Received: 17 October 2019 | Accepted: 24 December 2019 DOI: 10.1002/jmv.25659 RESEARCH ARTICLE A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts A. Hayes1 | D. Nguyen1 | M. Andersson2 | A. Antón3 | J.‐L. Bailly4,5 | S. Beard6 | K. S. M. Benschop7 | | S. Blomqvist9 | E. Cunningham10 | D. Davis11 N. Berginc8 | J. L. Dembinski12 | S. Diedrich13 | S. G. Dudman14 | R. Dyrdak15,16 | G. J. A. Eltringham17 | S. Gonzales‐Goggia18 | R. Gunson19 | H. C. Howson‐Wells20 | A. J. Jääskeläinen21 | F. X. López‐Labrador22,23 | M. Maier24 | M. Majumdar25 | S. Midgley26 | A. Mirand27 | U. Morley28 | S. A. Nordbø29,30 | S. Oikarinen31 | H. Osman32 | A. Papa33 | L. Pellegrinelli34 | A. Piralla35 | N. Rabella36 | J. Richter37 | M. Smith34,38 | A. Söderlund Strand39 | K. Templeton40 | B. Vipond41 | T. Vuorinen42 | C. Williams43 | E. Wollants44 | K. Zakikhany45 | T. K. Fischer23,26 | H. Harvala46 | P. Simmonds1 1 Nuffield Department of Medicine, University of Oxford, Oxford, UK 2 Microbiology Laboratory, John Radcliffe Hospital, Headley Way, Headington, Oxford, UK 3 Respiratory Viruses Unit, Virology Section, Microbiology Department, Hospital Universitari Vall d’Hebron, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron, Barcelona, Spain 4 Université Clermont Auvergne, LMGE UMR CNRS, UFR Médecine, Clermont‐Ferrand, France 5 CHU Clermont‐Ferrand, National Reference Center for EV and Parechovirus‐Associated Laboratory, Clermont‐Ferrand, France 6 Enteric Virus Unit, Virus Reference Department, National Infection Service, Public Health England, London, UK 7 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands 8 Department for Public Health Virology, National Laboratory of Health, Environment and Food, Ljubljana, Slovenia 9 National Institute for Health and Welfare, Mannerheimintie, Helsinki, Finland 10 Viapath Infection Sciences, St. Thomas’ Hospital, London, UK 11 Microbiology, Virology and infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK 12 Department of Virology, Norwegian Institute of Public Health, Oslo, Norway 13 National Reference Center for Poliomyelitis and Enteroviruses, Robert Koch Institute, Berlin, Germany 14 Department of Microbiology, Oslo University Hospital Rikshospitalet, Inst. Clinical Medicine, University of Oslo, Oslo, Norway 15 Department of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden 16 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden 17 Molecular Diagnostics Laboratory, Microbiology, Freeman Hospital, Newcastle Upon Tyne, UK 18 Public Health England Poliovirus Reference Laboratory, National Infection Service, Public Health England, London, UK 19 West of Scotland Specialist Virology Centre, Glasgow Royal Infirmary, Glasgow, UK 20 Nottingham University Hospitals NHS Trust, Clinical Microbiology, Queens Medical Centre, Nottingham, UK 21 University of Helsinki and Helsinki University Hospital, HUSLAB, Virology and Immunology, Helsinki, Finland ------------------------------------------------------------------------------------------------------ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Journal of Medical Virology published by Wiley Periodicals, Inc. J Med Virol. 2020;92:1065–1074. wileyonlinelibrary.com/journal/jmv | 1065 1066 | HAYES ET AL. 22 Virology Laboratory, Joint Units in Genomics and Health and Infection and Health, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO‐Public Health)/Universitat de València, Av. Catalunya, València, Spain 23 CIBEResp, Centro de Investigación Biomédica en Red en Epidemiología y Salud Pública, Instituto de Salud Carlos III, Madrid, Spain 24 Institute of Virology, Leipzig University Hospital, Leipzig, Germany 25 The National Institute for Biological Standards and Control, Hertfordshire, UK 26 Department of Virus and Special Microbiological Diagnostics, Virus Surveillance and Research Section, Statens Serum Institut, Copenhagen, Denmark 27 CHU Clermont‐Ferrand, Laboratoire de Virologie—Centre National de Référence des Entérovirus et Parechovirus, Laboratoire Associé—Clermont‐Ferrand, France 28 UCD National Virus Reference Laboratory, University College Dublin, Belfield, Dublin, Ireland 29 Department of Medical Microbiology, St. Olavs University Hospital, Trondheim, Norway 30 Department of Clinical and Molecular Medicine, Faculty of Medicine and Health Sciences, Norwegian University of Science and Technology, Trondheim, Norway 31 Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland 32 Public Health England Birmingham Public Health Laboratory, Heartlands Hospital, Birmingham, UK 33 Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece 34 Department of Biomedical Sciences for Health, University of Milan, Milan, Italy 35 Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 36 Virology Section, Santa Creu i Sant Pau University Hospital, Barcelona, Spain 37 Department of Molecular Virology, Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus 38 King’s College Hospital, Bessemer Wing, Denmark Hill, London, UK 39 Laboratory Medicine, Department of Clinical Microbiology, Lund University Hospital, Sölvegatan, Lund, Sweden 40 Edinburgh Specialist Virology, Royal Infirmary of Edinburgh, Edinburgh, UK 41 Public Health England, South West Regional Laboratory, Pathology Sciences Building, Science Quarter, Southmead Hospital, Bristol, UK 42 Clinical Microbiology, Turku University Hospital and Institute of Biomedicine University of Turku, Turku, Finland 43 Microbiology, Royal Oldham Hospital, Oldham, UK 44 Clinical and Epidemiological Virology, KU Leuven, REGA Institute, Clinical and Epidemiological Virology, Leuven, Belgium 45 Katherina Zakikhany‐Gilg, Public Health Agency of Sweden, Department of Microbiology, Unit of Laboratory Surveillance of Viral Pathogens and Vaccine Preventable Diseases, Stockholm, Sweden 46 NHS Blood and Transplant, Colindale, London, UK Correspondence Peter Simmonds, Nuffield Department of Abstract Medicine, University of Oxford, South Parks Polymerase chain reaction (PCR) detection has become the gold standard for Road, Oxford, OX1 3SY, UK. Email: Peter.Simmonds@ndm.ox.ac.uk diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high Funding information Wellcome Trust, Grant/Award Number: assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and ISSF204826/Z/16/Z sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in‐house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV‐A71, echovirus 30, coxsackie A virus 21, and EV‐D68), HPeV3, and specificity controls. Reported results from 48 in‐house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In‐house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10−5). EV‐specific PCRs showed low cross‐reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. HAYES ET AL. | 1067 The study provides reassurance of the performance of the many in‐house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point‐of‐care tests. KEYWORDS enterovirus, enterovirus A71, parechovirus, PCR, RNA transcripts 1 | INTRODUCTION enterovirus network (ENPEN) to members in diagnostic and reference laboratories for evaluation of the sensitivity of their Enteroviruses (EVs) belonging to the family Picornaviridae are routinely used assays for detection and typing of enteroviruses. currently classified into 116 serologically distinct enterovirus types, which can be assigned into four genetically distinct species, HEV‐A to D1,2 and over 250 human rhinovirus (HRV) types divided into three 2 | M A T E R I A L S AN D M E T H O D S species (A‐C). Species B EV types including echoviruses and coxsackie B viruses (CBVs) are the most frequently identified viral causes of 2.1 | RNA transcript synthesis meningitis and other central nervous system (CNS)‐associated infections in western countries, while the species A serotype, Available full‐length cDNA clones of EV species A (EV‐A71 EV‐A71 is an important cause of hand, foot, and mouth disease and genogroup B4 strain, accession number AF316321)11, B (Bastianni encephalitis in South‐East Asia and EV‐D68 within species D has prototype strain of echovirus 30 [E30], AF162711), C (Coe strain of recently emerged as a respiratory pathogen occasionally leading to coxsackivirus A 21 [CVA21], D00538), and D (Fermon strain of 3 acute flaccid myelitis (AFM). Infections with human parechoviruses EV‐D68, NC_038308), and parechovirus (HPeV3, GQ18302612) were (HPeVs) in the genus Parechovirus are enteric, usually asymptomatic selected for this study. For rhinovirus species C (HRV‐C49, apart from those of HPeV type 3, which is associated with sepsis‐like MF775365), a partial 5′‐UTR‐VP4‐VP2 clone was assembled from illness, meningitis, and encephalitis in young children.4-6 amplified sequences. All plasmids were transformed into DH5α Although there is no effective antiviral treatment available for EV competent cells by heat shock, with single colonies picked and grown infections, detection and identification of EV and HPeV infections are in liquid medium before plasmid extraction using the QIAprep Spin vital for informing other treatment options, supportive care and Miniprep Kit (Qiagen), according to the manufacturer’s instructions. prognosis of affected individuals. The reverse‐transcriptase polymer- Plasmids were linearized at the 3′ end, and RNA transcripts were ase chain reaction (RT‐PCR) is now the “gold standard” for diagnosing produced using the MEGAscript T7 Transcription Kit (Ambion), EV and HPeV infections due to its advantages of fast turn‐around followed by DNase treatment to remove template DNA. RNA was time and high sensitivity over virus isolation.7 Even in severe cases, purified using the RNAEasy Mini Kit (Qiagen), according to the viral loads are relatively low in cerebrospinal fluid (CSF) samples that manufacturer’s instructions. are typically tested in patients presenting with meningitis or encephalitis, and may be missed by less sensitive methods. To detect all EV types, RT‐PCR assays for the detection of EV 2.2 | RNA quantification and stability assessment RNA usually target the highly conserved 5′ non‐translated region (5′NTR). Depending on the primer and probe design, some molecular Quantification of RNA transcripts was carried out using the detection methods may fail to detect certain EV types such as NanoDrop ND‐1000 UV‐Vis Spectrophotometer (Thermo Fisher EV‐D68, whereas some assays may also detect HRVs (reviewed in Scientific) and the Qubit 4 Fluorometer (Thermo Fisher Scientific). Holm‐Hansen3). EV and HPeV serotypes are defined serologically The concentrations obtained were used to calculate copy numbers of and genetically by their capsid region sequences; virus typing, transcripts produced, assuming a mean molecular mass for each base therefore, requires amplification and sequencing of regions within of 330 g/mol. A serial dilution of RNA transcripts (105 to 10−1 8,9 this structural gene block, typically VP1. copies/µL) was prepared using the RNA storage solution (Thermo Evaluation of sensitivity and specificity of the diagnostic assays Fisher Scientific; 1 mM sodium citrate, pH 6.4) containing herring used for EV and HPeV detection and typing is essential. We have sperm carrier RNA (50 µg/mL) and RNasin (New England BioLabs UK, previously evaluated the use of RNA transcripts of several EV and 100 U/mL). Dilutions were aliquoted and stored at −80°C before HRV serotypes and HPeV1 for quality control purposes in six expert testing and distribution to the participating laboratories. clinical virology laboratories in Europe.10 Following this study, we EV species A (EV‐A71) and C (CVA21) transcripts were have now produced a further set of RNA transcript standards for investigated for stability at different temperatures. Transcripts selected representative serotypes from EV species A‐D and HPeV3. were incubated in storage solution for up to 30 days at The RNA standards were distributed via the European non‐polio ambient temperature, 4°C and 37°C. A further aliquot of each was 1068 | HAYES ET AL. freeze‐thawed three times. The amount of RNA was quantified by physicochemical methods (Nanodrop and Qubit). The values obtained RT‐PCR and values compared to those of the original preparations. by the two methods' values were similar (within a factor of two in all cases; data not shown) and the mean value was used for calibration. Serial dilutions of each RNA transcript ranging from 104 to 10−1 2.3 | Transcript amplification by real‐time RT‐PCR copies in 5 µL were assayed in replicate by quantitative EV and HPeV RT‐PCR assay. Amplification was highly reproducible between For quantification of RNA sequences before distribution, In‐house replicates and between transcripts of different EV species and quantitative real‐time RT‐PCR was carried out using the StepOne- HPeV3 (Figure 1). All samples were positive with an input of 10 Plus Real‐Time PCR System (Thermo Fisher Scientific), according to copies, while detection of single copies of RNA was stochastic the manufacturer’s instructions. The following reaction conditions (2/5 replicates detected). The end‐point detection of the RNA were used: 50°C for 30 minutes, 95°C for 15 minutes followed by 45 transcripts and their similarity in Ct values to each demonstrates cycles of 95°C for 10 seconds then 60°C for 1 minute. A total of that quantitation was reproducible between the transcripts of the 20 μL reaction volume containing 2 μL of the diluted transcript was five different viral species (all Ct values were within a factor of two used. PCRs used primers and probes as previously described for from each other), Finally, the measured gradients of the lines of best 13 14 15 EV, HRV, and HPeV (Table S1). fit between log viral load and Ct‐value were between 3.1 and 3.6, consistent with efficient amplification. To investigate the stability of RNA transcript, two representative 2.4 | Laboratory evaluation EV transcripts (EV‐A71 and CVA21) were subjected to a range of temperatures and freeze‐thaw cycles and their RNA content was RNA from EV species A‐D and HPeV was distributed in 200 μL volumes assessed by real‐time PCR (Figure 2). No or minimal changes in Ct of storage buffer at concentrations of 10 and 103 copies/5 μL. The HRV values (reflecting residual RNA concentrations) were observed on species C, as a negative transcript control, was distributed only at the freezing‐thawing or incubation for up to 30 days at 4°C or ambient higher concentration (10 copies in 5 μL) to investigate cross‐reactivity 3 temperate, while there was an approximately 10‐fold reduction in of EV assays with rhinoviruses. Storage buffer was included as another RNA levels on incubation at 37°C for 30 days No decline in Ct values negative transcript control. Sample labeling was coded, and the details was observed for any transcript stored at 4°C or room temperate of coding were provided to participating laboratories only after all results were received. Coded transcript panels were sent by standard registered post to the participating laboratories in February 2019. 2.5 | Participating laboratories The RNA transcripts were distributed via ENPEN to member diagnostic and reference laboratories for evaluation of the sensitivity of their routinely used assays for detection and typing of EV and HPeV. Laboratories were identified by a standardized code (L1, L3….). Coded transcripts panels were shipped by standard registered UK post in February 2019. Participating labs were asked to test 5ul of each sample sent using their routine detection and/or typing assay. Results were reported through an EU survey tool (https://ec.europa.eu/eusurvey/ runner/ENPEN_transcript_study_panel_A_2019). Collected information comprised the results of testing in terms of positivity or negativity, Ct values were obtained, virus type and further technical information such as volume tested, extraction method (if used) and additional information on the testing methods used (listed in Supplementary Methods). 3 | RES U LTS F I G U R E 1 Quantitative RT‐PCR of RNA transcripts of different EV species, HRV and HPeV. Ct values of replicate dilutions of EV, HRV and HPeV RNA transcripts used in the evaluation panel; data 3.1 | Validation of the transcript panel points indicate mean values of 3 technical replicate; error bars show standard errors of the mean. EV, enterovirus; HPeV, human RNA concentrations of EV‐A71, E30, CVA21, EV‐D68, HPeV3, parechovirus; HRV, human rhinovirus; RT‐PCR, reverse‐ and HRV‐C49 transcripts were determined by two different transcriptase polymerase chain reaction HAYES ET AL. | 1069 methods for EV and HPeV detection; in‐house assays were used for 19 of the 22 results sets provided by 12 reference laboratories for the study (86%), with only one reference laboratory located within a local hospital contributing results from three different commercial assays). This compares 29/41 (71%) of in‐house result sets provided by 27 diagnostic laboratories. Commercial kits used included assays from BioMérieux, Seegene, Progenie, Luminex, Fast Track Diagnostics, Elite Ingenius, Biofire, Altona, and AusDiagnostics (Table 1). A total of 37 sets of in‐house typing results were provided, from these 18 sets (14 for EV typing, 3 for HPeV, and 1 for both) were reported by reference laboratories and 12 sets by diagnostic laboratories from 7 different countries including Spain, Sweden, Greece, Italy, Germany, Norway, and UK. Most of these results were reported for EV typing (n = 29) but HPeV typing was also performed (n = 10). F I G U R E 2 Stability of RNA transcripts on incubation at different 3.3 | Sensitivity and specificity of screening temperatures and freeze/thawing. Fold changes in RNA detection of methods two representative RNA transcripts preparations of CAV21 (EV species C) at low copy number and EV‐A71 (species A, high copy The sensitivity and specificity of detection assay results were number) used for laboratory distribution. Transcripts were incubated calculated, and totals adjusted for the declared target range of the for various durations at different temperatures. Detected viral loads tests. Intended assay targets included combined EV and HPeV were compared to those of RNA transcripts stored at −80°C. RNA detection (n = 23), EV detection only (n = 17), HPeV detection only transcripts were additionally subjected to three freeze/thaw cycles (rapid cooling and thawing; right hand panel). Bar heights show fold (n = 10) and combined EV and HRV detection (n = 7) as well as mono‐ reductions of RNA relative to the starting amount; error bars show specific assays for EV‐D68 (n = 1) and EV‐A71 (n = 1). Two HRV‐only SEMS of three assay repeats assays were evaluated for specificity only. In general, laboratories reported high rates of detection (98% for over the month period, providing reassurance that transcripts CVA21; 100% for EV‐A71, E30, and EV‐D68) of the EV transcripts at received by the participating laboratories were not degraded. the higher concentration (103 RNA copies in 5 µL) (Figure 3). More A panel of 12 RNA transcripts was constructed containing two variable detection of the low concentration transcripts (10 RNA concentrations of EV‐A71, E30, CVA21, EV‐D68, and HPeV3 (10 and copies in 5 µL) was reported, ranging from 62% (EV‐D68) to 90% 1000 copies in 5 µL), HRV‐C49 (1000 copies in 5 µL) and a water (EV‐A71). Detection frequencies of the HPeV3 transcripts were control. These panels were sent to the participating laboratories comparable; 97% for higher concentration and 75% for lower using standard registered post at ambient temperature; delivery concentration. For assays reporting Ct values for the higher and times ranged from one to 4 weeks providing reassurance for the RNA lower concentration transcripts, values were compared to evaluate quality. viral load ratios (Figure S1). Although no assay produced quantitative results, reported results showed a 58 to 106 fold differences in geometric mean viral loads, close to the expected 100‐fold 3.2 | Participating laboratories difference. Assays were therefore reasonably quantitative in relative terms in this concentration range. A total of 39 laboratories from 17 European countries including Assays were also generally highly specific, with only 2 high Ct Belgium (number of participating laboratories = 1), Cyprus (1), value (weak positive) results reported falsely positive from the 62 Denmark (1), France (1), Finland (4), Germany (2), Greece (1), Italy tests performed. A larger number of tests specific for EVs reported (2), Ireland (1), Netherlands (1), Norway (3), Slovenia (1), Spain (3), HRV detection, with five tests designed for the detection of EV Sweden (3), and UK (14) participated to this study. From these, 12 (n = 2), EV and HPeV (n = 2) and EV‐D68 (n = 1) reporting positive were classified as national reference laboratories and the remaining results with the HRV‐C49 RNA transcript. 