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Correlation between Ocular Demodex Infestation and Serum
Immunoreactivity to Bacillus Proteins in Patients with Facial
Rosacea
Jianjing Li, MD,1,2,3, Niamh O'Reilly4, Hosam Sheha, MD, PhD,1,2, Raananah Katz, MD,1,2,
Vadrevu K. Raju, MD,1,2, Kevin Kavanagh, PhD,4, and Scheffer C. G. Tseng, MD, PhD1,2
1Ocular Surface Center, Miami, Florida 2Ocular Surface Research Education Foundation, Miami,
Florida 3The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, China 4Medical
Mycology Unit, Department of Biology, NUI Maynooth, Maynooth, County Kildare, Ireland
Abstract
Purpose—To investigate correlation between ocular Demodex infestation and serum.
Design—A prospective study to correlate clinical findings with laboratory data.
Participants—We consecutively enrolled 59 patients: 34 men and 25 women with a mean age of
60.4±17.6 years (range, 17–93).
Methods—Demodex counting was performed based on lash sampling. Serum immunoreactivity to
two 62-kDa and 83-kDa proteins derived from B oleronius was determined by Western blot analysis.
Facial rosacea, lid margin, and ocular surface inflammation were documented by photography and
graded in a masked fashion.
Main Outcome Measures—Statistical significance based on correlative analyses of clinical and
laboratory data.
Results—These 59 patients were age matched, but not gender matched, regarding serum
immunoreactivity, ocular Demodex infestation, or facial rosacea. There was a significant correlation
between serum immunoreactivity and facial rosacea (P = 0.009), lid margin inflammation (P =
0.040), and ocular Demodex infestation (P = 0.048), but not inferior bulbar conjunctival
inflammation (P = 0.573). The Demodex count was significantly higher in patients with positive
facial rosacea (6.6±9.0 vs. 1.9±2.2; P = 0.014). There was a significant correlation of facial rosacea
with lid margin inflammation (P = 0.016), but not with inferior bulbar conjunctival inflammation
(P = 0.728). Ocular Demodex infestation was less prevalent in patients with aqueous tear-deficiency
dry eye than those without (7/38 vs. 12/21; P = 0.002).
Conclusions—The strong correlation provides a better understanding of comorbidity between
Demodex mites and their symbiotic B oleronius in facial rosacea and blepharitis. Treatments directed
to both warrant future investigation.
© 2010 by the American Academy of Ophthalmology Published by Elsevier Inc.
Correspondence: Scheffer C. G. Tseng, MD, PhD, Ocular Surface Center, 7000 SW 97 Avenue, Suite 213, Miami, FL 33173.
stseng@ocularsurface.com..
NOR is the recipient of a Hume Scholarship.
Financial Disclosure(s): The authors have made the following disclosures: Scheffer C. G. Tseng has filed 2 patents for the use of tea tree
oil and its ingredients for treating demodicosis.
NIH Public Access
Author Manuscript
Ophthalmology. Author manuscript; available in PMC 2010 September 28.
Published in final edited form as:
Ophthalmology. 2010 May ; 117(5): 870–877.e1. doi:10.1016/j.ophtha.2009.09.057.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Demodex, a microscopic, obligate, and elongated mite, is the most common ectoparasite in
humans. In the skin, D folliculorum is found in hair follicles, whereas D brevis lives in
sebaceous glands. They often coexist in the same skin area such as the face, cheeks, forehead,
nose, and external ear tract, where active sebum excretion favors their habitation and breeding
(for reviews see Baima and Sticherling1 and Forton et al2). In the eye, D folliculorum can be
found in the lash follicle, whereas D brevis burrows deep into lash sebaceous glands and
meibomian glands.
