DOI: 10.1111/jdv.

13517 JEADV

REVIEW ARTICLE

Study of Demodex mites: Challenges and Solutions

N. Lacey, A. Russell-Hallinan, F.C. Powell*
The Charles Institute of Dermatology, University College Dublin, Belfield, Dublin 4, Ireland
*Correspondence: F.C. Powell. E-mail: frank.powell@ucd.ie

Abstract
Demodex mites are the largest and most complex organisms of the skin microflora. How they interact with the innate
and adaptive immune systems is unknown. Their potential to have a pathogenic role in the causation of human skin dis-
orders causes continued speculation. With growing interest in the microflora of human skin and its relevance to cuta-
neous health, the role of Demodex mites needs to be better understood. The main challenges facing scientists
investigating the role of these organisms and possible solutions are reviewed under the following headings: (1) Determin-
ing the mite population in skin, (2) Transporting, extracting and imaging live mites, (3) Maintaining mites viable ex vivo
and (4) Establishing methods to determine the immune response to Demodex mites and their internal contents.
Received: 30 June 2015; Accepted: 13 October 2015

Conflicts of interest
There are no conflicts of interest for this article. Galderma laboratories have sponsored research in our Institute
relating to Demodex. The work in this review was performed independently.

Funding sources
Aspects of this work were funded by grants from The Mater Foundation, the National Rosacea Society of
America, and in particular, the Irish Health Research Board, grant reference code: HRA_POR/2010/46.

Background Mites are, however, commonly found associated with eyelash

The microorganisms we now know as follicle mites were first
discovered in human ear wax in 1841.1 These mites were given
their own genus (Demodex, from the Greek ‘demo’ for lard and
‘dex’ for boring worm), which related to their worm-like mor-
phology and habitat in sebaceous follicles.2,3 Although more
than one hundred and seventy years have passed since they were
first identified the relevance of Demodex mites as a component
of the skin microflora remains unknown, their interaction with
the innate and adaptive immune systems is unclear, and their
potential as causative agents in the pathogenesis of human skin
disorders causes continued speculation.4
Demodex mites have been found in all areas of human skin,
but the largest numbers are present in facial skin.5–8 This proba-
bly reflects the higher density and greater activity of sebaceous
glands in this area of the skin. Mites are also found in the exter-
nal ear canal (in relation to the ceruminous wax-producing
glands), in sebaceous glands of the nasal vestibule, in Mont-
gomery’s glands of the breast areola and on eyelids related to
glands of Moll and the modified sebaceous meibomian
glands.1,5,6,9 Demodex mites are not usually found in sebaceous
glands associated with terminal hair growth of the scalp, but
with the onset of androgenetic alopecia and the conversion of
follicles to the vellus type, mites can be found in this region.10
hairs. Demodex mites have been found in all races of the human
adult population, but rarely in newborns and infrequently in
children.5,11–13 This has led to the presumption that mites are
transferred from the mother (or other adult) to the older child
by physical contact. The increasing maturity of the sebaceous
units in the maturing child probably provides the appropriate
environment for Demodex colonization.
Desch and Nutting in 197211 confirmed morphologically that
two distinctive species of Demodex reside in the human piloseba-
ceous unit. Demodex folliculorum (up to 440 lm in length) is
found predominantly in the upper infundibulum portion of the
unit in groups, while the smaller Demodex brevis (up to 240 lm
in length) is found deeper in contact with the glandular portion
of the unit. Molecular studies have confirmed the different spe-
cies based on their nuclear and mitochondrial gene markers.14–17
Demodex folliculorum and Demodex brevis male and female
mites can be identified morphologically by their size (female
mites are bigger) and their external sexual organs5,11 (Fig. 1A,B).
Unlike practically, all other species of mites, adult Demodex have
a cross-striated body.13 The adult mite body is composed of an
elongated striated vermiform opisthosoma, which can comprise
7/10 of the total length of Demodex folliculorum and 2/3 of
Demodex brevis. Four pairs of legs with claw-like appendices are

