- Email: [email protected]
Detection of Norovirus by BD MAX™, Xpert® Norovirus, and xTAG® Gastrointestinal Pathogen Panel in stool and vomit samples
Accepted Manuscript Title: Detection of Norovirus by BD MAXTM , Xpert® Norovirus, and xTAG® Gastrointestinal Pathogen Panel in Stool and Vomit Samples Authors: Martin P. McHugh, Daniel Guerendiain, Alison Hardie, Juliet Kenicer, Laura MacKenzie, Kate E. Templeton PII: DOI: Reference:
S1386-6532(18)30155-0 https://doi.org/10.1016/j.jcv.2018.06.005 JCV 4014
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
23-2-2018 9-5-2018 7-6-2018
Please cite this article as: McHugh MP, Guerendiain D, Hardie A, Kenicer J, MacKenzie L, Templeton KE, Detection of Norovirus by BD MAXTM , Xpertreg Norovirus, and xTAG® Gastrointestinal Pathogen Panel in Stool and Vomit Samples, Journal of Clinical Virology (2018), https://doi.org/10.1016/j.jcv.2018.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of Norovirus by BD MAXTM, Xpert® Norovirus, and xTAG® Gastrointestinal Pathogen Panel in Stool and Vomit Samples
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Running Title: Stool and Vomit Detection of Norovirus by Nucleic Acid Amplification Tests
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Martin P. McHugh1, Daniel Guerendiain1, Alison Hardie1,† , Juliet Kenicer1,* , Laura
Department of Molecular Microbiology, Royal Infirmary of Edinburgh, Edinburgh, UK
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MacKenzie1, Kate E. Templeton1, #
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preparation of this manuscript
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† Co-author Alison Hardie sadly passed away after a short illness during the
* Current address: Scottish Escherichia coli O157/VTEC Reference Laboratory, Royal
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Infirmary of Edinburgh, Edinburgh, UK
# Corresponding author
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Dr Kate E. Templeton
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Department of Molecular Microbiology Royal Infirmary of Edinburgh 51 Little France Crescent, Edinburgh, EH16 4SA, UK Email: [email protected]
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Abstract word count: 239/250 Text word count: 1880/2500
Highlights:
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Molecular assays reliably detect norovirus from stool and vomit Fast turnaround and simplicity of Xpert Norovirus suit it to near patient testing The BD-MAX System is useful for high-throughput norovirus testing xTAG GPP had lower norovirus sensitivity/specificity compared to other platforms
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Abstract
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Background: Norovirus is a leading cause of infectious gastroenteritis, characterized by
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outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests
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commercial platforms now available.
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allow rapid and sensitive laboratory diagnosis of norovirus, with a number of
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Objectives: Evaluate the performance of the Becton Dickinson BD-MAXTM System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus
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Assay and BD-MAXTM in vomit samples. Study design: 163 diarrhoeal stool samples were tested on four diagnostic systems
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(laboratory-defined real time RT-PCR (assigned as gold standard), BD MAXTM, Xpert®
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Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms. Results: In stool, sensitivity and specificity of the BD-MAXTM was 96.8 % and 100 %, for Xpert® Norovirus Assay was 91.9 % and 100 %, and for xTAG® GPP was 79.0 % and 87.1
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%. In vomit samples positive and negative percent agreement was 95.6 % and 92.0 %, between the BD-MAXTM and Xpert® Norovirus. Conclusions: The BD-MAXTM System with user defined settings and the Xpert® Norovirus
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Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a
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number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.
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Abbreviations: Gastrointestinal pathogen panel (GPP)
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Keywords: Norovirus; stool; vomit; Xpert Norovirus; xTAG Gastrointestinal Pathogen
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Panel; BD MAX
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1. Background
Norovirus is a small, non-enveloped, positive stranded RNA virus in the Caliciviridae family. Infection results in diarrhoea, vomiting, abdominal pain and fever lasting 1-3
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days, although only 10-12% of individuals seek medical attention [1]. Norovirus is the
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most common cause of acute gastroenteritis worldwide and causes outbreaks in hospitals, nursing homes, cruise ships, and other closed settings [2–5]. There are an
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estimated 19-21 million norovirus cases in the United States every year [6]. Outbreaks of norovirus not only confer significant morbidity but have also been shown to increase allcause mortality, particularly in elderly patients [7]. Norovirus transmission can be interrupted by environmental cleaning, good hand hygiene and isolation or cohorting of
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infected individuals [8,9]. However, in healthcare settings these measures can lead to ward closures, lost bed days, staff absence and increased costs [10,11]. It is essential to identify infected individuals rapidly and accurately so that infection control precautions
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can be used effectively, limiting the impact on patient services [12]. The introduction of real time reverse-transcriptase polymerase chain reaction (RT-PCR)
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improved the laboratory detection of norovirus by providing sensitive and specific
results in a clinically relevant timeframe [2,12]. New assays for norovirus detection must maintain the high sensitivity and specificity already achieved with real time RT-PCR while
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improving turnaround time [13]. The Xpert® Norovirus Assay (Cepheid, Sunnyvale, USA)
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is FDA and CE-IVD approved and provides rapid nucleic acid extraction, amplification,
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and detection with limited operator input, which lends well to near-patient testing
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[14,15]. The BD MAXTM System (Becton Dickinson, Franklin Lakes, USA) offers automated
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nucleic-acid extraction and thermocycling thus reducing laboratory hands-on time and can be run as an “open platform” with user-defined protocols. The xTAG® Gastrointestinal Pathogen Panel (GPP; Luminex, Austin, USA) is an FDA and CE-IVD
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approved multiplex nucleic acid test for detection of up to 15 gastrointestinal pathogens [16], this syndromic approach eliminates the need for multiple microscopic, culture or
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molecular tests. Direct comparison of the performance characteristics for these assays is
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not currently available.
2. Objectives
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Evaluate the performance of the Xpert® Norovirus Assay, BD MAXTM, and xTAG® GPP for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and
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BD-MAXTM in vomit samples.
3. Study Design
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3.1. Patient Samples. Samples from patients with symptoms of gastroenteritis (loose
stools, diarrhoea, vomiting) were identified, related clinical data and laboratory results were collated and samples were anonymised before inclusion in the study. To give a
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more representative collection, multiple samples from local outbreaks were not
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included. Stool samples were collected between October 2011 and November 2013,
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vomit samples were collected between November 2014 and February 2015. Samples
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were stored at -80°C long term, after thawing samples were stored at 2-8°C and tested
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within 24 hours.
3.2. Comparison between Commercial Platforms. The presence of norovirus was determined using a gold standard reference real time RT-PCR assay and results with
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three commercial assays were compared to the reference result. For the reference real time RT-PCR and xTAG-GPP, a 10% suspension of sample prepared in 1 ml sterile water
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(Promega, Madison, USA) was vortexed briefly before being centrifuged (20,000 g, 5
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minutes). Nucleic acid was extracted from 200 µl of supernatant into 100 µl elution buffer by NucliSENS® easyMAG® (bioMérieux, Mary-l’Étoile, France) automated extraction. Real time RT-PCR was performed on the ABI 7500 Fast instrument (Applied Biosystems, Foster City, USA) in a reaction comprised of 10 µl EXPRESS qPCR Supermix
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Universal (Invitrogen, Carlsbad, CA), 2 µl EXPRESS SuperScript® Mix (Invitrogen), 2 µl oligonucleotide mix (as described by Kageyama et al [17]) and 6 µl nucleic acid extract. RNA was reverse transcribed at 50°C for 15 minutes, denatured at 95°C for 20 seconds, then PCR was conducted for 45 cycles of 95°C for 3 seconds and 60°C for 30 seconds.
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The BD MAXTM System is an automated platform for hands-off nucleic acid
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extraction, thermocycling and real-time product detection. Sample suspensions were
prepared as defined above, then 250 µl supernatant loaded into Sample Buffer Tube for extraction with BD MAXTM RNA Extraction Kit RNA-3 (Becton Dickinson). Cartridges were
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loaded into the BD MAXTM System and RNA was reverse transcribed at 50°C for 15
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minutes and after denaturation at 98°C for 20 seconds the PCR was conducted for 45
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cycles of 98°C for 12 seconds and 60°C for 25 seconds. Samples were tested with the
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Xpert® Norovirus Assay (with integrated nucleic acid extraction and real-time RT-PCR)
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and xTAG® GPP (analysed with the MAGPIX® analyser) following manufacturer’s instructions [18,19]. Positive (Norovirus GI and GII) and no-template controls were included with every run of the batch-based assays, and daily with the Xpert assay.
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3.3. Xpert® Norovirus Assay Performance with Vomit Samples. Vomit samples were tested with the BD MAXTM and Xpert® Norovirus Assay. A 10% suspension was made in 1
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ml sterile water (PROMEGA), centrifuged (20,000 g, 5 minutes) and supernatant was
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used for testing on both platforms as described above. Discrepancies between the two platforms were resolved by re-testing with the reference real time RT-PCR described above.
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4. Results 4.1. Comparison between Commercial Platforms. Unformed stool samples from 163 patients were tested with the reference real time RT-PCR assay prior to testing with the
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BD MAXTM, xTAG® GPP and Xpert® Norovirus Assay. Most patients were hospital inpatients (88.3%, n = 144) and the median age was 78 years old (interquartile range 51 to 87 years). In total 62 (38.0%) samples were positive for norovirus by the reference
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assay, 35 were GI and 27 GII. BD MAXTM correctly identified 161 (98.7%) samples, xTAG® GPP 137 (84.0%) and Xpert® Norovirus Assay 158 (96.9%; Table 1). The two false
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negative samples on BD MAXTM were GII by the reference assay (cycle threshold [Ct] not
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available), while all five false negatives on the Xpert® Norovirus Assay were GI (median
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reference Ct 36, range 30 – 38). One sample gave an invalid result on the Xpert®
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Norovirus Assay which on repeat was GI positive, and seven samples generated errors
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which on repeat were negative. Nine GI (median reference Ct 33, range 28 – 39) and four GII samples (median reference Ct 27, range 21-32) were false negative on xTAG® GPP, with an additional 13 norovirus false positives (xTAG® GPP does not report
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genogroup). A comparison of the practical aspects of the assays evaluated here is presented in Table 2.
