Association of spirochetal infection with Morgellons disease

Patient selection and dermatological samples

Representative non-biopsy dermatological specimens were collected from four randomly-selected patients who met the key clinical criterion for MD, namely that filaments visible with a hand-held microscope at 60X magnification must be present under unbroken skin or projecting from spontaneously appearing skin lesions. Patients 1 and 2 are Americans residing in Texas while patients 3 and 4 are Canadians residing in Alberta, Canada (Table 1). Written informed consent for submission of clinical samples and publication of clinical details and clinical images was obtained from each study subject. Patient anonymity and confidentiality were strictly maintained. The study was exempt from Institutional Review Board approval because all testing was performed as part of routine clinical care, and patient anonymity and confidentiality were strictly maintained.

The detailed histopathological findings in these patients were reported previously¹⁷. All patients were seroreactive to Borrelia burgdorferi antigens (strains B31 and 297, IGeneX Laboratory, Palo Alto, CA) and negative on rapid plasma reagin (RPR) testing (RPR Card Test Kit, BD Diagnostic Systems, Sparks, MD). Patient 1 was on doxycycline therapy for Lyme disease at the time of the study, while patient 2 had previously been treated with doxycycline for Lyme disease but had been off treatment for several years at the time of the study. Patients 3 and 4 were not on antibiotic therapy at the time of the study. None of the study patients had evidence of a delusional disorder, as determined by standard neuropsychiatric testing using the Rorschach, Minnesota Multiphasic Personality Inventory (MMPI), Millon Clinical Multiaxial Inventory (MCMI) and Wechsler Adult Intelligence Scale (WAIS) formats.

The late-stage BDD biopsies used for comparison were kindly provided by Dr. Dorte Döpfer, Faculty of Veterinary Medicine, University of Wisconsin, Madison, WI. Biopsies were taken as part of an intervention study conducted by the University of Wisconsin¹⁵. The diagnostic criterion for late-stage BDD was the presence of pronounced keratin projections from ulcerative lesions that were at least two centimeters in diameter and located above the heel bulb of the hind feet of cattle. Biopsy samples were stored and shipped in a fixative of 1.5% glutaraldehyde/1.0% formaldehyde in Sorensen’s Buffer at pH 7.35 (Tousimis Research Corporation, Rockville, MD). Duplicate samples were used for each of the light and electron microscopic studies described below.

Light microscopy

The gross morphology of dermatological specimens collected from Patients 1–4 was observed at 8X, 40X, and 100X magnification with illumination superior to the specimen, thus verifying the presence of filaments within and protruding from epithelial tissue. BDD biopsy material was examined at 8X to observe gross morphological characteristics.

Morgellons samples were formalin-fixed and embedded in paraffin, sectioned, and stained with Warthin-Starry and/or Dieterle silver nitrate-based staining for the light microscopic detection of spirochetes under oil immersion at 1000X magnification. Warthin-Starry staining and Dieterle staining were performed by Interscope Pathology Medical Group, Canoga Park, CA, and McClain Laboratories LLC, Smithtown, NY, respectively.

BDD biopsies were formalin-fixed and embedded in paraffin, sectioned, and stained for the detection of spirochetes by Warthin-Faulkner silver nitrate-based staining at Prairie Diagnostics, University of Saskatchewan, Saskatoon, Saskatchewan.

Formalin-fixed paraffin-embedded MD sections were processed for immunofluorescent anti-Borrelia staining and imaging as previously described²⁰ at the University of New Haven, West Haven, CT, by the following protocol: fixed specimens were pre-incubated with 10% normal goat serum (Thermo Fisher Scientific, Waltham, MA) in PBS containing 0.5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) for 30 minutes to block non-specific binding of the secondary antibody. The slides were washed with PBS containing 0.5% BSA and then incubated for 1 hour with fluorescein isothiocyanate (FITC)-labelled Borrelia-specific polyclonal antibody (Thermo Fisher Scientific, #73005) at a 1:50 dilution in PBS containing 1% BSA pH 7.4. The slides were washed and then counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) for 10 minutes. In negative control samples, anti-specifically targeted antibody was replaced with normal rabbit IgG (Vector Laboratories, Burlingame, CA, #I-1000). Mounted slides were imaged using fluorescent microscopy.