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Demodex: Challenges and Solutions
A A
B
Figure 1 Male and Female Demodex folliculorum. Live Demodex
mites were mounted in 20 lL glycerol on a coverslip inverted over
a chamber slide cavity. Images were recorded on a compound
microscope using a canon EOS 6D digital camera. (A) Male Demo-
dex folliculorum with black arrow indicating the male reproduction
organ (penis) on the dorsal side of the podosoma. (B) Female
Demodex folliculorum with black arrow indicating the female repro-
duction opening (vulva) on the ventral side of the podosoma.
found on the podosoma, the gnathosoma (mouth) has seg-
mented palps each with palpal claws and an oral needle for feed-
ing (Fig. 2M). Externally, the mite body is covered with a hard
exoskeleton. Mites do not appear to have an anus, and waste
material seems to be stored in a crystalline form within the
opisthosoma. Following copulation (which presumably takes
place within the pilosebaceous follicle), it is thought that the
female mites of both species deposit their ova in the deeper areas
of the pilosebaceous unit where they evolve through the further
stages of the lifecycle; larva (a triangular-like podosoma is evi-
dent in early development in D. folliculorum, with three pairs of
underdeveloped legs, and the opisthosoma developing in length
with age) to protonymph (opisthosoma is longer again, with
three pairs of more developed legs) to nymph (four pairs of legs
have developed with the longest opisthosoma) and finally the
adult form. An in vitro behavioural study by Spickett14 estimated
the reproductive lifecycle of human Demodex folliculorum to be
14.5 days through all life stages, but these studies have proved
difficult to replicate because mites rapidly desiccate and die
when removed from the skin. It has also not been possible to
date to maintain mites alive outside the skin for more than a few
days. Mites are occasionally seen on the skin surface, and it is
thought that they can migrate from one follicle to another at a
speed of 16 mm/h.13 As mites are thought to be negatively pho-
totaxic, this migration is presumed to occur at night, although
mites have also been collected from the skin surface during day-
time.13
Demodex mites are frequently seen in histopathological sec-
tions of skin biopsies (mainly facial) from human adult
patients7, but are usually not related to any inflammatory
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765
B C D
E F G H
I J K L
Gnathosoma
Opisthosoma
Podosoma
Figure 2 Recording and imaging live Demodex using image
reconstruction software. This adult female Demodex was placed
on a small amount of glycerol on a coverslip (~20 lL) and inverted
over a chamber slide cavity. Images were recorded on a com-
pound microscope (940) using a canon EOS 6D digital camera.
The first image was taken when the mite gnathosoma (mouth was
in focus) and images were recorded at each plane of focus of the
mite. (A–L) showing 12 of 40 images that were then overlayed and
reconstructed with image software (Quick PHOTO MICRO 3.0) to
give the full structure of a live Demodex (M).
process and rarely reported as being relevant to the pathological
process being studied. Mites are semi-translucent organisms and
can be hard to visualize and are difficult to stain. Rose bengal,
merbromin, iodine, Congo red, trypan blue, acridine orange,
iodonitrotetrazolium, safranin and methylene blue stains have
been used with varying success5,13 while fluorescein dye has been
used to improve the identification of mites on epilated eye-
lashes18 and skin scrapings.19 However, mite take-up of most of
these dyes does not appear to be compatible with their contin-
ued viability. Using dark field microscopy, autofluorescence of
living mites improves their visualization.
Demodex species are part of the normal fauna of the skin of
most mammals without causing disease. The potential for
Demodex mites to behave as pathogens in animals in certain cir-
cumstances is well recognized.20 The change in role from normal
fauna to pathogenic activity in animals appears to correlate with
an increase in the normal mite population. When vastly
increased numbers of mites develop on animal’s skin, it is often
reflected in cutaneous disease. The best documented animal
cutaneous disorder caused by increased cutaneous mite popula-
tion is the demodectic mange of dogs.20,21
© 2015 European Academy of Dermatology and Venereology
766
Increased numbers of Demodex mites have been identified in
many human dermatological disorders including pityriasis fol-
liculorum,22,23 pustular folliculitis of the face,24 scalp erup-
tions,25 blepharitis,26–28 spinulosis of the face,29 granulomatous
and papulopustular rosacea,30–34 androgenetic alopecia and peri-
oral dermatitis.10,35 Increased numbers of mites have also been
found in the skin of patient’s immunosuppressed by disease or
by immunosuppressive therapies.36–40 Increased mite counts can
also be found in some women during pregnancy.41
Various pharmaceutical agents have been used to reduce the
Demodex mite population in humans including topical tretinoin,
gamma benzene hexachloride lotion, 1% permethrin cream
rinse,3710% povidone–iodine, 75% and 100% alcohol, 50% baby
shampoo, 4% pilocarpine, 100% tea tree oil, 100% caraway oil,
and 100% dill weed oil,42 crotamiton 10% cream,34 Iver-
mectin.43 Photodynamic therapy has also been used for this pur-
pose.19 The relative effectiveness of these agents in killing
Demodex mites can be difficult to evaluate in the laboratory as
viability of mites is currently based on their movement, an inter-
mittent and at times difficult to visualize parameter.
Rosacea is the skin condition that is most commonly postu-
lated to be related to Demodex overpopulation of the skin. The
initial observation was made in 1933 when Ayres and Ander-
son44 reported that patients with Acne Rosacea responded clini-
cally to topical treatment, which was assumed to be toxic to
Demodex, suggesting an etiological link. Since then, there have
been numerous studies which have shown that the mite popula-
tion is increased in patients with rosacea,31–34 and that treat-
ments that appear toxic to mites lead to a clinical improvement
in the skin disorder.45,46 However, the reason why the mite pop-
ulation is increased in patients with rosacea is unknown and
although acaricide therapy is effective in clearing the clinical fea-
tures of this disorder, their mode of action in rosacea has yet to
be clearly defined as anti-inflammatory or other actions have
not yet been excluded.
With growing interest in the microflora of the human skin
and its relevance to cutaneous health and disease, the role of
this complex organism needs to be better understood. Demo-
dex mites appear to have a unique relationship with the innate
immune system in human skin. Their presence within the
pilosebaceous units is tolerated without reaction in most cases.
This could imply that Demodex mites may have an as yet
unidentified beneficial role for the human host in the adaptive
immune system of the skin. A potential pathogenic role in
certain diseases such as rosacea could occur when the mite
population increases beyond a certain critical point possibly
leading to damage of the follicular unit and triggering a host
response.
The main challenges facing the dermatologist/scientist investi-
gating the role and relevance of these organisms in the
biobalance of the human skin can be summarized under the fol-
lowing headings:
JEADV 2016, 30, 764–775
Powell et al.
1 Determining the mite population in skin
