A 49-year-old Indo-Aryan female presented at a Calgary clinic in early March 2015 complaining of vaginal itching of several weeks duration. At the time of presentation she had been living part time in the province of Alberta and part time in the province of Manitoba. Examination revealed an external vaginal and vulvar papular red rash, and an area surrounding the vagina displayed skin loss, hypopigmentation, cracks, fissures, and ulceration. Similar hypopigmented patches of skin occurred on her breasts. She was treated with oral fluconazole for 3 days while awaiting swab and culture results. Multiple vaginal/cervical swabs were obtained and the differential diagnosis included Trichomonas infection, yeast infection, bacterial vaginosis, Chlamydia infection, gonorrhea, varicella-zoster virus and herpes simplex virus (HSV). Testing was positive for HSV type 1. Based on these results she was treated with valacyclovir. By late March 2015 her symptoms had improved but were unresolved with genital fissures and ulceration still present. Test results from March 2015 were positive for IgG antibodies to HSV type 1 or 2. The possibility of lichen planus (LP) was explored but a genital biopsy was not performed.
In June 2015, she presented with unresolved gynecological symptoms and, in addition, had developed ulcerated, punctate erosions with ragged edges on her back (Figure 1). Microscopic examination of the back lesions at 50X magnification revealed unusual black fibers embedded in skin tissue, meeting the key diagnostic criterion for MD. Due to the association between MD and LD, the possibility of LD was explored. Although she did not recall a tick bite or characteristic EM rash, she recalled having an unidentified arthropod bite on her right forearm in the summer of 2014, and she admitted to having taken frequent walks through a large local wooded park in Alberta at the time when the bite occurred. She recalled a bite that was red, itchy, deeper than a typical mosquito bite, and that took longer than a month to disappear.
Serum, whole blood, and skin specimens were forwarded to IGeneX Laboratories for serological and PCR testing in June 2015. Antibodies to both Bbsl and RFB species and Bb DNA were detected in the serum sample. Vaginal Borrelia culture samples were forwarded to Universite de Moncton for Bb PCR testing, and the samples yielded positive results for the presence of Bbsl genes when run with appropriate positive and negative controls, as described in Materials and Methods.
In August 2016, the patient presented with pustular open sores on her shoulders and arms. As her symptoms had progressed she was treated with doxycycline 100mg BID for 3 months. Treatment with doxycycline resulted in clinical improvement but not resolution, so treatment was extended to 6 months. In January 2017, antibiotic treatment was discontinued because it had failed to completely resolve her symptoms. Serological tests were positive for Bb, Babesia duncani and Ehrlichia chaffeensis (Table 1). Based on her clinical symptoms and test results she was treated with azithromycin 500mg QD and nitazoxanide 500mg BID. By July 2017 her symptoms were much improved, and her lesions had mostly resolved, although a few minor lesions containing fibers were still present. Everyday function improved and she maintained full-time employment as an accountant and cared for her two children. She reported flare-ups of skin lesions if she consumed sugar or processed foods or became stressed. 
In October 2017, she had an outbreak of acne-like lesions on her chin consistent with perioral dermatitis. Azithromycin and nitazoxanide treatment was discontinued and treatment with doxycycline was resumed. By December 2017, her skin condition and other symptoms had worsened, so doxycycline treatment was switched back to azithromycin and nitazoxanide. Repeat serological testing performed in December 2017 was negative for Ehrlichia chaffeensis but positive for Bb, Babesia duncani, and Bartonella henselae (Table 1). Treatment with azithromycin and nitazoxanide was continued as she had previously done well on this regimen.
In December 2018, antibiotic treatment was terminated. The patient then relapsed with the development of annular rashes on her hands, legs, back and abdomen (Figure 2A-C). These lesions started at less than 1 cm and increased up to 6-7 cm in diameter, and the advancing border was raised and erythematous consistent with secondary EM rashes. Microscopic examination of the rashes at 100X magnification revealed blue embedded fibers consistent with MD (Figure 3). A biopsy taken from the advancing edge was formalin-fixed and forwarded to McClains Laboratories for histological staining (Figure 4). Sections stained with hematoxylin and eosin (H&E) exhibited parakeratotic hyperkeratosis, spongiosis, and hemorrhage. The stratum spinosum and stratum basale demonstrated disarray and some degeneration with the formation of vacuoles and apoptotic bodies (Figure 5). Perivascular lymphocytic infiltrate was present in the stratum basale and dermis, indicating chronicity. Gomori trichrome staining revealed that the fibers contained collagen. Dieterle staining revealed bacterial aggregates within the hyperkeratotic stratum corneum with both long extracellular spirochetes and intracellular organisms among the keratinocytes in the stratum basale (Figure 6A). Lymphocytes and glandular tissue within the dermis also displayed positive Dieterle intracellular staining. Anti-Bb immunostaining was positive with strongly staining bacterial aggregates in the hyperkeratotic stratum corneum. These aggregates displayed both positive and negative staining, indicating that the aggregates were composed of a mixture of different bacterial species (Figure 6B).
Aggregates generally displayed a central positively stained core surrounded by a sheath of negatively stained bacteria. Positively stained Borrelia organisms consistent with cystic morphology were visible within many of the bacterial aggregates. Positive intracellular and extracellular staining was prominent within the keratinocytes of the stratum basale and stratum spinosum (Figure 7A). The lymphocytes in dermal infiltrates and cells within dermal glandular tissue revealed positive anti-Bb intracellular staining (Figure 7B and C). Anti-Bb staining of fungal-infected skin served as a negative control (Figure 7D). Gram staining revealed Gram-positive cocci scattered on the superficial stratum corneum and in bacterial aggregates within the hyperkeratotic stratum corneum (Figure 8A). The deeper layers of the epidermis and dermis were devoid of Gram-positive organisms (Figure 8B). These findings indicate Borrelia organisms were present in mixed-bacterial aggregates, and both intra- and extracellularly among the keratinocytes of the stratum basale, consistent with the different morphological variants of Bb described previously. Gram-positive cocci were limited to the upper layers of the epidermis only.
Based on clinical observation and test results, treatment with azithromycin and nitazoxanide was resumed along with a short course of prednisone. By June 2018 the patient reported feeling very well, but she reported that her skin would still break out if she consumed sugar or processed foods. Her current treatment consists of a cycling maintenance regimen of tinidazole 500mg BID and trimethoprim/sulfamethoxazole DS BID. She reports her status at present as being well and functional.