International Journal of Clinical Medicine, 2013, 4, 577-581
Published Online December 2013 (
Open Access IJCM
Antimicrobial Assay of Chlorhexidine-Wetted Textile
Napkins for Surgical Site Disinfection in Ocular Surgery
Amir Reza Daneshmand Eslami
Department of Ophthalmology, National Medical Academy of Postgraduate Education, Kiev, Ukraine.
Received October 29th, 2013; revised November 25th, 2013; accepted December 6th, 2013
Copyright © 2013 Amir Reza Daneshmand Eslami. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: As a new intraoperative disinfection method, chlorhexidine-wetted textile napkins have been employed
in order to cover the upper and lower eyelid edges, eyelid skin, eyelashes, lid margins and palpebral conjun ctiva during
phacoemulsification cataract extraction. This study was conducted to compare the antimicrobial activity of textile nap-
kins before and after their use. Methods: This study evaluated 80 textile napkins wetted with 0.02% aqueous solution
of chlorhexidine. All textile napkins were divided into groups. The study group consisted of 60 used textile napkins
which were collected from 29 patients (30 eyes) at the end of phacoemulsification, and the control group included 20
unused sterile textile napkins. Antimicrobial assay was performed by means of measuring the growth inhibition zones
of the standard or clinical isolate strains under the textile napkins on the surface of agar media. Results: The number of
textile napkins and th e diameter of the growth inh ibition zones (mm) in the study group and in the control group relat-
ing to gram-positive, gram-negative, and fungi were: 24/31 vs. 8/31, 32/30 vs. 8/30, and 4/30 vs. 4/30. The diameter of
the growth inhibition zones of gram-positive bacteria was more than other investigated microorganisms. In the growth
inhibition zones, exog enou s microorganism colon ies were not fo und. Conclusion: Antimicrobial activity of textile nap-
kins wetted with 0.02% aqueous solution of chlorhexidine against gram-positive bacteria is more than gram-negative
bacteria and fungi, and is preserved to the end of the phacoemulsification.
Keywords: Chlorhexidine; En dophthalmitis; Levofloxacin; Phaco emulsification; Surgical Site Disinfectio n
1. Introduction
Post-operative endophthalmitis after cataract surgery is a
rare but serious and potentially blinding complication
[1,2]. The sources of ocular bacterial contamination are
commonly the conjunctival sac and the eyelids [3,4].
Different methods of antimicrobial prophylaxis are used
to prevent the post-operative infectious complications.
The main idea of these methods is to eliminate the tran-
sient organism and reduce the resident flora to as low
level as possible [5].
Since 2005, a new disinfection method in order to
prevent microbial contamination of surgical wound dur-
ing intraocular surgery has been employed in Ukraine.
The components of the method are sterile textile napkins,
and an antiseptic for in stance, 0.02% aqueous solution of
chlorhexidine (CHG) [6].
The purpose of this work is to compare the antimicro-
bial activity of textile napkins wetted with 0.02% CHG
before and after their use during phacoemulsification
cataract extraction.
2. Materials and Methods
2.1. Patients
Thirty eyes of 29 patients with cataract were included in
the study and operated by phacoemulsification cataract
extraction and intraocular lens implantation. Patients un-
derwent day surgery at the Central Kiev Ophthalmic Hos-
pital “Eye Microsurgery Centre” by one surgeon. The ex-
clusion criteria included systemic or local infection, con-
junctivitis, blepharitis, dacryocystitis, meibomian gland
dysfunction and diab etes mellitus. The eye scheduled for
surgery received one drop of 0.5% levofloxacin oph-
thalmic solution and, 0.1% dexamethasone ophthalmic
solution five times per day for four days (the last drop for
an hour before surgery were administered by nurses).
One hour before the surgery, the patients received the
Antimicrobial Assay of Chlorhexidine-Wetted Textile Napkins for Surgical Site Disinfection in Ocular Surgery
standard eye drops to dilate the pupil. Retrobulbar anes-
thesia was performed in addition to the preoperative ap-
plication of ocular compression.
