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ABSTRACTTwo-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferistrain B31 and various recombinant antigens, and subsequently, the number of Borrelia-specific interferon gamma (IFN-?)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferiB31-specific IFN-?-secreting T cells/2.5 × 105PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) (P= 1.000); however, the median number of B. burgdorferiB31-specific IFN-?-secreting T cells/2.5 × 105PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) (P= 0.016). We conclude that the BorreliaELISpot assay, measuring the number of B. burgdorferiB31-specific IFN-?-secreting T cells/2.5 × 105PBMCs, correlates with exposure to the Borreliabacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis.