNOWLEDGMENTS

We express our appreciation to the volunteer subjects for their cooperation.

The technical assistance of Cynthia Arnold, Elizabeth Fogarty, Diane McTaggart, Christine Yezierski, and Dianne Mills is gratefully acknowledged.

LITERATURE CITED

1. Gross, A., L. Cofone, and M. B. Huff. 1973. Iodine inactivating agent in surgical scrub testing. Arch. Surg. 106:175-178.

2. Kundsin,R. B., and C. W. Walter. 1973. The surgical scrub-practical consideration. Arch. Surg.

107:75-77.

3. Lowbury,E.J.L., and H. A. Lilly.1960.Disinfection of the hands of surgeons and nurses. Br. Med. J. 1:1445-1450.

4. Michaud, R. N., M. B. McGrath, and W. A. Goss. 1972. Improved experimental model for measuring skin de-germing activity on the human hand. Antimicrob. AgentsChemother. 2:8-15.

5. Peterson, A. F. 1973. The microbiology of the hands: evaluatingthe effects ofsurgicalscrubs. Dev. Ind. Microbiol. 14:125-130.

6. Peterson, A. F. 1974. The microbiology of the hands: assessing new scrubbing devices and procedures. Dev.Ind. Microbiol. 15:417-419.

7. Reid,D.E., C. W. Walter, and A. S. Buck. 1950. Surgi-calscrubbingwith pHisoderm G-11 as applied to a maternity hospital. Surg. Gynecol. Obstet.

91:537-544.

8. Walter, C. W., and R. B. Kundsin. 1969. The bacterio-logic study of surgical gloves from 250 operations. Surg. Gynecol.Obstet. 129:949-952.

9. Winer,B. J.1971.Statistical principlesinexperimental design,2nded. McGraw-Hill, New York.

VOL. 3, 1976

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