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.
Highlights
Lyme disease, caused by the tick-borne bacteria Borrelia burgdorferi, is the most commonly reported vector-borne infectious disease in North America
To detect the low levels of Borrelia genomic DNA in whole blood samples from early Lyme disease patients we developed an isothermal amplification (IA) assay that amplifies the seven B. burgdorferi target regions used with a previously described Borrelia broad-range PCR electrospray ionization mass spectrometry detection and genotyping assay (PCR/ESI-MS)[16]
Our results demonstrate an improved strategy for detecting B. burgdorferi infection from whole blood in patients with erythema migrans and acute Lyme disease
Summary
Lyme disease, caused by the tick-borne bacteria Borrelia burgdorferi, is the most commonly reported vector-borne infectious disease in North America. Direct Borrelia molecular tests, such as PCR, from blood can detect and identify active infection sooner than serologic tests but typically these tests have suffered from low assay sensitivity for clinical use. For example early studies using PCR to detect Borrelia in the blood during active infection had limited success with sensitivities of only 18.4% and 26.1% [3,4]. Studies in recent years have reported a higher detection rate down to 10 genome copies through sampling larger blood volumes, in some cases culturing prior to PCR, and by using different PCR techniques such as qPCR or nested PCR [5,6,7]
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