British Journal of Dermatology {199i) 128,650-659.

Density of Demodex foUiculorum in rosacea:a case-control study using standardizedskin-surface biopsy

F.FORTON AND B.SEYS*Clinic of Dermatology. Saint Pierre University Hospital, llniversite Libre de Bruxelles. Brussels. Belgium'Unit of Medical Sciences Pedagogy, Faculty of Medicine. Universite C^tholique de iMuvain. Brussels, Belgium

Accepted for publication 26 June 1992

Summary A standardized skin-surface biopsy (1 cm^) ofthe cheek was performed in 49 patients with rosacea[15 with erythematotelangiectatic rosacea (ETR). three with squamous rosacea (SR), 33 withpapulopustular rosacea (PPR)]. and 45 controls.

A mean density of 0-7 Demorf(;x/o//i('u/orum/cm^ was found in controls. 98% of whom had less thanfive Demodex/cm^. When all clinical types of rosacea were considered collectively, the density ofDemodex was significantly higher in patients with rosacea than in controls (mean=10-8/cm^:P<O-()()1). When the various clinical types of rosacea were considered separately. Demodex densitywas statistically significantly higher than in controls only in the PPR patients (mean—12-8/cm-:P<0001) .

The same type of comparison was also made for three other groups of subjects—patients witbisolated inflammatory papules (n = 4). rhinophyma (n = 3). and HIV infection {n = 21). respectively: inthese groups, the Demodex density did not differ significantly from controls.

The present study demonstrates a high density oW.fotlicuhrwn in PPR, and supports its pathogenicrole in the papulopustular phase of rosacea. The study suggests that standardized surface biopsy couldbe a useful diagnostic tool for PPR. with a 98% specificity when Demodex density is higher than 5/cm-.

Demodex/oHicuiorum is a transparent mite. 0-3 mm long,which asymptomatically parasitizes the human piloseh-aceous follicles (Fig. l).^"" The proportion of Demodexcarriers (prevalence) increases with age.''^'' and thereported prevalence is also determined by the fastidious-ness of the detection method used.'' '" A variety ofprevalence rates in different age-groups have beenreported in a number of studies (Table i).5-''ii '^

The pathogenic role of Demodex is still a matter ofdebate. To date, it has been implicated in the occurrenceof the dry. 'pityriasis-like' erythematosquamous rosa-cea."* in papulopustular and/or granulomatous rosa-j,g3 19-22 including localization on the bald scalp,^' inisolated inflammatory papules. '̂'̂ ^-^^ and in some casesof blepharitis.^^

It is difficult to establish the pathogenicity of D.foUiculorum in rosacea because of three factors: thelocalization of the disease, the obligate character of theparasite, and its ubiquity in man. Indeed, because

Correspondence: Dr F.Forton, rue F, Binje 8. B-l()30 BruxelJes,Belgique.

rosacea is a benign dermatosis mainly Iocali2ed to theface, it may be difficult to justify a standard skin biopsy tostudy the disease. In addition, as D. foUiculorum is a veryhost-specific obligate parasite, it cannot at present begrown in vitro, which makes a massive experimentalinfestation impossible. The mere detection of its presenceis no proof of pathogenicity because it is so ubiquitous. Itis usually considered as playing a pathogenic role whenpresent in 'very large' numbers.̂ ** and in an intradermallocation.''*"-''^'*

The principal aim of the present study was to deter-mine the pathogenic relevance of D. foUiculorum inrosacea by comparing the mite density in healthysubjects with that in patients suffering from rosacea. Weconsidered the various clinical types of rosacea separa-tely, i.e. erythematotelangiectatic (ETR). squamous orpityriasis-like rosacea (SR). and papulopustular rosacea(PPR).

In addition, the same comparison was made inpatients suffering from rhinophyma or isolated inflam-matory papules (IIP), and in individuals suffering fromhuman immunodeficiency virus (HIV) infection.

