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

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.

Effect of genomic drift of influenza PCR tests.

BACKGROUND: Nucleic acid amplification assays have become the method of choice for influenza (Flu) testing due to superior accuracy and faster turnaround time. Although assays are designed to detect highly conserved genomic targets, mutations can influence test sensitivity. Most of the circulating viruses in the United States during the 2014-2015 season were associated with significant genetic drift; however, the effect on testing was unknown. OBJECTIVES AND STUDY DESIGN: We compared the performance of Prodesse ProFlu+/ProFAST+ (PFlu/PFAST), FilmArray Respiratory Panel (RP), cobas® Influenza A/B test (cIAB), and Xpert® Flu (Xpt) in a retrospective analysis of consecutive nasopharyngeal specimens received for a two-week period during the winter of 2015. Furthermore, limits of detection (LOD) were determined with six isolates of Flu. RESULTS: Of the 275 specimens, 63 were positive for FluA by PFAST, 60 were positive by RP, 58 were positive by cIAB and 52 were positive by Xpt. Only a subset of 135 specimens was tested by PFlu, of which 32 were positive. The sensitivity/specificity for PFAST, RP, cIAB, Xpt and PFlu was 100/99.1%, 96.7/99.5%, 91.8/99.1%, 85.2%/100%, and 75.6%/98.9%, respectively. LOD analyses demonstrated assay performance variations were strain associated. Specifically, PFlu's and cIAB's LODs were higher with A/Texas/50/2012-like and A/Switzerland/9715293/2013-like strains, while Xpt's highest LOD was with the Swiss strain. CONCLUSIONS: Strain-associated assay performance variation is known to occur with other Flu test methods; hence, it is not surprising that such variation would be observed with molecular tests. Careful monitoring and reporting for strain-associated variances are warranted for all test methods.