【病毒外文文献】2018 Proficiency testing for the detection of Middle East respiratory syndrome-coronavirus demonstrates global capacity

This article has been accepted for publication and undergone full peer review but has not been through the copyediting typesetting pagination and proofreading process which may lead to differences between this version and the Version of Record Please cite this article as doi 10 1002 jmv 25266 This article is protected by copyright All rights reserved Accep ted Art i c l e Gregory Walker ORCID iD 0000 0003 0044 1394 Proficiency testing for the detection of Middle East respiratory syndrome coronavirus demonstrates global capacity to detect MERS CoV Running Title Proficiency testing for the detection of MERS CoV Authors T Theis 1 K A Lau 1 J L Gray 1 C J Oxenford 2 G J Walker 3 W D Rawlinson 4 5 1 RCPAQAP Biosecurity Sydney NSW Australia 2 Laboratory and Surveillance Strengthening Team Country Health Emergency Preparedness and IHR World Health Organization Lyon France 3 Virology Research Laboratory Prince of Wales Hospital Sydney NSW Australia 4 Serology and Virology Division SAViD NSW Health Pathology SOMS and BABS University of NSW Sydney NSW Australia 5 Biotechnology and Biomolecular Sciences Faculty of Science and School of Medicine Faculty of Medicine University of NSW Sydney NSW Australia Address correspondence to Professor William D Rawlinson Serology and Virology Division SAViD Microbiology NSW Health Pathology Level 4 Campus Centre Prince of Wales Hospital Randwick NSW 2031 Australia T 61 2 93829113 F 61 2 93829098 E w rawlinson unsw edu au This article is protected by copyright All rights reserved Accep ted Art i c l e ABSTRACT The first reported case of Middle East respiratory syndrome coronavirus MERS CoV infection was identified in Saudi Arabia in September 2012 since which time there have been over 2 000 laboratory confirmed cases including 750 deaths in 27 countries Nucleic acid testing NAT is the preferred method for the detection of MERS CoV A single round of a Proficiency Testing Program PTP was used to assess the capability of laboratories globally to accurately detect the presence of MERS CoV using NAT A panel of eleven lyophilized specimens containing different viral loads of MERS CoV common coronaviruses and in vitro RNA transcripts was distributed to laboratories in all six WHO Regions A total of 96 laboratories from 79 countries participating in the PTP with 76 96 79 2 reporting correct MERS CoV results for all nine scored specimens A further 10 laboratories 10 4 scored correctly in 8 9 specimens of the PTP The majority of laboratories demonstrated satisfactory performance in detecting presence of MERS CoV using NAT However some laboratories require improved assay sensitivity reduced cross contamination of samples and improved speciation of coronavirus subtypes for potentially complex clinical specimens Further PTP and enhanced links with expert laboratories globally may improve the laboratory performance Keywords MERS CoV quality assurance quality assessment nucleic acid testing WHO INTRODUCTION Middle East respiratory syndrome MERS is a viral respiratory disease first identified in Saudi Arabia in 2012 with cases continuing to be detected in the Middle East Cases detected elsewhere have either been infected in the Middle East or been part of a chain of transmission originating in the Middle East The disease is due to infection with the Middle East respiratory syndrome coronavirus MERS CoV previously known as Novel coronavirus 2012 or HCoV EMC 1 2 Human infection is thought to follow exposure to infected camels or human to human transmission primarily in healthcare settings As of March 2018 the World Health Organisation WHO had been notified of 2 143 laboratory confirmed cases of infection with MERS CoV globally including 750 deaths 3 Apart from epidemiologic clustering and virus specific diagnostic testing there is little clinically that distinguishes MERS CoV infection from other severe viral respiratory infections such as severe acute respiratory syndrome SARS or influenza Nucleic acid testing NAT is the preferred method for detecting MERS CoV Suitable specimens for testing include lower respiratory tract samples LRT nasopharyngeal swabs oropharyngeal swabs nasal washes and nasal aspirates The LRT samples have been shown to contain the highest viral loads possibly due to virus tropism for LRT cells 4 5 Confirmation using real time reverse transcription polymerase chain reaction RT PCR assays require a positive result for at least two different specific targets on the MERS CoV genome using a validated assay or a positive rRT PCR result for one specific target on the MERS CoV genome plus This article is protected by copyright All rights reserved Accep ted Art i c l e MERS CoV sequence confirmation from a separate viral genomic target 6 Nucleic acid sequencing of an amplicon has also been recommended when there are discordant results on different assays 6 7 confirming the specificity of the target It is important that laboratories develop technical capability to accurately and promptly identify MERS CoV in order to implement appropriate infection control and isolation procedures to reduce the potential for transmission and aid a rapid epidemiological investigation External quality assessment programs are an essential tool for monitoring the diagnostic proficiency of laboratories and providing results that allow implementation of improved testing thereby strengthening global capability in reducing spread The World Health Organization WHO initiated a single round Proficiency Testing Program PTP for the detection of MERS CoV by PCR which was conducted in the first half of 2015 by the Royal College of Pathologists of Australasia Quality Assurance Programs RCPAQAP METHODS Participation The WHO identified 133 laboratories from 98 countries in all six WHO regions as potential participants 102 laboratories confirmed their interest and 99 subsequently confirmed their ability to receive the panel A total of 96 laboratories located in 79 different countries returned results and participated in the PTP Table 1 The additional verification step was introduced to ensure laboratories had all the documentation and permits in place that were required to import the material into their country Panel description The proficiency testing PT panel consisted of 11 specimens This was made up of nine specimens A I of inactivated gamma irradiation 50 kGy MERS CoV human coronavirus OC43 human coronavirus 229E and a negative control Specimen I as well as two synthetic specimens J 500 nucleotides of the open reading frame 1a ORF1a starting at nucleotide 10 923 a 502 nucleotide region of the open reading frame 1b ORF1b starting at nucleotide 18 054 as well as two regions one from the RNA dependent RNA polymerase RdRp starting at nucleotide 14 994 and another from the nucleocapsid N protein gene starting at nucleotide 29 523 with a length of 392 and 491 nucleotides respectively The five RNA transcripts were designed to yield positive results in RT PCR assays that were originally published by Corman et al 6 7 which formed the basis of the This article is protected by copyright All rights reserved Accep ted Art i c l e WHO Interim guidance Laboratory Testing for Middle East Respiratory Syndrome Coronavirus 9 Specimen J contained all five RNA transcripts whilst Specimen K contained a single transcript covering upE This was designed to challenge participants with a sample K that would yield an equivocal result as confirmatory testing to initial screening would return negative All specimens were provided lyophilised and were tested for homogeneity and stability Homogeneity was confirmed and no significant sample degradation was detected after storage for seven days at 37 C and subsequent 21 days at 80 C The MERS CoV strain used was provided by Public Health England and all coronaviruses included in the panel were prepared at the Victorian Infectious Diseases Reference Laboratory VIDRL in Melbourne Australia Following gamma irradiation of the viruses inactivation was confirmed and viral RNA was quantified using real time PCR 8 The relative measure of the concentration of virus specific target was determined by generating standard curves using a set of MERS CoV and HCoV specific primers to quantify the GE copies mL of each specimen 6 7 Three external referee laboratories confirmed sample characteristics Assessment Criteria Participants were requested to test all specimens of the proficiency testing panel and 1 Rule out or confirm the presence of MERS CoV 2 Rule out or confirm the presence of a HCoV other than MERS CoV 3 Identify the HCoV if present Participating laboratories were assessed on their capacity to correctly analyse specimens A I using their existing PCR detection protocols and reagents This was performed qualitatively with correct responses assigned on the basis of reporting the expected result e g MERS CoV ruled out other HCoV confirmed present and identified as human coronavirus 229E Performance was assessed separately for the detection of MERS CoV and the detection and identification of other HCoVs Results submitted for synthetic specimens J and K were not scored as participants were likely to obtain varying results depending on the gene region and PCR marker used by the testing laboratory In particular participants performing MERS CoV specific testing according to the assay developed by the United States Centers for Disease Control and Prevention US CDC would expect a negative result for Specimen J as this assay targets a region of the N gene that is different to the region that was used to design the in vitro RNA transcript included in Specimen J 10 These specimens were included as they could provide interesting information in regards to how participating laboratories handle equivocal test results This article is protected by copyright All rights reserved Accep ted Art i c l e RESULTS Capability to detect MERS CoV and other HCoVs by PCR Overall 76 96 79 2 laboratories correctly reported the presence or absence of MERS CoV in all nine scored specimens Figure 1 An additional 10 10 4 laboratories correctly reported the presence or absence of MERS CoV in 8 9 scored specimens whilst 10 10 4 had at least 2 discordant results Table 2 The absence of MERS CoV in the negative control sample Specimen I was correctly reported by 88 96 91 7 participants Table 3 5 participants reported false positive results and 3 laboratories did not report a result for this specimen I for unknown reasons A large proportion of participants reported that they have no or limited capability to test for human coronaviruses other than MERS CoV In order to account for this variability performance was assessed separately for the detection of MERS CoV and the detection and identification of other HCoVs There were 69 96 71 9 laboratories that returned results regarding other HCoVs with 27 28 1 correctly confirming or ruling out the presence of other HCoVs in all nine specimens Table 2 and 29 30 2 correctly identifying the two other HCoVs included in Specimen B HCoV OC43 Specimen C HCoV 229E and Specimen H MERS CoV MERS Middle East Respiratory Syndrome MERS CoV Middle East Respiratory Syndrome coronavirus N nucleocapsid protein gene NAT nucleic acid testing ORF1a open reading frame 1a ORF1b open reading frame 1b PT proficiency testing PTP Proficiency Testing Program QA quality assurance RCPAQAP Royal College of Pathologists of Australasia Quality Assurance Programs RdRp RNA dependent RNA polymerase rRT PCR real time reverse transcription polymerase chain reaction upE upstream of the E protein gene US CDC United States Centers for Disease Control and This article is protected by copyright All rights reserved Accep ted Art i c l e Prevention VIDRL Victorian Infectious Diseases Reference Laboratory WHO World Health Organization REFERENCES 1 Woo PC Lau SK Huang Y Yuen KY Coronavirus diversity phylogeny and interspecies jumping Exp Biol Med Maywood 2009 234 10 1117 1127 2 Zaki AM van Boheemen S Bestebroer TM Osterhaus AD Fouchier RA Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia N Engl J Med 2012 367 19 1814 1820 3 WHO Middle East Respiratory Syndrome coronavirus MERS CoV 2018 http www who int emergencies mers cov en Accessed 01 March 2018 4 Palm D Pereyaslov D Vaz J et al