A highly specific rapid antigen detection assay for on-site diagnosis of MERS
Identifieur interne : 000872 ( Pmc/Corpus ); précédent : 000871; suivant : 000873A highly specific rapid antigen detection assay for on-site diagnosis of MERS
Auteurs : Yixin ChenSource :
- The Journal of Infection [ 0163-4453 ] ; 2016.
Url:
DOI: 10.1016/j.jinf.2016.04.014
PubMed: 27144915
PubMed Central: 7127149
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A highly specific rapid antigen detection assay for on-site diagnosis of MERS</title><author><name sortKey="Chen, Yixin" sort="Chen, Yixin" uniqKey="Chen Y" first="Yixin" last="Chen">Yixin Chen</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27144915</idno><idno type="pmc">7127149</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127149</idno><idno type="RBID">PMC:7127149</idno><idno type="doi">10.1016/j.jinf.2016.04.014</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000872</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000872</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A highly specific rapid antigen detection assay for on-site diagnosis of MERS</title><author><name sortKey="Chen, Yixin" sort="Chen, Yixin" uniqKey="Chen Y" first="Yixin" last="Chen">Yixin Chen</name></author></analytic><series><title level="j">The Journal of Infection</title><idno type="ISSN">0163-4453</idno><idno type="eISSN">1532-2742</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Gadsby, N J" uniqKey="Gadsby N">N.J. Gadsby</name></author><author><name sortKey="Helgason, K O" uniqKey="Helgason K">K.O. Helgason</name></author><author><name sortKey="Dickson, E M" uniqKey="Dickson E">E.M. Dickson</name></author><author><name sortKey="Mills, J M" uniqKey="Mills J">J.M. Mills</name></author><author><name sortKey="Lindsay, D S" uniqKey="Lindsay D">D.S. Lindsay</name></author><author><name sortKey="Edwards, G F" uniqKey="Edwards G">G.F. Edwards</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, J F" uniqKey="Chan J">J.F. Chan</name></author><author><name sortKey="Lau, S K" uniqKey="Lau S">S.K. Lau</name></author><author><name sortKey="To, K K" uniqKey="To K">K.K. To</name></author><author><name sortKey="Cheng, V C" uniqKey="Cheng V">V.C. Cheng</name></author><author><name sortKey="Woo, P C" uniqKey="Woo P">P.C. Woo</name></author><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K.Y. Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wernery, U" uniqKey="Wernery U">U. Wernery</name></author><author><name sortKey="Corman, V M" uniqKey="Corman V">V.M. Corman</name></author><author><name sortKey="Wong, E Y" uniqKey="Wong E">E.Y. Wong</name></author><author><name sortKey="Tsang, A K" uniqKey="Tsang A">A.K. Tsang</name></author><author><name sortKey="Muth, D" uniqKey="Muth D">D. Muth</name></author><author><name sortKey="Lau, S K" uniqKey="Lau S">S.K. Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Corman, V M" uniqKey="Corman V">V.M. Corman</name></author><author><name sortKey="Eckerle, I" uniqKey="Eckerle I">I. Eckerle</name></author><author><name sortKey="Bleicker, T" uniqKey="Bleicker T">T. Bleicker</name></author><author><name sortKey="Zaki, A" uniqKey="Zaki A">A. Zaki</name></author><author><name sortKey="Landt, O" uniqKey="Landt O">O. 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Chen</name></author><author><name sortKey="Luk, H K" uniqKey="Luk H">H.K. Luk</name></author><author><name sortKey="Poon, R W" uniqKey="Poon R">R.W. Poon</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Song, L W" uniqKey="Song L">L.W. Song</name></author><author><name sortKey="Wang, Y B" uniqKey="Wang Y">Y.B. Wang</name></author><author><name sortKey="Fang, L L" uniqKey="Fang L">L.L. Fang</name></author><author><name sortKey="Wu, Y" uniqKey="Wu Y">Y. Wu</name></author><author><name sortKey="Yang, L" uniqKey="Yang L">L. Yang</name></author><author><name sortKey="Chen, J Y" uniqKey="Chen J">J.Y. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, P C" uniqKey="Woo P">P.C. Woo</name></author><author><name sortKey="Lau, S K" uniqKey="Lau S">S.K. Lau</name></author><author><name sortKey="Wernery, U" uniqKey="Wernery U">U. Wernery</name></author><author><name sortKey="Wong, E Y" uniqKey="Wong E">E.Y. Wong</name></author><author><name sortKey="Tsang, A K" uniqKey="Tsang A">A.K. Tsang</name></author><author><name sortKey="Johnson, B" uniqKey="Johnson B">B. Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, K H" uniqKey="Chan K">K.H. Chan</name></author><author><name sortKey="To, K K" uniqKey="To K">K.K. To</name></author><author><name sortKey="Chan, J F" uniqKey="Chan J">J.F. Chan</name></author><author><name sortKey="Li, C P" uniqKey="Li C">C.P. Li</name></author><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K.Y. Yuen</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="letter"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Infect</journal-id><journal-id journal-id-type="iso-abbrev">J. Infect</journal-id><journal-title-group><journal-title>The Journal of Infection</journal-title></journal-title-group><issn pub-type="ppub">0163-4453</issn><issn pub-type="epub">1532-2742</issn><publisher><publisher-name>Published by Elsevier Ltd on behalf of The British Infection Association.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27144915</article-id><article-id pub-id-type="pmc">7127149</article-id><article-id pub-id-type="publisher-id">S0163-4453(16)30041-X</article-id><article-id pub-id-type="doi">10.1016/j.jinf.2016.04.014</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A highly specific rapid antigen detection assay for on-site diagnosis of MERS</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Chen</surname><given-names>Yixin</given-names></name></contrib></contrib-group><aff id="aff1">State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, School of Public Health, Xiamen University, Xiamen 361102, Fujian, China</aff><contrib-group><contrib contrib-type="author" id="au2"><name><surname>Chan</surname><given-names>Kwok-Hung</given-names></name></contrib></contrib-group><aff id="aff2">Department of Microbiology, The University of Hong Kong, Hong Kong</aff><contrib-group><contrib contrib-type="author" id="au3"><name><surname>Hong</surname><given-names>Congming</given-names></name></contrib><contrib contrib-type="author" id="au4"><name><surname>Kang</surname><given-names>Yahong</given-names></name></contrib><contrib contrib-type="author" id="au5"><name><surname>Ge</surname><given-names>Shengxiang</given-names></name></contrib></contrib-group><aff id="aff3">State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, School of Public Health, Xiamen University, Xiamen 361102, Fujian, China</aff><contrib-group><contrib contrib-type="author" id="au6"><name><surname>Chen</surname><given-names>Honglin</given-names></name></contrib></contrib-group><aff id="aff4">Department of Microbiology, The University of Hong Kong, Hong Kong</aff><aff id="aff5">State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong</aff><aff id="aff6">Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong</aff><aff id="aff7">Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong</aff><aff id="aff8">Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310006, China</aff><contrib-group><contrib contrib-type="author" id="au7"><name><surname>Wong</surname><given-names>Emily Y.M.</given-names></name></contrib></contrib-group><aff id="aff9">Department of Microbiology, The University of Hong Kong, Hong Kong</aff><contrib-group><contrib contrib-type="author" id="au8"><name><surname>Joseph</surname><given-names>Sunitha</given-names></name></contrib><contrib contrib-type="author" id="au9"><name><surname>Patteril</surname><given-names>Nissi Georgy</given-names></name></contrib><contrib contrib-type="author" id="au10"><name><surname>Wernery</surname><given-names>Ulrich</given-names></name></contrib></contrib-group><aff id="aff10">Central Veterinary Research Laboratory, Dubai, United Arab Emirates</aff><contrib-group><contrib contrib-type="author" id="au11"><name><surname>Xia</surname><given-names>Ningshao</given-names></name></contrib></contrib-group><aff id="aff11">State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, School of Public Health, Xiamen University, Xiamen 361102, Fujian, China</aff><contrib-group><contrib contrib-type="author" id="au12"><name><surname>Lau</surname><given-names>Susanna K.P.