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Seroepidemiological Investigation of Contacts of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Patients (WHO, November 19 2013)

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  • Seroepidemiological Investigation of Contacts of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Patients (WHO, November 19 2013)

    [Source: World Health Organization, full PDF document: (LINK). Edited.]

    Seroepidemiological Investigation of Contacts of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Patients

    Date: v1 19 November 2013



    This document was adapted from a protocol developed by the Consortium for the Standardization for Influenza Seroepidemiology (CONSISE), a global partnership aiming to develop influenza investigation protocols and standardize seroepidemiology to inform public health policy for pandemic, zoonotic and seasonal influenza. This international partnership was created out of a need, identified during the 2009 H1N1 pandemic, for better (standardized, validated) seroepidemiological data to estimate infection attack rates and severity of the pandemic virus and to inform policy decisions. More information on the CONSISE network can be found on their website:

    ? World Health Organization 2013

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    A cohort study design encompassing a comprehensive assessment of all contacts of confirmed and probable MERS-CoV cases, including household, familial, social, occupational and health care-associated contacts, is necessary to determine the spectrum of illness and risk factors associated with infection, routes and risk of transmission, and to guide efforts for prevention of transmission of the MERS-CoV virus. This protocol outlines 1) how to find and test all contacts of laboratory-confirmed and probable MERS-CoV patients, and 2) methods to assess risk factors for MERS-CoV infection.

    Health care personnel contacts can be investigated using a different protocol (see WHO website: ).

    This investigation will provide data to evaluate some of the key clinical, epidemiological and serological characteristics of MERS-CoV cases and their contacts to inform national and international policy, and improve guidance to reduce the spread of infection. This investigation will also provide data to evaluate risk factors for infection.

    Comments for the user?s consideration are provided in purple text throughout the document as the user may need to modify methods slightly because of the local context in which this study will be carried out.



    As of 3 October 2013, more than 139 laboratory-confirmed cases of human infection with novel coronavirus (MERS-CoV) have been reported to WHO[1]. It is suspected that MERS-CoV is a zoonotic virus, but information on exposures is limited and an animal reservoir is still unknown.

    Limited human-to-human transmission has occurred in several clusters in health-care facilities, households and in one work setting [1-6]. At this stage, however, as surveillance targets patients with severe disease, the total spectrum of disease and proportion of infections that may be mild or asymptomatic is unclear. In addition, the attack rate in family, and other social groups, and exposures and activities that result in infection in these settings is unknown.

    A comprehensive assessment of known contacts ? household, familial, social and occupational ? of confirmed and probable MERS-CoV cases can help to determine the extent of MERS-CoV infections, identify potential viral source(s), to understand transmission dynamics, and to guide public health prevention and control efforts to reduce human-to-human transmission of MERS-CoV. This investigation will provide data to evaluate some of the key clinical and epidemiological characteristics of cases and their contacts to inform the development of national and international policy to reduce the spread of MERS-CoV infection. This protocol outlines how to 1) identify and test ALL contacts (not limited to close contacts) for MERS-CoV infection using virologic and serologic testing and 2) provides methods to compare the exposures of infected and non-infected individuals to evaluate risk factors for infection.

    A separate protocol specifically for assessment of health-care personnel is available on the WHO MERS-CoV website.

    The data collected from this study will be used to characterize the key epidemiological transmission features of MERS-CoV virus, to help understand spread, severity, spectrum of disease, and impact on the community and to inform operational models for implementation of countermeasures such as case isolation, contact tracing and quarantine.

    The primary objectives of this study are to:
    • Estimate frequency of MERS-CoV infections (as measured by virologic and serologic tests) in relation to human and other exposures (i.e. evaluate determinants/risk factors [including sources] for infection) among contacts of confirmed MERS-CoV cases
    • Evaluate (modifiable) risk factors (e.g. exposures, behaviours, practices) for human MERS-CoV infection
    • Evaluate the extent of MERS-CoV transmission among contacts of confirmed and probable MERS-CoV patients
    • Describe the presentation and clinical course of disease with MERS-CoV infection

    Quantify the proportion of asymptomatic/sub-clinical MERS-CoV infections

    COMMENT: Comprehensive study investigations such as the one described below can provide rich data to assess a number of secondary outcomes. Many other secondary objectives can be investigated in terms of epidemiological, immunological, clinical, virological, economic, genetic, behavioural, environmental, and animal factors associated with risk of MERS-CoV infection or outcome of infection. These are not discussed in detail in this protocol.



