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BioFire FilmArray respiratory panel RP2.1 for SARS-CoV-2 detection: The pitfalls

Published:August 08, 2022DOI:https://doi.org/10.1016/j.jinf.2022.07.030
      Dear Editor,
      We read with great interest the Livingstone et al. article describing the results of testing 4,640 patients for SARS-CoV-2 through the acute medical admissions pathway with BioFire FilmArray Respiratory PCR Panel 2.1 plus (BioFire RP2.1 plus, BioFire Diagnostics, bioMérieux, Marcy l'Etoile, France).
      • Livingstone R.
      • Lin H.
      • Brendish N.J.
      • Poole S.
      • Tanner A.R.
      • Borca F.
      • et al.
      Routine molecular point-of-care testing for SARS-CoV-2 reduces hospital-acquired COVID-19.
      The authors concluded that the use of BioFire RP2.1 for COVID-19 significantly reduced the time to obtain results spent on assessment cohort wards and the proportion of healthcare-ssociated-COVID-19 infection.
      • Livingstone R.
      • Lin H.
      • Brendish N.J.
      • Poole S.
      • Tanner A.R.
      • Borca F.
      • et al.
      Routine molecular point-of-care testing for SARS-CoV-2 reduces hospital-acquired COVID-19.
      BioFire RP2.1 plus is a multiplex nested PCR allowing the simultaneous detection of four bacteria and 19 viruses, including SARS-CoV-2 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). BioFire RP2.1 (not including MERS-CoV) was launched for emergency use authorization (EUA) in Taiwan in May 2020 and introduced in the China Medical University Hospital (CMUH), a 2,100-bed university-affiliated hospital located in Taichung, Taiwan, to replace the BioFire RP panel in February 2021.
      From May 2021 to 5th July 5, 2022, a total of 3,710 nasopharyngeal swab specimens from 3,710 patients with respiratory tract infection or suspected COVID-19 were submitted for respiratory pathogen detection using the BioFire RP2.1 panel in the CMUH. Among these specimens, 561 (15.1%) were positive for one of the target pathogens in the panel, and 56 (10.0%) were positive for SARS-COV-2 (Table 1). Among the 56 SARS-CoV-2 positive specimens, 11 (19,6%) were also positive for other pathogens. The concomitant pathogens identified, along with SARS-CoV-2, were adenovirus plus human rhinovirus/enterovirus (n = 3), human rhinovirus/enterovirus plus parainfluenza virus (n = 2), human rhinovirus/enterovirus alone (n = 3), adenovirus alone (n = 2), and coronavirus HKU1 alone (n = 2). Among the 56 specimens positive for SARS-CoV-2 by BioFire RP2.1, 47 (83.9%) were rechecked by either cobas Liat or cobas 6800 systems (Roche Diagnostics Basel, Switzerland) due to the request of cycle threshold (Ct) values by the attending physicians (Table 1), and 20 (42.6%, 20/47) of them became negative by either system.
      Table 1Detection of pathogens from nasopharyngeal swab specimens using the BioFire FilmArray Respiratory PCR Panel 2.1 (BioFire RP2.1) and/or cobas Liat or cobas 6800 Systems and from 14th May 2021 to 5th July 5 2022.
      No.Age/sexDate of testBioFire RP2.1detectedAdditional tests
      cobas Liat

      System results for SARS-CoV-2 (cycle threshold value)
      cobas 6800System results for SARS-CoV-2 (cycle threshold value, orf1ab/E genes)
      127/F2021/5/20Coronavirus HKU1

      SARS-CoV-2
      NDPositive (33.20/33.73)
      272/M2021/5/20Coronavirus HKU1

      SARS-CoV-2
      Positive (28)Positive (-/36.71)
      375/M2021/5/22SARS-CoV-2NDPositive (18.89/18.62)
      444/M2021/5/23SARS-CoV-2Positive (13.4)ND
      537/F2021/5/29SARS-CoV-2NDPositive (30.97/32.22)
      644/F2021/6/1Human rhinovirus/enterovirus

