Evaluating Contact Lens Microbial Contamination related to Hand Hygiene Compliance
Ngoc Lam
Instructors: Mark Vaughan, Eunice Chin, Mahta Khosravi

Table of Contents
1. Introduction………………………………………………………………………………..2
2. Materials and Methods
2.1. Hand hygiene methods………………………………………………………………3
2.2. Handling of sterile CLs……………………………………………………………….3
2.3. Culturing of the contaminated CLs………………………………………………….3
2.4. Gram stain and biochemical tests on the isolated colonies………………………4
2.5. Statistical analysis of data……………………………………………………………4
3. Results
3.1. Microbial colony growth on the inoculated TSA media……………………………5
3.2. Bacterial growth on the inoculated MSA media……………………………………6
3.3. Fungal growth in the inoculated SDA media……………………………………….8
3.4. No bacterial growth on the inoculated MacConkey agar………………………….9
3.5. Gram stain and biochemical tests for the identification of unknowns…………...10
4. Discussion…………………………………………………………………………………13
References…………………………………………………………………………………19

1

1. Introduction
The use of contact lenses (CLs) has become popular due to the convenience that these
medical devices provide to wearers, especially to those who have active lifestyles. CLs
nowadays come in different prescription ranges, wearing modalities, or materials that wearers
can choose to suit their needs and lifestyles. CLs are primarily used for vision correction,
therapeutic, and cosmetic purposes with a significant evolving of the contact lens materials over
time to lessen negative side effects, support a normal corneal metabolism, and maintain the
stability of the tear film (Moreddu et al., 2019). Unlike conventional glasses which have frames
that might block the peripheral field of vision, CLs are placed directly on top of the eyes and
provide their wearers with a full field of vision (Kirkliauskienė et al., 2024). CLs thus come into
direct contact with the living cells and tissues of the eyes and require more care compared to
glasses.
Hand hygiene compliance is important in contact lens wear and care as it affects the
levels of contact lens contamination. Noncompliance with hand hygiene is the main source of
lens contamination and the level of lens contamination is significantly affected by techniques of
hand hygiene (Barlow et al., 1994; Ly et al., 1997). Poor hand hygiene prior to lens handling is a
risk factor for the development of microbial keratitis and inflammatory events of the corneal
(Fonn & Jones, 2019). Thus, it is hypothesized in this study that compliance with good hand
hygiene would statistically significantly reduce the levels of microbial contamination found on
handled CLs prior to lens insertion, further reducing risk of contact lens-related microbial
keratitis development.
Although there is not yet a known cause and effect relationship between a common
contact lens-related inflammation such as microbial keratitis and hand hygiene compliance, few
studies have been done on examining the efficacy of various hand hygiene protocols on the
changing levels of microbial contamination of CLs. This scientific study looks into how various

2

hand hygiene protocols affect the levels of contact lens microbial contamination associated with
the handling procedure of the lens insertion, with a focus on risk-related microbial keratitis
development.
2. Materials and Methods
2.1. Hand hygiene methods
Prior to handling the sterile contact lens, the experimenter performed each hand hygiene
protocol amongst a total of 4 hand hygiene methods include no handwashing; hand washing
with tap water and dry with paper towel; handwashing with soap (Softsoap brand), rinsing with
tap water, and dry with paper towel; and hand hygiene using an alcohol-based hand sanitizing
gel (Lifebuoy brand) (Ly et al., 1997; Morgan et al., 2011). Adapting from a study by Barlow et
al. (1994), each hand hygiene method was performed on different occasions of the experimental
days given a gap of at least 3 hours between each conducted hand hygiene method in order to
mitigate the mix-up effects of these hand hygiene methods; and a total of 4 replicates were
included for each hand hygiene procedure.
2.2. Handling of sterile contact lenses
All contact lenses used for this experiment were MyDay brand daily disposable, soft
contact lenses with -0.5D prescription. Each pre-unpacked lens was handled under a ducted
fume hood to minimize the potential contamination of the surrounding environment. All typical
lens manipulations prior to lens insertion were conducted in each replicate including eversion of
the lens, taco test, and placement of lens on the fingertips for at least 15 seconds (Barlow et al.,
1994).
2.3. Culturing of the contaminated CLs
These experimental methods are adapted from the studies by Ly et al. (1997) and
Mowrey-McKee et al. (1992) with modifications. Each handled contact lens was placed in a test
tube containing approximately 3.3 mL sterile 0.1% peptone water and then agitated at high
speed using a vortex mixer for at least 15 seconds. An approximate 750μL extract of the total
3

3.3mL the peptone water was inoculated onto tryptic soy agar (TSA), BD BBLTM MacConkey
agar, Mannitol salt agar (MSA), and Sabouraud dextrose agar (SDA) using a spread plate
method. The TSA, MacConkey agar, and MSA were incubated at 370C for 48 hours before
manual colony counting was performed and morphologies of colonies were recorded. The SDA
was incubated at 250C and checked daily for the visible growth of colonies before manual
colony counting and morphological recording of colonies were performed.
2.4. Gram stain and biochemical tests on the isolated colonies
Using a streak plate method, each colony from the inoculated TSA was further isolated
into a new TSA based on their characterized morphologies to ensure that isolates were pure
colonies. The inoculated TSA with presumptive isolates were incubated for 48 hours and then
stored in plastic bags in the refrigerator at 50C for a maximum of 2 weeks to keep the isolates
from dehydration and died off.
These methods are adapted from studies by Ly et al. (1997) and Kirkliauskienė et al.
(2024) with modifications.Gram staining was performed on these isolated colonies as well as
SDA colonies. Bacterial isolates were further characterized using biochemical tests where
catalase test, bacitracin susceptibility test (0.04U), mannitol salt, and coagulase test were
included for identification of the unknown Gram-positive bacteria while the 5% sheep blood agar
was performed to characterize the unknown Gram-negative bacteria.
2.5. Statistical analysis of data
Data are presented as mean ± standard deviation. The F-test for variance was
conducted to determine if the variance of each hand hygiene method’s replicate was equal or
unequal to the variance of the corresponding replicate of the no hand washing method. The two
independent sample t-test with either unequal variance or equal variance was then performed to
examine for statistically significant difference between the average number of colony growth in
each replicate of each hand hygiene method and no hand washing method. Statistical
significance was determined at p < 0.05 in all tests.
4

3. Results
3.1. Microbial colony growth on the inoculated TSA media
Table 1
Summary of manually counted number of colony growth on the inoculated TSA
Trial
No HW

1
26

Water & towel
>300 (TNTC)
Soap & water & towel
19
Sanitizing gel

5

2
27

3
82

4
133

113
53

128
53

38
64

13

24

101

Figure 1
Average number of microbial colony growth on the inoculated TSA

The manually counted numbers of microbial growth on the inoculated TSA in all
replicates were summarized in Table 1 and the morphologies of 4 identifiable microbial isolates
across experimental replicates were described in Table 2. The average number of microbial
growth was highest (93 ± 48.22 colonies) in the hand hygiene method where the experimenter’s
hands were washed with tap water and dried with a paper towel (Figure 1). The method of no
handwashing was recorded having the second highest average number of microbial growth (67
5

± 51.19 colonies) in all replicates (Figure 1). The method of handwashing using soap, water,
and dry with a paper towel had the second lowest mean number of microbial growth (47.25 ±
19.53 colonies) while the use of alcohol-based sanitizing gel had the lowest mean number of
microbial isolates (35.75 ± 44.19 colonies) throughout all replicates (Figure 1). The differences
in the average number of microbial colony growth between each method of hand hygiene
versus no handwashing was found to be not statistically significant using two independent
sample t-tests with equal variances (p > 0.05).
Table 2
Characterized morphologies of microbial isolates on the inoculated TSA

Unknown

Recorded morphologies of isolated colony

1

White, circular, entire edge, raised elevation, opaque, shiny

2

Grey, circular, entire edge, raised, opaque, dull

3

Grey-yellowish, circular, entire edge, umbonate, opaque, dull

4

White, rhizoid, raised, opaque

3.2. Bacterial growth on the inoculated MSA media
Table 3
Summary of manually counted number of colony growth on the inoculated MSA
Trial
No HW
Water & towel
Soap & water & towel
Sanitizing gel

1
29
117
6
3

2
19
50
16
4

6

3
15
117
11
3

4
76
23
23
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Figure 2
Average number of microbial colony growth on the inoculated MSA

The manually counted numbers of bacterial isolates on the inoculated MSA in all
replicates of experimental hand hygiene methods were summarized in Table 3 and a total of 3
different growth patterns of bacterial colonies on the inoculated MSA were recorded in Table 4.
There was the highest average number of bacterial growth in all replicated trials of hand
hygiene method using tap water and drying with a paper towel (76.75 ± 47.77 colonies),
followed by the method of no handwashing having the second highest average number of
bacterial growth (34.75 ± 28.12 colonies) (Figure 2). Hand sanitizing using an alcohol-based gel
showed the lowest mean number of bacterial isolates (5.5 ± 4.36 colonies), followed by the
handwashing using soap, water, and dry with a paper towel having the second lowest mean
number of bacterial growth (14 ± 7.26 colonies) in all replicates (Figure 2).
The two independent sample t-test with equal variances were performed for the group of
no hand washing versus hand washing using water and dry with a paper towel while two
independent sample t-tests with unequal variances were performed for the group of no hand
washing versus hand washing using soap, water and dry with a paper towel and hand sanitizing

7

gel. The differences in the average number of bacterial colony growth between every method of
hand hygiene versus no handwashing was found to be not statistically significant (p > 0.05).
Table 4
Growth patterns of bacterial colonies on the inoculated MSA

Unknown

Pattern of colony growth on the inoculated MSA media

1

Colony growth that turned agar into white/yellowish

2

Colony growth that turned agar into pink/reddish

3

Colony growth that did not change the agar’s color

3.3. Fungal growth in the inoculated SDA media
The manually counted numbers of fungal isolates on the inoculated SDA in all
experimental replicates were summarized in Table 5. A single type of fungal isolates are
identified on the inoculated SDA across the replicates which were morphologically described as
white, circular, entire edge, umbonate, opaque, and non-shiny. This type of fungal isolates had
a slow growth where it took an average 3-4 days to be visibly observable on the inoculated
SDA.
Table 5
Summary of manually counted number of colony growth on the inoculated SDA
Trial
No HW
Water & towel
Soap & water & towel
Sanitizing gel

1
13
33
11
2

2
8
70
25
9

8

3
6
16
11
7

4
68
15
31
45

The mean number of fungal growth was highest in the hand hygiene method using tap
water and drying with a paper towel (33.50 ± 25.70 colonies) and the method of no
handwashing showed the second highest mean number of of fungal growth (23.75 ± 29.65
colonies) (Figure 3). Hand sanitizing using an alcohol-based gel showed the lowest mean
number of fungal isolates (15.75 ± 19.72 colonies), followed by the handwashing using soap,
water, and dry with a paper towel having the second lowest mean number of fungal growth
(19.50 ± 10.12 colonies) in all replicates (Figure 3). The differences in the mean number of
fungal colony growth between each method of hand hygiene versus no handwashing was found
to be not statistically significant using two independent sample t-tests with equal variances (p >
0.05).
Figure 3
Average number of fungal colony growth on the inoculated SDA media

3.4. No bacterial growth on the inoculated MacConkey agar
There was no bacterial growth observed on the inoculated MacConkey media in all
replicates of 4 experimental hand hygiene methods and this can be explained by the use of
BBLTM MacConkey agar. In the Difco & BBL manual : manual of microbiological culture media
9

(2nd ed.) by Zimbro and Power (2009), BBLTM MacConkey agar does not support a wide range
of non-fastidious Gram negative bacilli.
Figure 4
No bacterial growth observed on an inoculated BBLTM MacConkey agar

3.5. Gram stain and biochemical tests for the identification of unknowns
Figure 5
Gram stain results of unknown microbial colonies isolated from the inoculated TSA

The isolated colonies that are listed as unknown 1, 2, and 3 in the Table 2 appeared to
be Gram-positive cocci under the Gram staining test while the unknown 4 listed in the Table 2
was characterized as Gram-negative bacilli under Gram-staining (Figure 5). The unknown SDA

10

isolates - characterized by the Gram-staining test, appeared to be larger than cocci-shaped
bacteria with many of them being in pairs as they were probably budding or reproducing, and
they were also not colored with resistance to take up a safranin counterstain (Figure 6).
Figure 6
Gram stain result of the unknown fungal isolates on the inoculated SDA

The unknown Gram-positive cocci number 1, 2, and 3 listed in the Table 2 all showed
positive results in the catalase tests. In the bacitracin susceptibility tests (0.04U), these
unknowns showed to be susceptible (or sensitive) to bacitracin and no hemolysis of the blood
agar (Figure 7). This result seems to be contradictory with the observations in the inoculated
MSA in the Table 4 where some colony growth turned the agar into pink/reddish or
white/yellowish, suggesting the presence of Staphylococcus spp. It was speculated that the
results of these bacitracin tests were false-positive due to the non-confluent growth of bacterial
isolates on the tested blood agar. The unknown Gram-negative bacilli number 4 listed in the
Table 2 showed resistance to bacitracin and no hemolysis of the blood agar.
The isolated Gram-positive, catalase-positive cocci unknowns were streaked onto the
mannitol salt agar media where their growth turned the agar either into pink/reddish or

11

white/yellowish (Figure 8). The subsequent coagulase tests of these unknowns did not show
visibly clear results with minor coagulations observed in some replicates of the tests.
Figure 7
Results of the bacitracin susceptibility test (0.04U)

Figure 8
The growth of Gram-positive, catalase-positive unknowns in the MSA

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4. Discussion
The main result of this research showed that there was a higher level of microbial,
bacterial, and fungal contamination on handled soft CLs when there was poor hand hygiene
practiced prior to lens wear. This result somewhat aligns with the previous study’s result by
Barlow et al. (1994) where it was found that the practice of hand washing with antibacterial
liquid soap prior to lens insertion was more effective than other hand hygiene methods including
the use of non-antibacterial soap bar, hand washing with water only, and no hand washing in
controlling the transferred amount of bacterial contamination from hands to the handled
hydrogel CLs. Fonn and Jones (2019) emphasized that hand hygiene is recommended by eye
care practitioners as it helps to minimize the level of microbial contamination transferred from
hands to contact lenses and contact lens wearers’ poor hand hygiene is specifically indicated by
inadequacy or absence of hand washing.
The absence of or inadequate hand hygiene in contact lens wear and care has been
statistically examined in several studies where a significant proportion of contact lens wearers
reported to have poor hand hygiene. An online survey conducted in the U.S. showed that 41%
of the total of 950 surveyed individuals who self-described themselves as daily disposable
contact lens wearers did not do hand washing with soap prior to inserting lenses and 15% of
this surveyed population hardly or did not wash their hand ever before lens insertion (Osborn et
al., 2017). Another internet-based survey conducted in the U.S. showed that just slightly more
than half of the participants who are non-daily soft contact lens wearers wash their hands with
soap prior to lens handling in the morning and in the evening (Hickson-Curran et al., 2011).
These statistics on hand washing behaviors of contact lens wearers emphasize the use of soap
in the hand washing procedure, so inadequate hand hygiene might include the lacking use of
soap in the hand washing procedure of contact lens wearers. A study on behaviors and
attitudes of contact lens wearers towards lens hygiene and care suggested that poor or
13

inadequate hand washing protocols should be worded with the no use of soap (Wu et al., 2010).
Other studies, however, showed contradictory results regarding how good hand hygiene
would reduce the microbial contamination of CLs. Ly et al. (1997) showed that the use of
alcohol wipes was associated with the least number of bacterial contaminants followed by no
hand washing where they proposed that various techniques of hand washing possibly dislodge
microbes from the hands’ finger nails and palms which later attached to the handled lenses.
Mowrey-McKee et al. (1992) found that hydrogel CLs which were handled by hand washing with
soap and water and dried with a paper towel had a higher level of microbial contamination
compared to lenses that were worn and later aseptically removed from the eyes. These
contradictory results potentially suggest the importance of hand drying in the hand washing
process to minimize the re-contamination of hands with pathogenic microbes. In this research,
hand drying was done using a paper towel obtained from the biological laboratory where there
were possible contaminations, highlighting a possibility for confounding effects of this factor on
the actual levels of microbial contamination transferred from the experimenter’s hands to lenses.
Hands are re-contaminated as microbes in the air blown by air blowing driers deposit on the
skin of wet hands, so air blowing driers are recommended for hand drying as an alternative if
single use paper towels are not available (McMonnies, 2012). However, the use of a possibly
contaminated paper towel in this research might reflect the fact that reusable textile towels that
are used to dry the lens wearers’ hands prior to lens wear are contaminated with daily use. Kato
et al. (2023) found that there was a statistically significant higher microbial load and area of
microbial film formation in textile towels used for a 6-month period compared to those used for a
2-month period.
The use of alcohol-based hand sanitizer in this research had the lowest levels of
microbial contamination transferred from hands to soft CLs which is supported by the study by
Ly et al. (1997) where hands sanitized using an alcohol wipe had the lowest level of bacterial
contamination transferred to hydrogel CLs. This result suggests that a certain amount of
14

microbial contamination was not transferred from a contaminated towel to hands and later from
hands to lens. In fact, the use of hand sanitizer is considered as an alternative to regular
handwashing with soap in studies. In an international online survey where roughly 40% of
surveyed individuals reported to practice proper handwashing, contact lens wearers’ hand
hygiene compliance includes hand washing with soap, or an alternative use of hand sanitizer or
wet wipe (Morgan et al., 2011). McMonnies (2012) also indicated that when hand washing is not
possible, alcohol-based solutions, gels or wipes can be alternatively used with a caution to not
leave any remaining alcohol on the hands before lens handling to prevent lens damage and eye
irritation.
It is important to reduce the levels of microbial contamination transferred from wearers’
hands to CLs because handled CLs are directly placed in the wearers’ eyes. Contact lens
handling is a source of contact lens-related microbial contamination where lens insertion
procedure involves the transfer of contaminants from the hands to lens and then directly from
lens to the eyes (Fonn & Jones, 2019; Szczotka-Flynn et al., 2010; Lievens et al., 2017).
Specifically, contact lens handling prior to lens insertion includes several manipulation steps
such as lens eversion, taco test, and the placement of lens on the fingertips to ensure lens'
rightness and free of defects, preventing discomfort and distorted vision resulting from the lens
wear (Barlow et al., 1994; Lievens et al., 2017). However, as contact lens wearers have different
habits in lens wear, they might not practice all these manipulation steps prior to lens insertion
and handled CLs are thus less or more contaminated with the transient flora of wearers’ hands.
Pathogens mainly attach to contact lens material and form a biofilm which weakens the defense
mechanism related to immunity in promoting the development of microbial keratitis (MK) (Maier
et al., 2022).
In 2019, about 140 million people use CLs to correct various types of refractive
impairments globally and MK is one of the most frequent adverse consequences induced by
contact lens use (Moreddu et al., 2019). MK is a sight-threatening corneal inflammation resulting
15

from scarring and perforation of patients’ cornea (Cheng et al., 1999). Causative organisms of
MK are commonly found in the environment including bacteria, fungi, and acanthamoebae
where bacteria are the most common causative pathogens and usually associated with more
severe consequences (Lievens et al., 2017; Cheng et al., 1999; Maier et al., 2022). At least two
circumstances should occur in the pathogenesis of contact lens-related MK include a defective
epithelial layer of the cornea and the presence of a sufficient amount of pathogens (Maier et al.,
2022). Contact lens wear interferes with defense mechanisms evolved by the corneal epithelium
in protecting the ocular surface against the invading of microbes such as P.aeruginosa
(Robertson & Cavanagh, 2008). Although differences in the levels of microbial contamination
was found to be not statistically significant in this research, a higher number of microbial
contamination transferred from the experimenter’s hands with poor hand hygiene to the lens
implies a possibility that the amount of microbes carried by the hands with poor hand hygiene
are sufficient and pathogenic to cause MK in cases with a defective corneal epithelia caused by
contact lens wear.
In cases of contact lens-related bacterial keratitis, the 3 most frequently isolated
bacterial species were Pseudomonas aeruginosa, Staphylococcus spp., and Serratia
marcescens (Hatami et al., 2021). This is aligned with the result of this research in which Gram
staining and catalase tests detected the presence of unknown species of Gram-positive,
catalase-positive cocci bacteria on handled CLs. Species of Staphylococcus are a part of
human skin’s microbiota and categorized as Gram-positive, catalase-positive bacteria (Moreddu
et al., 2019; Foster, 1996). Specifically, the results of Mannitol salt agar tests in this research
indicated the presence of unknown species of Gram-positive, catalase-positive cocci with high
salt-tolerance which either have the ability to ferment or non-ferment mannitol, suggesting the
presence of presumptive Staphylococcus aureus and coagulase-negative species such as
Staphylococcus epidermidis on handled CLs. Unlike other species of bacteria, Staphylococci
can withstand the osmotic pressure of 7.5% NaCl contained in Mannitol salt agar for their
16

growth in which the yellow agar indicates the breakdown of mannitol by presumptive
Staphylococcus aureus (Shields & Tsang, 2006). However, this interpretation should be
supported with the results of other biochemical tests with a possibility of repeated coagulase
tests.
Unfortunately, there was no growth of Gram-negative bacteria in all replicates of this
research. Gram-negative bacteria are predominant pathogens in contact lens-related MK with
the most common presence of P. aeruginosa (Stapleton & Carnt, 2012). This result can be
mainly explained by the use of BBLTM MacConkey agar. According to the Difco & BBL manual :
manual of microbiological culture media (2nd ed.) by Zimbro and Power (2009), BBLTM
MacConkey agar does not support the growth of a wide range of Gram-negative bacteria
including P. aeruginosa. Besides, the most common causative species of fungal keratitis are
Fusarium, Aspergillus, and Candida (Castano et al., 2024). While Fusarium and Aspergillus
genera appear branching under microscopic views, the Candida species do not show to resist
taking up stain in the Gram’s staining (Wacira et al., 2020; Aslam et al., 2015). Therefore,
staining and microscopic morphologies of the unknown fungal species in this research are not
sufficient to relate it to common causative species of fungal keratitis and it can be presumptively
categorized as an unknown yeast species of the human skin’s microbiota.
This research also comes with several limitations. There was no inoculated media used
as negative controls to test for the sterility of prepared media while the use of BD BBLTM
MacConkey agar does not favor the growth of many Gram-negative bacteria, negatively
affecting the measure of actual bacterial contamination levels on handled CLs. Several
confounding factors such as the use of contaminated paper towels might also be controlled in
the future research to examine for the actual microbial contamination that stemmed from
different hand hygiene methods. It is also worth noting that the interpretation of this research
might be constrained by a small number of replicates and time limit if there is growth of Gramnegative bacteria. Several experimental techniques such as the manual colony counting is
17

prone to error as some colonies grew small next to each other while biochemical tests for
bacterial identification are prone to either false positive and false negative indicated in the
results of this research’s bacitracin susceptibility tests.
Although differences in the mean levels of contact lens microbial contamination resulted
from poor and good hand hygiene techniques were found not to be statistically significant in this
study, the reduced average levels of contact lens microbial contamination related to good hand
hygiene methods including hand washing with soap and alternative using of alcohol-based gel
suggest that compliance with good hand hygiene should be practiced in the contact lens wear to
further reduce risk of developing contact lens-related ocular inflammations such as MK. A
repeated experiment of this project can aim to have more replicates to test for any statistical
significance of data. Contact lens-related MK can stem from different aspects of contact lens
use with modifiable factors such as lens wearing time, lens materials, and patients’
noncompliance with lens replacement schedule and hygiene guidelines (Stapleton & Carnt,
2012). Therefore, eye care professionals might also consider developing a detailed guideline for
hand hygiene protocols to raise awareness and better educate their patients. There are also
venues for future research to develop CLs with antimicrobial chemical properties. However, this
technology is still mostly in its research and developmental state described in a review by Khan
& Lee (2020).

18

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