Detection of Borreliae in Archived Sera from Patients with Clinically Suspect Lyme Disease

Abstract

The diagnoses of Lyme disease based on clinical manifestations, serological findings and detection of infectious agents often contradict each other. We tested 52 blind-coded serum samples, including 20 pre-treatment and 12 post-treatment sera from clinically suspect Lyme disease patients, for the presence of residual Lyme disease infectious agents, using nested PCR amplification of a signature segment of the borrelial 16S ribosomal RNA gene for detection and direct DNA sequencing of the PCR amplicon for molecular validation. These archived sera were split from the samples drawn for the 2-tier serology tests performed by a CDC-approved laboratory, and are used as reference materials for evaluating new diagnostic reagents. Of the 12 post-treatment serum samples, we found DNA evidence of a novel borrelia of uncertain significance in one, which was also positive for the 2-tier serology test. The rest of the post-treatment sera and all 20 control sera were PCR-negative. Of the 20 pre-treatment sera from clinically suspect early Lyme disease patients, we found Borrelia miyamotoi in one which was 2-tier serology-negative, and a Borrelia burgdorferi in two—one negative and one positive for 2-tier serology. We conclude that a sensitive and reliable DNA-based test is needed to support the diagnosis of Lyme disease and Lyme disease-like borreliosis.

Borrelia miyamotoi: a widespread tick-borne relapsing fever spirochete

“…in the general population in endemic areas (0.6-19.7%) [70][71][72][73][74][75][76]. Some studies have shown that standard serologic tests for Lyme borreliosis can fail to diagnose B. miyamotoi infections, and serology for B. miyamotoi is currently based on antibodies against the glycerophosphodiester phosphodiesterase (glpQ) gene, which is present in B. miyamotoi, but not in Lyme borreliosis spirochetes [46,77,78]. However, it is not specific for B. miyamotoi because homologs are present in other relapsing fever spirochetes [78,79].…”

DNA Sequencing Diagnosis of Off-Season Spirochetemia with Low Bacterial Density in Borrelia burgdorferi and Borrelia miyamotoi Infections

“…In a previous report, the M1/M2 primer pair has been shown to be able to amplify a correspondent 358-bp segment of the 16S rDNA of B. coriaceae and a correspondent 358-bp segment of the 16S rDNA of a novel Borrelia, both belonging to the relapsing fever Borrelia group, as the species of B. miyamotoi [25]. …”

Gene Amplification and Sequencing for Bacterial Identification

“…16S rRNA gene sequencing has also been successfully used to identify the etiological agents of other infections such as bacillary angiomatosis (caused by Bartonella henselae and Bartonella quintana; Regnery et al, 1992;Relman, Loutit, Schmidt, Falkow, & Tompkins, 1990) and human ehrlichiosis (caused by bacteria in the genera Ehrlichia and Anaplasma; Anderson, Dawson, Jones, & Wilson, 1991;Chen, Dumler, Bakken, & Walker, 1994;Maeda et al, 1987), which are difficult to isolate and grow in vitro. Direct PCR of the 16S rRNA gene has been used to detect borreliae in sera from patients with suspected Lyme disease (Lee, Vigliotti, Vigliotti, Jones, & Shearer, 2014), Mycoplasma and Chlamydia in patients with respiratory tract infections (Borel et al, 2008;Touati et al, 2010) and Mycobacterium species in patients with mycobacteriosis (Nakano et al, 2010;Syre, Myneedu, Arora, & Grewal, 2009). 16S rRNA gene sequencing is particularly useful for diagnosis of leprosy caused by the non-cultivable mycobacterium, Mycobacterium leprae, using skin biopsies of leprosy patients (Kurabachew, Wondimu, & Ryon, 1998;Phetsuksiri et al, 2006).…”