BioFire FilmArray Pneumonia Panel enhances detection of pathogens and antimicrobial resistance in lower respiratory tract specimens

Study design and samples

Sputum, tracheal aspirates, bronchoalveolar lavage fluid (BAL), and mini-BAL specimens, which were submitted to the clinical laboratory for microbiological evaluation, were collected at the Nagasaki University Hospital between October 2020 and January 2021. The hospital had 874 beds, and the average number of inpatients and outpatients were 669 and 1653 patients per day, respectively, during this period. The quality of sputum and tracheal aspirates was assessed according to the Miller and Jones classification [6]. Samples were randomly selected from specimens P1 (purulent, grade 1—pus amounting to less than one-third of the specimen), P2 (purulent, grade 2—pus amounting to one-third to two-thirds of the specimen), and P3 (purulent, grade 3—pus amounting to more than two-thirds of the specimen). De-duplication was performed if samples were repeatedly collected from a single patient, and samples from unique patients were included in this study. The specimens were stored at − 80 °C until assay using the PN panel.

Culture methods

Sputum and tracheal aspirate samples, which were prepared using Sputazyme (Kyokuto Pharmaceutical Industrial Co., Ltd.), and BAL and mini-BAL samples were streaked using 10 µL loop and cultured on Nissui Separated Plate Sheep Blood Agar/Chocolate Agar EXII (NISSUI PHARMACEUTICAL CO., LTD.), CHROMagar Candida/BTB agar (Prepared media) (KANTO CHEMICAL CO., INC.). Additionally, if the physician specifically requested testing for methicillin-resistant Staphylococcus aureus (MRSA), the samples were cultured on Pourmedia MRSA SELECTIVE AGAR II (Eiken Chemical Co., Ltd.). All bacteria that were considered pathogens, excluding normal flora, were picked up and identified even if they were cultured in rare or few quantities. After pure culture, isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), MALDI Biotyper system (Bruker Daltonik, GmbH). Streptococcus pneumoniae was identified using the optochin disc diffusion test.

Antimicrobial susceptibility was determined using a BD Phoenix M50 (Becton Dickinson). MRSA and extended-spectrum β-lactamase (ESBL) producers were automatically determined using BD Phoenix M50. Antimicrobial susceptibility of Haemophilus influenzae was determined using minimal inhibitory concentration (MIC) plates customized by Eiken Chemical Co., Ltd.

Analysis using the BioFire FilmArray Pneumonia Panel

The BioFire FilmArray Pneumonia Panel (bioMérieux Japan Ltd.), which has been approved by Food and Drug Administration for use in clinical settings in the United States, simultaneously detects 18 bacterial targets (Acinetobacter calcoaceticus-baumannii complex, Enterobacter cloacae complex, Escherichia coli, H. influenzae, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus species, Pseudomonas aeruginosa, Serratia marcescens, S. aureus, Streptococcus agalactiae, S. pneumoniae, Streptococcus pyogenes, Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae), eight viral targets (adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, influenza A, influenza B, parainfluenza virus, and respiratory syncytial virus), and seven targets related to antimicrobial resistance (mecA/mecC and staphylococcal cassette chromosome mec [SCCmec] right-extremity junction [MREJ], KPC, NDM, OXA-48-like, VIM, IMP, and CTX-M) on the FilmArray system.

The BioFire FilmArray Pneumonia Panel pouch, which is a closed and disposable system, contains all the reagents required for nucleic acid extraction and purification, reverse transcription, and PCR. The stored specimens were analyzed using the PN panel. Briefly, the pouch was hydrated by the hydration injection vial via the pouch hydration port. Approximately 200, 50, or 10 µL of the specimen was mixed with the sample buffer using a vortex mixer. After flashing in a centrifuge to remove foam, the mixture was transferred to the sample injection vial and injected into the pouch via the pouch sample port. The sample preparation process, including the sample volume used for the assay, was off-label. The prepared pouch was inserted into the instrument, and subsequent assay steps (nucleic acid extraction and purification, nested multiplex PCR, and detection of each target in the array) were automatically performed on the system. The system reports the detection results of pathogen and antimicrobial resistance markers and the semiquantification results of the detected bacterial targets excluding three atypical bacteria.

Comparison between the PN panel and bacterial culture-based methods

The detection results of the PN panel were compared with those of bacterial culture methods and AST, and positive and negative percent agreements (PPA and NPA) were calculated as follows: PPA, the number of concordant positive results divided by the number of all positive results by culture-based methods; NPA, the number of concordant negative results divided by the number of all negative results by culture-based methods. The 95% confidence intervals were calculated using R (version 3.5.2) [7].

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