27 as primary diagnostic laboratories (Table S2). Methodology differences contributed substantially to the sensi- Most laboratories (n = 36) participated in the evaluation of tivity of the screening assays (Figure 3B). In particular, commercial detection assays, some evaluating multiple assays; this produced a assays, often highly multiplexed for other viral targets in CSF, total of 63 sets of results for detection assays. Reference showed significantly reduced sensitivity for the detection of RNA Laboratories showed greater investment in in‐house detection transcripts at a lower concentration compared to in‐house methods 1070 | HAYES ET AL. T A B L E 1 Testing results from individual commercial assay platforms Detection1 Manufacturer Assay Ct 10 1000 HRV‐C Enterovirus assays Altona RealStar Y 4/4 4/4 P BioMerieux Enterovirus R Y 4/4 4/4 N EV/HPeV assays Ausdiagnostics Resp. viruses 16‐well N 0/5 4/52 N Ausdiagnostics Viral 8‐well version 3.0 N 2/5 5/5 N Ausdiagnostics Viral 8‐well version 01 N 1/5 4/52 N Biofire Film array ME panel v1.4 N 0/5 4/53 N Elite‐Ingenious Meningitis viral 2 MGB panel Y 4/5 5/5 N Fast Track FTD Viral Meningitis Y 5/5 5/5 N Fast Track FTD Neuro 9 Y 5/5 5/5 N Luminex NxTAG Resp. Pathogen Panel Y 5/5 5/5 N Progenie Real Cycler EVPA ‐ Version 4 Y 2/5 4/4 N Progenie Real Cycler Monotest Y 1/5 5/5 N HRV/RV assays BioMerieux Rhino/Entero R gene Y 1/3 ‐ P Seegene Allplex Resp. Panel 2‐ RP9802x Y 1/4 3/3 N Seegene Allplex Resp. Panel RV16 Y 3/4 4/4 P Parechovirus assays BioMerieux Parechovirus r‐gene Y 1/1 1/1 N 1 Detection frequencies in the 10 and 1000 copies/5 µL transcript dilutions. Insensitive results—low detection rate of the 10 copy/5 µL control—are underlined, unexpected results—detection failure of 1000 copy/5 µL controls are indicated in bold. 2 The 1000 copy/5 µL E30 (EV species B) transcript was undetected in both assays. 3 The 1000 copy/5 µL HPeV3 transcript was undetected. (56% detection rate compared to 81%; P = .0009; Figure 3B). There higher concentration EV RNA transcripts were successfully typed by was a comparable difference in HPeV detection rates, with 50% of 88% of reference laboratories (45 of 51) and 71% of diagnostic the lower concentration transcript detected by commercial and 86% laboratories (36 of 57; P = .02 by Fisher’s Exact Test) whereas the by in‐house assays. There were significant differences in assay lower concentration EV transcripts were successfully typed by 41% sensitivity in results reported by diagnostic and by reference of reference laboratories (21 of 51) and 31% of diagnostic laboratories (Figure 3C). This is largely accounted for by the greater laboratories (16 of 51; P = 0.2). High concentration HPeV RNA use of in‐house assays by reference laboratories. transcripts were successfully typed by all reference (3 of 3) and Commercial assays included a variety of platforms and assay diagnostic laboratories (6 of 6) but one of each failed detection of the specificities (Table 1); several including Biofire, AusDiagnostics, low copy HPeV transcript. Progenie and some assays from Seegene and bioMérieux were unable to detect the lower concentration RNA control (10 RNA copies in 5 µL), and in some cases, even the higher concentration 4 | D I S C U SS I O N RNA transcript (1000 copies in 5 µL) (Biofire, AusDiagnostics). We further investigated the sensitivity of the Biofire assay with This study describes a quality control exercise for EV and HPeV intermediate RNA concentrations; assay sensitivity lay between detection and typing in 43 virus diagnostic and reference labora- 400 and 1000 RNA copies for most of the EV transcripts and tories in 17 European countries. Intrinsic to the study design was the between 1000 and 40 000 copies for HPeV RNA (Table 2). use of stabilized RNA transcripts of representative serotypes and species of EV and HPeV. 3.4 | Sensitivity and accuracy of EV and HPeV typing methods 4.1 | The suitability of RNA transcripts for quality assurance EV and HPeV typing performed by 22 participating laboratories were based upon amplification by PCR and Sanger sequencing of VP1 and/ RNA transcripts were used to evaluate the sensitivity and specificity or VP3/VP1 (EV) or VP3/VP1 (HPeV) regions either using species‐ or of commercial and in‐house detection