DEMODEX FOLLICULORUM IN ROSACEA 651

Figure 1. Demodex follicuJorum as it can be seenon a skin surface biopsy, (a) Three-quartersright aspect { x 500): from top to bottom, thebody consists of a gnatbosome (g! or rostrummade of the oral parts, a podosome (p) bearinglour pairs of legs, and an opistbosome (o) orabdomen, transversely striated along its wholelength. |b) Six parasites grouped in the samepilosebaceous follicle ( x 250).

Table 1. Demodex prevalence according to sampling mctbods since 1910'

Subjects

Reference Method n

250

10

100—

29

1435

9

100400

50

23

100

1124668

206

88

J70

State

aaa

a

ac

a

a

c

ca

a

a

a

aa

a

a

a

Age(years)

< 55-U)> 1 0

30-57

1-82—

—

—

53-91

40-10040-69

44-96

21-60

27-87

0-900-90

0-80

19-58

0-90

Prevalence

%

050

100

90

100100

52

13

100

8948

98

70

84

1018

28

17

5 4 9

Kaufmann-Wolf"

I'uss''

Beerman and Stokes''^

iireckenridge''

Riechers and Kopf'"*

Nom''

1925

1933

1934

1953

1969

1970

Norn

Nutting and Green'^

Roth'

Ayleswnrtb and Vance"

Norn"'

Andrews'*

Sengbusch and Hauswirth'*^

1910 Nose, comedone expression

50 lanugo hairs, epilation

Comedone-extractor, 3 successive extractions

Comedones and superficial scrapings

1 slide/biopsy. I biopsy/case

16 biopsies/case

50-1 50 lashes/case, epilation8 lashes/ca.se. epilation

280 hairs, epiiation; or expression of materialfrom sebaceous glands

Biopsy

Eyelid biopsy, multiple step sections

Biopsy

Biopsy exhibiting follicles

Adhesive tape impression, nose and eyelids

1 single sebum extraction from nasolabial folds

1 single sebum extraction from nasolabial folds

1971

1976

1979

1982

1982

1982

1986

' For references prior to 1910. see Beennan and Stokes'-^: a, alive, c. acadaver.

652 F.FORTON AND B.SEYS

Figure 2, StandardIH'CI sKin-Mjriiu'f mop.sy. A slide with, on the skinside, a drop of cyanoacrylic adhesive, and, on the other side, a blackcircle enclosing a standard area of 1 cm~, is applied to the skin.

Methods

Sampling method: the standardized skin-surface biopsy

Skin-surface biopsy is a non-invasive sampling methodby which it is possible to collect the superficial part ofthehorny layer and the complete follicle contents. It consistsof placing a drop of cyanoacrylic adhesive (Loctite®) on amicroscope siide. applying the adhesive-bearing surfaceofthe slide to the skin, and removing it gently after it hasbeen allowed to dry (about 1 min).^'*'^'' Initially, a blackpreprinted circle. 11-5 mm in diameter (Decadry® No.223. Alphac Products), is placed on the slide, to facilitateexamination of a standard surface area of 1-03 cm^(Fig. 2). After removal from the skin, each sample isclarified with 2-3 drops of immersion oil. and thencovered with a cover slip.

The samples were studied microscopically at standardmagnifications ( X 40. x 100. x 400). and each speci-men was examined at least three times. The examinationwas performed as soon as possible after sampling, inorder to aid detection of the parasites, because theirmovements decrease with time, and they graduallydisintegrate. In the present study, a maximum of 4 helapsed between the time of sampling and examinationof specimens.

Only D. folliculorum clearly identified on the basis ofanatomical characteristics were counted.'' A detailedrecord of the number and distribution of the mites waskept on a chart in the patient's notes. The cheek was thesite used for skin surface biopsy in controls, and inpatients with rosacea. and HTV infection. In cases ofrhinophyma. the biopsy was on the nose, and in patientswith inflammatory papules on the face, samples were

taken from directly over the lesions. The clinical andmicroscopic examinations were performed by the sameinvestigator.

Patients

All patients in the study gave informed consent to skin-surface biopsy. They were recruited from in-patients andout-patients at the Saint Pierre and Brugmann Univer-sity Hospitals in Brussels (Belgium), from the staff ofthese hospitals, and from close relatives of the authors,between March 1987 and November 1988. Elevenpatients were excluded from the study at the time ofanalysis: four with ETR. five with PPR. one with IIP. andone HIV-seropositive patient. The reasons for exclusionwere as follows: the interval between sampling andmicroscopic examination exceeded 4 h or was notknown; microscopic examination had not been per-formed: one HIV-seropositive patient was excludedbecause his staging was not known.

Five groups of patients were analysed.

1 Forty-five controls with normal facial skin, withouttelangiectases (mean age 41 years: SEM 2- 3: range 2 1 -79). Thirty-four (75%) were women.2 Forty-nine patients with rosacea: 1 3 ETR. 3 SR. and33 PPR (mean age 49 years: SEM 1-8; range 22-78), Inthe subgroups, the mean age was 49, 58. and 48,respectively. Thirty-two patients (65%) were women.The proportion of women in the various subgroups wasalmost identical (77% HTR: 67% SR; 61% PPR). Thecontrols and the rosacea patients were matched for sex,but not for age (P<0 01).

In the 33 PPR patients, the nature and duration ofprevious therapy were noted. Eight patients had notbeen treated, or their previous therapy had been discon-tinued at least 2 months before the first visit: five werereceiving cosmetic treatments (moisturizing creams);seven were being treated with topical steroids: nine werereceiving other treatments, including oral tetracyclines,antibiotic creams, and topical epinephrine hydrochlor-ide: in four the type of treatment was not known.3 Four female patients sutfering from IIP (mean age 52years; SEM 6-4; range 35-65). The papules werelocalized periorally (one patient), on the cheek (onepatient), the chin (two patients), and the nose (onepatient).4 Three male patients with rhinophyma who were aged65. 71. and 77.5 Three factors led us to analyse the density of D.folliculorum in the patients with HIV infection: (a) the

DEMODEX FOLLICULORVM IN ROSACEA 653

Table 2. Demodex density tn the groupsstudied

Studied groups

Controls

Rosacea (all)Erythematotel angiectati cSquamousPapulopustular

Rhinophyma

Isolated inflammatory papules

HIV seropositiveStage 1Stage IiStage inStage IV

H

45

49133

33

3

4

214

14

Demodex density {Demodex/cm^)

Median

0

40

137

0

0

0000

03-01

1

181

2720

—

17

1. 1 1 •"

Mean ± SEM

O-7±O-25

1O8±2-O251 ±4-26

13 3±7 8012-8±2-36

0

6-3±4-05

000

1 8±O-81

Tests

—.

P<0-001NSNS

P<0-001

—

NS

NS

n. number of subjects; Q3-Q1, interquartile interval; tests, comparison with controls by robustrank-order test: NS. not signiScant; —. no computable value, i

development of opportunistic infections in immuno-depressed patients, including AIDS patients: (b) tworeports of an increase in numbers of D. foUiculorumduring corticosteroid therapyr""^ (c) the fortuitousfinding by a colleague of a large number oi D. follicttlorumin a sample of pityriasis versicolor scales taken from thethorax of an AIDS patient (this patient did not participatein the study).

Most ofthe 21 HIV-positive patients were male [n = 14(67%)]. which differentiates them from controls(P<n()05}. Their disease stage was determined duringthe clinical part ofthe study according to the nomencla-ture used at the time; two were asymptomatic carriers(stage I), one was in stage 11 (lymphadenopathic syn-drome), four in stage III [AIDS-related complex (ARC)],and 14 in stage IV (full-blown AIDS). The mean age ofthese patients was 37 (SEM. 2-4; range. 22-60).

Statistical analysis

Comparability of control and study groups for sex andage was assessed by means of the chi-squared test andStudent's f-test, respectively.

Because D. foUiculorum densities are counts, these arelikely to follow a priori a Poisson distribution, and thevariance will alter with the mean. This was actuallyobserved in control and study groups (Table 2). In such asituation, the usual parametric or non-parametric tech-niques which are sensitive to the equality of variances ofthe underlying distribution are not useful. Two methodswere used to overcome this difficulty, known as the

Behrens-Fischer problem.'" (i) The dependent variable(D. foUiculorum density) was applied a square-roottransformation in order to obtairi an equalization ofvariances. An ANOVA technique was then performed onthese data." This method was used for testing therelationship between D. foUiculorum density and pre-vious therapy in the PPR group, (ii) Because intragroupvariances remain too unequal, despite this transforma-tion, to test the main hypothesis of this study (Table 2)and relationship to age (Table 3), two non-parametrictechniques were used which require no assumption onthe underlying distribution of the data: the robust rank-order test for comparison of D. foUiculorum density

Table 3, Association between Demodex density and age tn controls androsacea groups

Age-groups(years)

20-2930-3940-4950-5960-6970-79

fs valueTest

n

1510

8705

Controls

Demodex densityMean ± SEM

l±0-70 1 ±0-11 3±0 5O-4±O-3

—0-8±O8

0028NS

n

46

1713

72

Rosacea

Demodex densityMean ± SEM

15-5±8-95-3±3-317±4-6

7-3±2 85-6±l-85-5±2-5

-0-066NS

fl, number of subjects: Demodex density. Demodex/cm^: r,. Spearmanrank-order correlation coefficient; —. no computable value.

654 F.FORTON AND B.SEYS

Figure 3. PPR in a 31'year-oid wunian in whom skin-suriacc biopsyshowed six Demodex/cm^.

between each ofthe studied groups and controls, and theSpearman rank-order correlation coefficient (r^) foranalysing the association of age and D. follicidorumdensity both in control and rosacea groups (Table 3).'"

Tests were performed at 0-05 significance level.Rejection region was two-tailed, except for analysis ofage (Table 3) in which it was one-tailed, in accordancewith the hypothesis of an increased Demodex densitywith age.

Mean values are given with the standard error ofthemean (SEM). When consistent, the median and theinterquartile interval (Q3-Q1) are given.

Results

The density of D. folliculorum showed no significantrelationship with age, either in the control group or inthe rosacea group (Table 3),

Table 4. Value ofthe criterion 'density of Demodex foUiciilorun) > 5/cm'to confirm a clinical diagnosis of papulopustLilar rosacea (PPR)

I!)ensity ofD. folliculoriim/cm' PPR Controls Total

>5<5

Total

1815

33

14445

1959

Diagnostic value: specificity 44/45 =0-98;sensitivity 18 /33=0 55:positive predictive value 18/19 = 0-95:negative predictive value 44/59 = 0 7 5 .

The density of D. foUivulorum in the group of rosaceapatients (mean 10-8; median 4) was significantly higherthan in controls (mean 0-7: median 0) [P<0-001 |(Table 2).

Demodex folliculorum density in the PPR patients(mean 12-8; median 7) was significantly higher thanin controls (mean 0-7; median 0) [P<0-0011 (Table 2:Figs 3 and 4).

In the ETR, SR. IIP and stage IV AIDS groups, thedensity was higher than in controls, although none ofthese differences was significant (Table 2}. In IIPpatients, the observed densities were 1 7, 8. 0 and 0. Thedensity was nil among rhinophyma and stage I, II, andIII AIDS patients.

When compared with controls, a density of D. follictt-loruni > 5 was very PPR-specific. This criterion had a98% specificity, a 95% positive predictive value, a55% sensitivity, and a 75% negative predictive value(Table 4).

The scattering of D. folliculorum densities variedmarkedly according to the group studied (Table 2). Theywere not very scattered in the controls and ETR groups,where 50% ofthe values around the median (interquar-tile interval) spread on an interval of only 1 Demodex/cm^. The values were more scattered in the PPR group,where the interquartile interval was 20. and in the SRgroup, where the interval was 27 (however, the smallnumber of findings in the latter group did not allow anyconclusion to be drawn). In the other groups. D,folliculorum densities showed an intermediate scatteringpattern.

