Performance evaluation of the Panther Fusion® respiratory tract panel

Highlights • Clinical specificity of Panther Fusion® is between 96 %–100 %, compared to LDT.• Clinical sensitivity Panther Fusion® is between 71.9 %–100 %, compared to LDT.• Overall linear regression showed good correlations between LDT and Panther Fusion® for all viruses, except RV and PIV-4.• The Panther Fusion® provides a random-access system with continuous loading and shorter sample-to-answer times compared to LDT.


Background
During the winter season, viral respiratory tract infections are among the most common infections and can be severe in children, elderly, and immunocompromised patients, often leading to hospitalization. Fast and reliable laboratory testing is essential for patient management [1][2][3]. Many laboratories have developed multiplex qRT-PCR assays to detect respiratory viruses, but more recently also commercial PCR assays have become available. Some of these assays can be used at point-of-care (POCT) and can consist of a limited panel (e.g. Influenza A and B viruses) [4][5][6] or a comprehensive panel detecting a large number of viruses and even atypical respiratory bacteria [7][8][9]. In contrast to some POCT respiratory virus PCR assays, which can be fast and easy to perform, most laboratory developed tests (LDT) require highly qualified personnel to perform and interpret the results, much longer times to result (up to ∼6 h) and more hands-on time because of, among others, separated nucleic acid extraction, amplification, analysis and QC.

Objectives
We have evaluated the Panther Fusion® and accessory respiratory virus panels. Panther Fusion® is an automated random access system for molecular detection with continuous loading, a turnaround time of 2.5 h and a throughput of up to 120 respiratory tract samples within 8.5 h.

Analytical performance evaluation
Analytical sensitivity and repeatability was performed using a log10 dilution series of cell culture isolates for influenza A virus (FluA/H1N1/ Netherlands/202/95), influenza B virus (FluB/Yamagata/Netherlands/ 138), respiratory syncytial virus(RSV)-A and RSV-B. Dilution series were prepared, aliquoted and stored at −80°C until used. was tested in 20 replicates in both the Panther Fusion® FLU A/B/RSV assay and a routine laboratory developed automated real-time RT-PCR (LDT) using Aurora FLOW (Roche, Almere, the Netherlands) as described before [4,10,11]. Relative analytical sensitivity was determined by probit analysis with 95 % positivity using IBM SPSS 24. Competitive interference for the FLU A/B/RSV assay was assessed using pairs of target viruses at different concentrations; a range of 0.5log10 serial dilutions were run to determine the sensitivity of these targets in the presence of a high amount of influenza A/B or RSV A/B. Repeatability of each assay was assessed using five replicates of (positive) process controls, containing corresponding virus. To determine the stability of samples in Panther specimens lysis tubes (STM), one replicate of each process control, was tested after 1 day and 6 weeks at 4°C.

Sanger sequencing
Samples with discrepant results and a Ct value < 30 were repeated in both Panther Fusion® and LDT. Confirmed discrepancies were analyzed by Sanger sequencing. For RV a 549 bp fragment of the VP4/VP2gene [12,13], and for AdV a 600bp fragment of the hexon gene [14], was amplified and sequenced using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and a 3130xl genetic analyzer (Applied Biosystems). Sequences were analyzed using SeqMan Pro lasergene 10 software (DNA star) and genotypes were determined using NCBI blastn database.

Table 1
Competitive interference within the FLUA/B/RSV assay.

Analytical performance evaluation
Relative Analytical sensitivity was determined by probit analyses at 95 % hitrate. Difference in relative sensitivity between Panther Fusion® and LDT was 0.53log10, 1.06log10, 0.36log10, 0.96log10, for FluA, FluB, RSV-A and RSV-B, respectively. The presence of two viruses in various concentrations was tested to establish the multiplex capacity of the assay. A high amount (Ct17) of influenza A virus resulted in an undetected internal control and a slightly less analytical sensitivity of the RSV-A and B viruses (Table 1). In contrast, high amounts of influenza B virus (Ct16) and RSV-A and B (Ct18-19) have no effect on the sensitivity.
Repeatability was determined by calculating %coefficient of variation (CV) of five replicates of the positive process controls. All replicates for all targets tested positive, with a mean Ct-value of 32.3 (31.2-35.2) and %CV < 4.78 %. Measurements after 6 weeks at 4°C were within 1.2 Ct-value difference compared to the initial results for all viruses except for PIV2 (Ct-value difference of 3.5).

