Graphical abstract
Highlights
- •
A novel diagnosis for Demodex using only a biomicroscope and forceps is presented.
- •
Removing the collarette and drawing eyelash to the side reveals highest mite numbers.
- •
Videos featuring mite tails at the eyelash follicle in situ are presented.
- •
The technique is rapid, simple and painless and thus convenient to patients and practitioners.
- •
Method may be easily incorporated into standard clinical examination routine.
Abstract
Purpose
To compare existing and novel diagnostic techniques for confirming ocular Demodex infestation and to recommend the most reliable method for routine use by eye care practitioners, based on yield and clinical applicability.
Methods
Fifteen participants with a prior Demodex blepharitis diagnosis or featuring typical cylindrical dandruff (CD) collarettes, and seven healthy controls were enrolled. Demodex presence was assessed using five techniques, applied consecutively, on a minimum of two different eyelashes on each eyelid of every participant, for each test, in situ : 1. using fine-point forceps and 25-40x biomicroscopy magnification, by eyelash rotation as proposed by Mastrota (ROT); 2. by removing cylindrical dandruff and exposing the eyelash insertion point at the lid margin (CDR); and 3. by laterally tensioning the eyelash (LET) following CDR. The typical appearance of cigar-shaped mite tails protruding from each assessed eyelash follicle was observed, and mite tails counted and averaged per participant for each assessment technique. 4. Lash epilation, and mite presence evaluated using bright-field microscopy at 10-40x magnification (EPI). 5. Finally, eyelash follicles were imaged using in vivo confocal microscopy (IVCM) and the images visually inspected for mite presence.
Results
In the Demodex group, the highest numbers of mites/eyelash were identified by LET (3.8 ± 1.4), versus CDR (2.4 ± 1.6) and ROT (1.1 ± 1.2), alone (all p < 0.002). An average of 1.0 ± 0.8 mites/lash was identified by EPI. IVCM failed to offer unequivocal evidence of Demodex presence even in confimed cases.
Conclusions
A novel technique for the clinical diagnosis and grading of Demodex in situ is described. By removing cylindrical dandruff and applying static, lateral tension to the eyelash without epilation, large numbers of mites are visible at the exposed eyelash follicle. The proposed method is convenient and clinically applicable, requiring only forceps and 25-40x biomicroscope magnification, and allowing rapid, efficient evaluation of large numbers of eyelashes.
1
Introduction
Demodex mites commonly reside in the sebaceous glands of the scalp, face, ears, and in the meibomian glands and eyelash follicles [ ]. Characteristic collarettes or cylindrical dandruff (CD) around the eyelash base are considered to be a sign ocular demodicosis [ ]. Demodex has a recognised association with anterior blepharitis and is believed to play a role in the perpetuation of ocular surface inflammation and dry eye disease [ ]. Recently, numerous treatment [ , ] and diagnostic [ , , ] options have emerged. While the topical application of tea tree oil ( Melaleuca alternifolia ) is the most commonly agreed management strategy [ ], a consensus on the optimal diagnostic technique for confirming mite presence, is lacking.
Traditionally, clinical diagnosis of Demodex infestation involves the epilation and microscopic evaluation of between 8 and 16 eyelashes per individual. Mites are visually identified by their characteristic morphology and motility [ ]. Despite subsequent refinements to the sampling and counting methods in an attempt to increase yield [ , ], it is acknowledged that many mites can remain within the orifice following eyelash removal [ , , ]. Mites embedded in the CD can be difficult to distinguish both in vivo and ex vivo , and addition of solvents to epilated lashes can cause mites to float away, decompose or perish, resulting in an underestimation of mite count. In the clinical setting, repeated eyelash removal ( e.g. for monitoring treatment efficacy) is uncomfortable and naturally undesirable to patients, and the time commitment and need for laboratory equipment (such as a 100-200x magnification light microscope, pipettes etc. ) limits widespread integration of this technique into standard clinical care.
Proposed diagnostic alternatives for easier assessment and better patient comfort include a technique by which an eyelash is rotated around its own axis using forceps [ ]. This motion reportedly “cores” Demodex from within the eyelash follicle, revealing mite tails at the insertion point of the lash. Using high biomicroscope magnification then allows the assessment of ocular Demodex infestation in situ . To date, adoption of the eyelash rotation technique in routine clinical applications appears not to be widespread, and no direct comparison between the various clinical diagnostic methods has been reported. Other diagnostic alternatives include in vivo confocal microscopy [ , , ] although this requires costly equipment unavailable to the majority of clinicians.
This study compares established and adapted techniques with the aim of optimising Demodex diagnosis in the clinical setting.
2
Methods
2.1
Participants
This study followed the tenets of the Declaration of Helsinki and was approved by the University of Auckland Human Participants Ethics Committee (UAHPEC 013430). Informed consent was obtained from all participants prior to study enrolment.