In the PPK group, there was no significant relation-ship between previous therapy and mean D.follicttloruwdensity, although the values varied from 6-9/cm- incases treated with topical corticosteroids to 19-6/cm-in those receiving no treatment. The values wereintermediate in cases receiving cosmetic treatment(16-4± 7-3/cm^) or other treatments ( l l -4±3-8/cm^).

Discussion

Demodex (Greek: demos = iaV. dex —woodworm) was firstdiscovered by Henle and Berger in 1841. and describedin detail by Simon in 1842. Two different species can befound in man: Demodex folliculorum. a long form whichlives in the pilosebaceous duct, and Demodex brevis. ashort form which inhabits the sebaceous and meibomianglands.'

The real prevalence of the mite is probably 100%.based on evidence from the most fastidious studies which

DEMODEX FOLLICULORVM IN ROSACEA 655

Tigurt- 4. Distribution of Dcmodrx density incontrol and PPR groups. • controls (H = 4 5 )

35 H

30

25

20

15

10

5

0

Upper normal limit of Demodex density

20 30Demodex density/cm^

40 50

have employed many samplings per individual(Table 1)."'" '"* However, we found only 15 Demodexcarriers in 45 controls (i.e. a prevalence of 3 3-3%). butthis was probably because we sampled only a small areaon each subject's face. Standardized skin-surface biopsyis not a method designed to study the mite prevalence inthe population, but to estimate Demodex density—or.more precisely, Demodex folUctdorum density—in eachsubject. The method collects the superficial part of thehorny layer and the whole follicle contents, and there-lore detects the few mites present on the skin surface andthe more numerous mites in the pilosebaceous duct.' *• *-i.e. almost exclusively D. folliculorum.^'^^ Demodex brevis.which is principally found in the sebaceous glands,' andthe occasional D. brevis and/or D. folliculorum whichhave penetrated into the dermis are not detected by thismethod.

Most published observations have shown that theprevalence oi Demodex increases with age (Table 1).' ''^In a more recent study, Sengbusch and Hauswirth founda pronounced increase in D. brevis prevalence with age,whereas the prevalence of D. folliculorum tended toremain more constant.'' In our study, we did notobserve an increased D. folUctdorum density with age,either in the control group or in the rosacea group (Table3). This stability of mite density with age could partlyexplain the stability of its prevalence.

The role of D. folliculorum in rosacea remains contro-versial. Marks and Harcourt-Webster in 1969,^^ andllamelet and Perrouiaz in 1988,'"* studied skin biopsiesfrom patients with rosacea, and found mites in only asmall proportion of cases (14/74 PPR" and 2/75rosaeea***). They suggested that these results arguedagainst a pathogenic role for Demodex in rosacea. The

discrepancy between these studies and our own observa-tions can be explained by the different methodologyemployed. Demodex is not as easily detected in histologi-cal preparations as in skin surface biopsy. In skin-surfacebiopsy specimens the mites are intact, alive, they move,and are easy to detect, whereas, according to Boge-Rasmussen, 'in histologic preparations, the mite shrinksrapidly and transforms into a translucent "ghost" sac ofchitin which it is impossible to identify in the prepara-tion'.-** However, with some experience, and a goodknowledge of its anatomy,'* Demodex is not very difficultto detect, and this is reflected by demonstration of themite in 10% of skin-biopsy specimens.** Another possibleexplanation would be that, aithough standard skinbiopsy allows the detection of Demodex. it is. however,limited by the small volume of material assessed. Astandard biopsy from a patient with rosacea is usuallysmall (< 1 cm') because it is taken from the face, andonly a limited number of sections are examined (forexample, four sections/case'*). In addition, the sectionsdo not necessarily contain a follicle, a necessary pre-requisite for detection of Demodex.^ Finally, because thethickness of the section is usually 5 /im. and the meaninterval between two sections is 2 5-50 /jm, D. follicu-lorum. the opisthosome of which has a width of 31-44jum.' and runs parallel to the axis ofthe hair, may bemissed because it is present between two sections.Performing serial sections at narrow intervals may. ofcourse, reduce the likelihood of this. In the studiesmentioned above,^^'^* serial sections were carried out inonly 24 of 108 biopsies (at 20-;im intervals)." and inseven of 75 cases.'* In contrast, the sampling obtainedwith skin-surface biopsy corresponds exactly with theusual location oiD. folliculorum. and makes it possible to

656 F.FORTON AND B.SEYS

analyse easily a large skin sample: a surface biopsy of1 cm^ allows detection of all, or nearly all, D. foUiculorumpresent in that area, because such a biopsy is composedof both superficial and deep tissue (hair infundibula).This does not, however, apply to D. brevis. and the veryfew Demodex which have penetrated into the dermis.which can only be detected by standard skin biopsy. Inour opinion, such a difference in the performance ofthemethods used to detect D. foUiculorum may largelyexplain the discrepancy between the studies mentionedabove and our own study.

In the studies of Marks and Harcourt-Webster,^^ andRamelet and Perroulaz,''* the mite had induced little orno local infiammation around the follicles, and had notpenetrated the dermis. hut it should be noted that theseobservations were made on only 14 and two mite-containing biopsies, respectively. However, perifollicularinflammation, and penetration of the dermis by mites,are phenomena which have been extensively docu-mented. Perifollicular inflammation occurring aroundinfested follicles has been noted by several authors, basedon systematic examination of skin biopsies (not only ofrosacea).''** '̂ '̂ ^ In 1979, Roth examined 100 palpebralskin biopsies, and found an infiltrate around 42% ofinfested follicles.''In 1982 Aylesworth and Vance exam-ined 1124 biopsies. 117 of which were infested hyDemodex. and found a lymphohistiocytic infiltrate around29% ofthe infested follicles." In 1986. we examined 69biopsies (selected only on the basis of the presence ofDemodex). We found 986 Demodex and 710 follicles, andestablished a statistically significant relationshipbetween the presence of Demodex and perifollicularlymphohistiocytic inflammation.*^ Other isolated histo-logical findings are in agreement with this, Demodexfeeds on epidermal cells,''' and thus creates breaches inthe superficial layers ofthe follicular epithelium, towardswhich the infiltrate makes its way. sometimes producinga true Demodex folficulitis.'^ The fact that Demodex hasbeen observed in biopsies from a variety of skin diseasesdoes not conflict with its involvement in rosacea.

The intradermal presence of Demodex has been docu-mented by histopathological studies of granulomatousand/or lupoid rosacea. In very rare instances. Demodexmay make its way through the follicular wall'^ andinduce in the dermis a granulomatous reaction withgiant cefls which will phagocytose the parasites.^""^'Grosshans found Demodex in the granulomas in 10 of 53patients with lupoid rosacea.'' In contrast, Ecker andWinkelmann could find it in only I of 30 cases; however,they did not specify whether they performed serialsections in their study.^'* The lymphocytes from the

Demodex granulomas and the perifollicular infiltrate ofrosacea are ofthe same type, i.e. T-helper lymphocytes. *''Moreover. anti-Demodex caprae antibodies can be foundin 22% of patients with PPR and granulomatousrosacea. These antibodies resemble those produced byrabbits sensitized by D, caprae antigens.''

In 1965, Robinson studied the mite population in3-mm punch biopsy specimens from the nasal crease,and did not observe any significant change after 28 daysof therapy with a 3% sulphur preparation applied to onehalf of the face, although there was clinical evidence of abeneficial effect from the treatment.'** As Robinsonstated, it was not possible to conclude from the study thattreatment with 3% sulphur did not affect the mite counts'as the method used may not be sufficiently sensitive todetect the difference", because the area sampled waslimited to the diameter of the punch (3 mm).