2.2. Surgical Site Disinfection
During the preoperative procedure used chlorhexidine
formulations were two-fold as follows. Firstly, 0.05%
alcohol-based chlorh exidine solution which was prep ared
by 1:400 dilution of 20% CHG (Chlorhexidine, 1,6-di(4’-
chlorophenyl-diguanido)hexane) using 70% ethanol for
disinfection. Secondly, 0.02% CHG which was prepared
by 1:1000 dilution of 20% CHG using sterile purified
water. The solutions were prepared in the hospital labo-
ratory with the potential concerns about quality control
and safety for these solution s as their sterility, stability of
pH, and shelf life. Antiseptic eye skin surface treatment
was carried out twice with two fresh sterile cotton swabs
soaked with 0.05% alcohol-based chlorhexidine in 2 min
interval by circular movements outwards (for the left
eye—clockwise, for the right—on the contrary). The
brow, upper and lower eyelids, eyelashes, and adjacent
forehead, nose, cheek and temporal orbital area were
scrubbed for 5 min before surgery and meticulous drap-
ing was used to isolate the effected eye. Prior to begin-
ning of surgery, the upper and lower eyelid edges, eyelid
skin, eyelashes, lid margins and palpebral conjunctiva
were covered with two textile napkins (Calico fabric),
prepared by a punch press and sterilized by autoclave in
the hospital laboratory by flushing them with 1 ml of
0.02% CHG. After two min the ocular surface was vig-
orously rinsed with 5 ml balanced salt solution of So-
dium Chloride (BSS).
Preventing CHG toxicity carried out according to the
following approaches: firstly, choosing Calico fabric
which CHG has ability to connect with [5], secondly,
preparing the textile napk in diameter 30 mm, thickness 1
mm that totally absorbs 6 drops of 0.02% CHG, thirdly,
meticulous batting, showering the ocular surface and the
textile napkins just before beginning the surgery with 5
ml BSS and lastly, using the constant flow of BSS from
an anterior chamber maintainer during phacoemulsifica-
tion. Thereby the expected residue of 0.02% CHG in the
operative area is much lower than toxicological dose of
CHG [7,8] likewise, the emergence of entry residual
CHG into the anterior chamber is avoided.
2.3. Surgical Technique
Phacoemulsification cataract extraction was performed
through a superior clear corneal incision with implanta-
tion intraocu lar lens using an injector system. During the
operations additional antimicrobial treatment was not
given. On completion of the surgery after sealing corneal
incisions, two textile napkins were withdrawn from each
eye by sterile forceps and placed in separate sterile Petri
dish with slightly opened lid. Then the lid was secured
with scotch tape and immediately sent to the Microbiol-
ogy Laboratory. After a subconjunctival injection of 0.5
ml dexamethasone and ceftriaxone, the conjunctival cav-
ity was washed with 2 ml BSS, this was followed by in-
stilling one drop of 0.5% levofloxacin ophthalmic solu-
tion into the inferior culs-de-sac and the eye was closed
with aseptic eye gauze pad. The duration of operation
ranged from 20 ± 5 min. Operations were completed
without surgery-related complications.
2.4. Study Sample
60 used textile napk ins collected at the end of the surg ery
were employed as study group. 20 unused sterile textile
napkins wetted with 6 drops of 0.02% CHG and em-
ployed as control group. Microorganism strains were
used to compare antimicrobial activity of unused textile
napkins with used ones wetted with 0.02% CHG. The
source of the microorganism strains for this work was
formed by pure clinical patterns isolated from ocular
post-operative infection, laboratory strains and standard
culture collection types of microorganisms. The micro-
organism strains were employed for the successful ac-
complishment of the study viz. gram-positive bacteria,
gram-negative bacteria and fungal species.
2.5. Antimicrobial Activity Assay
Antimicrobial activity assay was performed particularly
on the principle of Baue r-Kirby disc-diffusion sensitivity
method [9]. Relevant nutrient agars were used for micro-
bial cultures: Muller-Hinton agar media, some of them
were supplemented by 10% fetal calf serum and Sabou-
raud’s dextrose agar. More than one textile napkin was
examined for each microorganism strain. Determination
of antibacterial activity was carried out using standard
microbial suspension containing 106 CFU/ml by optical
turbidity standard. 1 ml of obtained standard microbial
suspension of each microbial strain was deposited on the
surface of the relevant microorganism nutrient agar in
Petri dishes, spread with a sterile spatula and covered all
surface of medium evenly to achieve uniform spread.