The pathogenic potential of these 2 mites remains arguable because a low number of
Demodex can be found in the skin and lashes of asymptomatic individuals. To probe into the
pathogenic role Demodex has in the external eye, we have improved the accuracy of the method
of lash sampling and mite detection3,4 and reported that the clinical sign of cylindrical dandruff
(CD) in the lash root is pathognomic for ocular Demodex infestation.3 Subsequently, using a
method of monitoring in vitro killing of adult Demodex mites, we have discovered that lid
scrubs with tea tree oil (TTO), but not baby shampoo, can successfully eradicate ocular mite
infestations.5 Because previously refractory ocular surface inflammation resolved after a
significant reduction of mite counts, we concluded that ocular Demodex infestation in some
patients might lead to ocular surface inflammation in lashes (trichiasis and madarosis), lid
(blepharitis), meibomian glands (lipid tear deficiency and tear film disturbance), conjunctiva
(conjunctivitis), and various corneal lesions.6,7 Intriguingly, 4 of the 6 patients with
Demodex blepharitis that manifested corneal lesions also suffered from facial rosacea and their
ocular surface inflammation was notably reduced by lid scrubs with TTO, but not by
conventional treatments such as lid hygiene with baby shampoo, topical steroids and
antibiotics, and systemic doxycycline.7 Nevertheless, it remains unclear whether the
aforementioned clinical improvement is derived solely from eradication of mite infestation or
from other known antibacterial,8–10 antifungal,11–13 and anti-inflammatory14,15 actions of
TTO. Even if we assume that the aforementioned clinical improvement is causatively linked
to the therapeutic action of TTO for killing mites, we still cannot rule out whether there is
concomitant microbial involvement in mite infestation.
Papulopustular rosacea is a chronic inflammatory dermatosis of the convexities of the central
face characterized by the presence of multiple small, dome-shaped erythematous papules and
papulopustules arising on a background of fixed inflammatory erythema.16 Although the
diagnostic criteria, classification, and grading of rosacea have recently been outlined,17,18 its
etiology and pathogenesis are poorly understood.19 The D folliculorum mites are frequently
seen in the follicles of skin of patients with rosacea.20 Using a skin surface biopsy technique,
which extracts mites from epidermal follicular canals, several investigators have shown a
significantly increased density of D folliculorum mites in the facial skin of patients with rosacea
when compared with control subjects,21,22 but the relevance of this finding to the pathogenesis
of the condition is disputed.
To reconcile the apparently disparate findings of increased numbers of D folliculorum mites,
perifollicular inflammation, and response of papulopustular rosacea to selective antibiotic
therapy in some patients, Kavanagh et al23 recently isolated Bacillus oleronius inside of
Demodex mites from 1 patient with papulopustular rosacea. They discovered that this
bacterium produced antigens capable of stimulating proliferation of peripheral blood
mononuclear cells in 16 of 22 (73%) patients with rosacea but only 5 of 17 (29%) control
subjects (P=0.0105). Furthermore, they pooled serum from 6 patients with papulopustular
rosacea and identified serum immunoreactivity to 2 proinflammatory 62-kDa and 83-kDa
proteins produced by this bacterium.23
As a first step toward addressing the aforementioned questions, we carried out a prospective
study of 59 patients and correlated their serum immunoreactivity to these 2 bacillus proteins
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with ocular Demodex infestation as well as with their clinical presentation of facial rosacea,
ocular surface inflammation, aqueous tear-deficiency dry eye, and conjunctivochalasis. Our
results show a strong correlation among positive serum immunoreactivity and ocular
Demodex infestation in facial rosacea and lid margin inflammation. The significance of
comorbidity of both microbial infection and mite infestation in ocular surface inflammation is
further discussed.
Patients and Methods
Patients
This study followed the tenets of the Helsinki Declaration of Human Studies and has been
approved by the ethics committee of the Ocular Surface Research and Education Foundation.
A total of 59 patients who presented with a number of ocular surface diseases (Table 1; available
online at http://aaojournal.org) were consecutively enrolled in the Ocular Surface Center
(Miami, FL) from September 2008 to April 2009. For all patients, in addition to routine
complete eye examinations, photographs were taken to document findings in the face and lid
margins and bulbar conjunctiva of both eyes. The fluorescein clearance test was performed to
determine if there was aqueous tear-deficiency dry eye as previously published.24,25
Fluorescein staining and maneuvers such as frequent blinking and digital pressure against the
lid to the globe were used to rule out conjunctivochalasis as another common cause of dry eye.
26,27 Afterward, lashes were epilated for microscopic detection and counting of Demodex mites
and the blood was drawn for detection of serum immunoreactivity to microbial proteins.
Clinical Grading
Clinical grading of facial rosacea, lid margin inflammation, and inferior bulbar conjunctival
inflammation was performed by 2 independent masked readers (HS and VKR). Both mite
counting and clinical grading were performed by examiners who had no knowledge about each
patient's clinical information and serum immunoreactivity. Standard photographs for grading
these three lesions were prepared by JL and SCGT. Facial rosacea was graded according to
erythema, flushing, telangiectasia, dome-shaped erythematous papules, pustules in a nasal or
central-facial distribution, and thickened skin with prominent pores (Fig 1). Lid margin
inflammation was graded according to the presence and extent of blood vessels and redness,
and scored as 0 for none or trace, 1 for mild, and 2 for severe (Fig 2, left). Because inflammation
of the bulbar conjunctiva was not uniformly distributed in all 4 quadrants, the inferior bulbar
conjunctiva was chosen and scored in a similar fashion as 0, 1, and 2 (Fig 2, right). After
reviewing these standard photographs, the 2 masked readers were asked to grade photographs
of all patients that were labeled only by an enrolled case serial number. Photographs of all 4
lids were reviewed at once and the score of the most severe grade among all 4 lids was chosen
for comparison. Any inconsistent grading between the 2 masked readers was arbitrated by
SCGT, who had no knowledge about the result of each patient's serum immunoreactivity at
the time.
Lash Sampling and Demodex Counting
Demodex mites were counted by microscopic examination of epilated lashes as previously
reported3 with recent modification.4 Briefly, 2 lashes with CD per lid were removed by fine
forceps under slit-lamp examination. Under a light microscope, 1 drop of saline was applied
by a pipette to the edge of the coverslip for lashes without retained CD, and 1 drop of fluorescein
solution was added for those with retained CD to allow embedded Demodex to migrate out.4
The total number of mites was used for comparison. In addition, D brevis was recorded
separately.
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Extraction of Bacterial Antigens
The Nutrient Broth (Oxoid) in the volume of 250 ml, pH 7, was inoculated with a loopful of
B oleronius and incubated for 48 hours at 30°C and 200 rpm. Late stationary phase cells were
sedimented by centrifugation at 4000×g for 20 minutes (Beckman GS-6). The supernatant was
discarded and cells were washed twice with phosphate-buffered saline (pH 7.2). Cells were
then resuspended in 0.2% Triton-X 100 in Lamberts Breaks buffer (pH 7) with added protease
inhibitors (10 μg/ml each of leupeptin, pepstatin A, aprotonin, and tosyl lysyl chlormethyl
ketone). The suspension was inverted for 1 hour at 4°C before sonication at 20% power for
three 10-second blasts using a soniprobe sonicator (HD 2200; Bandelin Sonopuls, Berlin,
Germany). The supernatant was centrifuged at 6000×g at 4°C for 2 minutes to separate the
protein preparation. Protein concentration was assessed by Bradford assay and if necessary
was precipitated with 3 times the volume of ice cold acetone.
Western Blot Analysis of Each Patient's Serum
The Western blot was based on 12.5% (wt/vol) sodium dodecyl sulphate-polyacrylamide gel
electrophoresis performed in a discontinuous buffer system, in which each well was loaded
with 20-μL samples, which consisted of 20 μgof B oleronius protein extract. For each patient,
a total of 5 ml of peripheral blood was drawn and centrifuged at 3500×g for 10 minutes. The
serum was transferred to a sterile, 7-ml bottle under a lamellar flow hood, labeled with a case
serial number and the sampling date, and stored in a −80°C freezer. After collection, a number
of serum samples were shipped from Miami to Ireland, where each serum sample was diluted
1/150 (V/V) with the antibody-diluting buffer (50 mmol/L Tris-HCl, 150 mmol/l NaCl
containing 0.05% Tween-20, 1% bovine serum albumin, and 3% nonfat dried milk) and used
as the primary antibody. The secondary antibody was anti-human immunoglobulin G-
horseradish peroxidase-linked whole antibody (Sigma Aldrich Chemical Co. Ltd, Poole, UK),
which was diluted 1/1000 with the antibody-diluting buffer. The immunoreactive protein bands
were visualized by incubating for 10 minutes the membranes in 10 mg of diaminobenzidine
tetrahydrochloride in 15 ml of 100 mmol/l Tris-HCl (pH 7) containing 10 μl of hydrogen
peroxide before washing in distilled water and drying. Standard positive and negative controls
of immunoreactive bands to either 62-kDa and 83-kDa proteins are demonstrated in Figure 3.