</given-names></name></contrib><contrib contrib-type="author" id="au13"><name><surname>Woo</surname><given-names>Patrick C.Y.</given-names></name><email>pcywoo@hku.hk</email><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff12">Department of Microbiology, The University of Hong Kong, Hong Kong</aff><aff id="aff13">State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong</aff><aff id="aff14">Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong</aff><aff id="aff15">Carol Yu Centre for Infection, The University of Hong Kong, Hong Kong</aff><aff id="aff16">Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou 310006, China</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author. State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital Compound, Pokfulam Road, Hong Kong. Tel.: +852 22554892; fax: +852 28551241. <email>pcywoo@hku.hk</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>1</day><month>5</month><year>2016</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>7</month><year>2016</year></pub-date><pub-date pub-type="epub"><day>1</day><month>5</month><year>2016</year></pub-date><volume>73</volume><issue>1</issue><fpage>82</fpage><lpage>84</lpage><history><date date-type="accepted"><day>24</day><month>4</month><year>2016</year></date></history><permissions><copyright-statement>© 2016 Published by Elsevier Ltd on behalf of The British Infection Association.</copyright-statement><copyright-year>2016</copyright-year><copyright-holder></copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><related-article related-article-type="article-reference" id="d32e302" ext-link-type="doi" xlink:href="10.1016/j.jinf.2015.10.011"></related-article></article-meta></front><body><p content-type="salutation">Dear Editors,</p><p id="p0010">Gadsby et al. in this Journal, recently described the clinical utility of molecular diagnostics in identifying <italic>Legionella</italic> infection in patients with acute LRTI.<xref rid="bib1" ref-type="bibr"><sup>1</sup></xref> We developed a rapid antigen detection assay for on-site diagnosis of MERS, which can be finished in 30 min.</p><p id="p0015">Since its first appearance in 2012, MERS has affected >25 countries in four continents with >1300 cases and a high fatality rate of >30%.<xref rid="bib2" ref-type="bibr"><sup>2</sup></xref> Detection of MERS coronavirus (MERS-CoV) and its antibodies in dromedaries implied that these animals are probably its reservoir.<xref rid="bib3" ref-type="bibr"><sup>3</sup></xref> Rapid laboratory diagnosis is essential for commencement of infection control measures. Although MERS-CoV can be cultured and antibody detection methods available, laboratory diagnosis of MERS-CoV infections is mainly achieved by real-time quantitative RT-PCR (qRT-PCR).<xref rid="bib4" ref-type="bibr"><sup>4</sup></xref> Recently, we have described the development of a monoclonal antibody (MAb)-based capture ELISA for MERS-CoV N protein (NP) detection and its use in field study for dromedaries.<xref rid="bib5" ref-type="bibr">5</xref>, <xref rid="bib6" ref-type="bibr">6</xref> However, both real-time qRT-PCR and NP capture ELISA take at least several hours to complete and require specific equipments in a clinical laboratory. In this study, we developed a MAb-based rapid NP detection assay for MERS.</p><p id="p0020">Cloning and purification of (His)<sub>6</sub>-tagged recombinant NP (rNP) of MERS-CoV was performed as described previously,<xref rid="bib6" ref-type="bibr"><sup>6</sup></xref> using a MERS-CoV strain (D789.1/14) isolated from a patient in Dubai. Production and selection of specific MAbs against the MERS-CoV NP was performed as described previously.