Clinical Evaluation of the BIOFIRE SPOTFIRE Respiratory Panel

The BIOFIRE SPOTFIRE Respiratory (R) Panel is a novel, in vitro diagnostic PCR assay with 15 pathogen targets. The runtime is about 15 min which is the shortest among similar panels in the market. We evaluated the performance of the SPOTFIRE R Panel with 151 specimens, including 133 collected from the upper respiratory tract (URT), 13 from the lower respiratory tract (LRT) and 5 external quality assessment program (EQAP) samples. The respiratory specimens were enrolled throughout the first two post-COVID-19 influenza seasons in Hong Kong (March to December 2023). For URT specimens, full concordance was observed between the SPOTFIRE R Panel and the standard-of-care FilmArray Respiratory 2.1 plus Panel (RP2.1plus) for 109 specimens (109/133, 81.95%). After discrepant analysis, the SPOTFIRE R Panel identified more pathogens than the RP2.1plus in 15 specimens and vice versa in 3 specimens. The per-target negative and positive percentage agreement (NPA and PPA) were 92.86–100% except the PPA of adenovirus (88.24%). For LRT and EQAP samples, all results were fully concordant. To conclude, the performance of the SPOTFIRE R Panel was comparable to the RP2.1plus.


Introduction
Respiratory infection is among the leading causes of disease burden and mortality worldwide [1][2][3].More than a health crisis, the socioeconomic impact brought by a highly transmissible pathogenic respiratory virus can be catastrophic, which is evident by the recent coronavirus disease 2019 (COVID-19) pandemic [4].The initial clinical manifestations of upper respiratory tract infection are usually indistinguishable among different etiologies.Albeit most of the symptoms are self-limiting for healthy adults, the sequelae can be serious for children, the elderly and the immunocompromised [5][6][7].Rapid identification of the culprit may guide optimal patient management and timely infection control measures.
Recent advances in molecular methods have revolutionized infectious disease diagnostics.The paradigm has been shifting from tedious culture-based methods to rapid nucleic acid amplification-based testing.For instance, there are several commercial in vitro diagnostic assays in the market, which can detect more than 20 respiratory pathogen targets in 45 to 90 min [8][9][10].Recently, the BIOFIRE SPOTFIRE Respiratory (R) Panel has been cleared by the U.S. Food and Drug Administration (FDA) 510(k) and waived by the Clinical Laboratory Improvement Amendments [11,12].Its principle of operation is very similar to that of the FilmArray Respiratory Panel from the same company, which automates and integrates quality-controlled nucleic acid extraction, reverse transcription, nested amplification of pathogen targets, fluorescent signal detection and melt curve analysis of amplified targets in a closed, disposable pouch, with data interpreted automatically.A comparison between the SPOTFIRE R Panel and the latest version of FilmArray Respiratory Panel (RP2.1plus) is shown in Figure 1.The SPOTFIRE R Panel is compatible with the novel SPOTFIRE System, whereas the RP2.1plus is run on FilmArray 2.0 or TORCH.The   In the literature, there are limited evaluation data on the clinical performance of the SPOTFIRE R Panel.In the light of this, we evaluated the performance of the SPOTFIRE R Panel in a clinical laboratory setting.The standard-of-care method which uses the RP2.1plus was the primary comparator.

Specimens
A total of 151 specimens were enrolled in this study, encompassing 133 upper respiratory tract (URT) specimens (swabs of nasal cavity/nasopharynx/throat and posterior oropharyngeal saliva), 13 lower respiratory tract (LRT) specimens (bronchoalveolar lavage fluid, endotracheal aspirate and sputum) and 5 external quality assessment program (EQAP) samples.The human respiratory specimens were referred to the Department of Pathology, Hong Kong Sanatorium & Hospital for standard-of-care testing of respiratory pathogens from March to December 2023, using the RP2.1plus on FilmArray 2.0 or TORCH (BIOFIRE Diagnostics, Salt Lake City, UT, USA).Aliquots of residual specimens were kept at 4 • C for 1 month and −80 • C for long-term storage.The specimens were selected to cover all the pathogen targets of the SPOTFIRE R Panel.The RP2.1plus quality controls of all enrolled specimens were valid.The minimum sample volume was 0.5 mL.

