Evaluation of a Novel High-Definition PCR Multiplex Assay for Simultaneous Detection of Tick-Borne Pathogens in Human Clinical Specimens

The incidence of tick-borne infections in the United States has risen significantly in the past decade. Ticks can transmit a variety of pathogens, including bacteria, protozoa, and viruses, that can cause serious illnesses. Therefore, the use of rapid, sensitive, and specific multiplex tests is important to identify the pathogen(s) in the acute phase and determine appropriate treatment to minimize the severity of the disease. The purpose of this study was to evaluate ChromaCode’s research use only (RUO) nine-target high-definition PCR (HDPCR) tick-borne pathogen (TBP) panel using 379 retrospective, remnant whole-blood and synovial fluid specimens previously submitted to Associated Regional and University Pathologists (ARUP) Laboratories and tested by clinically validated real-time PCR assays for Ehrlichia spp., Anaplasma phagocytophilum, Babesia spp., or Lyme Borrelia spp. The performance characteristics evaluated included positive percent agreement (PPA) and negative percent agreement (NPA) with the ARUP laboratory-developed tests (LDTs). All tested targets had an initial PPA greater than 97.0%, except Ehrlichia ewingii, with a PPA of 88.9%. The NPAs for all targets were between 98.8% and 100%. The TBP panel detected three coinfections, with two of Babesia microti and A. phagocytophilum and one of B. microti and E. chaffeensis, which were confirmed by the LDTs. There were 16 samples with discordant results compared to the LDT results, five of which were resolved by repeat testing on the TBP panel and bidirectional sequencing. Following discrepant resolution, the final PPA and NPA for the TBP panel were 97.7% (95% confidence interval [CI], 95.2% to 99.0%) and 99.6% (95% CI, 99.3% to 99.8%), respectively, with an overall agreement of 99.5% (95% CI, 99.2% to 99.7%) with the LDTs.