Laboratory capability for molecular detection and confirmation of novel coronavirus in Europe November 2012 Euro Surveill 2012 17 49 20335 5 Pebody RG Chand MA Thomas HL et al The United Kingdom public health response to an imported laboratory confirmed case of a novel coronavirus in September 2012 Euro Surveill 2012 17 40 20292 6 Corman VM Muller MA Costabel U et al Assays for laboratory confirmation of novel human coronavirus hCoV EMC infections Euro Surveill 2012 17 49 20334 7 Corman VM Eckerle I Bleicker T et al Detection of a novel human coronavirus by real time reverse transcription polymerase chain reaction Euro Surveill 2012 17 39 20285 8 Lau KA Theis T Gray J Rawlinson WD Ebola Preparedness Diagnosis Improvement Using Rapid Approaches for Proficiency Testing J Clin Microbiol 2017 55 783 790 9 WHO Laboratory Testing for Middle East Respiratory Syndrome Coronavirus Interim guidance 2015 http www who int csr disease coronavirus infections mers laboratory testing en Accessed March 1 2018 10 Lu X Whitaker B Sakthivel SKK et al Real Time Reverse Transcription PCR Assay Panel for Middle East Respiratory Syndrome Coronavirus J Clin Microbiol 2014 52 1 67 75 11 Zhang L Hao M Zhang K et al External quality assessment for the molecular detection of MERS CoV in China J Clin Virol 2016 75 5 9 This article is protected by copyright All rights reserved Accep ted Art i c l e 12 Pas SD Patelb P Reusken C et al First international external quality assessment of molecular diagnostics for Mers CoV J Clin Virol 2015 69 81 85 13 Seong M W Lee SJ Cho SI et al External Quality Assessment of MERS CoV Molecular Diagnostics During the 2015 Korean Outbreak Ann Lab Med 2016 36 3 230 234 Figure 1 Participant performance in the nine scored specimens for MERS CoV Figure 2 Average number of PCR assays performed per specimen per laboratory This article is protected by copyright All rights reserved Accep ted Art i c l e Table 1 Invitation and participation of laboratories worldwide WHO Region Invited Responded Agreed to participate Received samples Reported results La bs Countr ies La bs Countr ies Labs Countrie s Lab s Countri es La bs Countr ies African Region 13 13 7 7 7 7 7 7 6 6 Eastern Mediterranean Region 23 17 19 16 18 15 18 15 17 14 European Region 63 40 45 33 45 33 45 33 45 33 Region of the Americas 9 9 6 6 6 6 5 5 5 5 South East Asia Region 4 4 4 4 4 4 4 4 4 4 Western Pacific Region 21 15 21 15 19 14 19 14 19 14 Total 133 98 102 81 99 79 98 78 96 76 This article is protected by copyright All rights reserved Accep ted Art i c l e Table 2 Participant performance in PT panel Number of correct results No of participants n 96 correctly reporting MERS CoV detection Other HCoV detection Other HCoV identification 9 76 79 2 27 28 1 N A 8 10 10 4 15 15 6 N A 7 7 7 3 2 2 1 N A 6 2 2 1 2 2 1 N A 5 0 0 0 1 1 0 N A 4 0 0 0 4 4 2 N A 3 1 1 0 8 8 3 29 30 2 2 0 0 0 6 6 3 26 27 1 1 0 0 0 4 4 2 4 4 2 0 0 0 0 27 28 1 37 38 5 This article is protected by copyright All rights reserved Accep ted Art i c l e Table 3 Specimen characteristics and performance of participants Specimen Virus Genome equivalents L a No of participants n 96 with MERS CoV correct Other HCoV correct Other HCoV not specified Other HCoV ID correct Other HCoV ID not specified D i f f e r e n t i a t i o n A MERS CoV 4 3 x 10 2 94 97 9 47 49 0 48 50 0 N A N A B HCoV OC43 1 8 x 10 2 90 93 8 60 62 5 27 28 1 55 57 3 41 42 7 C HCoV 229E 1 6 x 10 2 87 90 6 61 66 7 27 28 1 55 57 3 41 42 7 H MERS CoV b ND not determined Table 4 Target genes used by participants for MERS CoV detection Target No of participants n 96 testing for a MERS CoV specific target in each specimen a A B C D E F G H I J K Mean SD upE 94 94 92 93 93 93 93 93 92 94 95 93 3 0 90 97 2 orf1a 49 45 46 49 49 49 49 49 46 51 53 48 6 2 29 50 7 orf1b 32 31 32 32 32 32 32 32 32 32 36 32 3 1 27 33 6 N 21 20 21 21 21 21 21 21 19 22 32 21 8 3 46 22 7 NCV N2 20 20 19 18 20 19 19 19 19 19 19 19 2 0 60 20 0 NCV N3 19 13 12 22 19 19 19 19 12 19 18 17 4 3 38 18 1 RdRp 9 12 10 9 10 10 10 9 9 10 16 10 4 2 06 10 8 a Assays include RT PCR conventional PCR and sequencing assays for N and RdRp