2 Transporting, extracting and imaging live mites
3 Maintaining mites viable ex vivo
4 Establishing methods to determine the immune response to
Demodex mites and their internal contents
Determining the mite population in skin
Demodex mites reside within the human pilosebaceous follicles
and while they may migrate on the skin surface are not found
there in large numbers unless there is an abnormal proliferation
of the mite population. In patients with the skin condition pityr-
iasis folliculorum, there is an enormous proliferation of the mite
population with associated scaling (but paradoxically little
inflammation), and in these circumstances, simple skin scrap-
ings of the skin surface with a glass slide will reveal multiple
mites within the scrapings of the skin surface scale.22,23 This
gives an indication of the increased skin mite population. In a
recent study of the mitochondrial genome of Demodex mites,
investigators obtained mites specimens by ‘drawing the curved
end of a bobby pin across the forehead of each participant’.17
However, this method is likely to yield small numbers of surface
or follicular orifical mites and would not be suitable for deter-
mining mite density in a given skin area.
Methods such as high-definition optical coherence tomogra-
phy47 and confocal laser scanning microscopy48 can identify
Demodex mites in vivo by the presence of their opisthosoma pro-
jecting from follicular orifices and give an indication of the pop-
ulation when compared to a similar area of skin in a control
subject. However, this does not necessarily reflect the intrafollic-
ular Demodex population as the number of projecting opistho-
soma may be influenced by other intrafollicular factors.49 In
order to quantify the number of mites in follicles and thereby
develop an estimate of the mite population in a designated skin
area, the current optimal method appears to be to repeatedly
extract the mites from within the pilosebaceous unit and then to
count them.49 Usually, the facial skin is the site chosen for study
of Demodex numbers. This is because the density and activity of
the sebaceous glands in this area is reflected in a higher Demodex
population relative to other areas and because most cutaneous
diseases with a postulated Demodex causation have facial mani-
festations.
Many techniques of Demodex extraction have been used in
the past. Squeezing or using a comedone extractor to sample
sebum from dilated follicles would seem a logical way of extract-
ing mites. Mites can be found with this technique, but it is diffi-
cult to standardize and can sometimes be painful for the patient
and lead to bruising. Mites are not easily visualized in extracted
sebum and they can be difficult to remove intact from it. Adhe-
sive tape (cellotape) stripping of the skin provides a sample of
the skin surface and material at the follicular orifices. This
method can be useful to detect mites on the skin surface as the
tape can be left on for several hours thus capturing emerging
© 2015 European Academy of Dermatology and Venereology
Demodex: Challenges and Solutions
and migrating mites. However, this technique provides only a
superficial sample of the pilosebaceous contents. Mites detected
by this method are difficult to count and to remove from the
sticky tape, and probably do not provide a representative sample
of the Demodex population. Epilating eyelashes is a useful and
reproducible method to detect eyelid mites as the ciliary follicles
frequently harbour these. Mites are usually seen closely adherent
to the hair shaft often enclosed in a cylindrical cuff of keratinous
material. However, although the number of mites on the cilia
may be compared within groups (patients and control subjects),
this method does not provide information relating to the Demo-
dex population within the meibomian or other modified eyelid
sebaceous glands.
The skin surface biopsy method of Marks and Dawber50 is
currently the method most commonly used for extracting mites
from human skin. This technique involves the application of a
glass slide to the skin on which a small drop of glue has been
applied. The glue spreads over a limited area of the skin surface
and enters the pilosebaceous follicular openings. The slide is
kept in contact with the skin for a designated period (usually
1 min) and then gently removed from the skin surface. By this
stage, the glue on the slide has dried and as the slide is lifted off
the skin, it extracts the contents of the upper pilosebaceous
canals. By repeating this procedure several times in precisely the
same location, sequential samples can be extracted from progres-
sively deeper levels in the same follicular canals. To provide con-
sistency, a standardized area of 1 cm2 is outlined on the slide to
facilitate counting of Demodex extracted from a specific area of
the skin.31,32,34,51 The standardized skin surface biopsy (SSSB),
even if repeated several times in the same location, is usually
completely painless and very well tolerated by the patient. Occa-
sionally, the fumes of the glue cause a mild stinging sensation of
the eyes (the patient can avoid this effect by closing their eyes
during the procedure), and rarely (particularly in elderly
patients), if the skin is very fragile or inflamed, there may be
minute spots of bleeding after the procedure. Sometimes, a resi-
due of the glue remains in contact with the skin after the proce-
dure as a fine dry film. This can be easily lifted off an hour or so
later as the adhesive bond diminishes. Scarring has never been a
consequence of our repeated use of this procedure in several
thousand patients and controls over a period of more than
25 years. Contact dermatitis has not been a problem in our
study groups, several of whom have been repeatedly exposed to
the glue. This method is well documented in the relevant litera-
ture and although the sampling technique and number of
repeated skin surface biopsies taken vary in reported studies, the
SSSB allows an estimate of the Demodex population in a desig-
nated area and a reasonable comparison of Demodex numbers
between patients and controls if taken in the same location with
the same frequency and the same technique.
We re-evaluated the SSSB to improve its efficacy and modi-
fied the technique to increase the accuracy of Demodex counts,
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767
to identify and observe Demodex life stages, and to facilitate
extraction of live Demodex mites from samples.
Method of determining the mite population in skin
In the first instance, different types of glue were evaluated to find
the most suitable one for repeated use. The tissue adhesive EPI-
glu (ethyl-2-cyanoacrylate) provided good skin adhesion, but
required refrigeration and did not have a long shelf life. This
restricted its repeated use in the clinical setting. The second glue
tested Dermabond (octyl-2-cyanoacrylate) had a purple colour
which obscured mite visualization. It was more liquid than other
glues tested and frequently ran over the side of the SSSB slide on
to the surrounding skin. It did not produce firm adhesion
between the slide and the skin, and paradoxically was sometimes
difficult to remove from the skin when adhesion occurred. Like
other studies31–33,51 standard superglue (Loctite) provided the
best qualities for taking a SSSB. A drop of this glue had a thick
consistency and remained centred in the 1 cm2 grid of a SSSB,
allowing easy orientation and application of the SSSB to the
skin. It formed a firm bond with the skin in the designated
1 min time period and was easily removed from the skin leaving
little residue behind. Using superglue, several consecutive SSSB’s
could be taken at the same skin site with little discomfort to the
subject. There seemed to be little antigenic stimulation by the
glue and no example of contact dermatitis was recorded in our
repeated use of this substance in the same subjects. Under
microscopic investigations, this glue was colourless and translu-
cent, which facilitated visualization of follicular casts and mite
identification and isolation (Fig. 3A,B). A negative observation
was a slight odour from the glue and minor transient ocular dis-
comfort (a stinging sensation) in subjects presumably due to
fumes from the glue being applied to the skin of the adjacent
cheek skin. This was largely avoided by instructing the subjects
to keep their eyes closed during the test procedure and by prefer-
entially using the forehead as a sample site.
Secondly, in order to further standardize the area of skin
being sampled by the SSSB and to repeatedly identify individual
mites on extracted samples, we printed and adhered transparent
adhesive strips to the reverse side of otherwise normal glass
slides. These strips had a fine black outline with a defined 1 cm2
area. This area was subdivided by a grid, with letter and number
markings outside the grid, defining nine small squares within
the one square centimeter (Fig. 3C). This subdivision facilitated
not only counting the number of Demodex and locating specific
mites and mite life stages at certain precise points on the sample,
but also allowed recording of mite movement over time. A small
drop (~250 lL) of glue was placed in the centre of the 1 cm2
sticker grid (on the side of the slide opposite the adhesive grid)
and allowed to disperse to cover 1 cm2. The forehead was chosen
as the preferential site for repeated sampling and provided a
consistently good supply of mites. In addition, this site mini-
mized the exposure of the eyes to the potentially irritating effect
© 2015 European Academy of Dermatology and Venereology
768
A the same facial site in individual subjects with the purpose of
100 µm
B C
Figure 3 Identifying Demodex in standardized skin surface biop-
sies. (A) Image of a skin surface biopsy using a stereomicroscope
(Olympus SZX16 model) at 639 magnification. Samples were not
clarified with oil and Demodex mites present in the samples are
indicated with red arrows. (B) Arrows indicate Demodex in a skin
surface biopsy, clarified with immersion oil and a coverslip, under a
compound microscope (9200). (C) The Modified Standardized
Skin Surface Biopsy slides, showing the letter and number markers
to aid sample orientation in the 1 cm2 9 piece grid. Image was
taken using a stereomicroscope (Olympus SZX16 model).
of the fumes from the glue and those patients with rosacea
whose inflamed skin was sampled had fewer telangiectasias at
this site. This reduced the potential to traumatize these by the
SSSB and avoid resultant fine bleeding points. In order to select
a defined location on the forehead for repeated and comparative
sampling, a fixed point was identified. This was achieved by
measuring 2 cm vertical to and 2 cm lateral to (either the right
or the left side) the centre point of the bridge of the individual’s
nose (Fig. 4a). This location was marked with a skin surface
marker. It provided a fixed point for consistent alignment with
each consecutive SSSB slide in that subject. This technique was
also used to identify a similar site for sample selection in com-
parison studies between rosacea patients and control subjects.
The side of the slide to which the glue had been applied was
firmly pressed on to the subject’s skin within 5 s of the glue
being dispersed. The lower marked point of the slide’s gridded
square was aligned to the marked point on the skin. The glue
was allowed to adhere to the skin surface for a timed period of
1 min. The skin on both sides of the slide was then firmly
pressed taut with the fingers of one hand, while one side of the
now adherent slide was gently levered upwards in an uninter-
rupted motion from the skin using the fingers of the other hand
(Fig. 4B). A small semi-transparent film of glue surface scales
and material extracted from follicles was then readily visible to
the naked eye on the previously adherent side of the slide
(Fig. 4c). By carefully and repeatedly using this technique, the
loss of follicular casts was minimized during the extraction pro-
cess. Up to five, consecutive SSSB’s were taken from precisely
JEADV 2016, 30, 764–775
Powell et al.
retrieving mites progressively deeper within the same follicles.
For consistency, all samples were evaluated within 4 h of
removal from the skin and mites were extracted from samples
for subsequent experiments within 24 h of sampling.
In initial studies, the adhesive SSSB samples were clarified
with a drop of immersion oil, covered with a coverslip and
viewed by standard compound microscope at 9200, 9400 mag-
nification (Fig. 3A). This process allowed clear visualization and
counting of mites. However, the duration of mite viability and
their movement appeared to be reduced in the presence of the
immersion oil, possibly due to heating of the sample by the halo-
gen light source. In addition, our ability to extract mites from
the oil saturated samples was impaired. Finally, extracted mites
were contaminated by the immersion oil and difficult to work
with. This procedure was therefore changed and samples were
subsequently examined without the use of immersion oil or cov-
erslip. Mites and their life stages were recorded using a stereomi-
croscope (Olympus SZX16, Fig. 5b). With experience, mites
were easily identified in these samples and could be counted in
the 1 cm2 grid of each SSSB using the A–C and 1–3 grid for ori-
entation. We called this adaptation of the SSSB the modified
SSSB or MSSSB. This modification had the further advantage
that movement of live mites could be observed and recorded
within the grid, and mites were easily extracted from the samples
for further study.
Transporting, extracting and imaging live mites
The adaptation of the MSSSB as described above facilitated the
study of live mites. As previously indicated, mites rapidly desic-
cate and die when removed from their follicular environment.
To overcome this and to enable their study in vitro, we devel-
oped a simple humidity chamber to maintain mite viability in
our MSSSBs in the short period between extraction of mites in
the clinic and the examination of samples in the laboratory.
Mite humidity chamber
A small flat cotton wool pad was placed in a standard petri dish
to cover the bottom of the dish (90 mm, 15.9 mm high). This
was moistened with 9 mL of sterile dH20. Petri dishes contain-
ing the moist cotton pads were preheated (named a humidity
chamber) and maintained in an incubator at 28°C in the clinic.
When the first MSSSB was taken, it was placed on the warm
moist cotton wool with the sample side facing upwards away
from the saturated cotton (grid downwards). The second and
third MSSSB’s were overlaid on this slide, each at an angle of
33°. This ensured that the lower samples were not contami-
nated or damaged by the overlying slides. If more than three
MSSSBs were taken from a subject, a second mite humidity
chamber was used. In order to prevent sliding movement of the
slides during transportation, the slides were secured by placing
a (3 cm wide, 1 cm high) ring-shaped foam stopper on the
© 2015 European Academy of Dermatology and Venereology
Demodex: Challenges and Solutions 769