<xref rid="bib6" ref-type="bibr"><sup>6</sup></xref> The rapid MERS-CoV NP detection assay, in the format of a lateral flow immunoassay (LFIA), was developed using a similar approach as described previously,<xref rid="bib7" ref-type="bibr"><sup>7</sup></xref> using LFIA test strip (Millipore) and two highly specific MAbs against the MERS-CoV rNP (MD3E9 for coating and MD8B6 for conjugating). Testing is carried out by mixing 380 μl of each sample with 20 μl lysis buffer for 10 s at room temperature. Then, 80 μl of the pre-treated sample was transferred into the sample well of the test card slowly at room temperature. Results are read visually in 30 min. Result is considered negative when the control line is positive but the test line negative, and considered positive when both the control and test lines are positive (<xref rid="fig1" ref-type="fig">Fig. 1</xref>). If the control line is negative, the test is considered invalid.<fig id="fig1"><label>Figure 1</label><caption><p>Rapid MERS-CoV assay showing negative, positive and weakly positive results.</p></caption><graphic xlink:href="gr1_lrg"></graphic></fig></p><p id="p0025">Initial assessment of the detection limit was performed using a human nasopharyngeal aspirate (NPA) spiked with various concentrations of MERS-CoV. First round of evaluation was performed using 56 human NPAs positive for various respiratory viruses and 10 human NPAs spiked with 10<sup>5.2</sup> TCID<sub>50</sub>/ml MERS-CoV. All human NPAs were collected from the clinical microbiology laboratory in Queen Mary Hospital, Hong Kong. Second round of evaluation was performed using 81 dromedary nasal swabs/tonsils collected during necropsies at the Central Veterinary Research Laboratory in Dubai. These dromedary respiratory samples were tested by real-time qRT-PCR and the rapid MERS-CoV assay. All tests were performed in duplicate.</p><p id="p0030">Initial assessment showed that the rapid MERS-CoV assay was consistently positive for NPA spiked with ≥10<sup>4.2</sup> TCID<sub>50</sub>/ml of MERS-CoV (<xref rid="tbl1" ref-type="table">Table 1</xref>). Evaluation using human NPAs showed that all 56 NPAs positive for various respiratory viruses were negative for the rapid assay and all the 10 NPAs spiked with 10<sup>5.2</sup> TCID<sub>50</sub>/ml of MERS-CoV were positive for the rapid assay. Further evaluation using dromedary nasal swabs/tonsils showed that all 65 samples negative by real-time qRT-PCR were also negative for by the rapid assay, indicating a specificity of 100%. For the 16 nasal swabs/tonsils positive by real-time qRT-PCR, 13 were also positive by rapid assay, indicating a sensitivity of 81%. All the 13 rapid MERS-CoV assay-positive samples had viral loads of ≥7.36 × 10<sup>7</sup> copies/ml, whereas the three rapid MERS-CoV assay-negative samples had viral loads of ≤2.14 × 10<sup>7</sup> copies/ml.<table-wrap position="float" id="tbl1"><label>Table 1</label><caption><p>Evaluation of rapid MERS-CoV assay.</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Evaluation</th><th>Rapid MERS-CoV assay results</th></tr></thead><tbody><tr><td colspan="2">Initial evaluation [Concentration of spiked MERS-CoV (log<sub>10</sub> TCID<sub>50</sub>/ml)]</td></tr><tr><td> 5.2</td><td>Positive</td></tr><tr><td> 4.2</td><td>Positive</td></tr><tr><td> 3.7</td><td>Positive</td></tr><tr><td> 3.2</td><td>Negative</td></tr><tr><td> 2.2</td><td>Negative</td></tr><tr><td colspan="2">Evaluation using human NPAs</td></tr><tr><td> NPAs positive for various respiratory viruses (n = 56)<xref rid="tbl1fna" ref-type="table-fn">a</xref></td><td>100% negative</td></tr><tr><td> NPAs spiked with MERS-CoV (n = 10)</td><td>100% positive</td></tr><tr><td colspan="2">Evaluation using dromedary nasal swabs/tonsils</td></tr><tr><td> RT-PCR positive (n = 16)</td><td>81% positive</td></tr><tr><td> RT-PCR negative (n = 65)</td><td>100% negative</td></tr></tbody></table><table-wrap-foot><fn id="tbl1fna"><label>a</label><p id="ntpara0010">Include influenza A virus (n = 5), influenza B virus (n = 5), influenza C virus (n = 2), parainfluenza virus 1 (n = 5), parainfluenza virus 2 (n = 5), parainfluenza virus 3 (n = 5), parainfluenza virus 4 (n = 5), human rhinoviruses (n = 5), human metapneumovirus (n = 5), human adenovirus (n = 2), respiratory syncytial virus (n = 2), human bocavirus (n = 2), human CoV HKU1 (HCoV-HKU1) (n = 2), HCoV-OC43 (n = 2), HCoV-229E (n = 2) and HCoV-NL63 (n = 2).</p></fn></table-wrap-foot></table-wrap></p><p id="p0035">We developed a rapid MERS-CoV assay with high specificity. Since the emergence of MERS, the diagnosis of almost all cases was confirmed by real-time qRT-PCR. However, its turn-around-time is about 4 h and the test requires expensive equipment, stringent laboratory set-up and personal attention to prevent cross contamination. As for the antigen capture ELISA that we developed recently, although the main equipments involved are less expensive ELISA plate washer and reader, it still requires technical expertise and 3 h to finish the test.<xref rid="bib6" ref-type="bibr"><sup>6</sup></xref> However, for the rapid MERS-CoV assay developed in the present study, it is extremely simple to perform by any health care worker with minimal training and can be finished in 30 min. Furthermore, the test does not require any specific equipment and the samples need only minimal preparation and pretreatment. In addition to being user-friendly and rapid, the test is highly specific. All human NPAs positive for other respiratory viruses, including all the four human CoVs, or dromedary nasal swabs/tonsils negative for MERS-CoV showed negative results using the rapid MERS-CoV test. In addition, results were also negative for dromedary respiratory/fecal samples spiked with our recently sequenced lineage A betacoronavirus found in dromedaries<xref rid="bib8" ref-type="bibr"><sup>8</sup></xref> (data not shown). All these confirm that this rapid MERS-CoV test has a very high specificity.</p><p id="p0040">The present rapid MERS-CoV test can be used as a rapid screening test for MERS-CoV infection. Despite its high specificity, the rapid MERS-CoV test has a moderate sensitivity. Our results showed that the detection limit of this rapid MERS-CoV test is around 10<sup>3.7</sup>–10<sup>4.2</sup> TCID<sub>50</sub>/ml of MERS-CoV, which is about 25–100 times less sensitive than the antigen capture ELISA with detection limit of about 10<sup>2.3</sup>. A moderately high sensitivity of 81% was confirmed using dromedary respiratory samples positive for MERS-CoV by real-time qRT-PCR. It is quite well-known that similar rapid tests for antigen detection in other viruses are less sensitive than ELISA or other antigen detection methods.<xref rid="bib9" ref-type="bibr"><sup>9</sup></xref> This is because a higher antigen load is required to for these rapid tests to produce a band visible to the naked eye. Since its sensitivity is just moderately high, this rapid MERS-CoV test can be used for initial rapid screening for suspected case of MERS. If the result is positive, the patient can be isolated with other cases of MERS; but if the result is negative, the patient should be isolated in single room and further tests such as real-time qRT-PCR or antigen capture ELISA should be performed.</p><sec id="sec1"><title>Contributions</title><p id="p0045">YC, HC, SKPL and PCYW conceived the study. UW, NX, SKPL and PCYW supervised the study. YC, KHC, CH, YK, SG and EYMW performed the experiments. KHC, EYMW, SJ and NGP collected the samples. YC, KHC, EYMW, SKPL and PCYW analyzed the data. PCYW wrote the manuscript. All authors corrected the manuscript.</p></sec><sec id="sec2"><title>Competing interests</title><p id="p0050">No author has conflict of interest.</p></sec><sec id="sec3"><title>Funding</title><p id="p0055">This work is partly supported by the Theme-based Research Scheme (Project No. T11/707/15), <funding-source id="gs1">University Grant Committee</funding-source>; <funding-source id="gs2">HKSAR Health and Medical Research Fund</funding-source>; <funding-source id="gs3">University Development Fund and Strategic Research Theme Fund, The University of Hong Kong</funding-source>; <funding-source id="gs4">National Natural Science Foundation of China</funding-source> (81371817); and <funding-source id="gs5">The National Scientific and Technological Major Project of China</funding-source> (2013ZX10004804-002).</p></sec></body><back><ref-list id="cebib0010"><title>References</title><ref id="bib1"><label>1</label><element-citation publication-type="journal" id="sref1"><person-group 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