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.
Highlights
In North America, Lyme disease is most often caused by Borrelia burgdorferi sensu stricto [1].According to the guidelines of the Centers for Disease Control and Prevention (CDC), its diagnosis is primarily based on symptoms, physical findings, and the possibility of exposure to infected ticks; laboratory testing is helpful if used correctly and performed with validated methods [2].the clinical manifestations of Lyme disease are highly variable and often not distinguished from those caused by other illnesses in clinical practice [3]
If residual infectious agents can be found in the archived serum samples of Lyme disease patients, a concurrent DNA sequencing-based test for borrelial cells in the whole blood at the time of the serology assay may be warranted as a supplementary diagnostic tool
Under a Material Transfer Agreement with the CDC of the United States, the authors received from the latter agency 32 blind-coded serum samples (100 μL each), including 12 serum samples collected from patients with clinically suspect Lyme disease according to the CDC criteria and had been treated with antibiotics for the infection, and 20 control serum samples collected from patients, two diagnosed with fibromyalgia, two with rheumatoid arthritis, two with multiple sclerosis, two with infectious mononucleosis, two with syphilis and two with severe periodontitis; and from healthy persons, four living in Lyme disease-endemic regions, and four living in non-endemic regions
Summary
In North America, Lyme disease is most often caused by Borrelia burgdorferi sensu stricto [1]. This study was designed to use 16S ribosomal RNA gene (16S rDNA) sequencing to determine if residual Lyme disease bacteria or bacterial fragments could be detected in the archived serum samples which were drawn from patients with a diagnosis of clinically suspect Lyme disease and had been tested by the 2-tier serology assay. If residual infectious agents can be found in the archived serum samples of Lyme disease patients, a concurrent DNA sequencing-based test for borrelial cells in the whole blood at the time of the serology assay may be warranted as a supplementary diagnostic tool. Since the currently used 2-tier serology test is unlikely to be helpful in the diagnosis of B. miyamotoi infections [16], the experimental design is aimed at detection of possible presence of relapsing fever borreliae in these archived serum samples
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