Results
Histological Examination – Dermatological Specimens
All patients were clinically diagnosed with MD by a healthcare provider based on the presence of skin lesions and/or skin crawling sensations with intradermal filaments that were visible with a hand-held microscope, as described in previous publications.[4–9] The dermatological material that met our diagnostic criterion was mainly in the form of calluses embedded with filaments, many of which were blue or red. MD calluses were easily removed from patients as they were composed of thickened skin that has separated from the dermis at the stratum basale. Histological examination of cross sections revealed epidermal layers from the stratum basale to the stratum corneum. In MD calluses, collagen and keratin filaments could be seen distributed throughout the epidermal tissue or projecting down from the stratum basale towards the dermis.
Dermatological specimens consisting of callus material from the following patients were submitted for histological sectioning and examination: 1–4, 8, 10–13, 15, 16, 18–20, 22, 23, 25. Biopsy sections were submitted for histological examination for patient 24. Sections of hair follicular bulbs and attached follicular sheaths rather than sectioned calluses were submitted for patient 5. Samples from patients 6, 7, 9, 14, 17 and 21 were used for culture and/or PCR detection only.
All histological sections were examined at 400X and 1000X magnification. In slides stained with Dieterle silver nitrate stain and/or anti-Bb antibodies, sectioned filaments embedded in epithelial cells were observed in samples of all callus material and in the biopsy material from patient 24. All filaments demonstrated the same morphology: a hollow medulla surrounded by a solid cortex. Filamentous material demonstrated no characteristics consistent with fungal elements such as hyphae, or any characteristics of known parasites such as microfilariae.
Dieterle silver nitrate staining revealed bacteria morphologically consistent with Borrelia spirochetes in 17/19 patients (Table 2). Positively-stained spirochetes were observed in dermatological sections from the specimens submitted for patients 1–4, 8, 10–13, 15, 16, 18, 19, 20, 22 and 23 (Figure 2A). No spirochetes were observed for patients 5 or 24. Patient 5 had only sectioned follicular bulbs and sheaths submitted and patient 24 had biopsy sections submitted. These biopsy sections demonstrated bacterial forms consistent with cystic variants of Bb.
Figure 2.
Evaluation of skin samples from Morgellons disease patients. A, Dieterle silver stain of skin sample from MD Patient 15 showing dark-staining spirochetes (1000x). B, Immunostaining of spirochete with anti-Bb antibody in skin sample from MD Patient 4 (1000x). C, Scanning electron micrograph of skin culture sample from MD Patient 6 showing wavy spirochetes (arrows). D, Hybridization of Bb-specific molecular beacon FlaB with spirochetes in skin sample from MD Patient 3 (400x).
Anti-Bb immunohistochemical staining was reactive in all the histological sections of dermatological specimens submitted including the follicles and follicular sheaths from patient 5 and the two biopsy sections from patients 24 and 25 (100%). Individual spirochetes were discernible in many of these specimens (Figure 2B). Staining of Bb spirochetes was positive in infected mouse liver samples. There was no significant Bb immunostaining of the Bb-negative mouse liver control, the mixed Gram-positive bacterial pellet or normal human skin. Staining of sections of mixed Gram-negative bacteria from the Gram-negative culture pellet showed only weak background staining. In control studies of the anti-Bb immunostaining performed at the University of New Haven, the antibody reacted with Bb but not with Treponema denticola.
Bb spirochetes in a callus specimen from patient 2 were detected by SEM and on sectioned calluses from patients 1 and 2 by TEM as previously reported.[6]
Culture – General Observations
Cultures were performed on skin, blood and/or vaginal specimens taken directly from patients 3, 4, 6–9, 13, 14, 17, 21 and 24. Motile viable organisms were observed in all cultures at 4 weeks of incubation except for the skin culture taken from patient number 6. Motile organisms displayed morphological variation, ranging from spherules to longer helical-shaped bacteria. None of the specimens could be subcultured onto blood agar, even in anaerobic conditions, and there was no contamination by commonly encountered aerobic Gram-positive bacteria. Some of the cultures had very few organisms, and documentation of growth by photography was difficult because of morphological variation. Some cultures were therefore concentrated by centrifugation and stained with Dieterle silver nitrate stain and anti-Bb immunostain for further characterization.
Skin Culture. Dermatological specimens were submitted for culture from patients 3, 4, 6, 9, 13 and 21. Examination of culture fluid at 4 weeks incubation revealed motile spirochetes, except for the culture from patient 6. The culture from patient 9 revealed little growth, but one long motile spirochete was observed. Dieterle silver nitrate staining of skin cultures was performed on specimens from patients 3, 6, and 13. These specimens demonstrated positive staining of both spherules and spirochetes consistent with morphological forms of Borrelia. All specimens demonstrated strongly positive Bb polyclonal immunostaining of the bacteria present as well as the surrounding cellular debris. This may have been due to secreted exoantigens, antigens released by lysed Bb, or the presence of Bb intracellular infection. Data is summarized in Table 3.
The culture fluid from patient 6 did not reveal motile spirochetes, so to ascertain the presence of spirochetes a centrifuged culture pellet was submitted for SEM. SEM revealed that spirochetes with morphological features consistent with Bb were present in the culture pellet (Figure 2C).
Blood Culture. Blood specimens for culture were taken from patients 3, 4, 7, 8, 13, 14, 17 and 24. Motile bacteria with spirochetal morphology were observed in culture fluid from the eight patients. Dieterle silver nitrate staining and anti-Bb immunostaining was performed on blood cultures concentrated by centrifugation from these patients. Patients 3, 7, 8, 13 and 17 demonstrated positive staining of both spirochetes and spherules consistent with morphological forms of Borrelia with Dieterle silver nitrate staining. These patients also demonstrated strongly positive Bb immunostaining of the bacteria in the samples as well as the surrounding cellular debris, as described above. Data is summarized in Table 3.
Vaginal Culture. Swabs of vaginal secretions were submitted for culture from patients 8, 14, 21 and 24. Motile bacteria were visible in culture fluid from all four patients. Staining of vaginal cultures concentrated by centrifugation followed by Dieterle silver nitrate staining and anti-Bb immunostaining was performed for specimens taken from patients 8 and 21. Both specimens demonstrated positive staining of both spherules and spirochetes consistent with morphological forms of Borrelia with Dieterle silver nitrate stain. Both specimens demonstrated strongly positive Bb immunostaining of the bacteria in the samples as well as the surrounding cellular debris, as described above. Data is summarized in Table 3.
Molecular Testing
A. PCR Detection of Borrelia. Various sample types from 20 patients were submitted for PCR detection of Borrelia at three independent laboratories. These samples included whole dermatological calluses, histological skin sections, skin culture, blood culture, vaginal cultures, and one specimen of intestinal epithelial tissue that had sloughed off in the patient's feces during an intestinal cleanse. Borrelia genes were detected in 18 of the patients whose samples were submitted, and results were equivocal for 2 patients. Amplicon sequences consistent with Borrelia DNA were obtained for the PCR products from 14 patients. Bb sensu stricto sequence was confirmed in 12 patients, while patient 23 was found to have an amplicon sequence consistent with B. miyamotoi and patient 24 had a sequence consistent with B. garinii. The latter patient had contracted Lyme disease in Europe. Positive PCR results are summarized in Table 4.
Skin cultures from patients 6, 9, 13 and 21 were subjected to PCR testing, and three of the four samples tested positive, confirming the presence of Borrelia in the cultures. The fourth culture sample (patient 21) had equivocal PCR testing using the 16S rRNA probe but tested positive using the FlaB molecular probe (see below). Thus molecular testing confirmed the presence of viable Borrelia spirochetes in all four skin culture samples.
Treponema denticola was detected in 5/16 scab/callus samples sent to Australian Biologics. T. denticola was not detected in any blood, skin or vaginal cultures (data not shown).
B. In-situ DNA Hybridization. Bb DNA was detected by staining with the fluorescent molecular probes FlaB and 740. Histological sections of callus material from patients 2, 3, 10, 11, 13, 15, 19, 20, 22 and sections of skin and/or vaginal culture pellets from patients 21 and 24 were stained with the FlaB probe. All these specimens were positively stained (Figure 2D). Histological sections of callus material from patients 1–4, 8, 10–13, 15, 16, and 18–20, and sections of the skin culture from patient 6 and blood culture from patient 7 were stained with probe 740. All of these specimens were positively stained. The results are summarized in Table 5.
BMC Dermatol. 2015;15(1) © 2015 BioMed Central, Ltd.