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4.2. Xpert® Norovirus Assay Performance with Vomit Samples. Based on their
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favourable performance in stool samples, we then evaluated the performance of the BD MAXTM and Xpert® Norovirus assays with vomit samples. Seventy vomit samples were included in the study, 45 (64.3%) were positive for norovirus (1 GI and 44 GII) with the BD MAXTM. Two (2.8%) samples returned invalid Xpert® Norovirus Assay results due to
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no amplification of the internal control, on repeat these were norovirus negative. Positive and negative percent agreement was 96% and 92%, respectively (Table 3). Two samples were false negative and two false positive on the Xpert® Norovirus Assay
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compared to BD MAXTM. Post-discrepant analysis after testing with the reference assay showed that Xpert® Norovirus Assay had correctly identified all 70 samples, while the
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BD MAXTM had two false negative and two false positive calls.
5. Discussion
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This study found norovirus detection from stool was largely comparable with the
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reference assay, the Xpert® Norovirus Assay, and the BD MAXTM open platform (Table 1).
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All assays detected norovirus with higher sensitivity and specificity than the xTAG® GPP
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(Table 1). We also show the Xpert® Norovirus Assay and BD-MAXTM can successfully
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detect norovirus from vomit samples (Table 3). This finding increases the types of samples available for diagnosis of norovirus infection [20]. Our results are comparable to other published studies of the Xpert® Norovirus
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Assay in stool. Gonzalez et al (2016) tested 1,403 stool samples from multiple study sites [14]. By a composite reference test, norovirus GI was present in 115 samples and GII in
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174; the Xpert® Norovirus Assay had sensitivity and specificity >98 % for both
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genogroups. Further studies have shown the Xpert Norovirus assay to have >95% sensitivity and specificity for detection of Norovirus [15,21–23]. The high sensitivity/specificity, rapid run time of 90 minutes, random access loading and low complexity of the Xpert® Norovirus Assay suggest that it could be used in a routine
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laboratory to reduce turnaround time of norovirus detection (Table 2). We also believe the assay could be used as a point of care test in care of the elderly units, emergency departments, and other high-incidence areas.
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The xTAG® GPP is a syndromic diagnostic assay that can detect fifteen enteropathogens in a single reaction. Interestingly, the xTAG® GPP detected 22 other
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enteropathogens in the faecal samples tested here (17 samples were norovirus negative by the reference assay). It was not possible to ascertain the clinical relevance of these calls for other enteropathogens, although it is possible that these were causative agents
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of diarrhoea. We found the xTAG® GPP to have sensitivity of 79% and specificity of 87%
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for norovirus detection, the literature includes some studies reporting poor norovirus
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detection [24–27] as well as studies reporting sensitivity and specificity >90% for
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norovirus [16,28–30]. This discrepancy may reflect the high complexity of the test and
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suggest the need for confirmation of xTAG® GPP norovirus results with another method. Recently, Zhuo et al [31] reported false negative xTAG® GPP results for norovirus GII.2. In our study the four GII false negative samples were positive on all platforms except the
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xTAG® GPP, unfortunately subtype data were not available. Of the nine GI xTAG® GPP false negatives five were also negative with the Xpert® Norovirus Assay, suggesting a
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possible primer/probe mismatch in both these assays, or a failure to detect low viral
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loads (median reference Ct 36, range 30 – 38). We note our reference assay does not include an internal control so the 13 xTAG false positive calls could actually be genuine in the presence of a sample inhibitory in the reference assay. However, none of the xTAG GPP false positives were positive with the BD MAXTM or Xpert® Norovirus Assay,
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the latter of which does include an internal control. We ran the xTAG® GPP assay in batches of 96, using the full capacity of the MAGPIX reader which resulted in a turnaround time of 24 hours including extraction. Others have reported lower
turnaround time would have to be made for routine use of the xTAG® GPP.
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turnaround times of around 6-8 hours, a balance between costs per sample and
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Although there is a CE-IVD approved enteric bacterial assay available for the BD MAXTM system, at time of writing there is no commercial test for this platform that can detect norovirus. Therefore, we used the BD MAXTM as an open system with user-defined real
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time RT-PCR reactions. We believe that this flexibility is a major strength of the BD
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MAXTM System, as it allows rapid alteration of the assay in response to changes in the
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circulating norovirus genotypes. Due to the moderate batch size, cost, complexity, and
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quick turnaround time the BD MAXTM open system is suited to high-throughput
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diagnostic laboratories (Table 2). However, user-defined assays are not approved by external bodies so require significant internal validation prior to clinical use. This study does not include all available commercial assays for norovirus detection [32].
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Recently, the RIDA®GENE Norovirus GI/GII assay was evaluated in a panel of 719 stool samples from adults and children in the USA [33]. The RIDA®GENE assay showed
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sensitivity and specificity of 94.2% and 98.5%, respectively. Subtyping showed the assay
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detected a range of norovirus GI and GII subtypes, but also highlighted possible sequence variants missed for GI.2, GI.3, GI.5, GII.1, GII.3, GII.4, and GII.13. In recent years, syndromic panels have become increasingly common with norovirus an important component of any gastrointestinal panel [34–36]. A study by Huang et al [27] compared
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the Verigene® enteric pathogen test (Nanosphere Inc, Illinois, USA), FilmArray gastrointestinal panel (Biofire Diagnostics, Utah, USA), and the xTAG GPP in 152 stool samples. Sensitivity was reported as 89% for the Verigene, 90% for xTAG GPP, and 95%
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for FilmArray. Specificity for all platforms was ≥99%. However, only 19 norovirus positive samples were included. Chhabra et al [26] compare FilmArray, xTAG GPP, and a custom
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Taqman array card (TAC) for detection of gastroenteritis viruses in stool. A panel of 300
samples were tested, 41 contained norovirus covering 7 GI and 12 GII subtypes. All three platforms had specificity >99%, sensitivity for FilmArray was 88%, xTAG GPP was 79%,
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and TAC 88%. Unfortunately, the norovirus subtypes generating false negative results
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were not reported. Further studies comparing norovirus detection between assays are
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needed, our study provides useful data for comparing platforms.
