Middle East Respiratory Syndrome Coronavirus Infection Dynamics and Antibody Responses among Clinically Diverse Patients, Saudi Arabia Hail M. Al-Abdely,1 Claire M. Midgley,1 Abdulrahim M. Alkhamis, Glen R. Abedi, Xiaoyan Lu, Alison M. Binder, Khalid H. Alanazi, Azaibi Tamin, Weam M. Banjar, Sandra Lester, Osman Abdalla, Rebecca M. Dahl, Mutaz Mohammed, Suvang Trivedi, Homoud S. Algarni, Senthilkumar K. Sakthivel, Abdullah Algwizani, Fahad Bafaqeeh, Abdullah Alzahrani, Ali Abraheem Alsharef, Raafat F. Alhakeem, Hani A. Aziz Jokhdar, Sameeh S. Ghazal, Natalie J. Thornburg, Dean D. Erdman, Abdullah M. Assiri, John T. Watson, Susan I. Gerber Middle East respiratory syndrome coronavirus (MERS-CoV) Infection with Middle East respiratory syndrome (MERS) shedding and antibody responses are not fully understood, coronavirus (MERS-CoV) results in a wide range of particularly in relation to underlying medical conditions, clini- clinical manifestations, from mild or asymptomatic illness cal manifestations, and mortality. We enrolled MERS-CoV– to severe respiratory failure (1–8); infection has a reported positive patients at a hospital in Saudi Arabia and periodically mortality rate of 35% (9). Most MERS cases have been collected specimens from multiple sites for real-time reverse reported in older adults with underlying medical condi- transcription PCR and serologic testing. We conducted inter- tions (4,7). Asymptomatic or mild infections are typically views and chart abstractions to collect clinical, epidemiologic, reported in younger, healthy adults, including healthcare and laboratory information. We found that diabetes mellitus personnel (2,4). MERS-CoV transmission is commonly as- among survivors was associated with prolonged MERS-CoV RNA detection in the respiratory tract. Among case-patients sociated with exposure to symptomatic patients in health- who died, development of robust neutralizing serum anti- care (1,2,10,11) or household (12) settings or with direct body responses during the second and third weeks of illness exposure to dromedary camels (13). was not sufficient for patient recovery or virus clearance. Infection prevention and control guidance for MERS- Fever and cough among mildly ill patients typically aligned CoV in humans is partially based on severe acute respiratory with RNA detection in the upper respiratory tract; RNA levels syndrome (SARS) coronavirus infection dynamics (14,15); peaked during the first week of illness. These findings should MERS-specific recommendations are incomplete. Investiga- be considered in the development of infection control poli- tions of virus shedding in MERS patients have demonstrated cies, vaccines, and antibody therapeutics. that MERS-CoV RNA can be detected in the respiratory tract for >1 month from illness onset (16,17); lower respira- Author affiliations: Ministry of Health, Riyadh, Saudi Arabia tory tract (LRT) specimens have higher (18–23) and often (H.M. Al-Abdely, A.M. Alkhamis, K.H. Alanazi, W.M. Banjar, more prolonged RNA levels (17,18) than upper respiratory O. Abdalla, M. Mohammed, H.S. Algarni, A. Alzahrani, tract (URT) specimens; more severely ill patients typically A.A. Alsharef, R.F. Alhakeem, H.A.A. Jokhdar, A.M. Assiri); have higher (18,21) and more prolonged (18) RNA levels; Centers for Disease Control and Prevention, Atlanta, Georgia, and MERS-CoV RNA is detected in the blood (17,22,24), USA (C.M. Midgley, G.R. Abedi, X. Lu, A.M. Binder, A. Tamin, serum (18,19,24), plasma (22,25,26), stool (19,23,27), and S. Lester, R.M. Dahl, S.K. Sakthivel, N.J. Thornburg, urine (17,19,23) of some patients. However, important D.D. Erdman, J.T. Watson, S.I. Gerber); Princess Nourah Bint knowledge gaps remain, particularly regarding shedding in Abdulrahman University, Riyadh (W.M. Banjar); Prince association with clinical manifestations and host factors (4). Mohammed Bin Abdulaziz Hospital, Riyadh (A. Algwizani, Serologic responses among MERS patients are incom- F. Bafaqeeh, S.S. Ghazal) pletely understood; such data are critical for the development DOI: https://doi.org/10.3201/eid2504.181595 1These first authors contributed equally to this article. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 753 RESEARCH of vaccines, antibody therapeutics, and diagnostics. Investi- Laboratory Investigation gations of MERS survivors have demonstrated that antibody In addition to retrospectively reviewing clinical MERS-CoV titers are higher and longer-lived in more severely ill patients test results, we periodically collected specimens throughout than in mildly ill patients (28), some of whom do not de- hospitalization for molecular and serologic testing at the US velop a detectable response (28,29). Antibodies are usually Centers for Disease Control and Prevention (CDC). Speci- detected by day 21 after illness onset (30,31) and can persist mens were collected from respiratory and nonrespiratory for >34 months after infection (32). Data on case-patients sites, frozen at <–70°C, and shipped on dry ice. Available who died, however, are limited (19,25,29). specimens were URT (nasopharyngeal, oropharyngeal swab, To address gaps in viral and antibody kinetics, we lon- or combined), LRT (sputum or tracheal aspirate), whole gitudinally assessed 33 hospitalized MERS-CoV–infected blood, serum, stool, and urine. Specimens were collected patients. Our aim was to characterize MERS-CoV infection during days 1–42 postenrollment and additionally at 1 year dynamics and antibody responses in relation to outcome, for serum. clinical manifestations, underlying medical conditions, and preillness exposures. Molecular Assays Specimens were processed and screened by upE and N2 Methods rRT-PCR. Specimens positive by only 1 RT-PCR were con- firmed by N3 assay as previously described (35). MERS- Patient Enrollment CoV isolation was performed as previously described (36). The study population was drawn from a MERS referral We attempted full genome sequencing, as previously de- hospital in Riyadh, Saudi Arabia. All patients testing posi- scribed (36), on the earliest available respiratory specimen tive for MERS-CoV locally by real-time reverse transcrip- (or serum, if not available) for each patient. tion PCR (rRT-PCR) assay and admitted to this hospital during August 1, 2015–August 31, 2016, were eligible for Serologic Assays participation. All enrolled patients provided informed writ- Serum specimens with sufficient volume were tested us- ten consent. ing 4 CDC serologic assays: 1) microneutralization (MN) assay (37); 2) spike (S)–specific pseudoparticle neutral- Data Collection ization assay (VSV-MERS-S); 3) S ELISA (Ig-specific) We reviewed epidemiologic interviews conducted at the (38); and 4) nucleocapsid (N) ELISA (Ig-specific) time of case identification to include patient demograph- (37,38). Additional description is available in Appendix 1 ics, symptom history, and relevant exposures during the 2 (https://wwwnc.cdc.gov/EID/article/25/4/18-1595-App1.pdf). weeks before onset. After patient death or discharge, we performed comprehensive medical chart reviews to collect Data Analysis medical history; symptoms before hospitalization; and dai- ly information regarding symptoms during hospitalization, Definitions clinical course, treatments, medications, patient vital signs, We defined illness onset as the first day of reported symp- diagnostic tests, and clinical outcome. toms consistent with MERS; for asymptomatic patients To assess MERS-CoV infection status, we retrospec- identified through routine contact investigations, we used tively reviewed 3 data sources (as available) containing the date of the first positive MERS-CoV test. We analyzed information on clinical diagnostic testing: 1) rRT-PCR re- data relative to the date of illness onset (day 0). Patients quest forms submitted to a regional testing facility; 2) hos- were classified as having diabetes mellitus (DM) if there pital copies of corresponding results; and 3) if the hospi- was a documented medical history of DM. Patients with tal’s clinical series was incomplete, rRT-PCR results from multiple periods of hyperglycemia during hospitalization the Health Electronic Surveillance Network (33), a national (random glucose readings >200 mg/dL), but with no docu- platform for reporting notifiable diseases in Saudi Arabia. mented medical history of