    The first stage of this investigation focuses on finding and testing (virologically and serologically) all contacts of laboratory-confirmed MERS-CoV patients. The cases and contacts identified during this investigation will make up the study cohort. The second stage of the study will be to conduct a nested case-control study among cases (virologically and serologically positive study subjects) and controls (serologically negative study subjects) found within your study cohort to evaluate risk factors for infection.

    Note that for the purposes of monitoring for the appearance of disease in persons at very high risk of infection, it is generally recommended to observe individuals with very close physical contact with a case for a period of up to 14 days after exposure. However, the goal of this study is different. Therefore, for the purposes of this study, it is presumed that individuals in the social sphere of the known case may have had other exposures, similar to those of the case, that could have resulted in primary infection from the source. Therefore, the approach used in this study is to follow the larger social sphere of ?contacts?, including those with relatively limited contact with the case but who might have been exposed to the same environment. For example, if the case owns a farm, all of the workers on the farm should be included for study as ?contacts? regardless of whether they had close physical contact with the known case. One might also include office colleagues, classmates, persons belonging to a social group that the case attends, etc., as additional cohorts of contacts, regardless of the degree of contact with the known case.


    Ethical approval will be sought in accordance with local, regional and national authorities.

    COMMENT: It is strongly recommended that ethical approval be obtained in advance from relevant ethical or institutional review boards (e.g. national Ministries of Health, Agriculture, etc.) using a generic protocol such as this one before an outbreak occurs. Once an outbreak occurs, the study design, questionnaires, sampling and consent forms can be modified rapidly to the actual situation. This may still have to be resubmitted for ethical approval, but as the generic protocol including this final step has already been approved, this could be a very rapid process, without substantial delay to the start of the investigations.



    Case definitions for reporting are provided by WHO and are subject to change as more information becomes available. On 3 July 2013 revised case definitions were published by WHO.

    COMMENT: Check for the most recent case definition at PROBABLE CASE

    COMMENT: The definition of a probable case may vary depending on the volume of cases in the local population. Currently, the definition of a probable case is narrow and intended to maximize contact tracing of laboratory-confirmed cases. During the course of the study, it is possible that the definition of a probable case will change.

    As of 3 July, three combinations of clinical, epidemiological and laboratory criteria can define a probable case:
    • A person with a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or Acute Respiratory Distress Syndrome) AND Testing for MERS-CoV is unavailable or negative on a single inadequate specimen1 AND The patient has a direct epidemiologic-link with a confirmed MERS-CoV case2.
    • A person with a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or Acute Respiratory Distress Syndrome) AND An inconclusive MERS-CoV laboratory test (that is, a positive screening test without confirmation)3 AND A resident of, or traveller to Middle Eastern countries where MERS-CoV virus is believed to be circulating in the 14 days before onset of illness.
    • A person with an acute febrile respiratory illness of any severity AND An inconclusive MERS-CoV laboratory test (that is, a positive screening test without confirmation)3 AND The patient has a direct epidemiologic-link with a confirmed MERS-CoV case2


    (1) An inadequate specimen would include a nasopharyngeal swab without an accompanying lower respiratory specimen, a specimen that has had improper handling, is judged to be of poor quality by the testing laboratory, or was taken too late in the course of illness.

    (2) A direct epidemiological link may include:
    • Close physical contact
    • Working together in close proximity or sharing the same classroom environment
    • Traveling together in any kind of conveyance
    • Living in the same household
    • The epidemiological link may have occurred within a 14-day period before or after the onset of illness in the case under consideration.

    (3) Inconclusive tests may include:
    • A positive screening test without further confirmation, such as testing positive on a single PCR target
    • A serological assay considered positive by the testing laboratory.

    (4) Currently, confirmatory testing requires molecular diagnostics, including either a positive PCR on at least two specific genomic targets or a single positive target with sequencing on a second. However, the interim recommendations for laboratory testing for MERS-CoV should be consulted for the most recent standard for laboratory confirmation ( ). See also notes on asymptomatic cases in this document. CONFIRMED CASE

    The most recent case definitions are available from WHO at

    A confirmed case of MERS-CoV is a person with laboratory confirmation of infection with MERS-CoV, either through direct detection of the virus (by viral isolation) or viral RNA (by molecular methods such as RT-PCR) in clinical specimens.


    The first stage of this investigation will be to identify all contacts of confirmed and probable MERS-CoV patients.