Densitometry was performed using a Typhoon computer package with Quant-L imaging
system to quantify the intensity of these 2 protein bands, and a value < 10 000–12 000 for the
62-kDa band or <10 000 for the 83-kDa protein band was considered positive.
Statistical Analysis
Numerical data were reported as the mean values ± SD, and nonnumerical data were recorded
as the presence (yes) or absence (no). We used the independent samples t-test to determine age
matching and to compare the Demodex counts between 2 groups. We used the Pearson chi-
square analysis to determine gender matching and the correlation of any variable between 2
groups. We used Pearson bivariate correlation to determine whether the Demodex counts were
influenced by age in different groups. All these statistical analyses were performed using SPSS
software version 11.5 (SPSS, Inc., Chicago, IL), and reported as 2-tailed probabilities, with
P<0.05 considered significant.
Results
The 59 patients consisted of 34 men and 25 women with a mean age of 60.4±17.6 years (range,
17–93). Their age distribution was not different when they were subdivided into 2 groups
according to the presence or absence of serum immunoreactivity, ocular Demodex infestation,
facial rosacea, or aqueous tear-deficiency dry eye (P = 0.151, 0.805, 0.868, and 0.706,
respectively; Table 2). When the Demodex counts were analyzed, we did not note any
significant correlation in the age distribution regardless of whether it was analyzed as a whole
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group (P=0.153), with or without serum immunoreactivity (P=0.159 and 0.847, respectively),
or with or without facial rosacea (P=0.163 and 0.988, respectively). Although there was a trend
toward older ages having higher Demodex counts in patients with ocular Demodex infestation
(P=0.099), such a difference did not reach significance.
Regarding the gender, there was no difference in these 59 patients with or without aqueous
tear deficiency (P=0.977). However, there was a significant difference between 2 groups with
or without serum immunoreactivity, ocular Demodex infestation, or facial rosacea (P=0.012,
0.024, and 0.008, respectively). Specifically, more men were found in the group with positive
serum immunoreactivity or facial rosacea, but more women were in the group without ocular
Demodex infestation. Such a gender difference suggested that higher ocular Demodex
infestation was correlated with higher serum immunoreactivity and facial rosacea in male
patients.
Clinical Grading
After reviewing the standard photographs illustrated in Figures 1 and 2, the 2 masked readers
had no difficulty in grading coded photographs in all 59 patients without knowing patients'
clinical information and serum immunoreactivity. The agreement rate between them reached
47/59 (80.0%), 44/59 (74.6%), and 46/59 (78.0%) in grading facial rosacea, lid margin
inflammation, and inferior bulbar inflammation, respectively. For those patients presented with
discrepant grading, the final score was arbitrated by the third reader.
Correlative Analyses
Table 1 (available online at http://aaojournal.org) lists the final grading of all relevant clinical
information of 59 patients, who were subdivided into 2 groups according to positive (n = 21)
or negative (n = 38) serum immunoreactivity. Further comparison of these 2 groups showed
that positive serum immunoreactivity was significantly correlated with ocular Demodex
infestation (P=0.048), facial rosacea (P=0.009), and lid margin inflammation (P = 0.040), but
not with inferior bulbar conjunctival inflammation (P=0.573).
When these 59 patients were subdivided into 2 groups with (n = 38) or without (n = 21) ocular
Demodex infestation, our analyses showed a significant correlation between the presence of
ocular Demodex infestation and serum immunoreactivity (P=0.048). However, ocular
Demodex infestation only had a borderline but not significant correlation with facial rosacea
(P=0.075) and lid margin inflammation (P=0.073), but had no correlation with inferior bulbar
conjunctival inflammation (P=0.599). The Demodex count in patients with facial rosacea (6.6
±9.0) was significantly higher than that in patients without (1.9±2.2; P=0.014). However, the
Demodex count was not different between the 2 groups with or without positive serum
immunoreactivity (P=0.178). Consistent with what has previously been reported,3,6,7 D
folliculorum was the main species found in epilated lashes, and was detected in 37 of 38 patients
with positive ocular Demodex infestation. In contrast, D brevis was found in 4 of 38 patients
(Table 1, available online at http://aaojournal.org; group I, case nos. 1 and 18 and group II,
case nos. 16 and 31). Although the detection of D brevis was not correlated with serum
immunoreactivity, 3 of 4 such patients also had facial rosacea.
