Exploring the Association Between Morgellons Disease and Lyme Disease

Identification of Borrelia Burgdorferi in Morgellons Disease Patients

Marianne J Middelveen; Cheryl Bandoski; Jennie Burke; Eva Sapi; Katherine R Filush; Yean Wang; Agustin Franco; Peter J Mayne; Raphael B Stricker

Disclosures

BMC Dermatol. 2015;15(1) 

In This Article

Methods

Patient Selection

All patients included in this study met the key diagnostic criterion documented by a healthcare provider: the presence of fibers that were visible underneath unbroken skin or that were embedded in or projecting from skin. Patients were selected from across Canada and the USA, and they were included in the chronological order in which they volunteered. No patients were excluded from study participation provided that they had sample material that was suitable for study and provided that they met the diagnostic criterion. Written informed consent for participation in the study was obtained from all participants. Approval for sample collection was obtained from the Western Institutional Review Board, Olympia, WA. Further approval for sample testing was obtained from the Institutional Review Board of the University of New Haven, West Haven, CT.

A diagnosis of Lyme disease or positive Lyme serologic testing prior to study participation was not a requirement. Some patients had a prior Lyme diagnosis or serologic testing for Lyme disease while others did not, as shown in Table 1. Those who were not tested with Lyme serology prior to the study were encouraged to be tested, but did so after samples for the study had been collected. Some patients declined to have Lyme serologic testing performed. Some of the subjects had received antibiotic therapy for Lyme disease but were not receiving treatment during the time of sample collection. Two subjects, 1 and 12, were currently taking antibiotics during the time of sample collection.

A total of 25 patients were included in the study. Patients 1–5 were previously presented as case studies.[6,7,9] Clinical patient data is shown in Table 1. All patient samples were deidentified and coded, and all laboratory testing was performed in a blinded fashion.

Cultures

Borrelia culture medium was prepared using Barbour–Stoner–Kelly H (BSK-H) complete medium with 6% rabbit serum (Sigma Aldrich, #B8291) and the following antibiotics: phosphomycin (0.02 mg/ml) (Sigma Aldrich), rifampicin (0.05 mg/ml) (Sigma Aldrich), and amphotericin B (2.5 μg/ml) (Sigma-Aldrich), as described previously.[11] Inocula for blood cultures were prepared as follows: Ten milliliters of whole blood were collected by venipuncture and left at room temperature for 10 to 15 minutes to allow clotting, followed by low speed centrifugation to separate red blood cells. Serum and some blood cells just below the serum layer were used as inocula. The serum/blood cell preparation was inoculated into the BSK medium.

For other cultures, whole scabs removed from the patient, skin scrapings from small lesions removed by a scalpel blade, or vaginal samples collected by swabbing inside the vagina with a sterile cotton-tipped swab were inoculated into the BSK medium. Cultures were incubated in an Oxoid anaerobic jar (Thermo Scientific) containing an AnaeroGen sachet (Thermo Scientific) to provide an anaerobic environment at 32°C. Culture fluid samples were examined by bright-field and/or dark-field microscopy for visible motile spirochetes weekly for up to 4 weeks. Cultures were processed for imaging and PCR by centrifuging the fluid at 15,000 g for 20 minutes to concentrate spirochetes, retaining the pellet and discarding the supernatant.

Dieterle and Anti-Bb Immunostaining

Dermatological specimens and/or culture pellets from patients were processed for specialized staining at either McClain Laboratories LLC, Smithtown, NY; Interscope Laboratories, Interscope Pathology Medical Group, Canoga Park, CA; or the University of New Haven, West Haven, CT. Dieterle silver-nitrate staining was performed at either Interscope Laboratories or McClain Laboratories. Anti-Bb immunostaining was performed at McClain Laboratories or the University of New Haven.