    For the purposes of this study, contacts of a confirmed or probable human MERS-CoV case are defined as all individuals who are associated with some sphere of activity of the case and who may have similar environmental or other exposures as the case. Contacts can include household members, other family contacts, visitors, neighbours, colleagues, teachers, classmates, co-workers, servants, members of a social group, or others, and do not have to have had close personal contact with the case. Each group of contacts will form a separate, though sometimes overlapping, cohort of contacts. For example, one cohort may include all of the schoolmates who sit in the same classroom as the case if the case is a student, or office colleague of an office worker, even if they have not had recent close contact with the case. Alternatively, if the case owns a farm, or works on a farm, all of the workers on that farm could be included as contacts regardless of whether or not they had close physical contact with the case. The goal is to include a broad range of people with different types of exposures who have been part of the same environments as the case in order to be able to link type of exposure to evidence of infection.

    COMMENT: Identification of contacts should be the first stage of your investigation.

    COMMENT: The user of this protocol will need to undertake some preliminary investigations to understand who could be a potential contact of the case(s). The nature and number of contacts will depend on the context of the situation. Contacts should be grouped according to the social sphere of the case in which they belong, e.g. household, workplace, farm, etc.

    COMMENT: The definition of a contact for the purposes of this study is different from contacts as defined for identifying probable cases.

    COMMENT: Health-care personnel exposed to the case should be investigated using a separate protocol. See WHO MERS-CoV HCP Protocol on the WHO coronavirus website:



    Primary study subjects are all contacts of a confirmed MERS-CoV case who are associated with some sphere of activity of the case. All cases and contacts identified in this study will compose the study population cohort.

    All efforts will be made to identify the contact cohort at the initial recruitment, including infants and children, to generate the sampling frame for follow up. Details of the contacts will be kept in a line list by the investigation team (Appendix A). At the time of recruitment, combined nasal and throat swabs for virologic confirmation if individuals are symptomatic, blood for serologic testing, and exposure data will be collected (see Questionnaire in Appendix B). The recommended clinical specimens may change in the future as more information is learned about the optimal specimens and appropriate timing after exposure to detect MERS-CoV infection.

    Active follow-up of all contacts will take place ideally through daily face-to-face or telephone interview as soon as possible after identification of a confirmed case to ask about the possible development of illness. Contacts will be monitored daily for 30 days after the onset of illness in the case. A baseline blood sample will be taken at time of first interview and sent to a reference laboratory.

    Any contact who is symptomatic (fever and cough) at initial visit or by the end of the study period should be tested virologically according to current standards for laboratory confirmation of MERS-CoV. A second ?convalescent? serum sample should be collected from all subjects at least 14-21 days after an acute baseline sample is collected.

    Ill contacts found to have evidence of MERS-CoV infection will be re-classified as confirmed cases and reported as such to WHO. Each newly confirmed MERS-CoV case will initiate a new contact investigation as outlined above.

    COMMENT: If feasible, consider serial sera collection of all recruited subjects at baseline, 2-week, 4-week or 6-week intervals, including for the confirmed case. This will allow study of antibody kinetics in infected individuals.


    During the site visit, the purpose of the study will be explained to all eligible subjects (all contacts) and their consent obtained by a trained member of the investigation team. Consent for children under the age of 18 years old will be obtained from their parents or guardians. Verbal assent will also be obtained for children under 17 years old.

    COMMENT: The age of consent and assent may vary by country. Check with local IRB requirements.


    After enrolment and informed consent, a standardized baseline data set will be collected with any specimens for MERS-CoV testing (and date of specimen collection). Baseline data to be collected include: age, gender, location, relationship to confirmed case-patient, occupation, signs and symptoms, and underlying conditions. In addition, detailed questions will be asked to evaluate risk factors for human infection with MERS-CoV (Appendix B). These are included in the data collection form in Appendix B under the section for ?exposures? and include specific aspects of timing, frequency and duration of exposure(s). The questionnaire should be administered each time sera are collected. The study questionnaire for the use of all cases and contacts can be found in Appendix B.


    Before study implementation, front-line staff, including all study personnel, will be trained in infection control procedures (standard, contact, droplet or airborne precautions), including proper hand hygiene and the correct use of surgical or respiratory face masks, if necessary, not only to minimize their own risk of infection when in close contact with patients during home visits and elsewhere, but also to minimize the risk of staff becoming a vector of MERS-CoV transmission between subjects or households.



    There is now increasing evidence that lower respiratory tract specimens such as bronchoalveolar lavage, sputum and tracheal aspirates contain the highest viral loads and these should be collected when possible. However, published case series also demonstrate the importance of upper respiratory tract specimens such as nasopharyngeal/oropharyngeal swabs for detecting the virus. It is recommended that both upper and lower respiratory tract specimens be collected whenever possible.

    To increase the likelihood of detecting the virus, multiple samples from multiple sites should be collected over the course of the illness. Even after the initial detection of the virus, continued sampling and testing will add to current knowledge about the duration of virus shedding and is strongly encouraged. Virus has been detected in blood, urine and faeces but at levels below those found in the lower respiratory tract.

    Serum samples should be collected. Paired samples are preferred but single samples are also of value. Paired serum samples should be collected 14-21 days apart, with the first being taken during the first week of illness. If only a single sample is to be collected, it should be done at least 14 days after onset of symptoms.

    Specimens should reach the laboratory as soon as possible after collection. The importance of proper handling during transportation cannot be overemphasized. When there is likely to be a delay in the laboratory receiving respiratory tract specimens, it is strongly recommended that the specimens be frozen, preferably to -80?C, and shipped on dry ice. It is, however, important to avoid repeated freezing and thawing of specimens.

    Serum should be separated from whole blood and can be stored and shipped at 4?C or frozen to -20?C or lower and shipped on dry ice. The storage of respiratory and serum specimens in domestic frost-free freezers should be avoided, owing to their wide temperature fluctuations.

    Transport of specimens within national borders should comply with applicable national regulations.

    International transport of MERS-CoV specimens should follow applicable international regulations as described in the WHO Guidance on Regulations for the Transport of Infectious Substances 2013-2014 available at:

    Additional records should be kept for each biological sample, including the time of collection, the conditions for transportation, personnel involved in collection and transport, and the time of arrival at the study laboratory.

    COMMENT: You may consider that specimens will be aliquotted so that specimens remain in country and only aliquots are sent to a reference lab. Some serologic assays may become available to be done in country.


    MERS-CoV case definitions are described above and can be found at:

    As of 6 June, to consider a case as laboratory-confirmed, one of the following conditions must be met:
    • positive RT-PCR or other validated molecular assays for at least two different specific targets on the MERS-CoV genome
      • OR

    • one positive RT-PCR assay for a specific target on the MERS-CoV genome and an additional different PCR product sequenced, confirming identity to known sequences of the new virus.

    A positive PCR assay for a single specific target without further testing is considered presumptive evidence of MERS-CoV infection. Final classification of cases will depend on clinical and epidemiological information, combined with laboratory data. Member States are requested to immediately notify WHO of any confirmed or probable case.

    Full details for virologic laboratory testing of MERS-CoV can be found here: SEROLOGIC METHODS

    COMMENT: Several serological assays are currently available, having been developed by different laboratories using different platforms, including fluorescent antibodies, enzyme-linked immunosorbent assays, luciferase immunoprecipitation systems, and virus neutralisation. The assays each have advantages and disadvantages but appear to have similar utility. Until their interoperability and comparability are better understood, more than one assay should be performed for each serum sample. An algorithm will be needed to decide the combinations of results considered ?positive? for the purpose of comparative analysis.

    COMMENT: Only a limited number of laboratories have the facilities for MERS-CoV serologic testing and therefore collaboration between countries without current capacity and designated reference laboratories is possible.

    Collaboration is at the discretion of Member States carrying out the investigation, but WHO strongly supports such collaboration and would willingly facilitate collaboration and possible shipment elsewhere for testing.


    The following section discusses the endpoints ? that is, what will be measured and calculated using the data that are collected in this study ? for the primary objectives, including statistical advice.



    The following will be assessed as study endpoints corresponding to the study?s primary objectives:
    1. Evaluate source(s), risk exposures, and (modifiable) risk factors for human infection with MERS-CoV
    2. Estimate the age-specific frequency of MERS-CoV infection (as measured by virologic and serologic tests) in relation to human and other exposures
    3. Describe the full spectrum of clinical disease associated with MERS-CoV infection
    4. Quantify proportion of asymptomatic/sub-clinical; mild illness; and severe illness (hospitalization/ICU/death) with MERS-CoV infection.



    One way to measure risk factors for infection is to compare the characteristics and exposures (behaviours and practices) of infected individuals (i.e. laboratory-confirmed and sero-positive individuals) versus non-infected (i.e. sero-negative contacts).

    To assess risk factors for transmission: To evaluate human-to-human transmission, a comparison of human exposures of infected (seropositive and laboratory-confirmed patients) vs non-infected (seronegative contacts) will be made.

    The reported practices among cases and (possibly matched, see above) contacts should be compared using appropriate statistical tests, e.g. bivariate associations between risk factors and (asymptomatic) infection will be determined by chi-square statistics or 2-sided Fisher?s exact test and expressed as odds ratios with 95% confidence intervals. Multivariable logistic regression will be used to further analyse the associations.

