Streamlining Borrelia burgdorferi cultivation using quantitative PCR screening - Greiter, B. M.; Sidorov, S.; Osuna, E.; Schalch, A.; Greiter, L. M.; Robinson, E.; Seiler, M.; Bressan, M.; Imkamp, F.; Beer, O. S.; Ens, L.; Nolte, O.; et al. (2026)
Introduction. Direct detection of Borrelia burgdorferi by culture is considered the gold standard for confirming Lyme disease (LD). However, B. burgdorferi culture is not routinely used in clinical practice or research due to its lengthy protocol and low success rate. This study aimed to streamline the process by integrating a specific quantitative PCR (qPCR) screening early into the B. burgdorferi culture workflow for identification of cultures that are likely to yield viable spirochetes.Methods. Thirty-two blood plasma and 11 cerebrospinal fluid (CSF) samples were collected from 32 children with serologically confirmed LD and incubated in modified Kelly-Pettenkofer medium for up to 9 weeks, with weekly assessments for viable spirochetes using microscopy. After 3 weeks, the presence of B. burgdorferi DNA in culture was assessed by qPCR targeting the B. burgdorferi flagellin B gene. The estimated copy number of the target template was compared to the assay's 95% limit of detection (LOD).Results. After 9 weeks of incubation, viable spirochetes were observed in 2 (n=2/32, 6.3%) plasma cultures and 3 (n=3/11, 27.3%) CSF cultures. These were only observed in cultures showing copy numbers above 95% LOD in qPCR testing at week 3 (n=2/3 plasma cultures, 66.7%; n=3/3 CSF cultures, 100.0%).Conclusion. Culturing B. burgdorferi is challenging and, despite a high workload, often not successful. qPCR may serve as an effective screening tool for B. burgdorferi cultures, enabling the culturing process to be streamlined by prioritizing cultures with target copy numbers exceeding the 95% LOD of the qPCR assay.
Keywords: journalArticle; *Bacteriological Techniques/methods; *Borrelia burgdorferi/isolation & purification/genetics/growth & development; *Lyme Disease/diagnosis/microbiology/blood; *Real-Time Polymerase Chain Reaction/methods; Borrelia burgdorferi; Child; Child, Preschool; DNA, Bacterial/genetics; Female; Flagellin/genetics; Humans; Lyme disease; Male; Sensitivity and Specificity; culture; phase-contrast microscopy; spirochete