For those samples submitted to McClain Laboratories for anti-Bb immunostaining, the following protocol was used: formalin-fixed, paraffin-embedded dermatological tissue and culture pellets were sectioned and immunostained using an unconjugated rabbit anti-Bb polyclonal antibody (Abcam ab20950) and then incubated with an alkaline phosphatase probe (Biocare Medical #UP536L) followed by a chromogen substrate (Biocare Medical #FR805CHC) and counterstained with hematoxylin. Positive and negative controls of both Dieterle and anti-Bb immunostains were prepared for comparison purposes using liver sections from uninfected mice and mice experimentally inoculated with Bb, as previously described.[12] Staining was titrated to define optimal antibody dilutions. For comparison purposes, controls of culture pellets from mixed Gram-positive bacteria and mixed Gram-negative bacteria, and sections of normal human skin were also examined to determine possible cross-reactivity with commonly encountered dermatological microorganisms.

For those samples submitted to the University of New Haven, anti-Bb immunofluorescent staining was performed as follows: formalin-fixed paraffin-embedded MD sections were processed for staining and imaging as previously described.[13] Dermatological specimens were formalin-fixed, blocked in paraffin and sectioned by McClain Laboratories. Culture fluid was fixed with acetone at -20°C onto SuperFrost™ slides (Thermo Fisher Scientific, Waltham, MA). Fixed specimens, both dermatological sections and fixed culture fluid, were pre-incubated with 10% normal goat serum (Thermo Fisher Scientific) 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 then washed with PBS containing 0.5% BSA and then incubated for 1 hour with fluorescein isothiocyanate (FITC)-labeled Borrelia-specific polyclonal antibody (Thermo Fisher Scientific, #73005) at a 1:50 dilution in PBS containing 1% BSA pH 7.4 followed by washing and then counterstained with 4', 6-diamidino-2-phenylindole (DAPI) for 10 minutes. For the negative control samples, anti-specifically targeted antibody was replaced with normal rabbit IgG (Vector Laboratories, Burlingame, CA, #I–1000). Images were obtained using fluorescent microscopy.

Electron Microscopy

Samples for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were forwarded to the Electron Microscopy Facility, Department of Materials Science and Engineering, Clemson University, Anderson, South Carolina. Procedures were performed as previously described.[6,7]

For SEM, culture pellets, fixed in buffered 2.5% glutaraldehyde were washed in buffer and dehydrated in a graded series of ethanol concentrations, followed by immersion in hexamethyldisilazane for 5–15 minutes and then air-dried at room temperature. Dry samples were mounted on Al mounts and were not coated but placed into a Hitachi TM3000 microscope (Tokyo, Japan) and imaged in the variable pressure mode.

For TEM, glutaraldehyde-fixed samples were washed in buffer and dehydrated in a graded series of ethanol concentrations. Samples were immersed in a 50:50 mixture of LR White embedding resin and 100% ethanol for 30 minutes, followed by pure LR White resin until they settled on the bottom of the vial. Resin-immersed samples were placed into pure resin in beam capsules and polymerized at 60°C overnight. Sections were cut on an Ultracut E microtome (Leica Microsystems, Wetzlar, Germany) producing sections 60–90 nm thick, and were placed onto copper grids then stained in uranyl acetate for 20 minutes. Imaging was performed using a Hitachi 7600 microscope.

Molecular Testing

PCR - University of New Haven. DNA was extracted from culture pellets and/or dermatological tissue by lysing overnight in 180 μl tissue lysis buffer (Qiagen) and 20 μl Proteinase K (Qiagen) at 56°C in a shaking water bath and phenol:chloroform extraction the following day. DNA was resuspended in 50–100 μl 1X TE buffer.

A published TaqMan assay targeting a 139-bp fragment of the gene encoding the Borrelia 16S rRNA was used for the detection of Borrelia in DNA extracted from patient samples.[14] Reactions were carried out in a final volume of 20 μl and consisted of 900 nM of each primer, 200 nM of probe, and 10 μl of 2X TaqMan Universal PCR Master Mix (Applied Biosystems). Amplifications were carried out on a CFX96 Real-Time System (Bio-Rad) and cycling conditions consisted of 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Fluorescent signals were recorded using CFX96 Real-Time software and Cq threshold was set automatically. Reactions were performed in triplicate. Positive and negative controls were run simultaneously. The positive control was Bb sensu stricto strain B-31. Four negative controls were used: water, normal human foreskin, normal skin from two Lyme patients, and normal skin from one Morgellons patient. Borrelia DNA was not detected in any of the negative controls.

