Persistent Lyme disease with cutaneous Borrelia biofilm formation.

dear editor, Lyme borreliosis, the most common tick‐borne infectious disease, is caused by Borrelia burgdorferi sensu lato (Bb) and can affect various organs. Erythema migrans (EM) of the skin is the most frequent clinical diagnosis. Sapi et al. previously showed that Bb spirochetes accumulate to form cutaneous biofilm aggregates expressing specific markers, including the well‐established biofilm marker alginate.

A 54‐year‐old woman was referred to our clinic with chronic, linear nonpalpable erythemata of the gluteal region (Figure 1a) and the left breast. Additionally, the patient reported fatigue and hip pain. In the preceding months, the patient had been treated for acute EM with doxycycline over 3 weeks. The patient lived in a tick‐endemic area in rural Austria and recalled multiple tick bites over the past year. Primary laboratory findings revealed positive Bb‐specific IgG antibodies in enzyme immune assay (156 rU mL−1; negative < 16 rU mL−1) and immunoblot (both Euroimmun, Lübeck, Germany) and negative IgM antibodies (4 rU mL−1) in enzyme‐linked immunosorbent assay. Histological examination showed typical signs of EM, consisting of a perivascular lymphocytic infiltrate with admixed plasma cells. Polymerase chain reaction (PCR) tested positive for Borrelia afzelii. Neuroborreliosis was excluded by the analysis of cerebrospinal fluid. Based on these findings, intravenous ceftriaxone (1 × 4 g) was administered over 4 weeks without symptom improvement. Therapy was switched to cefuroxime (4 × 500 mg) for 4 weeks, without effect. PCR and multilocus sequence typing revealed persistent skin infection with the same B. afzelii strain in two body sites (Figure 1c). The lack of response to antibiotics led us to suspect Bb biofilm formation, which we confirmed by antialginate (Thermo Fisher MA5‐2757; Thermo Fisher Scientific, Waltham, MA, USA) and anti‐Bb immunostaining (Anti‐Bb‐Flagellin, APC‐conjugated, US Biologicals B2570‐26F1‐APC; United States Biological, Salem, MA, USA) of a lesional skin biopsy (Figure 1d). A diagnosis of therapy‐refractory lyme disease (LD) with biofilm formation was made and intensified antibiotic therapy was initiated (intravenous azithromycin 1.5 g single injection and oral cephalexin 1000 mg twice daily for 3 weeks). After treatment, erythema subsided for the first time and joint pain improved. Histological examination of a skin biopsy of the formerly affected area showed an inconspicuous result and a negative Bb PCR.

Figure 1

(a) Initial presentation, marginal nonpalpable erythemas in the gluteal region. (B) Late presentation, sharply defined urticarial erythemas. (c) Genetic Borrelia afzelii strain characterization. Phylogenetic tree obtained by the neighbour‐joining method using the Kimura two‐parameter method and 1000 bootstrap replicates. The analysis involved 330 sequences comprising all 328 B. afzelii sequence types (ST) from the multilocus sequence typing database and Borrelia burgdorferi sensu stricto (ST1) as outgroup. Arrow indicates patient isolate. (d) Alginate‐positive Bb biofilm formations in a patient skin biopsy. Immunofluorescence labelling of antialginate positive aggregates (red), Bb‐specific antiflagellin (green) and 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). Yellow signal and arrows indicate overlap. Line indicates dermoepidermal border. [Colour figure can be viewed at wileyonlinelibrary.com]

After 1 month, the patient again reported severe fatigue and joint pain. Diffusely red, urticarial erythema appeared on the thighs (Figure 1b) and the upper left arm. Bb serology and histological examination of the skin lesion were unremarkable with negative Bb PCR. Further investigation revealed serum positivity for anticytosolic 5′‐nucleotidase 1A (cN1A) antibodies (106 U mL−1), elevated creatine kinase (358 U L−1, range 0–170) and progressive muscle weakness. Magnetic resonance imaging of the hips and shoulders revealed muscular oedema. A diagnosis of atypical post‐Lyme syndrome presenting as myositis was made. The patient was treated with lornoxicam and prednisolone (10 mg daily), which stabilized muscle symptoms, but did not improve erythema.

This is the first case in which combined Bb DNA and biofilm detection has led to a diagnosis of persistent LD progressing to myositis. The patient continued to show LD‐specific symptoms despite three empirical antibiotic regimens with full adherence. After the detection of biofilm formations, antibiotic therapy with the biofilm‐active antibiotic azithromycin proved effective for symptoms of acute LD, but did not prevent complications.

Approximately 10–20% of patients with EM treated with first‐line antibiotics (doxycycline and amoxicillin) experience persistent symptoms such as chronic fatigue and joint and muscle pain. When symptoms last 6 months or longer, the condition is referred to as post‐treatment LD syndrome (PTLDS). In the course of PTLDS, our patient developed symptoms of myositis including myalgia and muscle weakness. Several cases of LD‐related myositis were described and myositis with skin symptoms remains an exceedingly rare presentation of LD. Although the timeline and spatial distribution of symptoms suggest a causal relationship, coincidental myositis cannot be excluded. The pathomechanism of LD‐associated myositis remains unclear and a multifactorial pathogenicity is likely. In addition to immune dysregulation occurring during Bb infection, such as lymphopenia, decreased suppressor cell activity or polygonal B‐cell expansion, direct tissue invasion by Bb also appears to play a role. We propose that the formation of bacterial biofilms, which are known to ensure the survival of bacteria under severe conditions, is a contributing factor. Feng et al. found that aggregated biofilm‐like Bb microcolonies were more tolerant to antibiotics compared with other forms of Bb colonies. In addition, alginate‐expressing Bb biofilm formations were previously found in skin biopsies from borrelial lymphocytomas and autopsy tissue sections from the heart, brain, liver and kidney of a patient with LD. ,

In conclusion, the present case illustrates that biofilm formation in EM lesions may lead to persistent Bb infection and, as a consequence, PTLDS. Potential biofilm formation should be considered in therapy‐resistant LD in order to apply early calculated antibiotic treatment and prevent the occurrence of late complications.

Author contributions

Anna Gindl: Formal analysis (lead); investigation (equal); methodology (supporting); project administration (supporting); visualization (equal). Anna‐Margarita Schötta: Investigation (equal); methodology (equal); writing – review and editing (supporting). Sara Berent: Formal analysis (equal); investigation (supporting); methodology (supporting); visualization (supporting). Mateusz Markowicz: Investigation (supporting); project administration (supporting); supervision (supporting); writing – review and editing (equal). Florian Thalhammer: Investigation (supporting); supervision (supporting); writing – review and editing (equal). Hannes Stockinger: Conceptualization (supporting); supervision (supporting); writing – review and editing (equal). Georg Stary: Conceptualization (equal); investigation (equal); supervision (lead); writing – review and editing (equal). Johanna Strobl: Conceptualization (lead); investigation (equal); methodology (lead); project administration (lead); supervision (equal); writing – review and editing (equal).