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Evaluation of a 2% chlorhexidine gluconate

in 70% isopropyl alcohol skin disinfectant

D. Adamsa,*, M. Quayumb, T. Worthingtonb, P. Lambertb, T. Elliotta

aMicrobiology Research and Development Group, University Hospital Birmingham NHS Foundation Trust,

The Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UKbDepartment of Pharmaceutical and Biological Sciences, Aston University, Aston Triangle,

Birmingham B4 7ET, UK

Received 22 February 2005; accepted 17 May 2005Available online 10 October 2005

KEYWORDSChloraPrepw; Chlor-hexidine; Disinfec-tant; Povidone iodine;Isopropanol; Skinantisepsis

Summary The efficacy of a new skin disinfectant, 2% (w/v) chlorhexidinegluconate (CHG) in 70% (v/v) isopropyl alcohol (IPA) (ChloraPrepw), wascompared with five commonly used skin disinfectants againstStaphylococ-cus epidermidis RP62A in the presence or absence of protein, utilizingquantitative time-kill suspension and carrier tests. All six disinfectants [70%(v/v) IPA, 0.5% (w/v) aqueous CHG, 2% (w/v) aqueous CHG, 0.5% (w/v) CHGin 70% (v/v) IPA and 10% (w/v) aqueous povidone iodine (PI)] achieved a log10

reduction factor of 5, in colony-forming units/mL, in a suspension test(exposure time 30 s) in the presence and absence of 10% human serum.Subsequent challenges of S. epidermidis RP62A in a biofilm (with andwithout human serum) demonstrated reduced bactericidal activity. Overall,the most effective skin disinfectants tested against S. epidermidis RP62Awere 2% (w/v) CHG in 70% IPA and 10% (w/v) PI. These results suggest thatenhanced skin antisepsis may be achieved with 2% (w/v) CHG in 70% (v/v) IPAcompared with the three commonly used CHG preparations [0.5% (w/v)aqueous CHG, 2% (w/v) aqueous CHG and 0.5% (w/v) CHG in 70% (v/v) IPA].Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rightsreserved.

Introduction

Coagulase-negative staphylococci are frequentlyassociated with catheter-related bloodstream

infections.1,2 A characteristic feature of thesemicro-organisms is their ability to adhere andform biofilms on prosthetic devices, resulting inresistance to antimicrobial agents. In order toreduce the risk of microbial colonization andsubsequent sepsis of peripheral vascular catheters,it is recommended that the skin insertion siteshould bedisinfected for 30 s with an antimicrobialsolution.3 A chlorhexidine preparation is preferred;

Journal of Hospital Infection (2005)61, 287290

www.elsevierhealth.com/journals/jhin

0195-6701/$ - see front matter Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.jhin.2005.05.015

*Corresponding author. Tel.: C44 121 472 1311x3451; fax:C44 121 414 1682.

E-mail address:debbie.adams@uhb.nhs.uk

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however, povidone iodine (PI) or 70% isopropylalcohol (IPA) may be used.46 These agents usedifferent modes of action to achieve antisepsis,which may be reduced in the presence of organicmatter.7,8 Two percent chlorhexidine gluconate(CHG) preparations have not been universallyavailable in the UK. Recently, a 2% (w/v) CHG in

70% (v/v) IPA solution (ChloraPrepw

: Medi-Flexw

Incorporated; Kansas, USA) for skin decontamina-tion has been developed and is currently underreview for approval by the Medicines and Health-care Products Regulatory Agency (UK) for marketingauthorization. Clinical studies have demonstratedthat this skin disinfectant provided a significantlybetter and more persistent antimicrobial activitythan 70% (v/v) IPA or 2% (w/v) aqueous CHG at 24 hin patients receiving pre-operative skin antisepsison abdominal and inguinal sites (NZ106).9 Thisenhanced residual antimicrobial activity may alsopotentially reduce the risk of subsequent phlebitis

for patients requiring a peripheral vascularcatheter.

