Background: Nucleic acid amplification tests (NAATs), such as PCR, are preferred for respiratory virus testing, due to superior diagnostic accuracy and faster turnaround time. Panther Fusion® Respiratory Assays (Fusion), which includes FluA/B/RSV (FFABR), Paraflu and AdV/hMPV/RV, offers a modular approach to syndromic testing on a fully automated platform while improving gene targets and expanding the test menu. Objectives and study design: We evaluated Fusion using 275 consecutive nasopharyngeal specimens previously used in an analysis of five PCRs, as well as 225 archived specimens. Results: Of the combined 500 specimens, 134 were positive for influenza A (FluA), 54 for FluB, 65 for RSV, 64 for parainfluenza (PIV), 24 for adenovirus (AdV), 21 for humanmetapneumovirus (hMPV), and 40 for rhinovirus (RV) with Fusion. Of the positive samples Fusion correlated with historical results for all but one, despite multiple freeze-thaws cycles of this collection. Fusion was positive for an additional 33 samples, including 11 FluAs, 7 RSVs, 3 PIV3s, 3 AdV, 6 hMPV and 3 RVs. These samples were retested with corresponding Prodesse (Pro) assays using quadruple sample volume. This resolver test confirmed Fusion results for an additional 4 FluAs, 4 RSVs, 1 PIV3 and 3 AdVs. The sensitivity and specificity ranges of Fusion were 99-100% and 98-100%. Limit of detection (LOD) analyses were performed on a variety of Flu isolates. The LODs ranged from 2.69 to 2.99 log copies/ml and demonstrated superior LOD as compared to previously published data for some assays or to concurrent analyses with two new commercial tests.

To develop an effective Pseudomonas aeruginosa (PA) outer-membrane-vesicles (OMVs) vaccine, we eliminated multiple virulence factors from a wild-type P. aeruginosa PA103 strain (PA103) to generate a recombinant strain, PA-m14. The PA-m14 strain was tailored with a pSMV83 plasmid encoding the pcrV-hitA T fusion gene to produce OMVs. The recombinant OMVs enclosed increased amounts of PcrV-HitA T bivalent antigen (PH) (termed OMV-PH) and exhibited reduced toxicity compared to the OMVs from PA103. Intramuscular vaccination with OMV-PH from PA-m14(pSMV83) afforded 70% protection against intranasal challenge with 6.5 × 10 6 CFU (∼30 LD 50 ) of PA103, while immunization using OMVs without the PH antigen (termed OMV-NA) or the PH antigen alone failed to offer effective protection against the same challenge. Further immune analysis showed that the OMV-PH immunization significantly stimulated potent antigen-specific humoral and T-cell (Th1/Th17) responses in comparison to the PH or OMV-NA immunization in mice, which can effectively hinder PA infection. Undiluted anti-sera from OMV-PH-immunized mice displayed significant opsonophagocytic killing of WT PA103 compared to antisera from PH antigen- or OMV-NA-immunized mice. Moreover, the OMV-PH immunization afforded significant antibody-indentpednet cross-protection to mice against PAO1 and a clinical isolate AMC-PA10 strains. Collectively, the recombinant PA OMV delivering the PH bivalent antigen exhibits high immunogenicity and would be a promising next-generation vaccine candidate against PA infection.

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.