Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease

Abstract

Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion.

Borrelia burgdorferi Not Confirmed in Human-Biting Amblyomma americanum Ticks from the Southeastern United States

“…At Ibis Biosciences, further testing of PCR-suspect-positive samples was undertaken, using isothermal Borrelia enrichment performed as previously published (32). Enrichment of a negative control was also performed.…”

“…Enrichment of a negative control was also performed. Following isothermal Borrelia DNA enrichment, a broad-range PCR followed by electrospray ionization mass spectrometry (IA/PCR/ ESI-MS) was performed, in which each sample was subjected to a series of eight diagnostic PCRs (32,33). By the selection of Borrelia PCR targets that vary in sequence between Borrelia species and strains (genotypes), it is possible to use the IA/PCR/ESI-MS technology to identify the Borrelia species and distinguish its genotype even when present in mixtures of genotypes (32,33).…”

“…Following isothermal Borrelia DNA enrichment, a broad-range PCR followed by electrospray ionization mass spectrometry (IA/PCR/ ESI-MS) was performed, in which each sample was subjected to a series of eight diagnostic PCRs (32,33). By the selection of Borrelia PCR targets that vary in sequence between Borrelia species and strains (genotypes), it is possible to use the IA/PCR/ESI-MS technology to identify the Borrelia species and distinguish its genotype even when present in mixtures of genotypes (32,33). A volume of 10 l of isothermal amplified nucleic acid extract was used in each PCR, and all PCRs were analyzed using an electrospray ionization mass spectrometry system (Abbott Molecular, Des Plaines, IL) as previously described (32,33).…”

“…By the selection of Borrelia PCR targets that vary in sequence between Borrelia species and strains (genotypes), it is possible to use the IA/PCR/ESI-MS technology to identify the Borrelia species and distinguish its genotype even when present in mixtures of genotypes (32,33). A volume of 10 l of isothermal amplified nucleic acid extract was used in each PCR, and all PCRs were analyzed using an electrospray ionization mass spectrometry system (Abbott Molecular, Des Plaines, IL) as previously described (32,33). In silico analysis.…”

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome

“…Briefly, DNA was extracted from 300 µl of amniotic fluid using a method that combines beadbeating cell lysis with a magnetic-bead-based extraction method (38,39). The extracted DNA was amplified by the previously described broad bacteria and candida detection assay, according to the manufacturer's instructions.…”