The Panther Fusion System with Open Access Functionality for Laboratory-Developed Tests for Influenza A Virus Subtyping

Abstract

Nucleic acid amplification tests, such as PCR, are the method of choice for respiratory virus testing, due to their superior diagnostic accuracy and fast turnaround time. The Panther Fusion (Fusion; Hologic) system has an array of highly sensitive in vitro diagnostic (IVD) real-time PCR assays for respiratory viruses, including an assay for influenza A (FluA) virus, influenza B (FluB) virus, and respiratory syncytial virus (RSV) (FFABR assay). The Fusion system has Open Access functionality to perform laboratory-developed tests (LDTs) alongside IVD assays. We developed two LDTs for FluA virus strain typing on the Panther Fusion instrument, enabling side-by-side testing with the FFABR assay. The LDT-FAST assay uses proprietary primers and probes designed by Hologic for the Prodesse ProFAST+ (PFAST) assay. The exWHO-FAST assay is an expanded redesign of the WHO-recommended reverse transcriptase PCRs (RT-PCRs). To evaluate the performance of these two LDTs, 110 FluA virus-positive samples were tested. Of these, 104 had been subtyped previously; 54 were H3, 46 were 09H1, and 4 were fsH1. All were appropriately subtyped by both LDTs. Of the untyped FluA virus samples, three were subtyped as H3 by both LDTs and two were subtyped as H3 by the LDT-FAST assay only. The sample not subtyped by either LDT was retested with the FFABR assay and was now negative. Limit-of-detection (LOD) analyses were performed with five FluA virus strains. The LDT-FAST LODs were similar to the FFABR assay LODs, while the exWHO-FAST LODs were higher for two H3N2 strains, findings that were explained by analysis of primer/probe homology. In conclusion, either FluA virus typing assay would be a valuable complement to the Panther Fusion respiratory menu given the performance of these LDTs, the system’s full automation, and the ability to split eluates for both IVD and LDT testing.

Optimization and Clinical Evaluation of an Automated Commercial Analyte-Specific Reagent Assay for Mycoplasmoides genitalium Macrolide Resistance Detection in Primary Clinical Specimens

“…The Panther Fusion system open access channel has previously been utilized in direct detection or characterization of bacterial ( 41 ), parasitic ( 41 ), and viral ( 42 – 46 ) agents of disease and was posited in this study as an automated option for MRM determination within primary specimens testing positive for M. genitalium rRNA. Initial experimentation revealed that incubation of IVT wild-type 23S rRNA with high-concentration MRM primer and detection probe sequences, as well as an increased concentration of MgCl 2 , resulted in a propensity toward false-detections of macrolide resistance.…”

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

“…Actually, it was surprising that Fusion didn’t enhance the sensitivity of the LDTs. Our previous experience with a Fusion LDT for influenza A subtyping, demonstrated the system provides superior sensitivity for the detection of the virus compared to many commercial assays ( 16 ). Other studies have also shown enhanced sensitivity with Fusion ( 17 , 18 ).…”

Analytical validation of quantitative SARS-CoV-2 subgenomic and viral load laboratory developed tests conducted on the Panther Fusion® (Hologic) with preliminary application to clinical samples

“…The potential utility of sgRNA to identify intermediates of replication (potentially indicative of actively replicating virus) prompted development of high throughput automated specimen-to-answer quantitative sgRNA and Viral Load (VL) assays (based on the E target) on the Hologic Panther Fusion ® platform using its Open Access Functionality. Several Laboratory Developed Tests (LDTs) targeting different pathogens including SARS-CoV-2 [ 16 ] were developed on the Panther Fusion ® to allow for simultaneous detection of norovirus and rotavirus [ 17 ], subtyping of Influenza A Virus (FluA) [ 18 ], characterization of respiratory tract infection with Influenza A virus (Flu) A, FluB, and Respiratory Syncytial Virus (RSV) [ 19 ], and detection/differentiation of pandemic and endemic coronaviruses (Human Coronaviruses (HCoV) NL63, 229E, HKU1, OC43 and SARS-CoV-2) [ 20 ]. Panther Fusion ® SARS-CoV-2 LDTs targets included ORF1a (Hologic Food and Drug Administration Emergency Use Authorization), the RNA-dependent polymerase gene [ 21 ] and E + N1 [ 22 ].…”