(a)

GL
UE
1 2 3

Label
Label
C
A B
B A
C
1 2 3

Label
A
B
C
1 2 3

Place a drop of glue

in the center to cover the full 1cm2

(b) (c)

(d) (e) (f) Sample packs

added at hospital
30 site

28

Temperature (°C)
26 Sample packs
arrive at laboratory
24 site
22
20 Pack
into incubator
18
16
0 1 2 3 4 5
Time (hr)

Figure 4 Standardized forehead location for consecutive standardized skin surface biopsies (SSSB’s) and transportation packs. (a) To
take consecutive SSSB’s from the same site we measured 2 cm up from the bridge of the nose and 2 cm left or right. A dot was made
with a skin marker. Using the dot on the SSSB slide, the slide was orientated to align the dot on the slide with the marked skin point. (b)
Slides were gently removed by holding skin taut with one hand and levering the slide slowly from the skin with the other hand. (c) example
of a SSSB slide when removed from the skin (a, b) was repeated for each consecutive SSSB. (d, e) Slides were sealed in a petri dish with
pre-wet cotton wool and temperature and humidity monitored by hydro-thermometer before placement in a travel thermapak. (f) Temper-
ature of the pack was monitored from time of addition to incubator until it reached optimal 28°C and during transit until it reached the lab-
oratory site. The pack maintained 28°C for the 2 h transit time.

uppermost slide. This was placed in position so that it did not
come into contact with the sample section of the third (top)
slide (Fig. 4d). The lid of the petri dish was then taped closed
pressing on the foam ring which, in turn, pressed firmly on the
underlying slides preventing movement. For transportation
times of more than a few minutes, mite humidity chambers
were placed in preheated transport packs (to 28°C). These con-
tained Constan Gel packs (Thermapak), which preserved the
temperature of the samples during transportation (Fig. 4e). A
temperature probe recorded the temperature during transporta-
tion. On arrival at the laboratory, the temperature of the trans-
port pack and samples, recorded by the temperature probe in
the pack (Fig. 4f) was noted. Mite humidity chambers were
then removed from the transport pack and placed in an incuba-
tor set at 28°C for further study.
Method of Extraction
To avoid further external contamination of samples, a stere-
omicroscope (Olympus, SZX16 model) and all equipment were
set up in a laminar flow hood using aseptic techniques. All
work was performed behind the airflow grid of the hood. Mite
humidity chambers containing MSSSB samples were removed
from the incubator when required and mite extraction was per-
formed in 4 minute time slots. This was to reduce the drying
effects as mites became rapidly desiccated (Fig. 5a). Samples
were replaced in the incubator when not in use. Mites in the