Testing with SPOTFIRE R Panel
The enrolled specimens were tested with the SPOTFIRE R Panel on the SPOTFIRE System (BIOFIRE Diagnostics, Salt Lake City, UT, USA) from September to December 2023, by 2 operators blinded to the RP2.1plus results.Briefly, archived specimens were brought to ambient temperature before testing.The Class II A2 biosafety cabinet work area and the pouch-loading station were cleaned with disinfectant wipes.A SPOTFIRE pouch was removed from the vacuum-sealed package and properly labelled.The pouch, the red-capped sample injection vial and the blue-capped hydration injection vial were inserted into the loading station.The hydration injection vial was unscrewed and inserted into the pouch hydration port to reconstitute the lyophilized reagents.For swabs in viral transport medium, each specimen was thoroughly homogenized and dispensed into the sample injection vial (containing sample buffer) using the transfer pipette provided (approximated 0.3 mL).For posterior oropharyngeal saliva and LRT specimens, 0.5 mL of each specimen was mixed with an equal volume of working Sputasol (Oxoid Ltd., Basingstoke, UK) at ambient temperature, and 0.3 mL of the liquefied specimen was dispensed into the sample injection vial containing sample buffer.After adding the specimen, the cap of sample injection vial was closed tightly and the specimen was mixed with sample buffer by gently inverting 3 times.The sample injection vial was unscrewed after 5 s and inserted into the pouch sample port, and the specimen was pulled into the pouch by vacuum.On the touchscreen of the SPOTFIRE System, an available module was selected.The pouch and specimen barcodes were scanned.The pouch was then inserted into the module with blinking blue light.The pouch was then pulled into the chamber, and the run started automatically.

Discrepant Analysis
The RP2.1plus was the primary comparator for evaluating the performance of the SPOTFIRE R Panel.Discrepant results were verified by alternative methods, as shown in Table 1.

Statistical Analysis
Positive and negative percent agreement (PPA and NPA) were calculated by using the following formulae: PPA = TP/(TP + FN) × 100% NPA = TN/(TN + FP) × 100% FN, FP, TN and TP stand for the number of false-negative, false-positive, true-negative and true-positive specimens, respectively.The 95% confidence intervals (CI) for PPA and NPA were calculated using the online version of GraphPad software (modified Wald method) [17].

Performance of the SPOTFIRE R Panel
A total of 153 runs were performed for 151 specimens, and the results of 2 were invalid due to internal process control failure (2/153, 1.31%).The repeated runs were valid.
Table 2 summarizes the frequency of pathogen detection for the URT specimens.The overall positive rate of the SPOTFIRE R and RP2.1plus were the same (121/133, 90.98%), and full concordance was observed for 109 specimens (81.95%).From the raw data, the SPOTFIRE R Panel detected more pathogens than the RP2.1plus in 18 specimens and vice versa in 5 specimens.

Not detected 12
From the raw data, SPOTFIRE R Panel detected more pathogens than RP2.1plus in 18 specimens, and vice versa in 5 specimens.
Table S3 summarizes the results of discrepant analysis for co-detections.From the raw data, the SPOTFIRE R Panel detected more pathogens than the RP2.1plus in 18 specimens and vice versa for 5 specimens.After discrepant analysis, the SPOTFIRE R Panel detected more pathogens than the RP2.1plus in 15 specimens and vice versa for 3 specimens.
Table 3 summarizes the per-target performance of the SPOTFIRE R Panel for the URT specimens after resolution of discrepancies.The PPAs ranged from 88.24 to 100% and NPAs ranged from 97.41 to 100%.For both the LRT specimens and EQAP samples, the results of the SPOTFIRE R Panel were fully concordant with the RP2.1plus (Tables S4 and S5).

Discussion
From the late COVID-19 pandemic, a rebound in the activities of non-SARS-CoV-2 respiratory pathogens has been observed worldwide [19][20][21][22][23][24].In clinical laboratories, the attention and resources have been shifting from SARS-CoV-2 detection to multiplex detection of SARS-CoV-2 plus other respiratory pathogens.At the time of writing, Hong Kong is experiencing the third influenza season since the end of the COVID-19 pandemic.The first post-COVID-19 influenza season lasted from April to May, and the second one lasted from August to November in 2023 [25].Compared with 2022, the average number of multiplex testings, the positive rate and the diversity of pathogens detected in our laboratory rose two-to four-fold in 2023.It can be speculated that a two-third reduction of the existing assay runtime may help alleviate the increasing test burden and streamline laboratory operations, and this warrants further cost-effectiveness analysis.
We enrolled respiratory specimens throughout the first two post-COVID-19 influenza seasons, for the sake of including the latest circulating strains.Owing to the scope of study, we did not perform strain characterization, albeit further genomic studies could be of epidemiological interest.
There are limited evaluation data on the clinical performance of the SPOTFIRE R Panel in the literature.The most comprehensive study was pursued by the manufacturer for substantial equivalence determination to other FDA-cleared comparator methods [26].A combination of prospectively collected and archived nasopharyngeal swabs (NPS) were used.The prospective study was conducted at four U.S. and one ex-U.S. clinical setting from December 2020 to June 2021.A total of 1120 NPS were enrolled, and the results were compared to a combination of two FDA-cleared panels.The PPA and NPA were 96.3-100% and 90.6-100%, respectively.On the other hand, 542 archived NPS from various clinical laboratories around the world were tested at the four U.S. sites.The PPA and NPA were 96.0-100% and 96.7-100%, respectively.The NPA of rhinovirus/enterovirus (RV/EV) for prospectively collected NPS was the lowest (695/767, 90.6%).Discrepant analysis confirmed the presence of RV/EV in 54 out of 72 discordant specimens (75%), and the NPA was 97.65% (749/767) after discrepant analysis.Similarly, the SPOTFIRE R Panel appeared to be more sensitive to rhinovirus in our study.Rhinovirus was identified by the SPOTFIRE R Panel only in 11 specimens and by the RP2.1plusonly in 1 specimen.Summing up our findings, the NPA and PPA of all pathogen targets were above 90% except the PPA of adenovirus (15/17, 88.24%).Discrepant analysis revealed that the Ct values of adenovirus were 40 in the two false-negative specimens.The PPA of adenovirus was 97% (prospectively collected NPS, 32/33) and 100% (archived NPS, 31/31) in the study by the manufacturer.Stochastic effects on the amplification of low-viral-load specimens and the relatively small number of adenovirus-positive specimens might account for the low PPA of adenovirus in our study.
For the RP2.1plus, a prospective clinical evaluation was pursued by the manufacturer to assess the performance for SARS-CoV-2 detection (details from the package insert of the RP2.1plus,BFR0000-8307-03 June 2022).The study was conducted at three geographically distinct sites in the U.S. from July to October 2020.A total of 524 NPS were enrolled, and the results were compared with a composite comparator of three tests with FDA emergency use authorization.The PPA and NPA were 98.4% (61/62) and 98.9% (457/462), respectively.For non-SARS-CoV-2 pathogens, the performance of the RP2.1plus was compared with its prior version, the RP2plus, using 220 archived specimens.The overall PPA and NPA were 97.6% and 99.8%, respectively, excluding MERS-CoV.
Notably, the SPOTFIRE R Panel identified more co-detections than the RP2.1plus.The additional targets co-detected were rhinovirus (n = 11), SARS-CoV-2 (n = 2), adenovirus (n = 2) and parainfluenza virus 4 (n = 1).From the literature, the clinical outcome of respiratory co-infection may vary for different pathogen combinations.For instance, Esper and co-authors reported that influenza patients co-infected with rhinovirus tended to have milder disease [27].On the other hand, co-infection of rhinovirus might be associated with refractory M. pneumoniae pneumonia and a higher frequency of B. pertussis LRT infection among hospitalized children [28,29].Furthermore, concomitant infection of M. pneumoniae or parainfluenza virus 4 in COVID-19 patients was associated with worse clinical outcomes [30,31].A more complete picture of microbial profiles with accumulated knowledge on the clinical outcomes of co-infection may aid etiologic diagnosis and prognosis.
In addition to URT specimens, we included 13 LRT specimens for compatibility testing.The results of both assays were fully concordant.A similar study was pursued by Hughes and co-authors to evaluate the performance of the RP2 for viral pathogen detection in LRT specimens, using the BIOFIRE Pneumonia Panel as comparator [32].A total of 200 LRT specimens were enrolled, encompassing bronchoalveolar lavage fluid, bronchial wash, sputum and tracheal aspirate.The overall PPA and NPA were 87% (71-95%) and 100% (99-100%), respectively.Further studies with larger and more representative sample pools are warranted to thoroughly assess the performance for LRT specimens.
This study was constrained by the small sample size of LRT specimens, negative specimens and specimens positive for rare pathogens, including Bordetella parapertussis, Bordetella pertussis and Chlamydia pneumoniae.An increased sample size might allow for better assessment of assay performance with consideration of local prevalence.For the SPOTFIRE R Panel, the spectrum of testing is narrower than that of the RP2.1plus.It does not include MERS-CoV and cannot distinguish between subtypes of seasonal coronavirus and parainfluenza virus.Concerning alternative methods for discrepant analysis, we did not have complete information on the detection targets and performance characteristics for some assay, and with different assay principles it is not easy to compare their sensitivity with the SPOTFIRE R Panel or RP2.1plus directly.Similar to other molecular assays, neither the SPOTFIRE R Panel nor RP2.1plus provides information on the viability or quantity of the pathogens detected or distinguishing between co-detection and co-infection.In this regard, the valuable data on target Ct values and melt curves may be useful to laboratorians and clinicians for better result interpretation and patient management.

