Helicobacter pylori infection, cagA status, and duodenal ulcer disease in children.

14 samples from noninoculated guinea pig brains as negative controls. All samples were assayed in a blinded fashion. Detection of the Borrelia and IC amplification products was performed using the DELFIA (dissociation-enhanced lanthanide fluoroimmunoassay) time-resolved fluorescence hybridization assay (Perkin-Elmer Wallac, Gaithersburg, MD), as described elsewhere [7]. Probe sequences were as follows: Borrelia: 5′-GATGCACACTTGGTGTTAATCAAAAG-3′; IC: 5′-GCGATGCTGTCGGAAACG-3′. To assess the sensitivity and reliability of our PCR assay, the Borrelia PCR target region was cloned into the pCR 2.1 vector. A dilution series was performed, with the addition of 1750 ng human genomic DNA per reaction, and the target region was then amplified, using our PCR assay. Time-resolved fluorescence was measured to determine positive amplification reactions. The quantification of b-actin genes has been used to calculate that each cell in the trigeminal ganglia contains 15.6 pg of DNA [8]. Therefore, we calculated that our PCR sensitivity was at least 2 Borrelia genomes per 112,000 cells. When our PCR method was used, no patients with AD or controls were positive for Borrelia species in the brain. Therefore, no brain sample in either group was positive by PCR analysis for Borrelia organisms, with a 95% confidence interval of 0%–20%. All 18 samples from guinea pig brains inoculated with B. burgdorferi were positive, whereas all 14 samples from noninoculated guinea pig brains were negative. In summary, using a very sensitive PCR assay that is able to amplify a Borrelia-specific DNA target sequence from all B. burgdorferi sensu lato species known to cause disease in humans, we found no evidence of Borrelia organisms in brains of patients with AD.