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770
A B
100 µm
C able practice, but once mastered was highly reproducible by
50 µm
50 µm
Figure 5 Skin surface biopsy (SSB). (a) This image shows mites
that have desiccated quickly in an SSB sample when removed
from their humidity chamber. Mite manipulation from samples must
be done quickly. We found four minute time slots were optimal to
maintain mite viability. (b) Demodex ecology of a follicle as seen
under a stereomicroscope (Olympus SZX16 model, 963). An adult
female and male Demodex folliculorum with a larval stage and an
ovum are indicated by red arrows (c) SEM images of a follicular
cast in a SSB. Red arrows indicate where Demodex folliculorum
has left impressions of it’s opisthosoma.
MSSSB sample were usually visualized clumped in groups, in
keratin-like material extracted from the follicles and referred to
as ‘follicular casts’. The extracted material from each follicle as
viewed was seen with the deepest part of the specimen project-
ing upwards. As Demodex are orientated head down in follicles
in vivo, the head, mouth parts and legs of the mites were visual-
ized uppermost in the extracted sample with the distinctive
waving motion of their legs attached to the upper body facili-
tating visualization. Adult mites in MSSSB’s that were left in
the humidity chambers in the incubator at 28°C for 2 h prior
to extraction often began to crawl out of their follicular casts,
which facilitated isolation and removal of mites. Many D. fol-
liculorum life stages (male and female adults, larva, ova)
(Fig. 5b) were visualized, particularly in deeper follicular sam-
ples. Scanning Electron Microscopic visualization of MSSSB
obtained follicular cast’s revealed indentations of Demodex
opisthosoma (Fig. 5c) in apparently empty follicular casts, indi-
cating that mites had the capacity to extract themselves from
these casts and were not prevented from doing so by the glue.
Adult mite specimens with their rigid exoskeletons proved to
be quite robust when extracted from casts provided they were
kept in a moist atmosphere at the appropriate temperature.
The LED light source in the stereomicroscope did not gen-
erate heat and the viability of the mites did not appear to be
adversely affected by this method of their visualization. Several
methods of extracting mites from the follicular casts were tried
before a satisfactory solution was achieved. These included the
use of fine insect needles, the hair of fine paint brush and
even floating mites free by washing the MSSSB in culture
solution. Each of these methods was found to be unsatisfac-
JEADV 2016, 30, 764–775
Powell et al.
tory for various reasons. Gently teasing mites from follicular
casts using a Moria fine forceps (FST, Germany) with a tip
diameter of 0.1 mm was found to be optimal and using this
method the maximum number of undamaged live mites were
obtained from follicular casts by microdissection under the
stereomicroscope (Fig. 6). This technique required consider-
the same operator. Isolated live mites were pooled in a desig-
nated area of the MSSSB on a vellus hair (to which they
adhered) before the slide was returned into the mite humidity
chamber and then the incubator at 28°C. Using the procedure
outlined above and with increasing operator experience,
almost all live mites could be retrieved from each MSSSB slide
without affecting their viability and without visible mite con-
tamination by the glue adhesive.
Method of Imaging
For detailed observation of live mites, mites were transferred
individually by Moria forceps into a thin layer of glycerol
(20 lL), which was placed on the centre of a coverslip. This was
then inverted over the cavity of a cavity slide so that the glyc-
erol-containing mites were retained within the cavity of the slide.
Mites were visualized using a compound light microscope with a
non-heat-generating light source (LED) (Olympus, CX22LED
model). Multiple images were recorded using a Canon EOS 6D
digital camera at each plane of focus of a mite. The images were
subsequently overlaid, reconstructed and compressed into a final
image by image software system (Quick PHOTO MICRO 3.0)
(Fig. 2A–M). The same procedure was undertaken for samples
viewed under stereomicroscope.
Maintaining mites viable ex vivo
Several investigators have attempted without success to maintain
Demodex mites viable ex vivo. Spickett in 1961 studied extracted
mites from human skin using a comedone expresser. Using a
finely drawn glass needle, he placed mites in sebaceous material
containing penicillin spread thinly on a coverslip and inverted
this over a cavity slide.14 Mites were kept in dark petri dishes
with moistened filter paper at 37°C. He investigated mite reac-
tion to heat, light and humidity. Although mite numbers were
low in this study by combining his in vitro studies with histologi-
cal data, he estimated the life span of Demodex folliculorum to be
14 days from ovum to adult, with adult females living for 5 days
and males for half this time. Grosshans et al.,52 found that sur-
vival of human Demodex mites could not be obtained, but did
not specify the methods used. Zhao et al.,53 extracted mites
using the cellophane tape method and studied the effect of tem-
perature on their viability. They concluded that the optimum
temperature for Demodex to survive in vitro was between 16 and
20 °C. At this temperature, they could maintain mites alive for
approximately 80 h (3.3 days). They maintained Demodex fol-
liculorum mites alive for 110 h (4.5 days) and Demodex brevis
© 2015 European Academy of Dermatology and Venereology
Demodex: Challenges and Solutions
Figure 6 Extraction of Live Demodex from Skin surface biopsies.
Steps of gentle manipulation of live Demodex by fine forceps from
follicular plugs under a stereomicroscope (Olympus SZX16 model).
Mites are indicated by red arrow.
for 145 h (5.8 days) at 6°C. In a subsequent study, they exam-
ined the capacity of various media to keep mites viable. These
included paroleine mineral oil, heated pig fat, human serum,
1640/seroculture solution and normal saline.54 They used a
moist chamber with 90% air humidity and placed mites in each
of the media outlined above. Mites survived longest (about
85 h) in the human serum medium. Tani et al.55 used xeno-
grafts of demodectic skin from dogs and hamsters onto
immunocompromised SCID mice skin. After 90 days, recipient
mice were killed and the mites were shown to be alive within the
grafted skin.
Evidence of mite viability in all these studies was based on
movement, either of the mite’s mouth parts, legs or body. The
limitations of this method of assessment are obvious as the
mites are not perpetually in motion, but in the absence of a
more robust assay method, this remains the only current way
of determining whether mites are alive or not. Provided that
repeated observations are taken over several hours with photo-
JEADV 2016, 30, 764–775
771
graphic recording of the position of the mite and its appen-
dages, it provides a reasonably reproducible method of
evaluation. Gaffer in 1964 studying Demodex canis noted that
living mites subjected to examination under a combination of
ultraviolet and blue violet light showed autofluorescence lim-
ited to the abdominal area of the body. After death, the aut-
ofluoresence extended to the thoracic area of the mites.56 The
possibility of using the autofluorescent quality of human
Demodex mites to assess viability has not been fully explored,
but it would appear from our preliminary investigations that it
might indeed have validity (Fig. 7b).