    COMMENT: Univariate statistical analysis by use of logistic regression for a case‐control study could be used to test the significance of each predictor on the outcome of infection. Multivariable logistic regression can be used to identify a combination of risk factors associated with the odds of infection.

    COMMENT: Alternatively, Mantel-Haenszel matched-pair analysis (McNemar test) can be used to estimate the strength and statistical significance of associations between exposures and infection. If matched analysis is to be performed, variables to be assessed as risk factors will have to be identified in the beginning of the study so these variables can be used as matching variables.


    The attack rate is defined as the proportion of a well-defined population that develops illness (e.g. signs and symptoms) over a particular period of time.

    List all forms of attack rates that may be estimated: clinical illness attack rates, infection attack rates, attack rate among children, subject type, etc., secondary attack rates.

    The secondary attack rate is a measure of the frequency of new cases of an illness among the contacts of known index cases in a defined period of time. Note that it may be very difficult to distinguish common exposure from secondary transmission.

    One may also calculate infection attack rate (IFR) using serologic results and can calculate attack rates by subject type (e.g. household contacts, occupational contacts) or by age, location (e.g. school, workplace, institutional setting [military, long-term care facility, etc.]).


    The seroprevalence of MERS-CoV antibodies (P) to the MERS-CoV virus can be determined for overall and if sample size allows, by each age group, or contact type (e.g. household, occupational, social or familial contacts) using MERS-CoV serologic results, as follows:
    P MERS-CoV serologic confirmation = (number of cases that test seropositive/ sample size of study population [all contacts recruited and tested]) X 100%
    COMMENT: You will not be able to extrapolate the seropositive rate of the study population to the general population as the contacts cannot be assumed to be a representative sample of the general population.


    The proportion of symptoms of subjects with evidence of infection (individuals with virologic confirmation per WHO guidelines or serologic positive results) should be calculated.


    Individuals in the study survey found to have serological evidence of acute MERS-CoV infection who do not recall having any defined symptoms during the period of the investigation period will be counted as probable asymptomatic infections. The lowest limit of asymptomatic infections is:


    The asymptomatic infection proportion = number of contacts who tested seropositive and had no history of symptoms ? total number of contacts testing seropositive



    Any deviations from the study methodologies should also be reported to aid in the interpretation of findings.


    Although not described as part of this investigation, we recommended that in conjunction with this outbreak investigation of familial, social and HCP contacts, environmental sampling, including testing of areas around the infected household, farms, markets and potential contaminated water sources, and retrospective and prospective animal mortality investigations should supplement these activities in collaboration with relevant parties (Figure 1), particularly if the objective includes identifying a zoonotic source of infection among index cases or contacts of the index case.


    Many people were involved in the creation and revision of this protocol as part of the CONSISE network. They include: Maria Van Kerkhove (Imperial College London, UK), Marianne van der Sande (RIVM; National Institute for Public Health and the Environment, The Netherlands), Anthony Mounts (WHO), Benjamin J Cowling (University of Hong Kong), Timothy Uyeki (USCDC), Richard Pebody (Public Health England), Julia Fitzner (WHO), Kaat Vandemaele (WHO), John Wood (formerly NIBSC), Othmar Engelhardt (NIBSC), Angus Nicoll (ECDC), Elizabeth Bancroft (ECDC), Cheryl Cohen (NICD South Africa), Susan I. Gerber (USCDC), Salah Al Awaidi (MoH Oman), and Jeffery Cutter (MoH Singapore).

    1. WHO, World Health Organization. Global Alert and Response. Coronavirus Infections. Available at: Last accessed 30 July 2013, 2012-2013.
    2. Assiri, A., et al., Hospital Outbreak of Middle East Respiratory Syndrome Coronavirus. New England Journal of Medicine, 2013. 2013 Jun 19. [Epub ahead of print].
    3. Guery, B., et al., Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission. The Lancet, 2013.
    4. Hijawi, B., et al., Novel coronavirus infections in Jordan, April 2012: epidemiological findings from a retrospective investigation. Eastern Mediterranean Health Journal, 2013. 19(Supplement 1): p. S12-S18.
    5. Memish, Z.A., et al., Family Cluster of Middle East Respiratory Syndrome Coronavirus Infections. NEJM, 2013. Published 29 May 2013: p. DOI: 10.1056/NEJMoa1303729.
    6. Pebody, R.G., 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): p. pii=20292. Available online: