The role of laboratory diagnostics in emerging viral infections: the example of the Middle East respiratory syndrome epidemic

Rapidly emerging infectious disease outbreaks place a great strain on laboratories to develop and implement sensitive and specific diagnostic tests for patient management and infection control in a timely manner. Furthermore, laboratories also play a role in real-time zoonotic, environmental, and epidemiological investigations to identify the ultimate source of the epidemic, facilitating measures to eventually control the outbreak. Each assay modality has unique pros and cons; therefore, incorporation of a battery of tests using traditional culture-based, molecular and serological diagnostics into diagnostic algorithms is often required. As such, laboratories face challenges in assay development, test evaluation, and subsequent quality assurance. In this review, we describe the different testing modalities available for the ongoing Middle East respiratory syndrome (MERS) epidemic including cell culture, nucleic acid amplification, antigen detection, and antibody detection assays. Applications of such tests in both acute clinical and epidemiological investigation settings are highlighted. Using the MERS epidemic as an example, we illustrate the various challenges faced by laboratories in test development and implementation in the setting of a rapidly emerging infectious disease. Future directions in the diagnosis of MERS and other emerging infectious disease investigations are also highlighted.

[1]  T. Wolff,et al.  Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections. , 2012, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[2]  Y. Guan,et al.  Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures , 2013, Journal of Virology.

[3]  Herman Tse,et al.  Interspecies transmission and emergence of novel viruses: lessons from bats and birds , 2013, Trends in Microbiology.

[4]  K. To,et al.  Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? , 2012, Journal of Infection.

[5]  Z. Memish,et al.  A family cluster of Middle East Respiratory Syndrome Coronavirus infections related to a likely unrecognized asymptomatic or mild case , 2013, International Journal of Infectious Diseases.

[6]  B. Bosch,et al.  Antibodies against MERS Coronavirus in Dromedary Camels, United Arab Emirates, 2003 and 2013 , 2014, Emerging infectious diseases.

[7]  Kevin M. Bradley,et al.  Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA , 2015, Analytical Biochemistry.

[8]  A. Hajeer,et al.  Severe neurologic syndrome associated with Middle East respiratory syndrome corona virus (MERS-CoV) , 2015, Infection.

[9]  Yee-Joo Tan,et al.  Use of Viral Lysate Antigen Combined with Recombinant Protein in Western Immunoblot Assay as Confirmatory Test for Serodiagnosis of Severe Acute Respiratory Syndrome , 2004, Clinical Diagnostic Laboratory Immunology.

[10]  Michael G. Katze,et al.  Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques , 2013, Proceedings of the National Academy of Sciences.

[11]  H. Feldmann,et al.  The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters , 2013, PloS one.

[12]  Andrew D. Ellington,et al.  Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) , 2015, PloS one.

[13]  C. Yip,et al.  Zika fever and congenital Zika syndrome: an unexpected emerging arboviral disease , 2016, BDJ.

[14]  K. To,et al.  Active Replication of Middle East Respiratory Syndrome Coronavirus and Aberrant Induction of Inflammatory Cytokines and Chemokines in Human Macrophages: Implications for Pathogenesis , 2013, The Journal of infectious diseases.

[15]  J. Chan,et al.  The emerging novel Middle East respiratory syndrome coronavirus: The “knowns” and “unknowns” , 2013, Journal of the Formosan Medical Association.

[16]  T. Bestebroer,et al.  MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment , 2013, The Journal of general virology.

[17]  Honglin Chen,et al.  An Animal Model of MERS Produced by Infection of Rhesus Macaques With MERS Coronavirus , 2013, The Journal of infectious diseases.

[18]  Tsugunori Notomi,et al.  Detection of Middle East respiratory syndrome coronavirus using reverse transcription loop-mediated isothermal amplification (RT-LAMP) , 2014, Virology Journal.

[19]  K. To,et al.  Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response , 2014, Virology.

[20]  Fekri Abroug,et al.  Family Cluster of Middle East Respiratory Syndrome Coronavirus Infections, Tunisia, 2013 , 2014, Emerging infectious diseases.

[21]  K. To,et al.  Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses , 2015, Journal of Clinical Microbiology.

[22]  P. Österlund,et al.  Middle East respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells. , 2016, The Journal of general virology.

[23]  Z. Memish,et al.  Viral Shedding and Antibody Response in 37 Patients With Middle East Respiratory Syndrome Coronavirus Infection , 2015, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[24]  A. Fontanet,et al.  Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission , 2013, The Lancet.

[25]  Wenling Wang,et al.  Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China , 2016, Emerging microbes & infections.

[26]  W. Tan,et al.  Serological Study of An Imported Case of Middle East Respiratory Syndrome and His Close Contacts in China, 2015 , 2016, Biomedical and Environmental Sciences.

[27]  U. Buchholz,et al.  Contact investigation of a case of human novel coronavirus infection treated in a German hospital, October-November 2012. , 2013, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[28]  L. Poon,et al.  Comparison of serological assays in human Middle East respiratory syndrome (MERS)-coronavirus infection. , 2015, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[29]  V. Cheng,et al.  Ebola virus disease: a highly fatal and panic-generating infectious disease reemerging in West Africa , 2014 .

[30]  M. Seong,et al.  Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR , 2016, Annals of laboratory medicine.

[31]  R. König,et al.  High Secretion of Interferons by Human Plasmacytoid Dendritic Cells upon Recognition of Middle East Respiratory Syndrome Coronavirus , 2015, Journal of Virology.

[32]  T. Bestebroer,et al.  Screening of an FDA-Approved Compound Library Identifies Four Small-Molecule Inhibitors of Middle East Respiratory Syndrome Coronavirus Replication in Cell Culture , 2014, Antimicrobial Agents and Chemotherapy.

[33]  Marion P G Koopmans,et al.  Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation , 2013, The Lancet Infectious Diseases.

[34]  Herman Tse,et al.  Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests , 2013, Journal of Infection.

[35]  Victor M Corman,et al.  Presence of Middle East respiratory syndrome coronavirus antibodies in Saudi Arabia: a nationwide, cross-sectional, serological study , 2015, The Lancet Infectious Diseases.

[36]  K. To,et al.  Cross-species transmission and emergence of novel viruses from birds , 2015, Current Opinion in Virology.

[37]  K. To,et al.  Differential Cell Line Susceptibility to the Emerging Novel Human Betacoronavirus 2c EMC/2012: Implications for Disease Pathogenesis and Clinical Manifestation , 2013, The Journal of infectious diseases.

[38]  V. Cheng,et al.  Emergence in China of human disease due to avian influenza A(H10N8)--cause for concern? , 2014, The Journal of infection.

[39]  P. Woo,et al.  A Systematic Approach to Novel Virus Discovery in Emerging Infectious Disease Outbreaks , 2015, The Journal of Molecular Diagnostics.

[40]  D. Dimitrov,et al.  Exceptionally Potent Neutralization of Middle East Respiratory Syndrome Coronavirus by Human Monoclonal Antibodies , 2014, Journal of Virology.

[41]  Christian Drosten,et al.  Performance and clinical validation of the RealStar® MERS-CoV Kit for detection of Middle East respiratory syndrome coronavirus RNA , 2014, Journal of Clinical Virology.

[42]  T. Bestebroer,et al.  MERS-coronavirus replication induces severe in vitro cytopathology and is strongly 3 inhibited by cyclosporin A or interferon-alpha treatment 4 5 6 , 2013 .

[43]  T. Kuiken,et al.  Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits , 2015, Journal of Virology.

[44]  Yan Li,et al.  Bat Origins of MERS-CoV Supported by Bat Coronavirus HKU4 Usage of Human Receptor CD26 , 2014, Cell Host & Microbe.

[45]  Chuan Qin,et al.  MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2 , 2016, Nature Microbiology.

[46]  Susanna K. P. Lau,et al.  Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease , 2015, Clinical Microbiology Reviews.

[47]  Seung Jun Lee,et al.  External Quality Assessment of MERS-CoV Molecular Diagnostics During the 2015 Korean Outbreak , 2015, Annals of laboratory medicine.

[48]  Victor M Corman,et al.  Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection , 2013, The Lancet Infectious Diseases.

[49]  A. Osterhaus,et al.  Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. , 2012, The New England journal of medicine.

[50]  Alimuddin Zumla,et al.  Coronaviruses — drug discovery and therapeutic options , 2016, Nature Reviews Drug Discovery.

[51]  Seung Jun Lee,et al.  Viral RNA in Blood as Indicator of Severe Outcome in Middle East Respiratory Syndrome Coronavirus Infection , 2016, Emerging infectious diseases.

[52]  P. Woo,et al.  Differential Sensitivities of Severe Acute Respiratory Syndrome (SARS) Coronavirus Spike Polypeptide Enzyme-Linked Immunosorbent Assay (ELISA) and SARS Coronavirus Nucleocapsid Protein ELISA for Serodiagnosis of SARS Coronavirus Pneumonia , 2005, Journal of Clinical Microbiology.

[53]  Victor M Corman,et al.  Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study , 2013, The Lancet Infectious Diseases.

[54]  P. Woo,et al.  Longitudinal Profile of Immunoglobulin G (IgG), IgM, and IgA Antibodies against the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein in Patients with Pneumonia Due to the SARS Coronavirus , 2004, Clinical Diagnostic Laboratory Immunology.

[55]  Christina E Douglas,et al.  Real-time reverse transcriptase polymerase chain reaction assays for Middle East Respiratory Syndrome , 2015, Molecular and Cellular Probes.

[56]  H. Zeller,et al.  Laboratory capability for molecular detection and confirmation of novel coronavirus in Europe, November 2012. , 2012, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[57]  G. Lin,et al.  External quality assessment for the molecular detection of MERS-CoV in China , 2015, Journal of Clinical Virology.

[58]  Z. Memish,et al.  Investigation of Anti–Middle East Respiratory Syndrome Antibodies in Blood Donors and Slaughterhouse Workers in Jeddah and Makkah, Saudi Arabia, Fall 2012 , 2013, The Journal of infectious diseases.

[59]  Christian Drosten,et al.  Serological assays for emerging coronaviruses: Challenges and pitfalls , 2014, Virus Research.

[60]  K. To,et al.  Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus , 2013, Journal of Infection.

[61]  Ik Joon Jo,et al.  MERS-CoV outbreak following a single patient exposure in an emergency room in South Korea: an epidemiological outbreak study , 2016, The Lancet.

[62]  P. Woo,et al.  Genetic relatedness of the novel human group C betacoronavirus to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5 , 2012, Emerging Microbes & Infections.

[63]  E. Lau,et al.  Kinetics of Serologic Responses to MERS Coronavirus Infection in Humans, South Korea , 2015, Emerging infectious diseases.

[64]  Z. Memish,et al.  Transmission of MERS-coronavirus in household contacts. , 2014, The New England journal of medicine.

[65]  T. Bestebroer,et al.  Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction. , 2012, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[66]  H. Woo,et al.  Surveillance of the Middle East respiratory syndrome (MERS) coronavirus (CoV) infection in healthcare workers after contact with confirmed MERS patients: incidence and risk factors of MERS-CoV seropositivity , 2016, Clinical Microbiology and Infection.

[67]  D. Cummings,et al.  Hospital outbreak of Middle East respiratory syndrome coronavirus. , 2013, The New England journal of medicine.

[68]  J. Peiris,et al.  Evaluation of the Directigen FluA+B Test for Rapid Diagnosis of Influenza Virus Type A and B Infections , 2002, Journal of Clinical Microbiology.

[69]  J. Chan,et al.  Middle East respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis , 2015, Virology Journal.

[70]  Kwok-Hung Chan,et al.  A highly specific rapid antigen detection assay for on-site diagnosis of MERS , 2016, Journal of Infection.

[71]  P. Woo,et al.  A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age , 2015, Emerging Microbes & Infections.

[72]  Z. Memish,et al.  Middle East respiratory syndrome coronavirus infections in health care workers. , 2013, The New England journal of medicine.

[73]  Julie Dyall,et al.  Repurposing of Clinically Developed Drugs for Treatment of Middle East Respiratory Syndrome Coronavirus Infection , 2014, Antimicrobial Agents and Chemotherapy.

[74]  Kevin M. Bradley,et al.  Standard and AEGIS nicking molecular beacons detect amplicons from the Middle East respiratory syndrome coronavirus , 2016, Journal of Virological Methods.

[75]  R. Baric,et al.  Rapid generation of a mouse model for Middle East respiratory syndrome , 2014, Proceedings of the National Academy of Sciences.

[76]  M. Müller,et al.  Replicative Capacity of MERS Coronavirus in Livestock Cell Lines , 2014, Emerging infectious diseases.

[77]  George F. Gao,et al.  Structure of the Fusion Core and Inhibition of Fusion by a Heptad Repeat Peptide Derived from the S Protein of Middle East Respiratory Syndrome Coronavirus , 2013, Journal of Virology.

[78]  B. Bosch,et al.  Antibodies against MERS Coronavirus in Dromedary Camels, Kenya, 1992–2013 , 2014, Emerging infectious diseases.

[79]  Z. Memish,et al.  Middle East Respiratory Syndrome Coronavirus Disease in Children , 2014, The Pediatric infectious disease journal.

[80]  K. Yuen,et al.  Potent Neutralization of MERS-CoV by Human Neutralizing Monoclonal Antibodies to the Viral Spike Glycoprotein , 2014, Science Translational Medicine.

[81]  P. Patel,et al.  Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus , 2013, PLoS currents.

[82]  P. Woo,et al.  Polyphyletic origin of MERS coronaviruses and isolation of a novel clade A strain from dromedary camels in the United Arab Emirates , 2016, Emerging Microbes & Infections.

[83]  Z. Memish,et al.  Infectious Middle East Respiratory Syndrome Coronavirus Excretion and Serotype Variability Based on Live Virus Isolates from Patients in Saudi Arabia , 2015, Journal of Clinical Microbiology.

[84]  B. Bosch,et al.  Specific serology for emerging human coronaviruses by protein microarray. , 2013, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[85]  Lu Lu,et al.  Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor , 2014, Nature Communications.

[86]  S. Park,et al.  Viral Load Kinetics of MERS Coronavirus Infection. , 2016, The New England journal of medicine.

[87]  P. Woo,et al.  Genetic Characterization of Betacoronavirus Lineage C Viruses in Bats Reveals Marked Sequence Divergence in the Spike Protein of Pipistrellus Bat Coronavirus HKU5 in Japanese Pipistrelle: Implications for the Origin of the Novel Middle East Respiratory Syndrome Coronavirus , 2013, Journal of Virology.

[88]  T. Uyeki,et al.  Development of Medical Countermeasures to Middle East Respiratory Syndrome Coronavirus , 2016, Emerging infectious diseases.

[89]  Xiaoyan Lu,et al.  Real-Time Reverse Transcription-PCR Assay Panel for Middle East Respiratory Syndrome Coronavirus , 2013, Journal of Clinical Microbiology.

[90]  J. Chan,et al.  The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities , 2013, The Lancet Infectious Diseases.

[91]  Xinxia Peng,et al.  Infection with MERS-CoV Causes Lethal Pneumonia in the Common Marmoset , 2014, PLoS pathogens.

[92]  Y. Guan,et al.  Detection of SARS Coronavirus in Patients with Suspected SARS , 2004, Emerging infectious diseases.

[93]  Z. Memish,et al.  Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study , 2013, The Lancet Infectious Diseases.

[94]  A. Tamin,et al.  Persistence of Antibodies against Middle East Respiratory Syndrome Coronavirus , 2016, Emerging infectious diseases.

[95]  Chuan Qin,et al.  Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways , 2015, The Journal of infectious diseases.

[96]  R. Couch,et al.  Generation of a Transgenic Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease , 2015, Journal of Virology.

[97]  Y. Guan,et al.  Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013. , 2013, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[98]  Ziad A Memish,et al.  Family cluster of Middle East respiratory syndrome coronavirus infections. , 2013, The New England journal of medicine.

[99]  G. Ha,et al.  Development and Validation of a Rapid Immunochromatographic Assay for Detection of Middle East Respiratory Syndrome Coronavirus Antigen in Dromedary Camels , 2015, Journal of Clinical Microbiology.

[100]  P. Woo,et al.  A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus , 2015, Emerging Microbes & Infections.

[101]  A. Ryo,et al.  Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen , 2016, Front. Microbiol..

[102]  H. Feldmann,et al.  Pneumonia from human coronavirus in a macaque model. , 2013, The New England journal of medicine.

[103]  Jian-Piao Cai,et al.  Treatment With Lopinavir/Ritonavir or Interferon-β1b Improves Outcome of MERS-CoV Infection in a Nonhuman Primate Model of Common Marmoset , 2015, The Journal of infectious diseases.

[104]  Z. Memish,et al.  Respiratory Tract Samples, Viral Load, and Genome Fraction Yield in Patients With Middle East Respiratory Syndrome , 2014, The Journal of infectious diseases.

[105]  Kwok-Hung Chan,et al.  Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment. , 2013, The Journal of general virology.

[106]  R. Favory,et al.  Kinetics and pattern of viral excretion in biological specimens of two MERS-CoV cases , 2014, Journal of Clinical Virology.

[107]  Krystal L. Matthews,et al.  Wild-type and innate immune-deficient mice are not susceptible to the Middle East respiratory syndrome coronavirus. , 2014, The Journal of general virology.

[108]  R. Baric,et al.  Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution , 2014, Proceedings of the National Academy of Sciences.

[109]  C. Ki,et al.  Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR , 2016, Annals of laboratory medicine.

[110]  M. Buchmeier,et al.  Human Coronavirus EMC Does Not Require the SARS-Coronavirus Receptor and Maintains Broad Replicative Capability in Mammalian Cell Lines , 2012, mBio.

[111]  Susan I Gerber,et al.  2014 MERS-CoV outbreak in Jeddah--a link to health care facilities. , 2015, The New England journal of medicine.

[112]  Victor M Corman,et al.  First international external quality assessment of molecular diagnostics for Mers-CoV , 2015, Journal of Clinical Virology.

[113]  Amit Kapoor,et al.  Middle East Respiratory Syndrome Coronavirus Infection in Dromedary Camels in Saudi Arabia , 2014, mBio.