BTCRCRCTD4D8CTCS C5CPD2D9D7CRD6CXD4D8 Serologic responses of 42 MERS coronavirus infected patients according to the disease severity Jae Hoon Ko Marcel A M uller Hyeri Seok Ga Eun Park Ji Yeon Lee Sun Young Cho Young Eun Ha Jin Yang Baek So Hyun Kim Ji Man Kang Yae Jean Kim Ik Joon Jo Chi Ryang Chung Myong Joon Hahn Christian Drosten Cheol In Kang Doo Ryeon Chung Jae Hoon Song Eun Suk Kang Kyong Ran Peck PII S0732 8893 17 30221 3 DOI doi 10 1016 j diagmicrobio 2017 07 006 Reference DMB 14391 To appear in Diagnostic Microbiology and Infectious Disease Received date 28 April 2017 Revised date 5 July 2017 Accepted date 10 July 2017 Please cite this article as Ko Jae Hoon M uller Marcel A Seok Hyeri Park Ga Eun Lee Ji Yeon Cho Sun Young Ha Young Eun Baek Jin Yang Kim So Hyun Kang Ji Man Kim Yae Jean Jo Ik Joon Chung Chi Ryang Hahn Myong Joon Drosten Christian Kang Cheol In Chung Doo Ryeon Song Jae Hoon Kang Eun Suk Peck Kyong Ran Serologic responses of 42 MERS coronavirus infected patients accord ingtothediseaseseverity Diagnostic Microbiology and Infectious Disease 2017 doi 10 1016 j diagmicrobio 2017 07 006 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting typesetting and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could a ect the content and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 Serologic responses of 42 MERS coronavirus infected patients according to the disease severity Jae Hoon Ko a 1 Marcel A M ller b c 1 Hyeri Seok a Ga Eun Park a Ji Yeon Lee a Sun Young Cho a Young Eun Ha a Jin Yang Baek d So Hyun Kim d Ji Man Kang e Yae Jean Kim e Ik Joon Jo f Chi Ryang Chung g Myong Joon Hahn h Christian Drosten b c Cheol In Kang a Doo Ryeon Chung a d Jae Hoon Song a d Eun Suk Kang i and Kyong Ran Peck a a Division of Infectious Diseases Department of Medicine Samsung Medical Center Sungkyunkwan University School of Medicine 81 Irwon ro Gangnam gu Seoul 135 710 Republic of Korea b Institute of Virology Charit Universit tsmedizin Berlin Berlin Germany c German Centre for Infection Research Germany d Asia Pacific Foundation for Infectious Diseases APFID Seoul Republic of Korea e Division of Infectious Diseases Department of Pediatrics Samsung Medical Center Sungkyunkwan University School of Medicine Seoul Republic of Korea f Department of Emergency Medicine Samsung Medical Center Sungkyunkwan University School of Medicine Seoul Republic of Korea g Department of Critical Care Medicine Samsung Medical Center Sungkyunkwan University School of Medicine Seoul Republic of Korea h Department of Molecular Cell Biology Center for Molecular Medicine Samsung Biomedical Research Institute Sungkyunkwan University School of Medicine Suwon 440 746 Korea i Department of Laboratory Medicine and Genetics Samsung Medical Center Sungkyunkwan University School of Medicine Seoul Republic of Korea 1 These authors contributed equally to this article as first authors Present address Division of Infectious Diseases Department of Internal Medicine Armed Forces Capital Hospital Seongnam Korea These corresponding authors contributed equally to this article ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 KEY WORDS Middle East respiratory syndrome coronavirus Prognosis Antibody Serologic response RUNNING TITLE Serologic response of MERS pneumonia WORD COUNTS Abstract 150 words Text body 3 075 words Correspondence to Kyong Ran Peck MD PhD Division of Infectious Diseases Department of Medicine Samsung Medical Center Sungkyunkwan University School of Medicine 81 Irwon ro Gangnam gu Seoul 135 710 Korea Tel 82 2 3410 0329 Fax 82 2 3410 0064 E mail krpeck skku edu AND Eun Suk Kang MD PhD Department of Laboratory Medicine and Genetics Samsung Medical Center Sungkyunkwan University School of Medicine 81 Irwon ro Gangnam gu Seoul 135 710 Korea Tel 82 2 3410 2703 Fax 82 2 3410 2719 E mail eskang skku edu ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3 ABSTRACT We evaluated serologic response of 42 Middle East respiratory syndrome coronavirus MERS CoV infected patients according to four severity groups asymptomatic infection group 0 symptomatic infection without pneumonia group 1 pneumonia without respiratory failure group 2 and pneumonia progressing to respiratory failure group 3 None of the group 0 patients showed seroconversion while the seroconversion rate gradually increased with increasing disease severity 0 0 60 0 93 8 and 100 in group 0 1 2 3 respectively P 0 001 Group 3 patients showed delayed increment of antibody titers during the 4th week while group 2 patients showed robust increment of antibody titer during the 3rd week Among patients having pneumonia 75 of deceased patients did not show seroconversion by the 3rd week while 100 of the survived patients were seroconverted P 0 003 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 1 INTRODUCTION Since the first reported case of Middle East respiratory syndrome coronavirus MERS CoV in 2012 Zaki et al 2012 small and large outbreaks have occurred resulting in 1 917 MERS CoV infections and 677 related deaths to date WHO 2017 To understand this fatal respiratory viral infection several serologic investigations have been conducted Corman et al 2016 Min et al 2016 Park et al 2015 Payne et al 2016 However practical analysis of serodiagnostic parameters for clinical usage was limited in previous studies due to insufficient sample size or clinical information We managed 45 MERS CoV infected patients which is the largest number of patients as a single center during the 2015 Korean MERS outbreak total 186 patients identified Cho et al 2016 Kim et al 2016 Park et al 2016 and reported that MERS CoV infected patients experienced four distinct clinical courses ranging from asymptomatic infection to severe pneumonia requiring mechanical ventilation Ko et al 2016 Based on these findings we evaluated serologic response of 42 MERS CoV infected patients according to the disease severity to investigate potential role of serodiagnostic parameters as prognostic markers 2 MATERIAL AND METHODS 2 1 Study population and samples Among 45 MERS CoV infected patients who were admitted to Samsung Medical Center a 1 950 bed tertiary care university hospital during the 2015 Korean MERS outbreak Ko et al 2016 we obtained sera from 42 patients MERS CoV infections were confirmed on the basis of real time reverse transcriptase polymerase chain reaction rRT PCR assays targeting upstream of the E gene upE and the open reading frame gene 1a ORF1a Corman et al 2012a Madani 2014 Epidemiologic investigation data and electronic medical records were reviewed to obtain exact exposure date symptom onset clinical course and outcome data for the patients One or two residual serum samples per week of illness were used for serologic testing during hospitalization periods Follow up serum samples obtained at outpatient clinics were also tested up to 6 months from ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 symptom onset The Institutional Review Board of Samsung Medical Center approved the present study 2 2 Patient grouping according to the disease severity The clinical course of MERS CoV infected patients was assessed six weeks after symptom onset and patients were divided into four disease severity groups asymptomatic infection group 0 symptomatic infection without pneumonia group 1 pneumonia without respiratory failure group 2 and pneumonia progressing to respiratory failure group 3 Ko et al 2016 For practical purposes respiratory failure was defined as the need for mechanical ventilation Only patients in group 3 experienced fatal outcomes 5 13 38 5 and interval from symptom onset to death was 27 days in median IQR 19 35 5 Proportion of underlying immunocompromising conditions including diabetes solid cancer or hematologic malignancies was not different between groups Ko et al 2016 The distinct clinical presentation of the four severity groups are presented in Supplementary Figure 1 and 2 and Supplementary Table 1 in addition to the previous report Ko et al 2016 2 3 Definitions Seroconversion status was determined based on neutralization activity if none of the serum samples from a MERS CoV infected patient necessarily including sera obtained after the 3rd week of illness showed neutralization activity the patient was considered to have negative seroconversion if none of the serum samples obtained by the end of the 3rd week of illness showed neutralization activity and no samples were available for neutralization tests thereafter the patient was considered to have an indeterminate response i e interpretation not applicable if any serum showed neutralization activity the patient was considered to have positive seroconversion Patients with an indeterminate response were excluded from calculation of the seroconversion rate This definition is based on the premise that no patients had previous exposure to MERS CoV as this was the first MERS outbreak in Korea as a non endemic country During the outbreak MERS CoV exposure dates and symptom onsets were clearly identified in ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 most patients owing to thorough contact investigation and monitoring of exposed individuals Cho et al 2016 Park et al 2016 MERS related symptoms included fever myalgia cough sputum and diarrhea To provide a common point of reference we used days post onset of illness dpoi to evaluate MERS CoV infected patients For asymptomatic patients the day of diagnosis of MERS CoV infection was considered as day of symptom onset Ko et al 2016 2 4 Serologic tests for MERS CoV antibody 2 4 1 Enzyme linked immunosorbent assay ELISA IgG and IgA Anti MERS CoV ELISA IgG and IgA Euroimmun L beck Germany were based on soluble MERS CoV spike protein S1 domain expressed in HEK 293T cells Muller et al 2014 Muller et al 2015 Muth et al 2015 Raj et al 2013 Sera were tested according to the manufacturer s instructions with 1 100 dilutions Secondary detection was done with peroxidase labelled anti human IgG and IgA Cut off values of OD ratio 0 4 for ELISA IgG and 0 2 for ELISA IgA were applied in the present study as these values exhibited optimal performance in predicting neutralization activity Ko et al under review 2 4 2 IFA IgM Anti MERS CoV IFA IgM Euroimmun was performed with slides carrying Vero cells infected with full MERS CoV Corman et al 2012b Meyer et al 2014 Muller et al 2014 Muller et al 2015 Sera were tested according to the manufacturer s instructions with 1 10 dilutions Weekly positive IFA intensity was considered cut off intensity value of IFA IgM which exhibited optimal performance in predicting neutralization activity Ko et al under review 2 4 3 PRNT MERS CoV PRNT was performed as previously described Meyer et al 2014 Muller et al 2014 Muller et al 2015 Pre dilution before setting up the log2 dilution series was 1 10 defining 1 20 as the lowest possible significant titer for categorizing a sample as positive Meyer et al 2014 2 5 Statistical analysis For comparison of clinical variables between groups one way analysis of variance ANOVA or ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 Kruskal Wallis test was used for continuous variables and Chi square or Fisher s exact tests was used for categorical variables Six week survival probability was calculated using the Kaplan Meier method The Cox proportional hazard model and log rank test were used to examine the association of seroconversion status with the six week mortality of MERS patients having pneumonia All P values were two tailed and those 0 05 were considered to be statistically significant R 3 3 1 for Windows RStudio Boston MA USA was used for all statistical analyses 3 RESULTS 3 1 Serologic response of MERS CoV infection according to the disease severity Seroconversion status of 42 MERS CoV infected patients is summarized in Table 1 None of the group 0 patients showed seroconversion and the seroconversion rate gradually increased with increasing disease severity 0 0 60 0 93 8 and 100 in groups 0 1 2 and 3 respectively P 0 001 Seroconversion was observed from 14 to 24 dpoi 18 dpoi in median mostly during the 3rd week of illness 88 0 of seroconverted patients with a known timeline Group 3 patients showed slightly delayed timing of seroconversion compared to group 2 patients 18 5 and 17 5 dpoi in median respectively without statistical significance and seroconversion during the 4th week of illness was exclusively observed in group 3 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 Table 1 Seroconversion status of MERS CoV infected patients according to the disease severity group Variables Classification by the disease severity Group 0 Asymptomatic n 3 Group 1 Symptomatic n 10 Group 2 Pneumonia n 18 Group 3 Resp failure n 11 Negative seroconversion 3 100 2 20 0 1 5 6 0 0 0 Indeterminate response 0 0 0 5 50 0 2 11 1 2 18 2 Positive seroconversion 0 0 0 3 30 0 15 83 3 9 81 8 Timing unknown N A 0 3 0 0 1 15 6 7 1 9 11 1 Second week of illness N A 0 3 0 0 1 14 7 1 0 8 0 0 Third week of illness N A 3 3 100 13 14 92 9 6 8 75 0 Fourth week of illness N A 0 3 0 0 0 14 0 0 2 8 25 0 dpoi N A 17 16 18 17 5 14 20 18 5 15 24 dpex N A 22 20 24 21 5 19 30 24 18 27 Seroconversion rate 0 0 0 0 3 5 60 0 15 16 93 8 9 9 100 Data are expressed as the number of patients or median range Seroconversion was confirmed by PRNT and a 1 20 dilution was defined as the lowest significant titer The timing of seroconversion was uncertain for two patients as the only sera available were collected after several months dpoi 79 and 140 at dpoi 14 Patients with an indeterminate response were excluded from seroconversion rate analysis Abbreviations MERS CoV Middle East respiratory syndrome coronavirus Resp respiratory dpoi days post onset of illness dpex days post exposure IQR interquartile range PRNT plaque reduction neutralization test Serologic responses of seroconverted patients are depicted according to the severity groups with 7 day intervals in Figure 1 Serologic response occurred from the 3rd week of illness and antibody response is weaker in patients with mild symptomatic patients group 1 than patients with pneumonia group 2 and 3 Group 2 patients showed robust increment of antibody titer during the 3rd week compared to the 2nd week the median OD ratios of ELISA IgG and IgA increased more than threefold and IFA IgM and PRNT increased from negative to 2 and 1 80 respectively and the titers did not significantly increase thereafter in comparison of the median values of 3rd week and 4th week no statistical significance was observed Meanwhile group 3 patients showed delayed and ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 9 continuous increment of antibody titers from the 3rd week the median values of each serologic test were significantly higher during the 4th week compared to those of the 3rd week in group 3 all P 0 05 In comparison between