Table 1 Inclusion and exclusion criteria

Patients with confirmed Lyme borreliosis

Inclusion criteria:

 Patient ≥18 years with confirmed proven or probable, early localized or disseminated Lyme borreliosis manifestation.^a

 In case of an EM reported at www.tekenradar.nl, the EM has been present < 3 months and the clinical diagnosis has been confirmed by the general practitioner.

 Subjects live or stay on the mainland of the Netherlands.

Exclusion criteria:

 Subjects unable to provide informed consent or do not have sufficient proficiency in the Dutch language.

 Subjects having started antibiotic treatment > 4 days before inclusion (for subjects included online) or > 7 days before inclusion (for subjects included through the clinical expert centers).

 Subjects having ongoing signs or symptoms attributed to a previous episode of Lyme borreliosis.

Healthy controls

Inclusion criterium:

 Participant ≥18 years old.

Exclusion criteria:

 Uncontrolled HIV-infection, if known. This is defined as an HIV-1 viral load > 40 copies/ml and/or CD4+ count < 500 × 10⁶ cells/liter in the past 12 months.

 Active syphilis or leptospirosis, an active infection with EBV/CMV, or an auto-immune disease, if known.

 Current LB with typical symptoms. A past episode or Borrelia seropositivity is not an exclusion criterium.

 Immunomodulating medication including > 7.5 mg prednisone daily, methotrexate, biologicals. Medication such as hydroxychloroquine, sulfazalazine or NSAIDs are accepted.

 Known immunodeficiency, hematologic malignancies in the medical history or chemotherapy during the past year.

 Subjects unable to provide informed consent or do not have sufficient proficiency in the Dutch language.

Potentially cross-reactive controls

Inclusion criteria:

 Participant ≥18 years old.

AND

1. Patients with syphilis:

 EITHER clinical symptoms suspected of secondary syphilis, in combination with a positive RPR card POCT result.

 OR early latent syphilis infection with VDRL/RPR ≥ 1:32.

2. Patients with Epstein Barr-virus or cytomegalovirus:

 Compatible signs and symptoms.

 Positive IgM or Paul-Bunnell of EBV/CMV on plasma.

3. Patients with leptospirosis:

 Compatible signs and symptoms.

 Positive serology (MAT), preferably confirmed by culture or PCR.

4. Patients with autoimmune diseases:

 EITHER confirmed rheumatoid arthritis.

 OR other autoimmune disorders diagnosed according to the leading guidelines.

Exclusion criteria:

 Uncontrolled HIV-infection, if known. This is defined as an HIV-1 viral load > 40 copies/ml and/or CD4+ count < 500 × 10⁶ cells/liter in the past 12 months.

 More than one of the listed potentially cross-reactive conditions, if known.

 Ever an episode of LB, ever treated for LB or a known tick bite in the past 6 months.

 Immunomodulating medication including > 7.5 mg prednisone daily, methotrexate, biologicals. Medication such as hydroxychloroquine, sulfazalazine or NSAIDs are accepted.

 Known immunodeficiency, hematologic malignancies in the medical history or chemotherapy during the past year.

 Subjects unable to provide informed consent or do not have sufficient proficiency in the Dutch language.

Observational cohort

Inclusion criteria:

 Participants ≥18 years old.

 Presenting at the specialized Lyme centers of the Amsterdam UMC or Radboudumc.

Exclusion criterium:

 Subjects unable to give informed consent or do not have sufficient proficiency in the Dutch language.