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We recognize several limitations. Our study was retrospective with samples
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being frozen before testing; this may have reduced the detectable amount of nucleic acid and impacted on the apparent sensitivity of the methods used. Another drawback of our study is that norovirus strain typing was not performed. Recent studies have
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shown the utility of norovirus subtyping when evaluating diagnostics assays [31,33]. During sample collection the predominant norovirus strain worldwide was GII.4 Sydney
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2012, and we believe our sample collection will reflect this [37,38]. However, multiple
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studies have shown the reference assay we used detects a wide range of GI and GII subtypes, so we are confident that samples negative by the reference assay did not contain norovirus [39–42]. It remains to be seen how well the evaluated assays will
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detect future epidemic strains of norovirus; collaboration between surveillance agencies and diagnostic laboratories is essential to identify and respond to novel strains [43,44]. In conclusion, we describe a laboratory evaluation of four NAATs for norovirus. A
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laboratory-defined real time RT-PCR assay, the BD-MAXTM System with user defined settings, and the Xpert® Norovirus Assay all showed excellent sensitivity and specificity
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for detection of norovirus GI and GII from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Each assay has their own strengths and weaknesses, clinical
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laboratories should consider skill mix, budget, and sample throughput to determine the
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best fit for their service.
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Funding
Ethical approval
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This research was funded by Becton Dickinson and Cepheid Inc.
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Study approval was granted by NHS Lothian SAHSC Bioresource (SR211), with ethical
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approval to test anonymised, excess patient samples (10/S1402/33).
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Competing interests The institution conducting this research received free consumables and reagents from Becton Dickinson and Cepheid Inc. The research was devised, performed and written up without any input from the funders.
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simultaneous detection of bacterial, viral, and parasitic causes of infectious gastroenteritis, J. Microbiol. Biotechnol. 23 (2013) 1041–1045. doi:10.4014/jmb.1212.12042. R. Zhuo, J. Cho, Y.Y. Qiu, B.D. Parsons, B.E. Lee, L. Chui, S.B. Freedman, X. Pang,
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gastroenteritis pathogens, Biomol. Detect. Quantif. 1 (2014) 3–7. doi:10.1016/j.bdq.2014.07.001. [37]
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and genetic analysis of a novel recombinant GII.P12-GII.3 among infants and children in Bangkok, Thailand between 2014 and 2016, Infect. Genet. Evol. 60 (2018) 133–139. doi:10.1016/j.meegid.2018.02.028. J. Li, T. Zhang, K. Cai, Y. Jiang, X. Guan, J. Zhan, W. Zou, Z. Yang, X. Xing, Y. Wu, Y.
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17 (2011) 1389–1395. doi:10.3201/eid1708.101837.
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TABLE 1. Norovirus detection comparison of commercial platforms to the reference real time RT-PCR in stool
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BD MAXTM
xTAG® GPP
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Positive Negative
Positive Negative
Reference Real Time RT-PCR Negative (n = 101)
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Reference Real Time RT-PCR Positive (n = 62)
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Commercial Assays
60 2b
49 13c
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Xpert® Norovirus Assay
Sensitivity [% (95% CI)]
Specificity [% (95% CI)]
PPVa [% (95% CI)]
NPVa [% (95% CI)]
96.8 (92.4 – 100)
100 (97.0 – 100)
100 (95.0 – 100)
98.1 (95.4 – 100)
79.0 (68.9 – 89.2)
87.1 (80.6 – 93.7)
79.0 (68.9 – 89.2)
87.1 (80.6 – 93.7)
91.9 (85.2 – 98.7)
100 (97.0 – 100)
100 (94.7 – 100)
95.3 (91.2 – 99.3)
0 101
13 88
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Positive 57 0 d Negative 5 101 a PPV, positive predictive value; NPV, negative predictive value b Both
GII by reference assay.
c Nine
GI and four GII by reference assay.
d
All GI by reference assay.
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Complexity
Advantages
Moderate
Can be modified rapidly
Moderate
Automation reduces hands-on time; can be modified rapidly Fast results with limited sample handling Syndromic testing gives results for multiple pathogens
Cepheid
FDA, CEIVD
NA
1.5 h
$$
Low
Luminex
FDA, CEIVD
Up to 96
24 h
$$$
High
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PT
Xpert Norovirus
ED
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TABLE 2. Comparison of molecular Norovirus detection assays used in this study Assay Manufacturer Approval Batch sizeb Turnaround Costc time Laboratory- NA NA Up to 96 <4 h $ defined real time RT-PCRa BD-MAX Becton NA Up to 24 <4 h $ open Dickinson system
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xTAG GPP
a
as described for reference assay number of samples per run c estimated cost per sample ($, low; $$, moderate; $$$, high) b
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Recommended setting Highthroughput central laboratories Highthroughput central laboratories
Near patient and rapid testing Reference centres
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TABLE 3. Norovirus detection in vomit
BD MAXTM BD MAXTM PPAa NPAa PPVa NPVa Norovirus Norovirus [% (95% CI)] [% (95% CI)] [% (95% CI)] [% (95% CI)] Positive (n = 45) Negative (n = 25) Positive 43 2 95.6 92.0 95.6 92.0 (89.5 – 100) (81.4 – 100) (89.5 – 100) (81.4 – 100) Negative 2 23 a PPA, positive percent agreement; NPA, negative percent agreement; PPV, positive predictive value; NPV, negative predictive value
A
CC E
PT
ED
M
A
Xpert® Norovirus Assay
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S1386-6532(18)30155-0 https://doi.org/10.1016/j.jcv.2018.06.005 JCV 4014
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
23-2-2018 9-5-2018 7-6-2018
Please cite this article as: McHugh MP, Guerendiain D, Hardie A, Kenicer J, MacKenzie L, Templeton KE, Detection of Norovirus by BD MAXTM , Xpertreg Norovirus, and xTAG® Gastrointestinal Pathogen Panel in Stool and Vomit Samples, Journal of Clinical Virology (2018), https://doi.org/10.1016/j.jcv.2018.06.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Detection of Norovirus by BD MAXTM, Xpert® Norovirus, and xTAG® Gastrointestinal Pathogen Panel in Stool and Vomit Samples
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Running Title: Stool and Vomit Detection of Norovirus by Nucleic Acid Amplification Tests
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Martin P. McHugh1, Daniel Guerendiain1, Alison Hardie1,† , Juliet Kenicer1,* , Laura
Department of Molecular Microbiology, Royal Infirmary of Edinburgh, Edinburgh, UK
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MacKenzie1, Kate E. Templeton1, #
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preparation of this manuscript
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† Co-author Alison Hardie sadly passed away after a short illness during the
* Current address: Scottish Escherichia coli O157/VTEC Reference Laboratory, Royal
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Infirmary of Edinburgh, Edinburgh, UK
# Corresponding author
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Dr Kate E. Templeton
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Department of Molecular Microbiology Royal Infirmary of Edinburgh 51 Little France Crescent, Edinburgh, EH16 4SA, UK Email: [email protected]
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Abstract word count: 239/250 Text word count: 1880/2500
Highlights:
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Molecular assays reliably detect norovirus from stool and vomit Fast turnaround and simplicity of Xpert Norovirus suit it to near patient testing The BD-MAX System is useful for high-throughput norovirus testing xTAG GPP had lower norovirus sensitivity/specificity compared to other platforms
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Abstract
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Background: Norovirus is a leading cause of infectious gastroenteritis, characterized by
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outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests
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commercial platforms now available.
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allow rapid and sensitive laboratory diagnosis of norovirus, with a number of
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Objectives: Evaluate the performance of the Becton Dickinson BD-MAXTM System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus
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Assay and BD-MAXTM in vomit samples. Study design: 163 diarrhoeal stool samples were tested on four diagnostic systems
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(laboratory-defined real time RT-PCR (assigned as gold standard), BD MAXTM, Xpert®
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Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms. Results: In stool, sensitivity and specificity of the BD-MAXTM was 96.8 % and 100 %, for Xpert® Norovirus Assay was 91.9 % and 100 %, and for xTAG® GPP was 79.0 % and 87.1
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%. In vomit samples positive and negative percent agreement was 95.6 % and 92.0 %, between the BD-MAXTM and Xpert® Norovirus. Conclusions: The BD-MAXTM System with user defined settings and the Xpert® Norovirus
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Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a
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number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.
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Abbreviations: Gastrointestinal pathogen panel (GPP)
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Keywords: Norovirus; stool; vomit; Xpert Norovirus; xTAG Gastrointestinal Pathogen
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Panel; BD MAX
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1. Background
Norovirus is a small, non-enveloped, positive stranded RNA virus in the Caliciviridae family. Infection results in diarrhoea, vomiting, abdominal pain and fever lasting 1-3
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days, although only 10-12% of individuals seek medical attention [1]. Norovirus is the
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most common cause of acute gastroenteritis worldwide and causes outbreaks in hospitals, nursing homes, cruise ships, and other closed settings [2–5]. There are an
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estimated 19-21 million norovirus cases in the United States every year [6]. Outbreaks of norovirus not only confer significant morbidity but have also been shown to increase allcause mortality, particularly in elderly patients [7]. Norovirus transmission can be interrupted by environmental cleaning, good hand hygiene and isolation or cohorting of
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infected individuals [8,9]. However, in healthcare settings these measures can lead to ward closures, lost bed days, staff absence and increased costs [10,11]. It is essential to identify infected individuals rapidly and accurately so that infection control precautions
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can be used effectively, limiting the impact on patient services [12]. The introduction of real time reverse-transcriptase polymerase chain reaction (RT-PCR)
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improved the laboratory detection of norovirus by providing sensitive and specific
results in a clinically relevant timeframe [2,12]. New assays for norovirus detection must maintain the high sensitivity and specificity already achieved with real time RT-PCR while
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improving turnaround time [13]. The Xpert® Norovirus Assay (Cepheid, Sunnyvale, USA)
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is FDA and CE-IVD approved and provides rapid nucleic acid extraction, amplification,
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and detection with limited operator input, which lends well to near-patient testing
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[14,15]. The BD MAXTM System (Becton Dickinson, Franklin Lakes, USA) offers automated
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nucleic-acid extraction and thermocycling thus reducing laboratory hands-on time and can be run as an “open platform” with user-defined protocols. The xTAG® Gastrointestinal Pathogen Panel (GPP; Luminex, Austin, USA) is an FDA and CE-IVD
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approved multiplex nucleic acid test for detection of up to 15 gastrointestinal pathogens [16], this syndromic approach eliminates the need for multiple microscopic, culture or
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molecular tests. Direct comparison of the performance characteristics for these assays is
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not currently available.
2. Objectives
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Evaluate the performance of the Xpert® Norovirus Assay, BD MAXTM, and xTAG® GPP for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and
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BD-MAXTM in vomit samples.
3. Study Design
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3.1. Patient Samples. Samples from patients with symptoms of gastroenteritis (loose
stools, diarrhoea, vomiting) were identified, related clinical data and laboratory results were collated and samples were anonymised before inclusion in the study. To give a
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more representative collection, multiple samples from local outbreaks were not
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included. Stool samples were collected between October 2011 and November 2013,
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vomit samples were collected between November 2014 and February 2015. Samples
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were stored at -80°C long term, after thawing samples were stored at 2-8°C and tested
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within 24 hours.
3.2. Comparison between Commercial Platforms. The presence of norovirus was determined using a gold standard reference real time RT-PCR assay and results with
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three commercial assays were compared to the reference result. For the reference real time RT-PCR and xTAG-GPP, a 10% suspension of sample prepared in 1 ml sterile water
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(Promega, Madison, USA) was vortexed briefly before being centrifuged (20,000 g, 5
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minutes). Nucleic acid was extracted from 200 µl of supernatant into 100 µl elution buffer by NucliSENS® easyMAG® (bioMérieux, Mary-l’Étoile, France) automated extraction. Real time RT-PCR was performed on the ABI 7500 Fast instrument (Applied Biosystems, Foster City, USA) in a reaction comprised of 10 µl EXPRESS qPCR Supermix
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Universal (Invitrogen, Carlsbad, CA), 2 µl EXPRESS SuperScript® Mix (Invitrogen), 2 µl oligonucleotide mix (as described by Kageyama et al [17]) and 6 µl nucleic acid extract. RNA was reverse transcribed at 50°C for 15 minutes, denatured at 95°C for 20 seconds, then PCR was conducted for 45 cycles of 95°C for 3 seconds and 60°C for 30 seconds.
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The BD MAXTM System is an automated platform for hands-off nucleic acid
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extraction, thermocycling and real-time product detection. Sample suspensions were
prepared as defined above, then 250 µl supernatant loaded into Sample Buffer Tube for extraction with BD MAXTM RNA Extraction Kit RNA-3 (Becton Dickinson). Cartridges were
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loaded into the BD MAXTM System and RNA was reverse transcribed at 50°C for 15
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minutes and after denaturation at 98°C for 20 seconds the PCR was conducted for 45
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cycles of 98°C for 12 seconds and 60°C for 25 seconds. Samples were tested with the
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Xpert® Norovirus Assay (with integrated nucleic acid extraction and real-time RT-PCR)
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and xTAG® GPP (analysed with the MAGPIX® analyser) following manufacturer’s instructions [18,19]. Positive (Norovirus GI and GII) and no-template controls were included with every run of the batch-based assays, and daily with the Xpert assay.
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3.3. Xpert® Norovirus Assay Performance with Vomit Samples. Vomit samples were tested with the BD MAXTM and Xpert® Norovirus Assay. A 10% suspension was made in 1
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ml sterile water (PROMEGA), centrifuged (20,000 g, 5 minutes) and supernatant was
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used for testing on both platforms as described above. Discrepancies between the two platforms were resolved by re-testing with the reference real time RT-PCR described above.
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4. Results 4.1. Comparison between Commercial Platforms. Unformed stool samples from 163 patients were tested with the reference real time RT-PCR assay prior to testing with the
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BD MAXTM, xTAG® GPP and Xpert® Norovirus Assay. Most patients were hospital inpatients (88.3%, n = 144) and the median age was 78 years old (interquartile range 51 to 87 years). In total 62 (38.0%) samples were positive for norovirus by the reference
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assay, 35 were GI and 27 GII. BD MAXTM correctly identified 161 (98.7%) samples, xTAG® GPP 137 (84.0%) and Xpert® Norovirus Assay 158 (96.9%; Table 1). The two false
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negative samples on BD MAXTM were GII by the reference assay (cycle threshold [Ct] not
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available), while all five false negatives on the Xpert® Norovirus Assay were GI (median
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reference Ct 36, range 30 – 38). One sample gave an invalid result on the Xpert®
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Norovirus Assay which on repeat was GI positive, and seven samples generated errors
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which on repeat were negative. Nine GI (median reference Ct 33, range 28 – 39) and four GII samples (median reference Ct 27, range 21-32) were false negative on xTAG® GPP, with an additional 13 norovirus false positives (xTAG® GPP does not report
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genogroup). A comparison of the practical aspects of the assays evaluated here is presented in Table 2.
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4.2. Xpert® Norovirus Assay Performance with Vomit Samples. Based on their
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favourable performance in stool samples, we then evaluated the performance of the BD MAXTM and Xpert® Norovirus assays with vomit samples. Seventy vomit samples were included in the study, 45 (64.3%) were positive for norovirus (1 GI and 44 GII) with the BD MAXTM. Two (2.8%) samples returned invalid Xpert® Norovirus Assay results due to
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no amplification of the internal control, on repeat these were norovirus negative. Positive and negative percent agreement was 96% and 92%, respectively (Table 3). Two samples were false negative and two false positive on the Xpert® Norovirus Assay
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compared to BD MAXTM. Post-discrepant analysis after testing with the reference assay showed that Xpert® Norovirus Assay had correctly identified all 70 samples, while the
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BD MAXTM had two false negative and two false positive calls.
5. Discussion
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This study found norovirus detection from stool was largely comparable with the
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reference assay, the Xpert® Norovirus Assay, and the BD MAXTM open platform (Table 1).
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All assays detected norovirus with higher sensitivity and specificity than the xTAG® GPP
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(Table 1). We also show the Xpert® Norovirus Assay and BD-MAXTM can successfully
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detect norovirus from vomit samples (Table 3). This finding increases the types of samples available for diagnosis of norovirus infection [20]. Our results are comparable to other published studies of the Xpert® Norovirus
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Assay in stool. Gonzalez et al (2016) tested 1,403 stool samples from multiple study sites [14]. By a composite reference test, norovirus GI was present in 115 samples and GII in
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174; the Xpert® Norovirus Assay had sensitivity and specificity >98 % for both
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genogroups. Further studies have shown the Xpert Norovirus assay to have >95% sensitivity and specificity for detection of Norovirus [15,21–23]. The high sensitivity/specificity, rapid run time of 90 minutes, random access loading and low complexity of the Xpert® Norovirus Assay suggest that it could be used in a routine
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laboratory to reduce turnaround time of norovirus detection (Table 2). We also believe the assay could be used as a point of care test in care of the elderly units, emergency departments, and other high-incidence areas.
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The xTAG® GPP is a syndromic diagnostic assay that can detect fifteen enteropathogens in a single reaction. Interestingly, the xTAG® GPP detected 22 other
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enteropathogens in the faecal samples tested here (17 samples were norovirus negative by the reference assay). It was not possible to ascertain the clinical relevance of these calls for other enteropathogens, although it is possible that these were causative agents
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of diarrhoea. We found the xTAG® GPP to have sensitivity of 79% and specificity of 87%
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for norovirus detection, the literature includes some studies reporting poor norovirus
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detection [24–27] as well as studies reporting sensitivity and specificity >90% for
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norovirus [16,28–30]. This discrepancy may reflect the high complexity of the test and
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suggest the need for confirmation of xTAG® GPP norovirus results with another method. Recently, Zhuo et al [31] reported false negative xTAG® GPP results for norovirus GII.2. In our study the four GII false negative samples were positive on all platforms except the
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xTAG® GPP, unfortunately subtype data were not available. Of the nine GI xTAG® GPP false negatives five were also negative with the Xpert® Norovirus Assay, suggesting a
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possible primer/probe mismatch in both these assays, or a failure to detect low viral
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loads (median reference Ct 36, range 30 – 38). We note our reference assay does not include an internal control so the 13 xTAG false positive calls could actually be genuine in the presence of a sample inhibitory in the reference assay. However, none of the xTAG GPP false positives were positive with the BD MAXTM or Xpert® Norovirus Assay,
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the latter of which does include an internal control. We ran the xTAG® GPP assay in batches of 96, using the full capacity of the MAGPIX reader which resulted in a turnaround time of 24 hours including extraction. Others have reported lower
turnaround time would have to be made for routine use of the xTAG® GPP.
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turnaround times of around 6-8 hours, a balance between costs per sample and
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Although there is a CE-IVD approved enteric bacterial assay available for the BD MAXTM system, at time of writing there is no commercial test for this platform that can detect norovirus. Therefore, we used the BD MAXTM as an open system with user-defined real
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time RT-PCR reactions. We believe that this flexibility is a major strength of the BD
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MAXTM System, as it allows rapid alteration of the assay in response to changes in the
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circulating norovirus genotypes. Due to the moderate batch size, cost, complexity, and
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quick turnaround time the BD MAXTM open system is suited to high-throughput
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diagnostic laboratories (Table 2). However, user-defined assays are not approved by external bodies so require significant internal validation prior to clinical use. This study does not include all available commercial assays for norovirus detection [32].
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Recently, the RIDA®GENE Norovirus GI/GII assay was evaluated in a panel of 719 stool samples from adults and children in the USA [33]. The RIDA®GENE assay showed
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sensitivity and specificity of 94.2% and 98.5%, respectively. Subtyping showed the assay
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detected a range of norovirus GI and GII subtypes, but also highlighted possible sequence variants missed for GI.2, GI.3, GI.5, GII.1, GII.3, GII.4, and GII.13. In recent years, syndromic panels have become increasingly common with norovirus an important component of any gastrointestinal panel [34–36]. A study by Huang et al [27] compared
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the Verigene® enteric pathogen test (Nanosphere Inc, Illinois, USA), FilmArray gastrointestinal panel (Biofire Diagnostics, Utah, USA), and the xTAG GPP in 152 stool samples. Sensitivity was reported as 89% for the Verigene, 90% for xTAG GPP, and 95%
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for FilmArray. Specificity for all platforms was ≥99%. However, only 19 norovirus positive samples were included. Chhabra et al [26] compare FilmArray, xTAG GPP, and a custom
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Taqman array card (TAC) for detection of gastroenteritis viruses in stool. A panel of 300
samples were tested, 41 contained norovirus covering 7 GI and 12 GII subtypes. All three platforms had specificity >99%, sensitivity for FilmArray was 88%, xTAG GPP was 79%,
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and TAC 88%. Unfortunately, the norovirus subtypes generating false negative results
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were not reported. Further studies comparing norovirus detection between assays are
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needed, our study provides useful data for comparing platforms.
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We recognize several limitations. Our study was retrospective with samples
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being frozen before testing; this may have reduced the detectable amount of nucleic acid and impacted on the apparent sensitivity of the methods used. Another drawback of our study is that norovirus strain typing was not performed. Recent studies have
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shown the utility of norovirus subtyping when evaluating diagnostics assays [31,33]. During sample collection the predominant norovirus strain worldwide was GII.4 Sydney
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2012, and we believe our sample collection will reflect this [37,38]. However, multiple
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studies have shown the reference assay we used detects a wide range of GI and GII subtypes, so we are confident that samples negative by the reference assay did not contain norovirus [39–42]. It remains to be seen how well the evaluated assays will
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detect future epidemic strains of norovirus; collaboration between surveillance agencies and diagnostic laboratories is essential to identify and respond to novel strains [43,44]. In conclusion, we describe a laboratory evaluation of four NAATs for norovirus. A
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laboratory-defined real time RT-PCR assay, the BD-MAXTM System with user defined settings, and the Xpert® Norovirus Assay all showed excellent sensitivity and specificity
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for detection of norovirus GI and GII from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Each assay has their own strengths and weaknesses, clinical
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laboratories should consider skill mix, budget, and sample throughput to determine the
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best fit for their service.
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Funding
Ethical approval
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This research was funded by Becton Dickinson and Cepheid Inc.
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Study approval was granted by NHS Lothian SAHSC Bioresource (SR211), with ethical
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approval to test anonymised, excess patient samples (10/S1402/33).
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Competing interests The institution conducting this research received free consumables and reagents from Becton Dickinson and Cepheid Inc. The research was devised, performed and written up without any input from the funders.
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I N U SC R
TABLE 1. Norovirus detection comparison of commercial platforms to the reference real time RT-PCR in stool
ED
BD MAXTM
xTAG® GPP
PT
Positive Negative
Positive Negative
Reference Real Time RT-PCR Negative (n = 101)
A
Reference Real Time RT-PCR Positive (n = 62)
M
Commercial Assays
60 2b
49 13c
CC E
Xpert® Norovirus Assay
Sensitivity [% (95% CI)]
Specificity [% (95% CI)]
PPVa [% (95% CI)]
NPVa [% (95% CI)]
96.8 (92.4 – 100)
100 (97.0 – 100)
100 (95.0 – 100)
98.1 (95.4 – 100)
79.0 (68.9 – 89.2)
87.1 (80.6 – 93.7)
79.0 (68.9 – 89.2)
87.1 (80.6 – 93.7)
91.9 (85.2 – 98.7)
100 (97.0 – 100)
100 (94.7 – 100)
95.3 (91.2 – 99.3)
0 101
13 88
A
Positive 57 0 d Negative 5 101 a PPV, positive predictive value; NPV, negative predictive value b Both
GII by reference assay.
c Nine
GI and four GII by reference assay.
d
All GI by reference assay.
21
I N U SC R
Complexity
Advantages
Moderate
Can be modified rapidly
Moderate
Automation reduces hands-on time; can be modified rapidly Fast results with limited sample handling Syndromic testing gives results for multiple pathogens
Cepheid
FDA, CEIVD
NA
1.5 h
$$
Low
Luminex
FDA, CEIVD
Up to 96
24 h
$$$
High
CC E
PT
Xpert Norovirus
ED
M
A
TABLE 2. Comparison of molecular Norovirus detection assays used in this study Assay Manufacturer Approval Batch sizeb Turnaround Costc time Laboratory- NA NA Up to 96 <4 h $ defined real time RT-PCRa BD-MAX Becton NA Up to 24 <4 h $ open Dickinson system
A
xTAG GPP
a
as described for reference assay number of samples per run c estimated cost per sample ($, low; $$, moderate; $$$, high) b
22
Recommended setting Highthroughput central laboratories Highthroughput central laboratories
Near patient and rapid testing Reference centres
I N U SC R
TABLE 3. Norovirus detection in vomit
BD MAXTM BD MAXTM PPAa NPAa PPVa NPVa Norovirus Norovirus [% (95% CI)] [% (95% CI)] [% (95% CI)] [% (95% CI)] Positive (n = 45) Negative (n = 25) Positive 43 2 95.6 92.0 95.6 92.0 (89.5 – 100) (81.4 – 100) (89.5 – 100) (81.4 – 100) Negative 2 23 a PPA, positive percent agreement; NPA, negative percent agreement; PPV, positive predictive value; NPV, negative predictive value
A
CC E
PT
ED
M
A
Xpert® Norovirus Assay
23