      SARS-CoV-2
      NDNegative
      732/F2022/4/20Human rhinovirus/enterovirus

      SARS-CoV-2
      NDNegative
      843/M2022/4/25SARS-CoV-2NegativeND
      91/M2022/5/5SARS-CoV-2NegativeND
      102/F2022/5/7SARS-CoV-2

      Adenovirus

      Human rhinovirus/enterovirus
      NDND
      1144/F2022/5/11SARS-CoV-2Positive (29.2)ND
      122/M2022/5/11SARS-CoV-2

      Human rhinovirus/enterovirus
      NDND
      131/M2022/5/14SARS-CoV-2Positive (11.7)ND
      143/F2022/5/16SARS-CoV-2

      Human rhinovirus/enterovirus

      Parainfluenza virus 4
      NegativeND
      1572/M2022/5/21SARS-CoV-2Positive (11.4)ND
      162/M2022/5/21SARS-CoV-2NegativeND
      172/M2022/5/23SARS-CoV-2NegativeND
      1817/M2022/5/24SARS-CoV-2NegativeND
      1972/F2022/5/24SARS-CoV-2NegativeND
      2081/F2022/5/24SARS-CoV-2Positive (34.2)ND
      2117/M2022/5/24SARS-CoV-2NegativeND
      228/M2022/5/25SARS-CoV-2NDND
      231/M2022/5/25SARS-CoV-2NDND
      246/F2022/5/25Adenovirus

      SARS-CoV-2

      H Human rhinovirus/enterovirus
      Positive (14.0)ND
      251/F2022/5/29SARS-CoV-2

      Parainfluenza virus 3
      NDND
      269/F2022/5/30SARS-CoV-2NDND
      272/M2022/6/5Adenovirus

      SARS-CoV-2

      Human rhinovirus/enterovirus
      NegativeND
      2864/M2022/6/7SARS-CoV-2NDNegative
      299/F2022/6/9Adenovirus

      SARS-CoV-2
      NDND
      3056/M2022/6/10SARS-CoV-2NDND
      311/F2022/6/10SARS-CoV-2NegativeND
      322/M2022/6/12SARS-CoV-2NDND
      3359/M2022/6/13SARS-CoV-2Positive (31.3)ND
      3478/F2022/6/20SARS-CoV-2Positive (16.4)ND
      353/F2022/6/21SARS-CoV-2Positive (15)ND
      365/M2022/6/22SARS-CoV-2Positive (32.6)ND
      371/M2022/6/22SARS-CoV-2Positive (29.8)ND
      3871/M2022/6/23SARS-CoV-2Positive (23.6)ND
      3970/F2022/6/23SARS-CoV-2Positive (29.5)ND
      403/F2022/6/23SARS-CoV-2Positive (33.0)ND
      414/F2022/6/24SARS-CoV-2Positive (36.2)ND
      423/F2022/6/24SARS-CoV-2Positive (31.2)ND
      4315/F2022/6/28SARS-CoV-2Positive (27.3)ND
      4469/M2022/6/29SARS-CoV-2Positive (14.7)ND
      4533/M2022/6/30SARS-CoV-2NegativeND
      465/M2022/6/30SARS-CoV-2Positive (20.3)ND
      4759/M2022/7/1SARS-CoV-2Positive (12.3)ND
      484/F2022/7/1SARS-CoV-2Positive (16.6)ND
      4932/M2022/7/2SARS-CoV-2NegativeND
      508/M2022/7/2SARS-CoV-2Positive (32.6)ND
      512/M2022/7/4SARS-CoV-2NegativeND
      521/M2022/7/5SARS-CoV-2NegativeND
      534/M2022/7/5SARS-CoV-2