This was followed by keeping the Petri dishes within 15 -
20 min at ambient temperature. Then the two textile nap-
kins from each eye (study group) or one textile napkin
(control group) were superimposed with flame sterilized
forceps gently onto the surface of the separate solid nu-
trient media and pressed them onto the surface of the
medium. After placing of the textile napkins, the study
and control Petri dishes with bacteria species were incu-
bated in thermostat at 37˚C during 24 h, Petri dishes with
Candida species for 48 h at 35˚C. The antimicrobial ac-
tivity was evaluated by measuring the diameter of the
Open Access IJCM
Antimicrobial Assay of Chlorhexidine-Wetted Textile Napkins for Surgical Site Disinfection in Ocular Surgery 579
growth inhibitio n zones of corresponding microorganism
under the textile napkins and around them in mm. A fur-
ther step was the identification of possible app earance of
microbial colonies in the growth inhibition zones of the
microorganisms. For this purpose the textile napkins
from the surface of nutrient media were removed, and
each Petri dish was re-incubated in thermostat at 37˚C
during 24 h (Petri dishes with Candida species for 48 h at
2.6. Ethics Approval
Approval for accessing the patient health records was
obtained from the local research ethics committee. In-
formed consent was obtained from each patient. The
study protocol and the safety and efficacy of the inter-
ventions were explained to all of the participants prior to
their enrolment.
2.7. Statistical Analysis
The Mann-Whitney U-test (for small samples) and the
Z-test were used to compare the studied variables. A P
value less than 0.05 was considered statistically signifi-
3. Results
The results of this study are summarised in Table 1.
The diameter of the growth inhibition zones of micro-
organism strains under the textile napkin and around
them varies for different microorganisms after incuba tion.
When removing the textile napkins from the surface of
nutrient agar media, no colony was seen on their inner
side. As well as after re-incubation of these media in the
field of the growth inhibition zones of the microorganism
strains identified at the first stage, no colonies of micro-
organism strain and exogenous microbial contamination
were formed. No ocular toxicity associated with CHG
was noted.
4. Discussion
Local preoperative antibiotic prophylaxis, sterile prepara-
tion of the skin surrounding the surgical eye with Povi-
done-Iodine 10%, meticulous draping of the lids and eye-
lashes [10], and instillation of Povidone-Iodine 5% onto
the ocular surface at least 3 - 5 min prior to surgery are
widely used in many countries. Many authors believe
these measures have the most long-standing and highest
quality which is the evidence of their efficacy [11-14].
With this approach, bacteria were isolated in the conjunc-
tival sac or in the anterior chamber at the beginning of
cataract extraction and on its completion [15,16]. Thus
the duration of action of these preoperative regimens is
disputed. Numerous studies have reported that after ap-
Table 1. Comparison of antimicrobial activity of textile
napkins wetted with 0.02% aqueous chlorhexidine.
Number of textile napkins/
The inhibition zone (mm)
Microbial strains Study group
(n = 60)*1 Control group
(n = 20)*2
Staphylococcus aureus 209p 8/31*3 2/31
Staphylococcus epidermid is 1534 8/31*3 2/31
Corynebacterium diphtheriae mitis 4 /31*3 2/31
Corynebacterium xerosis 4/31*3 2/31
Total Gram-positive strains 24/31*4 8/31*5
Salmonella typhimorium 79 8/30*3 2/30
Klebsiella pneumoniae 5758 8/30*3 2/30
Escherichia coli O111 8/30*3 2/30
Pseudomonas aeruginosa 8/30*3 2/30
Total Gram-negative strains 32/30 8/30
Candida albicans 2/30*3 2/30
Candida tropicalis 2/30*3 2/30
Total fungal strains 4/30 4/30
*1Used textile napkins, *2Unused textile napkins, *3P = 1 compared wi th the
control group. *4P = 0 compared with the total Gram-negative strains, *4P =
0 compared with the total fungal strains. *5P = 0 compared with the total
Gram-negative strains; *5P = 0.001 compared with the total fungal strains.
plying the various invasive techniques, including the
addition of antibiotics in the balanced salt solution, or
their infusion into the anterior chamber, the material
taken from anterior chamber at the conclusion of opera-
tions or postoperative endophthalmitis cases may release
bacteria sensitive to these antibiotics [17]. A disadvan-
tage of the intracameral antibiotics has potential risks,
such as toxic anterior segment syndrome secondary to
dilution errors [18].
In this study, antiseptic was presented in operation
area by noninvasive method to ensure sterile preparation
during ophthalmic surgery and its antimicrobial activity
was investigated directly by providing a comparative
analysis of activity of textile napkins wetted with 0.02%
CHG before and after their use. Main reasons for choos-
ing 0.02% CHG are as follows. Firstly, 0.02% CHG is
the most stable within the pH range 5 to 8 [5], in the pH
range of 0.5% levofloxacin ophthalmic solution [19].
Secondly, Staphylococcus aureus, Staphylococcus epi-
dermidis, Bacillus subtilis, Streptococcus pyogenes, Mi-
crococcus sp., Escherichia coli, Enterobacter aerogenes
and Pseudomonas aeruginosa are sensitive to it [20] and
lastly, methicillin-resistant Staphylococcus aureus exhib-
its low-level resistance to chlorhexidine [21]. Addition-
ally, as conducted research reveal, CHG as a cationic
molecule [5] binds to negatively charged cells of the oral
Open Access IJCM
Antimicrobial Assay of Chlorhexidine-Wetted Textile Napkins for Surgical Site Disinfection in Ocular Surgery
cavity. Thus, for a certain period of time, the oral cavity
becomes a CHG reservoir, which prolongs its chemical
activity in preparations [22]. The ocular surface (cornea
and conjunctiva) is also negatively charged [23,24], and
the paracellular space is more permeable to cations than
to anions at physiological pH [25,26] consistently the
ocular surface may also become a CHG reservoir, which
prolongs its chemical activity in preparations. This phe-
nomenon depathogenizes the ocular surface flora intra-
operatively and postoperatively but bacteriological cul-
ture from the ocular surface may continue to be positive.
Results showed that the use of BSS during cataract
surgery did not totally decrease th e antimicrobial activity
of the textile napkins due to CHG absorbed in the fibers
of certain textile, pa rticularly cotton, and consistently re-
sisted removal by washing [5]. Thereby, the textile nap-
kin virtually serves as a sustained release reservoir of
CHG during phacoemulsification. Moreover, equal di-
ameter of the growth inhibition zones before and after
phacoemulsification cataract extraction also indicates
that 0.5% levofloxacin ophthalmic solution instilled pre-
operatively is not an antagonist of 0.02% CHG. Addi-
tionally, antimicrobial activity of both unused textile nap-
kins and used textile napkins, wetted with 0.02% CHG
against gram-positive bacteria is more than gram-nega-
tive (Table 1), that is comparable to certain reports [5,
20]. Since microbial flora under the textile napkins
mixed with flora of the conjunctival sac and the lid mar-
gin, the result of microbial culture from the conjunctival
sac after withdrawing the textile napkins on completion
of the surgery is disputed. Test strains colonies were not
seen after incubation of Petri dishes on the surface and
inner side of used textile napkins. Likewise in th e growth
inhibition zones after re-incubation, exogenous microor-
ganism colonies that could contaminate used textile nap-
kins during surgery were not seen.
5. Conclusion
The antimicrobial activity assay of CHG-wetted textile
napkins indicates a persistent antimicrobial effect of a
residue of CHG in the textile napkins during phacoe-
mulsification cataract extraction. Intraoperatively, the
isolation of the lid edg es with 0.02% CHG-wetted textile
napkins in combination with a preoperative antibacterial
prophylaxis for instance 0.5% levofloxacin ophthalmic
solution reliably prevents microbial surface contamina-
tion during ophthalmic surgery.
6. Acknowledgements
The author appreciates Ms. Sepideh Elahi of INOVA
Fairfax Hospital, VA, USA for assistance with statistical
analysis. This paper was derived from the author’s doc-
toral dissertation.
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