Other analyses showed a significant correlation of facial rosacea with lid margin inflammation
(P=0.016), but not with inferior bulbar conjunctival inflammation (P=0.728). As a result, there
was also no correlation between lid margin inflammation and inferior bulbar conjunctival
inflammation (P=0.162). Aqueous tear-deficiency dry eye, defined by the fluorescein clearance
test, was detected in 19 of 59 (32%) patients. Interestingly, ocular Demodex infestation was
more commonly detected in patients without aqueous tear-deficiency dry eye than in patients
with (57.1% [12/21] vs 18.4% [7/38]; P=0.002). There is no difference in age (P=0.706) or
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gender (P=0.977) in patients with or without aqueous tear-deficiency. In contrast, the
prevalence of aqueous tear-deficiency dry eye was not different between the 2 groups of
patients with or without serum immunoreactivity (P=0.657) or with or without facial rosacea
(P=0.361). Conjunctivochalasis, which generates similar dry eye complaints and can clinically
be differentiated from aqueous tear-deficiency dry eye,27 was found in 16 of 59 (27%) patients.
The occurrence of conjunctivochalasis was also not different between 2 groups with or without
serum immunoreactivity (P=0.852), ocular Demodex infestation (P = 0.425), or facial rosacea
(P=0.535). These 59 patients also had other types of ocular surface diseases including floppy
eyelids (n = 9), pterygium (n = 9), chemical burns (n = 3), allergic conjunctivitis (n = 2), scleral
melt (n = 1), superior limbic keratoconjunctivitis (n = 1), conjunctival papilloma (n = 1), and
scleral melt (n = 1; Table 1, available online at http://aaojournal.org). Their occurrences did
not correlate with any of these analyses.
Representative Case Report
A 24-year-old man (group I, case no. 1) had complained of redness and blurred vision, more
so in the left eye than the right, since December 2006. Previously, he had worn monthly contact
lenses for 2 years without any problems. From February 2007, the left cornea developed lesions
prompting the suspicion of bacterial and viral infection. These problems persisted despite
discontinuation of contact lens wear, topical and systemic steroids, antibiotics, and antiviral
medication. Because of the progressive worsening of his vision, superficial keratectomy and
amniotic membrane transplantation were performed in a foreign country without success.
External examination showed mild rosacea in the face (Fig 4A). His best corrected visual acuity
was 20/20 in the right eye and 20/30 in the left eye. Lashes were largely clean without CD.
The Demodex count was zero per 8 lashes randomly sampled from both eyes. Lid margin
inflammation (graded as 1) and meibomian gland dysfunction evidenced by oil plugging of
the orifice were noted more in the left eye (Fig 4B). The left cornea showed prominent,
superficial neovascularization in the superior aspect and superficial haze in the central region
(Fig 4C). Furthermore, perilimbal (Fig 4C) and conjunctival inflammation (not shown), of
which the latter was graded as 1 in the inferior bulbar area, was also noted more in the left eye.
Because his condition did not improve despite mechanical cannulization of the meibomian
gland orifice and hot compresses, sampling of 16 lashes was repeated 5 months later and
revealed 1 D brevis. The Western blot analysis of his serum performed then showed strong
positive immunoreactivity to both 62-kDa and 83-kDa protein bands. A further inquiry of his
history revealed that he slept with a dog at night, and exhibited the strongest 4+ reaction to
dust mites among all allergens examined by the skin test. Because of the refractory nature, he
started lid scrub with 50% TTO solution every other day and lid massage with 5% TTO
ointment twice a day. Three months later, his visual acuity improved from 20/30 to 20/25 in
the left eye; lid margin inflammation (Fig 4D) as well as perilimbal conjunctival inflammation
and superficial corneal haze were notably reduced (Fig 4E).
Discussion
Lacey et al23 first linked skin mite infestation and microbial infection to explain why cutaneous
inflammation occurs in facial rosacea. Specifically, their experimental evidence supports the
notion that cutaneous inflammation might be exacerbated by strong host immune responses to
proteins produced by B oleronius living inside Demodex mites. Herein, our collaborative study
further established the comorbidity between Demodex infestation and Bacillus infection in
facial rosacea. This finding allows a better understanding of how ocular surface inflammation
might occur in some patients inflicted with ocular Demodex infestation.
Their previous study demonstrated 1 host immune response is positive immunoreactivity to
Bacillus 62-kDa and 83-kDa proteins that can be demonstrated by Western blot analysis using
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pooled patient sera.23 In the present correlative study, we looked into such immunoreactivity
in each individual patient serum. Furthermore, we measured Demodex infestation by sampling
eyelashes using our improved counting method.3,4 For the first time, our comprehensive
analyses disclosed a significant correlation among serum immunoreactivity to Bacillus
antigens, ocular Demodex infestation, and facial rosacea. In a total of 59 patients prospectively
and consecutively enrolled, positive serum immunoreactivity had a significant correlation with
facial rosacea (P=0.009) and ocular Demodex infestation (P=0.048). Patients with facial
rosacea had a significantly higher Demodex count than those without (P=0.014). Our
correlative analyses were age matched, but not gender matched. The lack of gender matching
could be explained in part by the finding that such correlations among positive serum
immunoreactivity, facial rosacea, and ocular Demodex infestation were greater in male
patients.
The strong correlation between serum immunoreactivity and ocular Demodex infestation was
linked not only to facial rosacea, but also ocular surface inflammation. Specifically, we noted
that lid margin inflammation had a significant correlation with facial rosacea (P=0.016) and
positive serum immunoreactivity (P=0.040) and a borderline correlation with ocular
Demodex infestation (P=0.075). This correlative result bodes well with a known fact that
patients with facial rosacea frequently manifested blepharitis. However, we did not observe
similar significant correlations with inferior bulbar conjunctival inflammation (Table 2). Of
course, the extent of conjunctival inflammation in our patients might have been influenced by
several factors such as the severity of facial rosacea and the association with other ocular
surface diseases. Another important consideration was that the host inflammatory response in
the conjunctiva might be much less as it is further away from the site of mite infestation, namely,
hair/lash follicles and glands. Along this vein of reasoning, it was worth noting that the rate of
detecting D brevis in 4 of 59 patients was notably lower than the 3 of 6 patients reported to
have developed conjunctivitis and keratitis.7 Because D brevis primarily resides in meibomian
glands, its detection would suggest the inflammation might more easily reach the conjunctiva
and the cornea. This scenario was illustrated in the representative case report (Fig 4). In this
representative case, we also learned that D brevis might require more lashes to be detected.
Interestingly, ocular Demodex infestation was significantly less prevalent in patients with
aqueous tear-deficiency dry eye (P=0.002), but not in patients with conjunctivochalasis
(P=0.425). Future studies are needed to determine whether the tear-deficient state and treatment
might interfere with ocular Demodex infestation. Collectively, such strong correlation reported
herein suggests that a point-of-care diagnostic tool might be developed to detect patient's serum
immunoreactivity.
If the strong correlation among serum immunoreactivity, ocular Demodex infestation, rosacea,
and blepharitis were causative, we speculate a new pathogenic paradigm in linking both
Demodex infestation and microbial infection by B oleronius in ocular surface inflammation.
The comorbidity of both Demodex mites and B oleronius rests in the symbiosis of the latter in
the mite, a scenario first reported in the hindgut of a termite.28 The final host inflammatory
response may be affected by a combination of factors such as whether such symbiosis exists
in ocular mite infestation, whether there is a sufficient load of released 83-kDa and 62-kDa
bacterial antigens, to which host elicits a proinflammatory response, and whether the host has
an increasing susceptibility to elicit such a response. For example, as shown in the
representative case, the host immune-inflammatory response might be modified and
exaggerated because of being allergic to dust mites. In this study, we cannot rule out the role
of other microbes that may have been colonized in the lid because of mites as the vector.
Because ocular surface inflammation is notably reduced by lid scrub with TTO, but not by
conventional treatments including lid hygiene with baby shampoo, topical steroids, and
antibiotics or systemic doxycycline in some rosacea patients that present with corneal lesions,
7 the mite's role in stirring up host inflammatory response cannot be ruled out. In short, the
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comorbidity based on a symbiotic relationship of B oleronius in Demodex mites also justifies
the consideration of a therapeutic strategy directed to killing the symbiotic bacterium via oral
antibiotics such as tetracycline and to killing and preventing mating/reinfestation of
Demodex mites, for example, lid scrub with TTO and general hygiene at the same time. Future
investigation into this comorbidity between mites and microbes may shed new light not only
on the understanding of the pathogenesis of this centuries-old common ailment of the skin and
eye, but also other similar unresolved human diseases.