      Human rhinovirus/enterovirus

      Parainfluenza virus 4
      NegativeND
      5413/F2022/7/5SARS-CoV-2NegativeND
      553/F2022/7/7SARS-CoV-2NegativeND
      5698/M2022/7/7SARS-CoV-2Positive (15.4)ND
      The results in boldface indicate the presence of negative results by either the cobas Liat or cobas 6800 system
      ND, not done.
      A multicenter evaluation of BioFire RP2.1 for the detection of SARS-CoV-2 in 524 nasopharyngeal swab samples was conducted by Berry et al. In this study, one or more targets on the panel were detected in 19.3% (n = 101) of specimens tested, with SARS-CoV-2 detected in 12.6% (n = 66) of specimens.
      • Creager H.M.
      • Cabrera B.
      • Schnaubelt A.
      • Cox J.L.
      • Cushman-Vokoun A.M.
      • et al.
      Clinical evaluation of the BioFire Respiratory Panel 2.1 and detection of SARS-CoV-2.
      Human rhinovirus/enterovirus was also detected in 32.7% (n = 33) and adenovirus in 3.0% (n = 3) of positive specimens, with one dual positive for both SARS-CoV-2 and adenovirus being detected. They revealed that SARS-CoV-2 results obtained from the BioFire RP2.1 were highly concordant with the composite reference results by three SARS-CoV-2 EUA tests, exhibiting 98.4% (61/62) positive percent agreement (PPA) and 98.9% (457/462) negative percent agreement (NPA).
      • Creager H.M.
      • Cabrera B.
      • Schnaubelt A.
      • Cox J.L.
      • Cushman-Vokoun A.M.
      • et al.
      Clinical evaluation of the BioFire Respiratory Panel 2.1 and detection of SARS-CoV-2.
      They concluded that the BioFire RP2.1 exhibited excellent performance in the detection of SARS-CoV-2.
      • Berry G.J.
      • Zhen W.
      • Smith E.
      • Manji R.
      • Silbert S.
      • Lima A.
      • et al.
      Multicenter evaluation of the BioFire Respiratory Panel 2.1 (RP2.1) for detection of SARS-CoV-2 in nasopharyngeal swab samples.
      In this study, the five false positive results by BioFire RP2.1 were further analyzed and the authors demonstrated that the concentration of SARS-CoV-2 in the specimens was near the limit of detection (LOD) for both the BioFire RP2.1 and the comparator assays.
      • Berry G.J.
      • Zhen W.
      • Smith E.
      • Manji R.
      • Silbert S.
      • Lima A.
      • et al.
      Multicenter evaluation of the BioFire Respiratory Panel 2.1 (RP2.1) for detection of SARS-CoV-2 in nasopharyngeal swab samples.
      Creager et al. evaluated the performance of the BioFire Respiratory Panel 2.1 (RP2.1) in the detection of SARS CoV-2 in comparison to three other SARS CoV-2 EUA assays.
      • Creager H.M.
      • Cabrera B.
      • Schnaubelt A.
      • Cox J.L.
      • Cushman-Vokoun A.M.
      • et al.
      Clinical evaluation of the BioFire Respiratory Panel 2.1 and detection of SARS-CoV-2.
      In the studies, the RP2.1 panel had 98 % PPA (48/49) and 100 % NPA (49/49), suggesting that the BioFire RP2.1 assay can be used to detect acute cases of SARS CoV2, even among patients with a low viral titer later in disease presentation.
      • Creager H.M.
      • Cabrera B.
      • Schnaubelt A.
      • Cox J.L.
      • Cushman-Vokoun A.M.
      • et al.
      Clinical evaluation of the BioFire Respiratory Panel 2.1 and detection of SARS-CoV-2.
      Eckbo et al. compared BioFire RP2.1 and the laboratory-developed test for 57 nasopharyngeal swab samples, including 30 clinical specimens (E gene Ct values <25 [n = 5], Ct 21-δ35 [n = 10], Ct >35-δ40 [n = 10], and negative [n = 5] and 27 tests for limit of detection.
      • Eckbo E.J.
      • Locher K.
      • Caza M.
      • Li L.
      • Lavergne V.
      • Charles M.
      Evaluation of the BioFire COVID-19 test and Respiratory Panel 2.1 for rapid identification of SARS-CoV-2 in nasopharyngeal swab samples.
      They demonstrated 100% concordance between the tests, and acceptable performance of BioFire RP2.1 at their stated limits of detection.