Clinical performance evaluation
A total of 1246 respiratory tract samples (sample type distribution, Table 2 Clinical sensitivity and specificity was determined using all retrospective and prospective samples, with the LDT as the gold standard (Table 3). This resulted in a specificity of > 99 % for all viruses except RV (96 %). Sensitivity of the Panther Fusion® varied from 71.9 %-100 %. Fig. 1  The discrepant samples negative by the Panther Fusion® were typed as AdV-2 and RV-A7. All other discrepant samples, with Ct > 30, were considered discrepant due to small differences in sensitivity between the assays and were not further investigated by sequencing.

Discussion
In this study, we evaluated the performance of the Panther Fusion® Flu A/B/RSV, Paraflu 1-4, AdV/hMPV/RV assays and compared this against a well-validated LDT assays. The relative analytical sensitivity of the Panther Fusion® system was slightly less for Flu A/B and RSV compared to the LDT, which corresponded with a slightly less clinical    PIV-4 (sensitivity 71.9 %) and AdV (sensitivity 78.9 %) compared to LDT. In addition, for Panther Fusion® Sam et al., found presumed false positive results for hMPV in 7 samples. In our study we also found 9 hMPV-positive samples in Panther Fusion® (Ct > 36.2) that were negative in our LDT. Amplification curves were re-evaluated and all exhibited low-fluorescence, which could result in ambiguous interpretation. It is not uncommon that multiplex syndromic PCR panels are less sensitive compared to well-established LDT assays [17,18]. These syndromic panels often are dependent on uniformed nucleic acid isolation and PCR procedures, which are a trade-off between sensitivity and consolidation. Linear regression showed good correlations between LDT and Panther Fusion® for all viruses, except RV and PIV-4. These findings could be explained by the large sequence diversity of RV. In addition, the RV LDT assay was not designed to detect all rhinovirus type C strains efficiently, which also contributes to the lower correlation. The number of PIV-4 positive samples in both assays was low (n = 23) and appear to be divided in two different subgroups. Whether this can be attributed to genetic differences between PIV-4A and PIV-4B strains and potential primer/probe mismatches could not be investigated. Primer and probe sequences are unknown, since commercial companies do not tend to share these data. Although for AdV a good correlation between both assays was demonstrated, Panther Fusion® missed 2 high positive AdV-2 samples in the retrospective study. Most likely this was not due to mismatch of primer/probe for AdV-2 as other AdV-2 samples included in the study were detected properly. Why these two AdV-2 samples with low Ct values were missed in the Panther Fusion® remains unclear.
The Panther Fusion® respiratory assays are officially only CE-IVD approved for nasopharyngeal swabs. However, in our diagnostic setting the most common respiratory tract samples are throat swabs, sputa, BAL and nasal washings. Therefore, those materials were added to the study. Sputa, BAL and nasal washings were pretreated according to our diagnostic standard protocol, direct use of these materials has not been tested. All internal controls where within the assay limits, according to the Panther Fusion® software. However, the software only declares a result as invalid if the internal control is negative and no other virus is tested positive. As a consequence a certain degree of PCR inhibition will be tolerated by the Panther Fusion®. In specific cases laboratories could formulate additional cut-off values for the internal control, but these would have to be monitored and interpreted by the user. In addition, in case of for instance a high positive influenza A virus result the internal control may be negative, presumably by competition, but the result will not be rejected by the Panther Fusion® software. Theoretically, co-infections with for instance influenza B virus or RSV at low amounts of virus could inadvertently be reported as negative (Table 1) due to PCR inhibition, that is not monitored correctly.
The Panther Fusion® respiratory panel does not include coronaviruses, enterovirus and bocavirus. Other syndromic respiratory panels such as ePlex (GenMark), Biofire® FilmArray® (bioMérieux), and xTAG RVP FASTv2 (Luminex) do. These panels do not distinguish between rhino-or enteroviruses but ePlex and FilmArray® do have quicker turnaround times of about 60 min, whereas the turnaround time for xTAG is about 6 h and requires more hands-on time [9]. Although the first Panther Fusion® results are produced after approximately 2.5 h, after every 15 min five additional, complete panel results are generated. Panther Fusion® also provides an open channel on which LDT tests can be adapted and run simultaneously with the respiratory panel (and other CE-IVD assays). Furthermore, from one extraction a total of three PCR reactions can be performed, allowing efficient use of samples with limited volume.
In conclusion, although slightly less sensitive as compared to an optimized LDT assay, Panther Fusion® and accessory respiratory virus panels offers a reliable, fully automated system with a minimum handson time, ease of use and short sample-to-answer times.

Ethical approval
The study was approved by the Medical Ethical Commission of the Erasmus MC (MEC-2016-702).

Declaration of Competing Interest
This study was supported by a research grant from Hologic Deutschland GmbH.