Automated Real-Time PCR Detection of Tickborne Diseases Using the Panther Fusion Open Access System

Abstract

Since the incidence of tickborne diseases has been accelerating in the United States, automation for testing has become essential in affected regions. Unfortunately, because the need is regional, commercial test manufacturers have not yet provided answers for clinical laboratories. The incidence of tickborne infections in the United States has risen significantly. Automation is needed for the increasing demand for testing. The Panther Fusion (Fusion) has an Open Access functionality to perform lab developed tests (LDTs) on a fully automated system. Our laboratory adapted two LDTs on Fusion; a multiplex real-time PCR for Anaplasma phagocytophilum and Ehrlichia chaffeensis (AP/EC) and a Babesia microti (BM) PCR. Limits of detection (LODs) were performed with target region plasmid panels spiked into whole blood. The LODs for AP, BM, and EC on the Fusion were 11, 17, and 10 copies/reaction, respectively. The performance of AP/EC was evaluated with 80 whole blood specimens, including 50 specimens previously positive for AP by our test of record (TOR) and 30 specimens (including 20 AP positive) spiked with EC plasmid. AP was detected in 49 out of 50 positive specimens and EC was detected in all 30 spiked specimens. BM PCR on Fusion was evaluated with 75 whole blood samples, including 16 specimens previously shown to be positive for BM and 59 negative specimens, of which 29 were spiked with BM plasmid DNA. BM was detected in 45 samples as expected. AP/EC and BM PCRs were successfully developed and optimized on the Panther Fusion with performance characteristics comparable to our TOR. These assays complement each other and allow for a modular testing approach for tickborne diseases which have differing clinical presentation. Furthermore, automation of these assays will help the lab meet the increasing demand for testing. IMPORTANCE Since the incidence of tickborne diseases has been accelerating in the United States, automation for testing has become essential in affected regions. Unfortunately, because the need is regional, commercial test manufacturers have not yet provided answers for clinical laboratories. Here, we describe the development of PCR tests on the highly automated Panther Fusion for three tickborne diseases. The Panther Fusion assays were evaluated using 155 archived whole blood (WB) specimens previously tested for Anaplasma phagocytophilum , Ehrlichia chaffeensis, and Babesia microti , while WB spiked with DNA from plasmid clones of the target regions were used for analytical sensitivity. We demonstrated that the Panther Fusion assays performed similar to the manual PCR tests used clinically in our laboratory and that automation of these tests had no adverse effect on the performance.