PMC:5968208 / 3339-8370 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/5968208","sourcedb":"PMC","sourceid":"5968208","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5968208","text":"MATERIALS AND METHODS\n\nSelection and participation of individuals\nThis survey was conducted between August 2015 and February 2016 after the last MERS case diagnosed in July 5, 2015. Based on a database of quarantined individuals provided by the Department of Epidemiologic Investigation of the Korea Centers for Disease Control and Prevention (KCDC), individuals from 4 regions with major outbreaks—Seoul, Gyeonggi, Chungcheong, and Jeonbuk —were selected. Individuals whose MERS status was diagnosed as positive using a polymerase chain reaction test were excluded from the analysis of this study. From the 14,831 quarantined individuals, 7,233 residents (48.8%) living in the 4 major MERS outbreak regions were selected. Of these individuals, calls requesting participation in this study were made to 3,291 individuals (45.5%) according to prioritization groupings. A total of 1,610 individuals (48.9%) ultimately participated in the study (Figure 1). Those who refused to participate have been described in another study [20].\nThe study individuals were prioritized in groups according to the transmission intensity of the MERS case they were exposed to, as follows: contact with super-spreading events (5 or more individuals infected) [21], contact with spreaders who infected 1 to 4 individuals, and contact with non-spreaders. We selected study subjects according to this prioritization of groups, and the selection rates were 48.8, 16.4, and 15.8%, respectively. We also categorized the subjects according to their exposure intensity (i.e., status when they were exposed to the MERS case), as follows: inpatients or outpatients at a MERS-affected hospital, cohabiting family members or paid caregivers of the MERS case, visitors of the hospitalized MERS case, healthcare workers employed at a MERS-affected hospital, and colleague of the MERS case. We selected more subjects from the categories of family, patients, and visitors (Table 1).\n\nStudy performance\nOnly individuals who consented to participate in the study through a telephone call and gave written consent at the time of study initiation underwent surveys at local public health centers. Each participant responded to a questionnaire on exposure and provided a blood sample. Following the first serologic test by an enzymelinked immunospecific assay (ELISA), positive or borderline cases were tested using an immunofluorescence assay (IFA) and a plaque reduction neutralization antibody test (PRNT).\n\nAntibody tests\nMERS-CoV antibody levels in all sera collected from contacts were measured using a recombinant S1 protein-coated human anti-MERS-CoV (immunoglobulin G [IgG]) ELISA kit (Euroimmun, Luebeck, Germany), which was used according to the manufacturer’s protocol. The results of the tested samples were determined by calculating the ratio of the optical density (OD) value of the sample to the OD value of the calibrator. Ratios ≥ 1.1 were considered positive, ratios ≥ 0.8 to \u003c 1.1 were considered borderline, and ratios \u003c 0.8 were considered negative.\nIFA slides were coated with MERS-CoV non-infected or infected Vero cells. Serum samples were diluted in phosphate-buffered saline (PBS) to 1:10, 1:100, 1:250, and 1:1,000, transferred to IFA slides, and incubated for 30 minutes at 37°C in a humidified chamber. The IFA slides were washed 3 times in PBS-T for 5 minutes each and incubated with fluorescein isothiocyanate-conjugated rabbit anti-human IgG (Abcam, Cambridge, MA, USA) diluted at 1:800 for 30 minutes at 37°C in a humidified chamber. After washing 3 times in PBS-T for 5 minutes, the slides were embedded with a mounting fluid, topped with a cover glass, and observed under a fluorescent microscope.\nThe neutralizing antibodies in the serum samples were measured by PRNT. The PRNT procedures were performed as follows. In brief, 1:10 diluted sera were heat-inactivated at 56°C for 30 minutes. Heat-inactivated sera were diluted serially 4-fold. After an equal volume of virus (MERS-CoV/KOR/KNIH/002_05_2015) was added to the volume of serum dilutions, these mixtures were incubated at 37°C for 2 hours. The mixtures were added to each well of the 24-well plate cultured with Vero cells. The plate was incubated at 37°C for 60 minutes, and 1 mL of 1.5% carboxymethylcellulose overlay medium was then added. After incubation for 3-4 days, cell staining was performed by crystal violet. The titer of neutralizing antibody by PRNT50 was calculated using the Kärber formula, as described previously [22]. A titer above 1:20 was interpreted as positive. All sera with a positive or borderline reaction in ELISA were tested by the IFA and PRNT assays for confirmation, and cases with positive results from any 2 assays were considered to be anti-MERS positive.\nThis study received approval from the bioethics committee of the KCDC (2015-08-EXP-03-P-A) and the institutional review board of the National Cancer Center (NCC 2016-0058). 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