Individuals of at least 18 years of age with a previous ocular demodicosis diagnosis by eyelash epilation, and/or signs of anterior blepharitis featuring typical cylindrical dandruff (CD) collarettes around the base of the eyelashes, were recruited. Additional age-matched participants free of anterior blepharitis were included as controls.
Participants presenting with major ocular or systemic disease, or reporting use of topical or systemic medications known to affect the eye, were excluded from the study.
2.2
Clinical measures
All participants completed the Ocular Surface Disease Index (OSDI) [ ] and the 5-item Dry Eye Questionnaire (DEQ-5) [ ].
A non-invasive tear film and ocular surface assessment was conducted using the Keratograph 5 M (Oculus, Wetzlar, Germany) and included measurement of the lower lid tear meniscus height, non-invasive tear break-up time (average of 3 measurements of first breakup detection), tear lipid layer quality [ ] and bulbar and limbal hyperaemia.
Anterior segment biomicroscopy assessment was conducted and the following features graded on a scale from zero to three (0=”none”; 1=”mild”; 2=”moderate”; 3=”severe”, unless otherwise noted): cylindrical cuffing (typical collarette shape), collarette height (0=<1 mm, 1 = 1 mm, 2 = 2 mm, 3=>2 mm), collarette number (0 = 0, 1=<4, 2=<9, 3=>10), madarosis, trichiasis, lid margin erythema, thickening, surface telangiectasia, and irregularity. The number of meibomian glands yielding lipid secretion were assessed with the Meibomian Gland Evaluator (TearScience, Morrisville, NC) [ ] and the expressed meibum quality graded.
Corneal staining was evaluated using sodium fluorescein (HUB Pharmaceuticals, Rancho Cucamonga, CA) with the aid of blue light and a yellow barrier filter, while conjunctival staining was assessed following application of lissamine green (HUB Pharmaceuticals, Rancho Cucamonga, CA) and white light. Both upper and lower lid margins were everted and lid wiper epitheliopathy graded according to the Korb scale [ ]. The clinical application and evaluation of stains was performed in accordance with the procedures established in the TFOS DEWS II Diagnostic Methodology report [ ].
2.3
Demodex assessment
Every participant underwent a 5-step eyelash assessment which was performed in the same test order each time, for least impact on the subsequent tests. Both upper and lower eyelids of both eyes of each participant were evaluated for Demodex presence using existing and refined clinical techniques for eyelash manipulation, epilation and in vivo confocal microscopy (IVCM).
The in situ techniques (1–3 below) were conducted under direct white light illumination and 25-40x biomicroscope magnification. Inspection of the upper lid eyelashes took place during participant downgaze, with the eyelids closed, while the lower eyelids were inspected during upgaze. A pair of sterilized, fine-tipped metal forceps (Altomed A5908 Jewellers Forceps No. 5 100 mm, Altomed, Bolton, UK) handled from the temporal side were used to manipulate the eyelashes during biomicroscopic observation. Mite tails protruding from the eyelash follicle resembling a typical cigar shape [ ] were observed and counted for each procedure, ensuring only mite tails that could be confidently identified were counted. To enable secondary confirmation, procedures were digitally recorded as still images and/or videos with a biomicroscope-mounted camera (Topcon DC-4, Topcon, Japan or Canon T1i DSLR, Canon, Japan).
2.3.1
Rotation (ROT)
From each lid, two eyelashes featuring prominent cylindrical dandruff (CD) were identified and successively rotated for 30 s, as described by Mastrota [ ]. During the rotation, the insertion point of the eyelash into the lid margin was continuously inspected for the characteristic cigar-shaped appearance of mite tails. Visible tails were counted and averaged across the 8 lashes to provide a mean count per lash.
2.3.2
Cylindrical dandruff removal (CDR)
Two different eyelashes with CD were selected. Using the tip of the forceps, the collarette base was secured and removed with a sliding motion along the eyelash. Where collarettes were fused with the adjacent cornified epidermis of the lid margin, removal typically resulted in (or required) separation of the fused tissue from the lid margin. By exposing the point of insertion of the eyelash into the lid margin in this way, protruding Demodex tails were counted (Video 1).
2.3.3
Lateral eyelash traction (LET)
The same two CD-free eyelashes were then subjected to a modified eyelash traction technique. In contrast to the technique described above (ROT), involving rotation of the lash around its own axis, the grasped eyelash this time was drawn only laterally, slowly alternating extension in nasal and temporal directions, under gentle constant tension, for 30 s. Mite tails emerging from the follicle during this lateral extension were counted ( Fig. 1 , Video 2).
2.3.4
Eyelash epilation and light microscopy (EPI)
Next, according to previously described methods [ , ], two different eyelashes per eyelid (eight eyelashes in total) were epilated and transferred to a glass slide. A standard laboratory bright-field microscope (Bresser, Rhede, Germany) at 100x, 200x and 400x magnification was used to count mites based on the characteristic morphological appearance and motility ( Fig. 2 ). Average mite count per eyelash was reported.