and characterization assays for genus‐specific assays. Half of the typing was performed at the EVs and HPeV. Multiple EV types were used in the panel to estimate reference laboratories and half at the diagnostic laboratories the range of serotypes and species that could be detected; resulting in the use of 37 different typing assays (Figure 4). The enteroviruses are substantially diverse genetically, and conserved HAYES ET AL. | 1071 F I G U R E 3 Detection frequencies of EV and HPeV transcripts. Frequencies of detection of the 10 and 1000 RNA copy/5 µL dilutions of each transcript by participant laboratories, divided by (A) transcript sequence, including detection frequencies of the HRV‐C and water negative controls. The detection frequency of the EV‐A71 transcript was significantly higher than achieved for the other EV species (B) Assay type—in‐ house for commercially available (results for individual commercial assays are shown in Table 1), and (C) laboratory type, diagnostic or national reference laboratory both shown separately for the EV and HPeV transcripts. P values above bars show frequency comparisons using Fisher’s Exact Test. EV, enterovirus; HPeV, human parechovirus; HRV‐C, human rhinovirus species C regions of the genome between EV species suitable for amplification highly stable at ambient temperature and on repeated freeze/ by PCR are largely confined to short motifs in the 5′‐UTR.13 EV and thawing (Figure 2). Finally, they are noninfectious, enabling their HPeV RNA transcripts possess a number of attributes that make international distribution by ordinary post, providing considerable them suitable as reagents for quality assurance (QA) and evaluation cost savings, assured biological safety and simple logistics. RNA of diagnostic assays. Most importantly, their content can be transcripts can indeed be very easily prepared on a large scale for quantified in absolute terms, so it is possible to express assay little cost—in the current study, the consumable costs for laboratory sensitivities in terms of RNA copies. This provides a stable production and packaging of the transcripts was approximately €120, quantitative standard for longer‐term evaluation of assays sensitiv- while postage cost to the participating laboratories was approxi- ities. Secondly, the transcripts are based on uniform, defined mately €390. This low cost enables participation in this and future sequences enabling assay failures to be investigated though RNA transcript‐based QA exercises to be uncharged. The wide comparisons of primer/probe and target sequences. Thirdly, un- availability and insignificant cost will facilitate the use of RNA limited amounts of RNA transcripts can be generated from cDNA transcripts in the development of screening and typing capacity in clones of each target guaranteeing long term stability and reprodu- Eastern Europe, where EV detection is largely restricted to virus cibility of the control materials. Fourthly, the RNA transcripts are isolation and neutralization assays.16 There are however also some potential disadvantages to the use of T A B L E 2 Sensitivity of biofire film array assay RNA transcript controls and limitations of the study. Firstly, the RNA controls were directly used without RNA/DNA extraction in some test RNA copies/5 µL formats, so the efficiency of this step, which may be critical in clinical Transcript 10 400 1000 40000 sample handling, and its downstream effects on overall assay EV‐A71 N1 P2 P P performance was not evaluated for all assays. In practice, however, E30 ‐3 N P P this can be evaluated through comparison of Ct values of transcript CAV21 ‐ N P P samples with and without an extraction step. The second potential EV‐D68 ‐ N P P disadvantage of RNA transcripts was the use of carrier RNA for HPeV3 ‐ N N P stabilization. Although the concentrations used were relatively low in 1 molecular terms (a 5 µL aliquot used for testing contained 250 ng of Negative in assay. 2 Positive in assay. RNA), its presence could interfere with high throughput sequencing 3 Not done. methods that may become increasingly used for virus typing in the 1072 | HAYES ET AL. F I G U R E 4 Frequencies of successful typing of EV and HPeV transcripts. Frequencies of successful typing of the 10 and 1000 RNA copy/5 µL dilutions of EV and HPeV transcripts divided by (A) transcript sequence and (B) laboratory type, diagnostic or national reference laboratory both shown separately for the EV and HPeV transcripts. P values above bars show frequency comparisons using Fisher’s Exact Test. EV, enterovirus; HPeV, human parechovirus future. However, the RNA amount used was well within the capacity of equated to an analytical sensitivity of greater than 50 EV/HPeV RNA current library preparation methods for Illumina and Nanopore genomes/mL for a standard 200 µL CSF extraction volumes. Almost methods, and indeed cDNA in these concentrations may be required all reference laboratories achieved this sensitivity using in‐house for efficient template preparation. We are currently investigating the PCR methods, but the 10 copy control was frequently negative in stabilizing properties of alternative carriers such as linear acrylamide testing with commercial assays. In the case of the biofire film array to avoid this currently theoretical problem in the future. Finally and assay, the mean limit of detection for the 4 EV species was greater more generally, RNA transcripts or cloned viral DNA sequences are than 2000‐4000 RNA copies/mL, with an even lower sensitivity for available from a relatively narrow range of viruses, typically those that HPeV3 (Table 2). These findings are consistent with previous possess relatively small, non‐segmented genomes, and for which full‐ evaluations of the Biofire film assay, with reported limits of detection length cDNA clones representative of currently circulating strains are for EV detection of >500 RNA copies/mL,18 and some reduction in available. Although RNA transcripts for EVs and HPeV used in the rates of detection of (unquantified) EV‐positive clinical samples current study can be readily derived from cloned sequences of a wide compared to conventional diagnostic assays.19-21 range of contemporary circulating virus strains, development of Viral loads in CSF are low, often at the limit of assay sensitivity of equivalent RNA (or DNA)‐based standards would be problematic or PCR, so variability in assay sensitivity could substantially influence impossible for many respiratory and enteric viruses, and for diagnostic target detection rates in diagnostic samples. The devel- adenoviruses, herpesviruses and other large DNA viruses with opment of guidelines for assay sensitivity for EV and HPeV RNA genomes that are too large for conventional cloning strategies. detection in CSF would be of considerable value in the future quality control of these assays. The legislation for the use of CE‐marked tests is driving the replacement of in‐house assays with commercial 4.2 | Analytical sensitivity of EV and HPeV tests, many of which offer syndromic testing with multiplexed detection assays detection of a large range of viruses and bacteria. Although such assays often have operational advantages as point‐of‐care tests in As described, the transcripts enabled assay sensitivity to be emergency rooms requiring rapid results, the effects of their determined in absolute RNA copy numbers, with almost all assays potentially reduced sensitivity on their clinical utility need to be able to detect 1000 copies of RNA transcripts but with more variable evaluated. Validation of their performance can be challenging for detection of 10 RNA copy controls for EVs and HPeV. There are no multiple analytes and existing studies typically do not include current statutory guidelines for EV or HPeV detection sensitivity for samples with defined viral loads or identified EV species or diagnostic assays although previous evaluations of widely used EV serotypes, nor investigation of samples that have failed detec- PCRs found detection limits of 10 to 50 copies for EV and tion.18-21 In the future, validation of such assays with calibrated RNA 17 HPeV. This corresponds to 50 to 200 RNA copies/mL using controls is of particular value for evaluation of their performance, standard 200 μL extraction volumes of CSF. In the current study, particularly when the availability of control material derived from positive results from the low copy number EV or HPeV controls traditional virus isolation methods becomes increasingly restricted. HAYES ET AL. | 1073 4.3 | Virus typing Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo and Department of Clinical, Surgical, In the current study, EV and HPeV typing were performed in both Diagnostic and Pediatric Sciences, University of Pavia, Pavia, Italy) reference and diagnostic laboratories (Figure 4). Although all results for assistance with the testing (L9); Margarita del Cuerpo for her reported the correct EV‐type identification and most laboratories excellent technical assistance and for support in the analysis and successfully typed the high concentration (1000 copies in 5 µL) interpretation of the results (L10); all members of the National transcripts, fewer than 50% of either reference or diagnostic Reference Center for Poliomyelitis and Enteroviruses: Berit Lenz, laboratories could successfully amplify and sequence the low Anja Schmidt, Sandy Sonntag, Claudia Zymelka (L11); Sandra Bergs concentration controls. The observed restriction in assay sensitivity for assistance with the testing (L12); Birgitta Holmgren and Claus underpins the importance of obtaining multiple samples including Bohn Chistiansen for scientific contributions, and Camilla Roseng- blood, respiratory samples and feces for EV diagnostic and typing ren for assistance with the testing (L13); Maija Lappalainen, Helsinki assays where viral loads are higher during acute infections.22-24 It is University Hospital, HUSLAB, Helsinki, Finland (L15); Jeroen also time to consider how EV and HPeV typing data can be centrally Cremer (National Institute for Public Health and the Environment collected and analyzed at the time when increasing numbers of (RIVM), Bilthoven, The Netherlands) for assistance with the testing diagnostic laboratories are starting to introduce typing within the (L17); Gemma Clark for assistance with the testing (L18); Paul Loy, hospital premises.7,16 Joel Paul, Fiona Shufflebottom, Royal Oldham Hospital for In conclusion, effective EV and HPeV detection and type assistance with the testing (L19); Steven Wilson and Helen Kirk identification are integral to clinical management, public health Granger for assistance with the testing (L20); Sheila Waugh for surveillance and outbreak preparedness for emerging strains. assistance with the testing (L22); technical assistance of Alisdair However, their genetic diversity, and often low viral loads in Maclean and Amanda Bradley‐Stewart (L24); Cristina Andrés for diagnostic specimens places stringent demands on the analytical assistance with the testing (L25); Elena Pariani and Sandro Binda for sensitivity and breadth of detection and typing assays. RNA assistance with the testing (L26); Sidsel Krokstad, Department of transcripts provide the means to independently evaluate these Medical Microbiology, St. Olavs University Hospital, 7006 Trond- aspects of their performance. In the future, they can provide heim, Norway for assistance with the testing (L27); Adeline Duard objective and fixed standards needed for a more critical assessment who tested and genotyped all the RNA transcripts in work of the effectiveness of the numerous, newly developed and currently supported by an annual grant to the Centre National de Référence largely unevaluated testing platforms for syndromic testing. We des Enterovirus‐Parechovirus from the French national public would be delighted to provide EV, HPeV and further RNA transcript health network (Santé publique France) (L29); Laura Cano (CIBER- controls for a wider range of viruses to laboratories for QA purposes esp, Centro de Investigación Biomédica en Red en Epidemiología y in the future. Salud Pública, Instituto de Salud Carlos III, Spain) for assistance with the testing; financial support was provided by CIBEResp, Instituto de Salud Carlos III, Spain. (L30); Shukriya Barzinci for the A C K N O W L E D GM E N T S laboratory work (L34); Cristian Celma, Krunal Polra, Karen Fuentes The authors are extremely grateful for Eeva Broberg (European and Jake Dunning (Unit Head) for assistance with the testing (L35); Centre for Disease Prevention and Control (ECDC) for considerable Mariann Nilsen and Zeidad Fernandez at the Virology laboratory at assistance in coordinating this study through the ENPEN network. Oslo University Hospital for excellent technical assistance (L36); The authors are also very grateful to Yoke‐Fun Chan, University Peter Muir (PHE, South West Regional Laboratory, Southmead Malaya for providing the EV‐A71 cDNA clone, and to David J. Evans Hospital, Bristol, UK) for assistance with the transcript testing and Martin Ryan for providing cDNA clones of E30, CAV21, and EV‐ (L37); Styliani Pappa for assistance with the testing in work D68. The work in Oxford was funded in part by a Wellcome ISSF financially supported by the EU Horizon 2020 research and grant (ISSF204826/Z/16/Z). The authors would like to acknowledge innovation program COMPARE (grant agreement No 643476) Prof. Jan Albert, MD (Department of Clinical Microbiology, (L38); Darja Duh and Katja Soršak at National Laboratory of Health, Karolinska University Hospital, and Department of Microbiology, Environment and Food, Department for Medical Microbiology, 2000 Tumor and Cell Biology, Karolinska Institute) for support in the Maribor, Slovenia for performing Sanger sequencing of the analysis, Annelie Bjerkner (Department of Clinical Microbiology, transcript panel for further genotype identification/analyses (L40); Karolinska University Hospital) for expert technical assistance (L1); Seb Cotton and Gina McAllister of the Edinburgh Specialist Virology Sanela Numanovic from the Norwegian Institute of Public Health Centre for assistance with the testing (L43); Svetlana Kaijalainen for her excellent technical assistance (L3); Christina Christodoulou and Päivi Hirttiö‐Tallbacka at the Expert Microbiology Unit, THL, for support in the analysis and interpretation the results (L4); Dr Helsinki (L44); Gaia Nebbia, Department of Infection, Guy’s and St Cillian De Gascun and Dr Jonathan Dean for assistance with the Thomas’ NHS Trust, London and Jane Mullen, Virology Laboratory testing (L5); Javier Martin for scientific support for the study (L6); Manager, Viapath for assistance with the testing (L45); Maria transcript testing by Laboratoriolääketieteen edistämissäätiö sr. 4 Zambon for scientific support for the study (L46) and Mandy (L7); Federica Giardina and Fausto Baldanti (Molecular Virology Bloemen for laboratory work (L99). 1074 | HAYES ET AL. 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