The criterion for determining the antimicrobialactivity of a disinfectant is usually the rate ofreduction of the number of viable micro-organ-isms when exposed to the antiseptic agent. Themost widely recognized definition with regards tobactericidal activity is a log10 reduction factor of5.10 Assessing the efficacy of a disinfectant maybe undertaken by various quantitative in vitromethods including suspension tests and carriertests.11

The aim of the present study was to determinethe antimicrobial efficacy of 2% CHG in 70% (v/v)IPA, which has recently become available in the UK,and to compare it with 70% (v/v) IPA, 10% (w/v)aqueous PI, 0.5% (w/v) aqueous CHG, 2% (w/v)aqueous CHG and 0.5% (w/v) CHG in 70% (v/v) IPAutilizing quantitative in vitro time-kill tests againstS. epidermidisRP62A at 30 s. Suspension tests wereused to determine the effectiveness of the disin-fectant in reducing the potential risk from impac-tion on insertion of vascular catheters. Althoughbiofilm formation develops following medicaldevice insertion, some disinfectants have residualactivity. Therefore, in addition to the suspensiontests, carrier tests were undertaken to evaluate thepotential inhibition of biofilms on disinfectantactivity.

Methods

Six skin disinfectants were evaluated: 70% (v/v) IPA(BDH; Poole, UK) was prepared by diluting 100%

(v/v) IPA in sterile distilled water; 0.5% (w/v) and2% (w/v) aqueous CHG (Sigma; St Louis, USA) wereprepared by diluting 20% (w/v) CHG in steriledistilled water; 0.5% (w/v) CHG in 70% (v/v) IPA(Adams Healthcare; Leeds, UK); 2% (w/v) CHG in70% (v/v) IPA (Medi-Flexw International; Kansas,USA) and 10% (w/v) aqueous PI (Seton Healthcare;

Oldham, UK).Evaluation of the efficacy of the antimicrobialagent s w as u nder tak en at 30 s; the rec-ommended time for disinfecting the intendedskin site of a peripheral vascular catheter priorto insertion.3

A neutralizing agent was prepared containing 2%(v/v) Tween 80 (BDH; Poole, UK), 1.17% (w/v)lecithin (Fisher Scientific; Loughborough, UK), 0.1%(v/v) Triton X-100 (Sigma; St Louis, USA) and 0.5%(w/v) sodium thiosulphate (BDH; Poole, UK) insterile distilled water. This was sterilized at 121 8Cfor 15 min and then stored at 4 8C until required.

Prior to commencing the antimicrobial time-killstudies, verification of the effectiveness and non-toxicity of the chosen neutralizing agent against therange of antimicrobial agents and the efficacy ofthe antimicrobial agents against the challengemicro-organisms were determined.

S. epidermidis RP62A stored on microbankbeads (Pro-Lab Diagnostics; Ontario, Canada)was revived by placing one bead in 3 mL brainheart infusion (BHI) broth (Oxoid; Basingstoke,UK) and incubating at 37 8C in air for 24 h. S.epidermidis RP62A is a reference biofilm-positive

strain and slime producer, which was confirmedunder current test conditions by Freeman et al.stechnique.12

In the suspension test, 10 mL broth containing 3!106 colony-forming units (cfu)S. epidermidisRP62Awas added to 990 mL disinfectant and mixed. After30 s contact time at room temperature, 100 mLsuspension was removed and added to 900 mLneutralizing agent, mixed and left to dwell for5 min. Serial dilutions were inoculated on to BHIagar plates which were incubated at 37 8C in air forup to 48 h. Further suspension tests were under-taken by adding 10% (v/v) human serum (Sigma; StLouis, USA) to the suspension prior to adding thedisinfectant. The evaluations were carried out intriplicate.

To evaluate the efficacy of the disinfectantsagainst a biofilm, a carrier test was undertaken witha 96-well polystyrene flat-bottomed microtitre tray(Immulonw 2HB Thermo Labsystems; Franklyn, MA,USA). A suspension of S. epidermidis RP62A wasdiluted in BHI to approximately 1!104. Two-hundred-microlitre aliquots of the suspensionwere inoculated into 16 wells of a sterile microtitre

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tray. This was then covered with a microplatesealer (Greiner-Bio-One; Gloucester, UK) andincubated at 37 8C in air for 24 h. Confirmation ofbiofilmproduction was undertaken by OToole andKolters13 technique. To determine the efficacy ofthe disinfectants against a biofilm in the presenceof protein, the carrier test was repeated; a

suspension of S. epidermidis RP62A was diluted inBHI to approximately 1!104 cfu/mL and 10%human (v/v) serum was added.