Electron microscopy

Morgellons and BDD samples were fixed in buffered 2.5% glutaraldehyde. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed by the Electron Microscopy Facility, Department of Materials Science and Engineering, Clemson University, Anderson, SC, according to the protocols below:

SEM. Glutaraldehyde-fixed samples for SEM were washed in buffer and dehydrated in a graded series of ethanol concentrations. Samples were then immersed in hexamethyldisilazane (Electron Microscopy Sciences, Hatfield, PA) for 5–15 minutes and air dried at room temperature. Dried samples were mounted on A-1 mounts. Samples were not coated but placed into a Hitachi TM3000 microscope and imaged in the variable pressure mode.

TEM. Glutaraldehyde-fixed samples were washed in buffer, followed by dehydration in a graded series of ethanol concentrations. Samples were then immersed in a 50:50 mixture of LR White™ embedding resin and 100% ethanol for 30 minutes, followed by pure LR White™ resin until samples settled on the bottom of the vial. The resin-immersed samples were then placed into pure resin in beam capsules and put into a 60°C oven overnight for polymerization. Sections were cut on an Ultracut E microtome to produce sections 60–90 nm thick, placed onto copper grids and stained in uranyl acetate for 20 minutes. Images were taken on a Hitachi 7600 microscope.

PCR

Morgellons calluses from patients 1–4 were forwarded to Australian Biologics (Sydney, Australia) for B. burgdorferi detection by PCR using the Eco™ Real-Time PCR system with software version 3.0.16.0. DNA was extracted from the tissue samples using the QIAamp DNA Mini Kit (QIAGEN). The four samples were analyzed in duplicate with positive and negative controls using primers AB-B1 for the Borrelia 16S rRNA gene target, as previously described²¹. The thermal profile for all analyses involved incubation for 2 mins at 50°C, polymerase activation for 10 mins at 95°C then PCR cycling for 40 cycles of 10 secs at 95°C dropping to 60°C sustained for 45 secs.

The magnitude of the PCR signal generated (∆R) for each sample was interpreted as positive or negative compared to positive and negative controls.

Borrelia spp. culture

Borrelial culture was performed as described previously²². B. burgdorferi was cultured in Barbour–Stoner–Kelly H (BSK-H) complete medium, with 6% rabbit serum (Sigma Aldrich, #B8291) and the following antibiotics: phosphomycin (0.02 mg/l), rifampicin (0.05 mg/l), and amphotericin B (2.5 µg/l) (Sigma-Aldrich) and incubated at 32°C with 5% CO₂. Cultured spirochetes were observed by dark-field microscopy and/or heat-fixed and stained with crystal violet (Dalynn Biologicals, Calgary, AB) under oil immersion at 1000X. For the immunofluorescence studies, cultured spirochetes (1×10⁷ individual spirochete cells) were centrifuged at 8,000×g for 10 minutes at room temperature, washed once with PBS pH 7.4, and then centrifuged again at 8,000×g for 10 minutes at room temperature. The pellet was resuspended in 100 µl of PBS pH 7.4, and then spread on microscope slides (SuperFrost+, Thermo Fisher Scientific). Spirochetes were fixed by incubating the slides in cold acetone for 10 minutes at -20ºC. Slides were then washed twice with PBS pH 7.4 at room temperature, and immunofluorescent staining with polyclonal anti-Borrelia antibodies was performed as described above.

Gross microscopic observations

Calluses from the four MD patients demonstrated white, red and blue filaments, alone or in any color combination, each 10 to 40 µm in diameter, embedded in or projecting from epithelial tissue (Figure 1A). BDD biopsies demonstrated pronounced, unusual keratin filament production typical of late-stage proliferative infection (Figure 1B).

Figure 1.

A) Morgellons disease filaments embedded in and projecting from epithelial tissue, 100X magnification. B) Proliferative bovine digital dermatitis (BDD) keratin filaments, 8x magnification.

A summary of the following histological, culture, electron microscopic and PCR results is shown in Table 2.

Light microscopy

Silver nitrate-based staining. Staining of dermatological tissue from patients 1–4 revealed visible black-stained spirochetes among keratinocytes and inflammatory cells (Figure 2A). These spiral or curved structures ranged from 0.1 µm to 0.5 µm in diameter and up to 30 µm long, and they were present mostly in the interior areas of the sections and not along the peripheral edge.

Figure 2.

A) Black-stained spirochetes in representative tissue sample from patient 2. Dieterle stain, 1000X oil immersion. B) Black-stained spirochetes in bovine digital dermatitis (BDD) tissue sample, Warthin-Faulkner stain, 1000X oil immersion. C) Distinct patches of anti-Borrelia fluorescence in histological section of callus from patient 1, 400X magnification. D) Distinct patches of anti-Borrelia fluorescence in histological section of callus from patient 2, 400X magnification.

Staining of BDD dermatological tissue revealed visible black-stained spirochetes among enlarged keratinocytes (Figure 2B). Spirochetes were approximately 0.1 µm to 0.2 µm in diameter and approximately 10 µm to 15 µm in length, and they varied in morphology from visibly spiral-shaped to straight or wavy in appearence.

Immunofluorescent anti-Borrelia staining. Immunofluorescent anti-Borrelia staining of fixed Treponema denticola spirochetes was performed as a negative control and immunofluorescence was not observed for these specimens (data not shown). Histological sections of dermatological material from patients 1–4 all demonstrated distinct patches of immunofluorescence at a magnification of 400X (Figures 2C and 2D). Patches of fluorescence appeared to occur most often in areas of sections corresponding to fibroblasts. Cultured spirochetes from patient 3 also demonstrated positive immunofluorescent staining with anti-Borrelia antibodies (see below and Figure 5C).

SEM and TEM

SEM revealed high-resolution surface imaging of a spirochete lying beneath a layer of dermatological tissue of a Morgellons callus and images consistent with morphological forms of Borrelia spp. (Figures 3A and 3B). TEM imaging of both Morgellons calluses and BDD biopsies revealed spirochetes in cross-section (Figures 3C and 3D).

Figure 3.

A) SEM from patient 2 tissue sample showing spirochete images that are consistent with morphological forms of Borrelia (arrows). B) SEM from patient 2 tissue sample showing single spirochete, upper middle right (long arrow) and morphological forms consistent with Borrelia, center (short arrow). C) TEM from patient 1 tissue sample showing sectioned spirochetes. D) TEM from BDD tissue sample showing sectioned spirochetes.

PCR

Real-time PCR analysis was positive for Borrelial DNA in tissue samples from MD patients 1–3 and negative in the sample from Patient 4. Samples from Patients 2 and 3 were clear positives and the sample from Patient 1 was a weak positive. The PCR profiles are shown in Figure 4.

Figure 4. Results of polymerase chain reaction (PCR) amplification in tissue samples from patients 1–4.

See text for PCR protocol. Samples from patients 2 and 3 were strongly positive, while the sample from patient 1 was weakly positive. The sample from patient 4 was negative.

Borrelia spp. culture

Motile spirochetes ranging from approximately 0.1 µm to 0.5 µm in diameter and up to 30 µm long were visible in cultures inoculated with dermatological tissue from both patients 3 and 4 (Figures 5A and 5B). Cultured spirochetes from patient 3 were identified as Borrelia by immunofluorescent staining with FITC-labelled polyclonal antibodies at 1000X magnification (Figure 5C). The culture obtained from the inoculum from patient 4 was lost due to contamination and generic identification was not obtained.

Figure 5.

A) Cultured spirochetes from patient 3 tissue samples, 1000X darkfield microscopy, oil immersion. B) Cultured spirochetes in clumps from patient 4 tissue sample, heat fixed and stained with crystal violet, 1000X oil immersion. C) Borrelial spirochetes demonstrating fluorescence from tissue culture of patient 3, 1000X oil immersion, enlarged and cropped.