Single Core Genome Sequencing for Detection of both Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia Species

Abstract

Lyme disease, initially described as Lyme arthritis, was reported before nucleic-acid based detection technologies were available. The most widely used diagnostic tests for Lyme disease are based on the serologic detection of antibodies produced against antigens derived from a single strain of Borrelia burgdorferi. The poor diagnostic accuracy of serological tests early in the infection process has been noted most recently in the 2018 Report to Congress issued by the U.S. Department of Health and Human Services Tick-Borne Disease Working Group. Clinical Lyme disease may be caused by a diversity of borreliae, including those classified as relapsing fever species, in the United States and in Europe. It is widely accepted that antibiotic treatment of Lyme disease is most successful during this critical early stage of infection. While genomic sequencing is recognized as an irrefutable direct detection method for laboratory diagnosis of Lyme borreliosis, development of a molecular diagnostic tool for all clinical forms of borreliosis is challenging because a “core genome” shared by all pathogenic borreliae has not yet been identified. After a diligent search of the GenBank database, we identified two highly conserved segments of DNA sequence among the borrelial 16S rRNA genes. We further developed a pair of Borrelia genus-specific PCR primers for amplification of a segment of borrelial 16S rRNA gene as a “core genome” to be used as the template for routine Sanger sequencing-based metagenomic direct detection test. This study presented examples of base-calling DNA sequencing electropherograms routinely generated in a clinical diagnostic laboratory on DNA extracts of human blood specimens and ticks collected from human skin bites and from the environment. Since some of the tick samples tested were collected in Ireland, borrelial species or strains not known to exist in the United States were also detected by analysis of this 16S rRNA “core genome”. We recommend that hospital laboratories located in Lyme disease endemic areas begin to use a “core genome” sequencing test to routinely diagnose spirochetemia caused by various species of borreliae for timely management of patients at the early stage of infection.

Large-Scale Sequencing of Borreliaceae for the Construction of Pan-Genomic-Based Diagnostics

“…The cfDNA sequencing method’s sensitivity was further improved by combining the results of the modified two-tiered serology testing to identify 86% of acute Lyme borreliosis cases. Additionally, a recent NGS detection study identified core genes within the Borreliella pangenome to increase the sensitivity of DNA-based Borreliaceae diagnostics [ 208 ]. Taken together, genomic Borreliella detection methods have significantly improved over the last decade.…”

“…Concurrently with the growth of Borreliellal/Borrelial genomics, a tentative preliminary pangenome, rather defined as the complete collection of all genes available within a species (or higher taxonomic unit), from existing sequenced and annotated Borrelia genomes was created. This research focused on B. burgforferi sensu stricto and the B. burgdorferi sensu lato complexes [ 208 , 274 ]. In this first pangenome study, several Lb spirochetes were chosen based on their host of origin, geo-locale origin, vector, and if they had been isolated from symptomatic individuals.…”

“…Subsequent studies on the B. burgdorferi pangenome were smaller in scale. One study centered on the design and application of a potential xenodiagnostic through the use of the pangenome by targeting a fragment of the B. burgdorferi 16S rRNA gene [ 208 ]. This was then applied with limited success on I. ricinius ticks [ 208 ].…”

“…One study centered on the design and application of a potential xenodiagnostic through the use of the pangenome by targeting a fragment of the B. burgdorferi 16S rRNA gene [ 208 ]. This was then applied with limited success on I. ricinius ticks [ 208 ]. This could stem from the limited sequences of the Borreliella 16S gene.…”

Polish Journal of Veterinary Sciences

“…The PCR reaction for Babesia species was carried out using a pair of primers, GF2 and GR2, which made it possible to amplify the DNA section with a 559 bp base-pair-long fragment of the 18S rRNA conservative gene . PCR for Borrelia was performed according to the method described by Lee et al (2019) with the primers M1 and M2 used to amplify the 16S rRNA gene fragment of B. burgdorferi s.l., with a length of 357 bp.…”