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False negative results caused by erroneous automated result interpretation algorithm on the FilmArray 2.0 instrument

Infections of the central nervous system (CNS) such as meningitis and encephalitis are potentially lifethreatening [1]. They can be caused by a variety of pathogens, which often share similar clinical presentations [2]. Moreover, cerebrospinal fluid (CSF) cultures are confounded by antibiotic pre-treatment prior to sample acquisition, resulting in frequent negative results for bacterial meningitis [3]. Fast and accurate laboratory diagnosis of CNS infection is critical for timely institution of appropriate therapy [4]. To address this clinical need, we conducted a verification study to evaluate the diagnostic performance of the FDAapproved FilmArray meningitis/encephalitis (ME) syndromic panel (adopted on the sample-to-result FilmArray 2.0 instrument, BioFire Diagnostics LLC, Salt Lake City, UT, USA), and subsequently applying it in routine clinical testing [5]. Here, we report the findings of an investigation into a series of false-negative results from the ME panel. Twenty-six de-identified residual CSF samples and two human-contrived CSF positive controls that have been previously tested by CSF culture and/or targetspecific polymerase chain reaction (PCR) tests were included in this study (Table 1). The mean testing time interval between the culture/PCR tests and the FilmArray ME panel was 4 days (95% Confidence interval [CI]: 2–6 days). The two human-contrived CSF positive controls were prepared from commercially-available NATtrol ME controls (ZeptoMetrix Corp., Franklin, MA, USA). The first positive control was comprised of cytomegalovirus, echovirus type 11, human herpesvirus 6, herpes simplex virus 2, varicella zoster virus, human parechovirus and Cryptococcus gattii. The second positive control was comprised of Escherichia coli K1, Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes and herpes simplex virus 1. All tests were performed according to the instructions of the manufacturer. The FilmArray 2.0 instrument is a multiplex PCR analyser with integrated sample preparation, and the ME panel is designed to detect 14 pathogens that are most commonly associated with community-acquired ME. All the reagents required for the analytical step are pre-packaged into the reagent storage compartments of the FilmArray pouch. Nucleic acid extraction, reverse-transcription PCR, target-specific nested PCR, and high resolution melting were all performed within the self-contained and disposable pouch. Target-specific nested PCR and high resolution melting were performed in three replicates. After completing the analytical run, the raw data were automatically interpreted by the onboard instrument software. At least two of the three melt curves must meet several analytical criteria in order for a target to be called as a positive detection by the FilmArray 2.0 software. First, the melt curve profiles need to be recognised as a PCR product by the software. It is unclear how the software achieves this. Second, the melting temperature (Tm) of the melt curves *Corresponding author: Chun Kiat Lee, Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, 5, Lower Kent Ridge Road, 119074, Singapore, Singapore, Phone: +65-6772-4175, Fax: +65-6772-4407, E-mail: chunkiatlee1983@gmail.com; chun_kiat_lee@nuhs.edu.sg Lily Chiu, Ka Lip Chew, Benedict Yan and Roland Jureen: Department of Laboratory Medicine, National University Health System, Singapore, Singapore Gabriel Yan: Department of Medicine, National University Health System, Singapore, Singapore Tze Ping Loh: Department of Laboratory Medicine, National University Health System, Singapore, Singapore; and Biomedical Institute for Global Health Research and Technology, National University of Singapore, Singapore, Singapore