It would appear from these studies that optimal conditions
for complete and sustained mite reproduction ex vivo would
require a pilosebaceous environment. In the absence of such an
in vitro model, the key to sustaining mite viability on removal of
mites from the skin was the maintenance of MSSSB’s in an
enclosed hydrated environment as described in the above sec-
tion, which preserved samples with 50% humidity and tempera-
ture at 28°C. These conditions were found to reduce desiccation
of follicular casts and retained the viability of mites they con-
tained. However, even in these conditions viable mite numbers
progressively diminished over time so that few mites remained
viable after 7 days in the moist chambers. While a female D. fol-
liculurom mite was observed apparently depositing an ovum in
the SSSB after removal from the skin, reproduction of mites did
not take place and morphosis of larval life stages to the next
stage was not recorded.
Using our isolation technique from an MSSSB, we placed
mites in different media in an attempt to prolong their viability
and facilitate reproduction and induce morphosis of larval–
nymphal stages already isolated from the MSSSB. Media assessed
included phosphate-buffered saline, olive oil, immersion oil,
glycerol, fetal bovine and human serum, commercial culture
media for skin cell lines. Placing mites directly into a small vol-
ume (20 lL) of human serum on a coverslip, inverted over a
cavity slide and stored in humidity chambers in an incubator at
28°C, allowed viable mites to be observed for more than 5 days.
Because the precise age of adult mites retrieved from the MSSSB
cannot be determined, and taking into account the short life
span of mites, a diminishing number of mites will survive even
in the most favourable of environments. The early life stages
with weaker exoskeletons were often hard to manipulate, very
fragile and although some development was observed, we were
not able to visualize any larval stage fully metamorphosing into
an adult mite. Mites did not demonstrate prolonged viability
when co-cultured with sebocyte or keratinocyte cell lines.
Establishing methods to determine cellular
immune response to Demodex mites and their
internal contents
Using a stereomicroscope under aseptic techniques in a laminar
flow hood and working beyond the airflow, mites were trans-
© 2015 European Academy of Dermatology and Venereology
772
A
Figure 7 Light- and dark-field examinations of Demodex. Light-
field (a) and Dark-field imaging (b) of Demodex mites showing aut-
ofluorescence of mite bodies on dark field examination. Note the
increased intensity of fluorescence at the chitinous plates (b yellow
arrow) and the dark staining predominantly limited to the mite
opisthosoma in the bright field image (a yellow arrows). This latter
staining was strongly associated with viability of mites and dimin-
ished as mites became less viable.
ferred into each cell culture system using a sterile Moria fine for-
ceps. Mites that were pooled initially in a MSSSB as described in
the above section were transferred in the number required for an
experimental set up. To record and confirm the transfer of live
mites into cell culture systems, live mites were detected by focus-
ing an inverted microscope through each plane of focus from
cell monolayer to top of the cell medium. This was performed in
a zigzag pattern working from top left of a culture well to the
bottom right corner until mites were detected and recorded.
Images and video segments were recorded using a Canon EOS
6D digital camera (Fig. 8a). Mites were difficult to transfer from
one medium to another without significant loss of mites. This
was due to the adhesive nature of the mite exoskeleton to the
forceps and other material surfaces and apparent electrostatic
forces, which caused mites to be attracted to these surfaces. Pre-
dominantly, mites were visualized floating in the medium, but
mites were also recorded at the monolayer level. Live mites at
different densities were applied to the following monolayer cell
culture systems; the immortalized human sebaceous gland cell
line (SZ95) and primary human epidermal keratinocytes (nHek,
Lonza). Both cell types were maintained in their respective com-
mercially available media (Sebomed medium supplemented with
10% FCS, human epidermal growth factor (5 ng/mL) for sebo-
cytes, keratinocyte basal medium supplemented with KGM-
Gold). Antibiotics were not added to the cell line media to avoid
any potential effect on Demodex viability or immune-modula-
tory potential. There was an almost universal absence of bacterial
contamination in our cell culture systems after the addition of
live mites taken from control subjects even after 48 h co-culture.
This might suggest either a lack of bacterial contamination of
JEADV 2016, 30, 764–775
Powell et al.
A B
Figure 8 Investigating an immune response to Demodex. (a) Red
arrows indicate groups of Demodex which had been isolated from
a skin surface biopsy and transferred into a cell culture system. (b)
Ruptured Demodex mite bodies achieved by physical rupture by
fine insect needle.
the mites or possibly an ability of viable mites to have an inhibi-
tory effect on bacterial growth in the culture medium. The cell
supernatants from cell lines challenged with Demodex were
removed over a time course and stored at 80°C for future
inflammatory mediator detection and analysis. RNA was also
isolated from the same challenged cells for correlating gene
expression studies (N. Lacey, A. Russell-Hallinan, C. C. Zoubou-
lis, F.C. Powell, manuscripts in preparation).
Our earlier work suggested that Demodex may harbour an
endosymbiont.57 A Gram negative staining bacterium, Bacillus
oleronius, isolated originally from the hindgut of a termite, was
cultured from a Demodex mite extracted from the skin of a rosa-
cea patient. Subsequent experiments using antigenic prepara-
tions of this bacterium induced an elevated PBMC response
(73%) in rosacea patients, in comparison to control subject
stimulated PBMC’s (29%). The antigens of this bacterium were
determined by MALDI-ToF mass spectrometry analysis to have
homology with common immunodominant antigens that elicit
humoral, cell mediated and innate immune responses.57
Although reported in the literature to be associated with Demo-
dex and blepharitis,58 and some serum reactivity studies,59 this
bacterium has not been directly cultured subsequently from live
Demodex mites and its true role in inducing the inflammatory
lesions in rosacea is unknown.
To study the possibility that mites contained endobacteria
(necessary for digestion or reproduction) that might, when
released from dead mites, have a role in initiating inflammatory
reactions in the host, we developed a reproducible method of
rupturing mites to release their internal contents. This proved
technically difficult because the mite exoskeleton was impervious
to several of the standard methods of disruption. Methods
evaluated included; (1) Demodex mites (n = 50) were trans-
ferred by fine forceps into a microfuge tube containing 200 lL
of sterile PBS. The tube was subjected to three rounds of LN2
freeze and thaw. However, fully intact mite bodies were visual-
ized in the tube subjected to this technique, (2) Demodex
(n = 50) in the same medium had sterile glass beads added and
were subjected to five rounds of 30 s vortexing. Intact mite bod-
© 2015 European Academy of Dermatology and Venereology
Demodex: Challenges and Solutions
ies were again visible after being subjected to this treatment.
