In the study from Norway, was detected in CSF from 16 out of 35 LNB cases, whereas the rest could not be completely identified . for studying epidemiological aspects of LNB, a tick-borne emerging disease. (sensu lato (s.l.) . In Europe, the species sensu stricto (s.s.) are all human pathogens, whereas in the USA, s.s. is the only species causing LB . is the predominant tick species in Sweden and the primary pathogen vector for both humans and animals [8, 9]. Lyme neuroborreliosis (LNB) in LHW090-A7 Europe is the second most frequent manifestation of LB after the skin manifestation erythema migrans (EM) [10, LHW090-A7 11]. In children, LNB most commonly presents as a facial nerve palsy or a subacute meningitis [12C14], but cases with only non-specific symptoms (headache, fatigue) occur and may cause troubles for the clinician [15, 16]. In some cases, LNB may be preceded by an EM in the head and neck region in the specific child . Other major clinical manifestations of LB in children are lymphocytoma and Lyme arthritis . There is no golden standard in the diagnostic assessments of LNB, either in adults or in children. The serological methods used in praxis for laboratory diagnostics in LNB are indirect methods [18C20]. They can demonstrate intrathecal production of anti-antibodies, one important criterion for the LNB diagnosis , but they may also be negative early in LNB . Other indirect diagnostic markers of LNB have also been studied, and the chemokine CXCL13 in CSF has shown promising results [23C27], also in pediatric LNB patients, where high sensitivity and specificity have been shown [23, 28]. Furthermore, an IL-10/CXCL1 ratio in CSF has been suggested to further improve LNB diagnostics in pediatric LNB patients . As a direct detection method, culture of spirochetes has been used, but the method has very low sensitivity and is technically difficult and tedious . Detection of spp. nucleic acids DNA in various clinical specimens, including the cerebrospinal fluid (CSF), has been tested in both adult and pediatric LNB patients, but sensitivity has been low [31C33]. Direct diagnostic methods based on polymerase chain reaction (PCR) are often in-house protocols and thus difficult to standardize . A PCR assay targeting the region and region of the genome detected LNB in adults with Tmem33 definite LNB and suspected LNB . Furthermore, using PCR as a complementary diagnostic method for LNB patients with short duration of symptoms or LNB patients with negative antibody index (AI) has been suggested [34, 35]. Additionally, identifying spp. with PCR technology might be epidemiologically useful, since new causative agents for LNB in humans may emerge . The aim of this study was to evaluate two different PCR technologies as diagnostic methods for direct detection of s.l. in CSF of well-characterized Swedish pediatric LNB patients. Material and methods Subjects This study was performed retrospectively on LHW090-A7 CSF and serum samples collected from children being evaluated for LNB (spp. PCR analyses were performed at the Division of Inflammation and Infection, Link?ping University, SE, and at the Clinical Microbiology Laboratory, Laboratory medicine, Region Sk?ne, SE. Clinical and laboratory data were collected from standardized questionnaires and medical records. Questionnaires included clinical information about character and duration of symptoms, tick bites, and previous antibiotic treatment. A 2-month follow-up visit was carried out at each pediatric department to evaluate the clinical recovery. Classification of patients and controls Children in the study were classified according to the European case-definitions for LNB (Table ?(Table1)1) . All children in the Definite LNB (AI+CCC Open in a separate window Lyme neuroborreliosis, cerebrospinal fluid, + yes, LHW090-A7 ? no, total cell count? ?5??106/L in CSF, antiantibody index, positive if ?0.3, showing an intrathecal production of anti-antibodies according to case-definition in European guidelines  Children not meeting the criteria for Definite LNB or Possible LNB  (i.e., initially having symptoms suspected for LNB but with no pleocytosis in CSF and negative anti-AI) were classified as Non-LNB (control group 1, AI. Children in control group 2 (AI in this study, as part of clinical routine evaluation of the patient and for classification of patient groups, was the flagellin-based IDEIA Lyme neuroborreliosis kit (IgM and IgG) (Oxoid Limited, former DAKO, Hampshire, UK) . Intrathecal production of anti-antibodies (i.e., positive anti-AI) was defined as AI? ?0.3. Pleocytosis in CSF was defined as total white blood cell count ?5??106/L in CSF . In a few patients, CXCL13 data were available (recomBead CXCL13 assay, Mikrogen Diagnostik, Germany) . Two different in-house real-time PCR assays, both targeting the rRNA gene of rRNA region.