      • Eckbo E.J.
      • Locher K.
      • Caza M.
      • Li L.
      • Lavergne V.
      • Charles M.
      Evaluation of the BioFire COVID-19 test and Respiratory Panel 2.1 for rapid identification of SARS-CoV-2 in nasopharyngeal swab samples.
      However, Tazi et al compared two PCR assays, BioFire RP2.1 plus and their laboratory's reference test, MAScIR SARS-CoV-2 M kit 2.0, a triplex real-time RT-PCR, using TaqMan technology, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and S genes.
      • Tazi S.
      • Kabbaj H.
      • Zirar J.
      • Zouaki A.
      • El Amin G.
      • El Himeur O.
      • et al.
      Comparative performance evaluation of FilmArray BioFire RP2.1 and MAScIR 2.0 assays for SARS-CoV-2 detection.
      The results were compared, and each discrepant sample with sufficient volume underwent a third test using ARGENE SARS-CoV-2 R-GENE kit, a triplex real-time RT-PCR, which also used TaqMan technology, targeting SARS-CoV-2 N (Nucleocapsid) and RdRp genes. Of the 80 specimens positive for BioFire RP2.1 Plus, 21 (26.3%) had discordant results on MAScIR, and only 11 could be tested on ARGENE, revealing negative results in five cases.
      • Eckbo E.J.
      • Locher K.
      • Caza M.
      • Li L.
      • Lavergne V.
      • Charles M.
      Evaluation of the BioFire COVID-19 test and Respiratory Panel 2.1 for rapid identification of SARS-CoV-2 in nasopharyngeal swab samples.
      These results led to them consequently retaining the SARS-CoV-2 positive results of these discordant samples on BioFire RP2.1 plus, regardless of the detection of one or both targets.
      • Tazi S.
      • Kabbaj H.
      • Zirar J.
      • Zouaki A.
      • El Amin G.
      • El Himeur O.
      • et al.
      Comparative performance evaluation of FilmArray BioFire RP2.1 and MAScIR 2.0 assays for SARS-CoV-2 detection.
      Although RT-PCR is the gold standard for the diagnosis of COVID-19, its diagnostic performance can vary widely owing to the lack of standardization of assays. The target genes (LOD, copies/mL) of BioFire RP2.1, cobas Liat and cobas 6800 were spike (S) and transmembrane glycoproteins (M) (160), orf 1ab and nucleocapsid protein (12), and orf 1ab and envelope protein (46), respectively. Additionally, for BioFire RP2.1, SARS-CoV-2 is reported qualitatively as detected if either the S or M gene assays are positive and Ct values are provided. As a result, it is difficult to conclude the false-negative or -positive results created by different assays because different gene targets and LODs are present in different assays. However, there is a clinical dilemma due to the change in the positive report by BioFire RP2.1 to negative results by another quantitative RT-PCR assay. In this study, 42.6% of BioFire RP2.1 SARS-CoV-2 positive results became negative by using other PER systems. Further studies are needed to investigate this discrepancy.
      In conclusion, we agree with Tazi et al. ’s recommendation that SARS-CoV-2 positive results by BioFire RP2.1, regardless of the detection of one or both targets, should be retained, and other quantitative RT-PCR assays should be performed to confirm the results.

      Funding

      The authors have not declared a specific grant for this research from any funding agency in the public, commercial, or not-for-profit sector.

      Patient and public involvement

      Patients and/or the public were not involved in the design, conduct, reporting, or dissemination of this research.

      Patient consent for publication

      Not required.

      Ethical approval information

      Not required.

      Data sharing statement

      Not required.

      Declaration of Competing Interest

      The authors declare no conflict of interest.

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