The cells in suspension in each well wereremoved by inversion of the plate; the wells werethen washed with 250 mL phosphate-buffered saline(PBS). Two-hundred microlitres of the selecteddisinfectant was added to each well and allowedto dwell for 30 s. The disinfectant was aspirated and250 mL neutralizing agent was added to each welland left for 5 min. The neutralizing agent wasremoved by inversion of the tray, and the microtitre

wells were washed with PBS. Removal of the biofilmfrom the microtitre well was undertaken by addinga 200-mL aliquot of BHI to each inoculated well.With a sterile pipette tip, the walls of the microtitrewells and base were scraped 10 times and the BHIwas removed from each well and collected. Thisprocedure was repeated a further three times andthe inoculum was mixed thoroughly. Previousstudies had demonstrated that four consecutivescrapes were required to remove O99% of themicro-organisms in a biofilm attached to a micro-titre well; successive scrapes failed to statistically

reduce this number further. The numbers of viableS. epidermidis RP62A in suspension were enumer-ated by serial dilutions, and 100 mL of each dilutionwas inoculated on to BHI agar plates. The plateswere then incubated at 37 8C in air for up to 48 h.Tests and controls were carried out 16 times.

Statistical analysis

Data were compared using the MannWhitneyU-test.Pvalues of equal to or less than 0.05 wereregarded as significant.

Results

In all tests, the controls containing no disinfectantresulted in a complete recovery of the initialinocula.

Table Ioutlines the results of the suspension andcarrier tests in both the presence and absence ofprotein. Efficacy of the disinfectant activity isrepresented as the log10 reduction factor of theinitial cfu/mL. None of the skin disinfectants testedachieved a log10 reduction factor O5 in all four

tests. Four disinfectants [70% (v/v) IPA, 0.5% (w/v)CHG in 70% (v/v) IPA, 2% (w/v) CHG in 70% (v/v) IPAand 10% (w/v) aqueous PI] achieved a log10reduction factor O5 at 30 s in the suspensiontests, both in the presence and absence of humanserum, and in the carrier test when challenged withS. epidermidisRP62A in a biofilm.

When evaluating the effectiveness of the sixdisinfectants against S. epidermidis RP62A in abiofilm enriched with 10% (v/v) human serum, 70%(v/v) IPA, 0.5% (w/v) aqueous CHG, 2% (w/v)aqueous CHG and 0.5% (w/v) CHG in 70% (v/v) IPA

achieved a log10reduction factor between 2 and 4at 30 s. In comparison, 2% (w/v) CHG in 70% (v/v)IPA and 10% (w/v) aqueous PI achieved a log10reduction factor of between 4 and 5. There was nostatistical difference between the two disinfectantson analysis (PZ0.28).

Table I Comparing the efficacy of 2% (w/v) chlorhexidine gluconate (CHG) in 70% (v/v) isopropyl alcohol (IPA)against five standard skin disinfectants on Staphylococcus epidermidis RP62A after 30 s of contact time utilizingsuspension and carrier tests

Antiseptic Log10reduction factor in cfu/mL of S. epidermidisRP62ASuspension

testSuspension testwith 10% human

serum

Carrier test:biofilm

Carrier test:biofilm enriched with

10% human serum

2% (w/v) CHG in 70% (v/v) IPA 6.5 6.3 5.3 4.770% (v/v) IPA 6.5 6.3 5.4 2.80.5% (w/v) aqueous CHG 6.5 6.3 4.1 2.32% (w/v) aqueous CHG 6.5 6.3 4.8 2.80.5% (w/v) CHG in 70% (v/v) IPA 6.5 6.3 5.8 3.610% (w/v) aqueous povidone iodine 6.5 6.3 5.9 4.4

cfu, colony-forming units. Bold type indicates a failure to achieve a log 10reduction factor of 5.

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