group 2 and 3 antibody titers of group 3 patients during the 3rd week were numerically lower than those of group 2 although only ELISA IgG showed statistically significant difference P 0 016 The antibody titers of group 3 patients continuously increased showing numerically higher titers compared to those of group 2 patients during the 4th week without statistical significance Detailed serologic test results for each patient are presented according to timeline and severity groups in Supplementary Tables 2 to 5 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 10 Figure 1 Serologic responses of seroconverted MERS CoV infected patients according to the severity groups with 7 day intervals The serologic responses of seroconverted MERS CoV infected patients are depicted according to the severity groups symptomatic infection without pneumonia group 1 pneumonia without respiratory failure group 2 and pneumonia progressing to respiratory failure group 3 The mean values of each serologic test for 7 day intervals are presented in box plots The antibody titers of symptomatic patients rise after the 2nd week Although PRNT titers were not statistically different between groups by the 3rd week of illness peak antibody response increased as severity increases a ELISA IgG in group 1 b ELISA IgG in group 2 c ELISA IgG in group 3 d ELISA IgA in group 1 e ELISA IgA in group 2 f ELISA IgA in group 3 g IFA IgM in group 1 h IFA IgM in group 2 i IFA IgM in group 3 j PRNT in group 1 k PRNT in group 2 l PRNT in group 3 Abbreviations MERS CoV Middle East respiratory syndrome coronavirus ELISA enzyme linked immunosorbent assay OD optical density IFA immunofluorescence assay PRNT plaque reduction neutralization test 3 2 Impaired serologic response of fatal MERS pneumonia As seroconversion rates were low in mild severity groups 0 in group 0 and 60 in group 1 outcome analysis was performed in patients having pneumonia group 2 and 3 Only 25 of deceased patients showed seroconversion by the end of the 3rd week of illness while 100 of survived patients seroconverted P 0 003 Table 2 This difference also could be discriminated by ELISA IgG with OD ratio cut off value of 0 4 P 0 003 and ELISA IgA with OD ratio cut off value of 0 2 P 0 010 IFA IgM response was not significantly different between survivors and non survivors with intensity cut off value of weakly positive P 0 135 In a Kaplan Meier analysis comparing seroconverted patients and non converted patients by the 3rd week of illness seroconverted patients showed significantly higher survival probability compared to patients with negative seroconversion Figure 2 P 0 001 by log rank test Negative seroconversion in pneumonia patients by the 3rd week of illness showed a hazard ratio of 27 83 95 CI 2 76 280 21 P 0 005 by the Cox proportional hazard model in predicting six week mortality ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 11 Table 2 Seroconversion rates by the end of 3rd week of illness according to outcome of MERS CoV infected patients having pneumonia group 2 and 3 Serologic tests Survived n 18 Deceased n 4 P value PRNT 1 20 dilution 18 100 1 25 0 003 ELISA IgG OD ratio cut off 0 4 18 100 1 25 0 003 ELISA IgA OD ratio cut off 0 2 17 94 4 1 25 0 010 IFA IgM Intensity cut off w 16 88 9 2 50 0 135 Data are expressed as the number of patients The population of this analysis is 22 MERS CoV infected patients with pneumonia group 2 and 3 whose sera were collected during the 3rd week of illness Abbreviation MERS CoV Middle East respiratory syndrome coronavirus PRNT plaque reduction neutralization test ELISA enzyme linked immunosorbent assay OD optical density IFA immunofluorescence assay w weak positive Figure 2 Survival probability of MERS CoV infected patients having pneumonia according to the seroconversion status by the 3rd week of illness Survival probability according to the seroconversion status was evaluated in MERS CoV infected patients having pneumonia whose seroconversion status during the 3rd week of illness is identifiable Seroconverted patients showed significantly higher survival probability compared to patients with negative seroconversion P 0 001 by log rank test Seroconversion was confirmed by PRNT and a ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 12 1 20 dilution was defined as the lowest significant titer Abbreviations MERS CoV Middle East respiratory syndrome coronavirus PRNT plaque reduction neutralization test 4 DISCUSSION Since previous hospital associated outbreaks of MERS occurred in endemic countries where primary infections flow from community into hospitals detailed clinical data of each patient were hard to obtain Corman et al 2016 However during the 2015 Korean MERS outbreak the first outbreak in a non endemic country epidemiologic links and entire clinical course of each patients could be clearly identified Ko et al 2016 Park et al 2016 Owing to the detailed epidemiologic and clinical informati