Intact mites were also noted to have become adhered to the glass
beads, (3) Live mites (n = 10) were transferred from MSSSB by
fine forceps into the cavity of a cavity slide containing 20 lL of
sterile PBS and individually physically ruptured under stereomi-
croscope by the operator using a fine insect pin and holder (FST,
Germany). This latter method proved to be optimal in obtaining
ruptured mites and their contents and was readily reproducible
by an experienced operator (Fig. 8b).
Mite body fragments and released internal components were
removed by pipette to a sterile microfuge tube for storage at
80°C for future stimulation experiments. Ruptured contents of
mites and mite bodies were also aspirated by pipette. The tip
and contents of pipettes were ejected into sterile culture tubes
containing nutrient broth (5 mL). Sterile PBS in a cavity slide
under the same conditions as mite rupturing acted as a control.
Tubes were sealed for subsequent microbiological study.
Conclusions
Determining the mite population in skin
Many methods have been used to study the mite population in
human skin as outlined above. The differing methodology, the
varying number of samples taken from the same or different sites
and the wide variation in the areas of facial skin sampled, have
created difficulties in interpreting results of publications from
different investigators. We propose that the Modified Standard
Skin Surface Biopsy (MSSSB) using the calibrated grid and sam-
ples taken repeatedly (at least three times) from a designated
facial skin site (the forehead at a measured distance from the
bridge of the nose as outlined earlier) be used in studies of facial
skin diseases. Several studies32–34 have confirmed that the fore-
head provides a source of mites that is constant and not signifi-
cantly different from other facial sites (e.g. the cheeks). In
addition, the skin of the forehead is fairly robust and unlikely to
be traumatized by this non-invasive procedure and the exposure
of the eyes to potentially irritating glue fumes is minimized.
Contact allergy (irritant or allergic) was minimal or absent in
our experience. Universal use of the MSSSB would facilitate
interstudy comparisons from different centres and allow scrutiny
of the mite population and its various life stages within their
biogeographic niche over time by examining a defined area of
follicles. Furthermore, it is anticipated that the use of this
method will allow investigator’s to test the efficacy of therapies
used to treat skin conditions thought to be related to Demodex
proliferation in vivo.37,40–44 Finally, using this method, the via-
bility of Demodex can be recorded as well as the mite numbers.
Transporting, extracting and imaging live mites
When mites are removed from their pilosebaceous habitat, they
desiccate rapidly. It is vital that extracted mites be maintained in
a moist environment. To study live Demodex in a laboratory set-
JEADV 2016, 30, 764–775
773
ting, we established mite humidity chambers and thermal trans-
port packs that maintained the viability of Demodex for many
hours after extraction from the skin before the transfer of the
humidity chambers to an incubator. Extracting mites from the
MSSSB required experience and patience. After assessing several
methods, we used a fine forceps that was effective and enabled
the great majority of mites to be retrieved.
High-power imaging of live mites using a compound micro-
scopic with a halogen light source was limited by the toxic effect
of the heat generated by the light source. We found that these
examinations were best carried out using a compound light
microscope with a non-heat-generating light source (LED)
(Olympus, CX22LED model). To record their 3D structure (as
the full mite body is difficult to achieve in one plane of focus),
we utilized an imaging programme to reconstruct each plane of
focus of a mite in a sample. This improved recording of Demo-
dex and their life stages, singly and in SSSB’s.
Maintaining mites viable ex vivo
The study of human Demodex has been hampered by the lack of
an in vitro or ex vivo culture model to sustain mite viability and
reproduce mites through all their life stages. Thus, the lifecycle
of Demodex is currently estimated based on few previous studies.
Our findings that adult Demodex frequently remained viable for
over 5 days and sometimes up to 10 days after extraction from
the skin and being maintained in a less than optimal environ-
ment, indicates that the proposed lifecycle of Demodex in its fol-
licular niche is possibly longer than at present estimated. This
would seem to be in keeping with other Demodex species such as
the Rat (Demodex ratticola) and hamster (Demodex aurati)
mites, with their full life cycles from ovum to adult taking
35 days and 19–24 days respectively.60 While we have shown
that viable mites can be studied over several days using a mite
humidity chamber as described above, it has not as yet proved
possible to establish an ex vivo environment that facilitates their
reproduction.
A further and recurring difficulty is the problem of estab-
lishing whether mites are in fact alive or not. As outlined
above, this is currently based on the mite’s motion or move-
ment, which is an unsatisfactory and crude evaluation, with
high variation between investigators. Further evaluation of the
extent of live vs. dead mites using dark-field autofluorescence
may prove helpful in this regard.
Methods to determine immune response to Demodex and
their internal contents
It is becoming clear that an aberrant innate immune response
is likely to be involved in the pathogenesis of rosacea.61 An
increase in TLR2 expression found in the epidermis of rosacea
patients, leads to increased serine protease activity, which
results in subsequent cleavage of abnormal pro-inflammatory
cathelicidin peptides.62,63 It has been suggested that the
© 2015 European Academy of Dermatology and Venereology
774
increased Demodex mite population could be considered as a
potential trigger for this innate immune cascade. Demodex
may produce serine proteases or may be coated with inflam-
matory mediators, which could also be involved in initiating
an inflammatory response.
The extraction techniques for the study of live Demodex
from MSSSB samples in this study, paves the way for more
intensive investigations on the biology of human Demodex
and its interaction with the host immune system. The ability
to insert and maintain live Demodex from SSSB’s in cell cul-
ture systems also provides a method of investigation of cellu-
lar immune response to mites to be established while the
established method of rupturing Demodex, described in this
review, should enhance studies on endobacteria and possible
endosymbionts of these mites.
Ethical approval
Ethical approval for this study was granted by the Research
Ethics Committee of the Mater Misericordiae University Hospi-
tal, Dublin, Ireland (ref. 1/373/638). This study was performed
in accordance with the declaration of Helsinki. Each participant
was given a study information leaflet before informed consent
was obtained.
Acknowledgements
The authors thank all participants in the study who provided
skin surface biopsy samples. We gratefully acknowledge the help
of Dr’s. Ruth Foley and Fiona Carter in developing our mite
transport packs and kits. The Electron Microscopy studies were
performed with the help and advice of Professor Dimitri Scholz.
We would also acknowledge with appreciation the generous
donation of the SZ95 sebocyte cell line from Prof. Christos C.
Zouboulis.
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