Conclusion
The performance of the SPOTFIRE R Panel was comparable to that of the RP2.1plus.
the SPOTFIRE R Panel is about 15 min which is one-third of the RP2.1plus.The pathogen targets of the SPOTFIRE R Panel are the same as those of the RP2.1plus except the Middle East respiratory syndrome coronavirus (MERS-CoV), and the subtype results of the seasonal coronavirus (229E, NL63, HKU1 and OC43) and parainfluenza virus (type 1 to 4) are not available.similar to that of the FilmArray Respiratory Panel from the same company, which automates and integrates quality-controlled nucleic acid extraction, reverse transcription, nested amplification of pathogen targets, fluorescent signal detection and melt curve analysis of amplified targets in a closed, disposable pouch, with data interpreted automatically.A comparison between the SPOTFIRE R Panel and the latest version of FilmArray Respiratory Panel (RP2.1plus) is shown in Figure 1.The SPOTFIRE R Panel is compatible with the novel SPOTFIRE System, whereas the RP2.1plus is run on FilmArray 2.0 or TORCH.The runtime of the SPOTFIRE R Panel is about 15 min which is one-third of the RP2.1plus.The pathogen targets of the SPOTFIRE R Panel are the same as those of the RP2.1plus except the Middle East respiratory syndrome coronavirus (MERS-CoV), and the subtype results of the seasonal coronavirus (229E, NL63, HKU1 and OC43) and parainfluenza virus (type 1 to 4) are not available.

Table 1 .
Methods used for discrepant analysis.

Table 2 .
Frequency of pathogen detection for upper respiratory tract specimens.
b SPOTFIRE R Panel detected more pathogens than RP2.1plus for 5 specimens, and vice versa.cCoronavirusNL63,parainfluenzavirus 2 and 4 were detected by RP2.1plus while seasonal CoV, PIV and rhinovirus/enterovirus were detected by SPOTFIRE R Panel.SPOTFIRE R Panel detected more pathogen types than RP2.1plus albeit the number of positive calls were the same.dRP2.1plusdetectedmore pathogens than SPOTFIRE R Panel for 5 specimens.For 1 specimen, the result of RP2.1plus was 'influenza A H1-2009' and that of SPOTFIRE R Panel was 'influenza A no subtype'.The result was classified as partial concordance with the same number of pathogens detected.eSPOTFIRERPanel detected more pathogens than RP2.1plus for all 18 specimens.For details of each pathogen target, please refer to TableS2.

Table 3 .
Per-target performance of the SPOTFIRE R Panel for upper respiratory tract specimens.