Contrary to the opinion of Marks and Harcourt-Webster.*' Ramelet and Perroulaz.*^ and Robinson.'^other authors are in favour of a ro\e for D. foUiculorum inrosacea. Large numbers of mites were found in pustulesin 50 patients with pustular rosacea/" in the drypityriasis-like erythematosquamous rosacea, and in adry type of PPR (10-15 DemtJdcv/'follicular scale').̂ ^^ in16 of 18 patients with rosacea.^"^ in 9 of 10 patients withrosacea (skin surface biopsies).-'^ in a pustular foUicuIltisof the face,̂ ^ and in a unilateral rosacea (1 5 Demodex/low-power field, in samples ohtained by scraping),"*"Several studies^ have demonstrated a decrease in thenumber of Demodex following local treatment (withiDanish ointment,'** with crotamiton.^^'^" or with hexa-chlorocyclohexane'**), and this decrease was associatedwith a dramatic clinical improvement.

In 1925, Kaufmann-Wolf studied 50 cases of pustularrosacea and found the mite in samples from the pustulesin all cases, but Demodex were not more numerous onhairs taken from the lateral area of the face.'" Thisfinding might be interpreted as indicating that, inpustular rosacea. mite proliferation is only superficial (atthe level of the pustule). However, our study demon-strates an increase of Demodex in the pilosebaceousfollicles, not merely in superficial pustules. This isprobably due to the fact that the hairs observed in ourskin surface biopsies were taken from areas of PPR, incontrast with those studied by Kaufmann-Woif.

In 1981 Varotticld.*^ found D./omcu/orum in afl theirpatients with rosacea (n = 25). whereas the mite wasabsent in all their healthy controls (n = 2 5). They used atape-stripping technique which consisted of scraping theskin several times, and collecting the scrapings by meansof double-sided adhesive tape stuck on to a microscope

DEMODEX EOLLICUWRUM IN ROSACEA 657

slide. Following treatment with oral metronidazole(500 mg/day for 20 days, followed by 250 mg/day for20 days), no D, folliculorum could be found on thepatients' cheeks. The authors suggested that theirmethod could detect the mite only when it was present inlarge numbers. They were in favour of a role for D.folliculorum in the pathogenesis of rosacea.

Our study confirms in a more accurate way that thedensity of D. foUiculorum in the rosacea group (10-8/cm') is greater than in the control group (0- 7/cm-). Thedifference is principally determined by the mite density inthe PPR group (12-8/cm^). The density among the KTRpatients ( 51 /cm^) is not statistically significantly differ-ent from that observed in controls. Sueh a result maypossibly be due to lack of power of the test [resulting froma small number of subjects («= 1 3)]. However, we areinclined to believe that Demodex density among the ETRpatients is normal, or intermediate between the controlsand the PPR patients, in view of the succession of theclinical stages of rosacea. Further studies involvinglarger numbers of subjects should clarify this point.

The SR patients apparently had larger numbers ofDemodex (13-3/cm^) than controls (0-7/cm-). butbecause ofthe limited number of cases we were not ableto demonstrate a statistically significant difference.

In practice, it is of value to know the thresholdbetween a 'normal' and 'abnormal' density of Demodex.to be able to confirm the clinical diagnosis of PPR.Therefore, on presentation ofthe results of our study, wearbitrarily chose a threshold of 5 Demodex/cm^ to obtaina very specific test, with few false-positive results (2%),which therefore becomes less sensitive, i.e. with manyfalse-negative results (45%) [Table 4: Fig. 4]. This is whya density greater than 5 Demodex/cm' allows us toconfirm the diagnosis of PPR with a positive predictivevalue of 9 5%. On the other hand, a density lower than, orequal to 5 Demodex/cm^. does not allow us to draw anyconclusion, because there is a 25% likelihood that thedisease is PPR anyway (negative predictive value = 75%).Some PPR cases have less than 5 Demodex/cm-, but itwould be too much to hope for a density lower than, orequal to, 5 Demodex/cm' in all the controls, and above 5Demodex/cm^ in all the cases of papulopustular rosacea,as there is no such perfect test in biology. This is probablydue to the multifactorial nature of rosacea. The clinicalpicture of rosacea depends not only on Demodex density,but also, for example, on the infiammatory reactioninduced by the mite: it is possible that this reaction variesfrom individual to individual, each subject responding totheir own Demodex density threshold.

