Abstract
A highly conserved 357-bp segment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.
Highlights
Reliable diagnosis of Lyme disease caused by Borrelia burgdorferi at its early stage of infection is important for the timely implementation of appropriate treatments to prevent tissue damages and to achieve a “cure” of the disease [1]
We introduce a pair of common polymerase chain reaction (PCR) primers for same-nested PCR amplification of a highly conserved segment with hypervariable regions of the 16S rDNA of B. burgdorferi and
PCR amplification of a 600-bp target DNA for direct DNA sequencing is technically challenging in a routine diagnostic laboratory because the sensitivity of 16S rDNA PCR amplification in diagnostic clinical microbiology is inversely related to the size of the PCR amplicon [22]
Summary
Reliable diagnosis of Lyme disease caused by Borrelia burgdorferi at its early stage of infection is important for the timely implementation of appropriate treatments to prevent tissue damages and to achieve a “cure” of the disease [1]. Using species-specific primers, such as the LD1 and LD2 [10,11], and TEC1 [12] primers to amplify a highly conserved segment of the 16S ribosomal RNA gene DNA (16S rDNA) of B. burgdorferi sensu lato for detection, followed by direct DNA sequencing of the nested PCR amplicon for validation, has been used to provide sensitive and specific molecular diagnosis of Lyme arthritis [13] and early Lyme disease at the spirochetemic stage [14]. B. miyamotoi followed by direct DNA sequencing of the PCR amplicon for the molecular diagnosis of these two borrelial infections in patients with spirochetemia Test can even diagnose off-season spirochetemias with low bacterial density in the deep winter months in the Northeast of the United States when human exposure to tick bites was very limited
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