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Figure 1.
Standard face photographs of rosacea (A, C) and normal subjects (B, D) of men (A, B) and
women (C, D).
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Figure 2.
Standard external photographs of eyelids (left column) and inferior bulbar conjunctiva (right
column). Inflammation of the lid margin and the inferior bulbar conjunctiva is graded as 0 for
no (A) to trace (D), as 1 for mild (B, E), and 2 for severe (C, F).
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Figure 3.
Western blot analyses. Representative examples of (A) positive serum immunoreactivity to
both 83-kDa and 62-kDa protein bands (marked by arrows; from group I case no. 8). B, Positive
to the 83-kDa protein band (from group I, case no. 20). C, Positive to the 62-kDa protein band
(from group I case no. 10). D, Negative to both protein bands (from group II, case no. 35). The
left lane shows the molecular weight standard.
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Figure 4.
Representative case (group I, no. 1). A 24 year-old man presented with mild facial rosacea
(A), grade 1 lid margin inflammation with meibomian gland dysfunction (B), and superior
corneal neovascularization, haze, and perilimbal injection (C). Three months after lid scrub
with tea tree oil, there was notable reduction of lid margin inflammation (D), perilimbal
injection, and corneal haze (E).
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Table 1
Key Patient Clinical Information Subdivided by Serum Immunoreactivity
Case No Age Gender Ocular Demodex Counts*Facial Rosacea Lid Margin Inflammation Inferior Conjunctiva Inflammation Dry Eye Cch Other Ocular Surface Diseases
Group I. Positive serum immunoreactivity
1 24 M 1/1 Y 1 1 N N —
2 35 M 3 Y 2 2 N N —
3 42 M 4 Y 0 1 N N Chemical burn
4 49 F 1 N 0 0 N N —
5 51 M 3 N 0 1 N N Scleral melt
6 52 M 2 Y 2 1 Y N Pterygium
7 60 F 0 N 0 0 Y Y —
8 61 F 0 N 0 0 Y N —
9 65 M 1 Y 0 1 Y N —
10 67 M 2 Y 2 0 N N Pterygium
11 67 M 5 N 2 1 N Y Floppy eye lids
12 69 M 10 Y 2 0 Y N Floppy eye lids
13 75 M 44 Y 2 2 N N —
14 75 M 0 Y 1 2 Y Y —
15 77 M 4 Y 0 0 N N —
16 77 M 2 N 0 0 N N —
17 78 M 11 Y 1 1 N Y Floppy eye lids
18 80 M 8/1 Y 1 0 N Y —
19 82 M 0 N 1 0 N N —
20 85 M 12 Y 0 1 N N —
21 90 F 11 Y 1 0 N Y Floppy eye lids
Group II. Negative serum immunoreactivity
1 17 M 4 Y 0 0 N N —
2 26 F 4 N 1 1 N N Pterygium
3 27 M 0 N 0 1 Y N Floppy eye lids, allergic
conjunctivitis
4 28 F 3 N 0 1 N N Pterygium
5 31 M 0 N 0 1 N N —
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Case No Age Gender Ocular Demodex Counts*Facial Rosacea Lid Margin Inflammation Inferior Conjunctiva Inflammation Dry Eye Cch Other Ocular Surface Diseases
6 35 M 0 Y 0 1 N N Floppy eye lids, pterygium
7 41 F 7 N 0 1 Y N —
8 43 M 12 Y 2 1 N N Pterygium
9 47 M 2 N 0 2 Y N Floppy eye lids
10 48 M 2 Y 0 0 N N —
11 49 F 0 N 0 0 N Y Allergic conjunctivitis
12 49 F 0 N 1 0 N N Superior limbic keratoconjunctivitis
13 53 F 0 Y 1 1 N N —
14 56 M 0 N 0 0 Y N Pterygium
15 56 M 0 N 1 2 Y N Chemical burn
16 58 M 12/9 Y 1 0 Y N Chemical burn
17 59 F 1 N 1 0 N N —
18 60 F 1 N 0 0 N Y —
19 62 F 14 Y 0 0 Y N —
20 62 F 1 N 0 0 Y N —
21 65 F 0 N 0 1 N N Membranous cicatricial pemphigoid
22 65 F 0 Y 0 0 N N —
23 66 F 4 N 1 1 N Y —