Nested PCR primers for the 16S rRNA, flagellin (Fla), OspC, uvrA and pyrG genes were used as previously described.[15–17] Reactions were carried out in a final volume of 50 μl using 10 μl template DNA. Final concentrations were 2X Buffer B (Promega), 2 mM MgCl2, 0.4 mM dNTP mix, 2 μM of each primer, and 2.5 U Taq polymerase (Invitrogen). "Outer" primers were used in the first reaction. "Inner" primers were used for the nested reaction, in which 1 μl of PCR product from the first reaction was used as template for the second. Cycling parameters were as follows: 94°C for 5 minutes followed by 40 cycles of denaturation at 94°C for 1 minute, annealing for 1 minute (temperature based on the primer set used), and extension at 72°C for 1 minute, with a final extension step at 72°C for 5 minutes. PCR products were visualized on 1–2% agarose gels.

Sanger sequencing was used for gene analysis. PCR products were extracted from the agarose gels using the QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's instructions. The eluates from each sample were sequenced in both directions using the primers that generated the products. Obtained sequences were compared by searching the GenBank database (National Center for Biotechnology Information) using BLAST analysis. Sequence alignment (Clustel W) and neighbor-joining phylogenetic analyses were conducted using MEGA version 5. Tree support was evaluated by bootstrapping with 500 replications.

PCR - Australian Biologics. Dermatological specimens and/or culture pellets were concentrated by centrifugation and stabilized with AL buffer (Qiagen). Samples were forwarded to Australian Biologics for Borrelia detection by real-time PCR targeting the 16S rRNA gene and endpoint PCR targeting the rpoC gene, as previously described.[8,18] The Eco™ Real-Time PCR system with software version 3.0.16.0 was used. DNA was extracted from the dermatological specimens and the culture pellets using the QIAamp DNA Mini Kit (Qiagen). Samples were analyzed in duplicate with positive and negative controls using primers for the Borrelia 16S rRNA and rpoC gene targets, as previously described.[8,18] Thermal profiles for all analyses were performed with incubation for 2 minutes at 50°C, polymerase activation for 10 minutes 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 (ΔR) for each sample was interpreted as either positive or negative as compared to positive and negative controls.

PCR products were visualized on 1–2% agarose gels and extracted from the gels using the QIAquick Gel Extraction kit (Qiagen) according to the manufacturer's instructions. Sanger sequencing was used for gene analysis, as described previously.[18]

Bb Molecular Beacons. Dr. Alan MacDonald designed the DNA sequences and generously donated the Bb molecular beacon DNA probes. Probe FlaB, a sequence of 23 nucleotides, was derived from the Bb open reading frame (ORF) BB0147 of the flagellin B gene that contains more than 1000 nucleotides. A nucleotide BLAST search of the 23 nucleotide probe sequence disclosed no matches other than that of Bb BB0147. Probe 740 was derived from the Bb ORF BB740 representing a Bb inner cell membrane protein, and a nucleotide BLAST search disclosed no matches other than that of the Bb ORF BB740.

Bb DNA staining and detection with molecular beacons was performed by the following protocol, as previously described.[12] Paraffin sections of dermatological specimens and culture pellets were completely dewaxed by baking at 60°C then immersed in serial 100% xylene baths, followed by serial immersion through 100% ethanol, 90% ethanol, 80% ethanol and distilled H2O, then air-dried. Fixed sections were immersed in 20 μl of working DNA beacon solution. Sectioned specimens were covered with plastic cut from a Ziploc® freezer bag then were heated at 90°C for 10 minutes to denature all DNA and RNA. Heat was reduced to 80°C for 10 minutes, then samples cooled gradually to room temperature. The stained slides were washed in PBS, and covered with 30% glycerol and a glass coverslip, then examined under an EPI Fluor microscope. Staining of research specimens was performed alongside staining of positive and negative controls. Positive controls consisted of a known Bb strain embedded in agarose, formalin-fixed and sectioned, as well as experimentally Bb-infected mouse liver sections. The negative control consisted of uninfected mouse liver sections.

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