Finally, such a test compares PPR patients with

healthy controls, and before it can be used as adifferential diagnostic tool in PPR. the Demodex densitiesin other facial dermatoses clinically similar to PPRrequire study, particularly lupus erythematosus, inorder to verify whether they equate with those ofcontrols. Moreover, it would be useful for the proposedthreshold density to be validated by other studies onlarger patient samples.

From a pathogenetic standpoint, the higher Demodexdensity observed only in PPR suggests, aithough it doesnot prove, that Demodex plays a role in the formation ofthe papules and pustules of rosacea only at this stage ofthe disease (stage III), and not before. This would becompatible with the present concept ofthe pathogenesisof rosacea, in which the disease is considered as a'primarily' venous vascular functional disease of theface, possibly originating in poor blood flow control bythe central nervous system.*'^*' The vascular changesprobably create an environment which favours themultiplication oi Demodex and/or their penetration intothe dermis. and this would then induce macroscopicallyvisible inflammation in the form of papulopustules: suchan hypothesis was suggested by Spickett in 1962.**However, the multiplication of Demodex and/or theirpenetration into the dermis may occur independently ofpredisposing vascular factors, thus leading to the forma-tion of IIP. Indeed, two ofthe four patients with IIP had ahigh Demodex density (17 and S/cm'^). which suggeststhat the mite may induce an infiammatory responsewhich falls outside the clinical scope of rosacea, as hasalready been reported.'''•^'•^^•^*^^ In our group of IIPpatients, however, the mean Demodex density was notstatistically different from that of controls: this is prob-ably due partly to the small size of the IIP sample, andpartly to the fact that IIP may have many other causesthan Demodex.

Demodex folliculorum density does act appear to behigher in cases of rhinophyma (mean = O/em^). butthere were only three cases of rhinophyma in our study.Clinically, rhinophyma differs markedly from rosacea,and it can develop in the absence of papulopustules.Moreover, it principally affects male patients. It ishowever, considered as stage IV ofthe disease. Perhaps itis a different, although closely related, disease. It is alsopossible that rhinophyma might be more readily as-sociated with a proliferation of D. brevis rather than D.foUiculorum: the absence of D. brevis in our study wouldthen be directly related to the technique used.

We did not find any increase oi Demodex density in thegroup of patients with HIV infection compared withcontrols.

658 F.FORTON AND B.SEYS

The findings ofthe present study should be confirmedin a larger patient group, particularly with regard topatients with ETR. SR, and controls from an older age-group. A similar study should also be carried out onother facial dermatoses. Standardized skin surface biopsycould be used to monitor the evolution of Demodexdensity in rosacea during treatment, for example withtopical metronidazole or crotamiton (Eurax®), eachpatient serving as their own control, by comparison oftreated and untreated sides of the face.

Standardized skin surface biopsy may also be utilizedfor the study of other dermatological diseases in which ithas been suggested that Demodex may play a pathogenicrole, for example perioral dermatitis, '''•**̂ -̂ ^ and Grover"sdisease.'*''

This study supports the pathogenic role of D, follicu-lorum in PPR and suggests the use of a standardized skin-surface biopsy as a diagnostic tool for the disease. Adiagnosis would be considered positive when the densityoi Demodex is above 5/cm-.

Acknowledgments

We thank Professor G.Achten. Professor M.Ledoux,Professor J.M.Lachapelle, Professor N.Clumeck. Dr ].DeMaubeuge. Dr D.Tennstedt, Dr J.Andre. Dr M.Bernard,Dr l.J.Stene, Dr S.De Rijcke, and Dr H.Lenain, for theirhelp and advice during the course of this study.

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