24 66 F 0 N 1 1 N N —
25 67 M 5 Y 1 1 Y Y Floppy eye lids
26 67 F 4 N 0 0 Y N Pterygium
27 67 M 0 N 0 0 N Y —
28 68 M 5 N 0 2 N N —
29 68 M 10 Y 0 0 N Y —
30 71 M 7 N 2 1 Y N Conjunctival papilloma
31 72 M 3/1 N 1 0 N Y —
32 72 F 5 N 0 0 Y Y Pterygium
33 72 F 0 N 0 0 N N —
34 75 F 0 Y 1 0 N N Floppy eye lids
35 76 F 0 Y 1 0 N N —
36 77 F 0 N 0 0 Y Y —
37 87 F 0 N 0 1 N N —
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Case No Age Gender Ocular Demodex Counts*Facial Rosacea Lid Margin Inflammation Inferior Conjunctiva Inflammation Dry Eye Cch Other Ocular Surface Diseases
38 93 M 3 N 0 0 N Y —
Cch = conjunctivochalasis; F = female; M = male; N = no; Y = yes.
*A total Demodex count is reported. For those with D brevis, its count is reported after/if detected.
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Table 2
Summary of Statistical Analyses
No. Analysis Statistical Methods P Value
I. Age-matched with or without
1 Serum immunoreactivity Independent-samples t-test 0.151
2 Ocular Demodex infestation Independent-samples t-test 0.805
3 Facial rosacea Independent-samples t-test 0.868
4 Dry eye Independent-samples t-test 0.706
II. Age correlation between Demodex counts in
5 All patients Pearson bivariate correlation 0.153
6 Positive serum immunoreactivity Pearson bivariate correlation 0.159
7 Negative serum immunoreactivity Pearson bivariate correlation 0.847
8 Ocular Demodex infestation Pearson bivariate correlation 0.099
9 Negative facial rosacea Pearson bivariate correlation 0.163
10 Negative facial rosacea Pearson bivariate correlation 0.988
III. Gender-matched with or without
11 Serum immunoreactivity Pearson chi-square analysis 0.012
12 Ocular Demodex infestation Pearson chi-square analysis 0.024
13 Facial rosacea Pearson chi-square analysis 0.008
14 Dry eye Pearson chi-square analysis 0.977
IV. Serum immunoreactivity correlation with
15 Ocular Demodex infestation Pearson chi-square analysis 0.048
16 Facial rosacea Pearson chi-square analysis 0.009
17 Lid margin inflammation Pearson chi-square analysis 0.040
18 Inferior bulbar conjunctival inflammation Pearson chi-square analysis 0.573
V. Ocular Demodex infestation/counts correlation with
19 Facial rosacea Pearson chi-square analysis 0.075
20 Lid margin inflammation Pearson chi-square analysis 0.073
21 Inferior bulbar conjunctival inflammation Pearson chi-square analysis 0.599
22 Counts with serum immunoreactivity Independent-samples t-test 0.014
23 Counts with facial rosacea Independent-samples t-test 0.014
VI. Other correlations
24 Dry Eye vs serum immunoreactivity Pearson chi-square analysis 0.657
25 Dry Eye vs ocular Demodex infestation Pearson chi-square analysis 0.002
26 Dry Eye vs facial rosacea Pearson chi-square analysis 0.361
27 Facial rosacea vs lid margin inflammation Pearson chi-square analysis 0.016
28 Facial rosacea vs inferior bulbar conjunctival inflammation Pearson chi-square analysis 0.728
29 Lid margin inflammation vs inferior bulbar conjunctival inflammation Pearson chi-square analysis 0.162
30 Cch vs serum immunoreactivity Pearson chi-square analysis 0.852
31 Cch vs ocular Demodex infestation Pearson chi-square analysis 0.425
32 Cch vs facial rosacea Pearson chi-square analysis 0.535
Cch = conjunctivochalasis.
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