DM, were considered as pos- MERS-CoV clinical diagnostic testing had been performed sible DM status. on URT or LRT specimens typically collected every other Cardiac disease included congestive heart failure, cor- day throughout hospitalization. Healthcare personnel col- onary artery disease, or a history of myocardial infarction; lected LRT specimens from intubated patients and URT reports of isolated hypertension were not included. Pulmo- specimens otherwise. MERS-CoV results were positive, nary disease included chronic obstructive pulmonary dis- probable, or negative and, if available, cycle threshold (C) ease, asthma or reactive airway disease, or use of supple- t values for MERS-CoV upstream of the envelope E (upE) mental oxygen at home. Renal disease included reports of or open reading frame (ORF) 1a (34); a probable finding chronic kidney disease. Secondary exposure was defined as indicated that only 1 of these 2 targets was detected. contact with MERS-CoV–infected persons in the 2 weeks 754 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 MERS-CoV, Saudi Arabia before illness onset. Primary exposure was defined as ei- on either URT or LRT specimens. Because mildly ill patients ther reported direct camel contact or no known contact with did not provide LRT specimens, we only assessed detection MERS-CoV–infected persons. in URT specimens when comparing severity groups. Illness Severity Prolonged MERS-CoV Detection We retrospectively categorized patients into 3 groups on To assess prolonged MERS-CoV detection, we expressed the basis of the need for supplemental oxygen, ventila- time to negativity as a binary variable: patients with time to tion, and clinical outcome. Group 1 (G1) received room air negativity <11 versus >11 days. We chose this cutoff to re- throughout hospitalization; group 2 (G2) required ventila- flect the median time to negativity among survivors. Given tor support (mechanical or nonmechanical) and survived; the low numbers of patients in our cohort, we also assessed and group 3 (G3) required ventilator support and died. 2 additional cut-offs for prolonged shedding to strengthen statistically significant findings: <14 versus >14 days and MERS-CoV Detection Period <21 versus >21 days. To analyze duration of detectable MERS-CoV among sur- vivors, we assessed the number of days from illness onset Viral Load to negativity in clinical respiratory specimens tested at the To approximate viral load in clinical results, we assessed regional testing facility, based on reports from the hospital or MERS-CoV upE rRT-PCR C values determined at the re- t the Health Electronic Surveillance Network. We defined the gional testing facility. We used C values from LRT speci- t day of MERS-CoV negativity as the first of >2 consecutive mens to assess mechanically ventilated patients. We were negative tests before discharge. These variables were based able to identify the minimum C value (or peak RNA level) t Figure 1. Timeline of clinical course and MERS-CoV detection, by patient, Saudi Arabia, August 1, 2015–August 31, 2016. Findings are presented by time since illness onset (day 0). Patients are grouped by illness severity and outcome. For each patient, day of admission, discharge or death, period of mechanical ventilation (if applicable), and MERS-CoV detection are depicted. For a subset of patients with sufficient data, the peak RNA level (or the minimum upstream of the envelope cycle threshold value) is depicted. Peak RNA was based on upper respiratory tract specimen results among group 1 patients and Pt 29, and lower respiratory tract specimen results in group 2 and group 3 patients. The date of death is shown for group 3 patients. Pt 11 and Pt 32 did not report any symptoms throughout their hospitalization. Pt 30 was hospitalized and mechanically ventilated before MERS onset because of a road traffic accident; this patient was excluded from severity and clinical course analyses. Pt 23 has been described previously (36). The first positive MERS-CoV rRT- PCR for Pt 20 was collected 1 day before symptom onset. BiPAP, bilevel positive airway pressure; CoV, coronavirus; MERS, Middle East respiratory syndrome; Pt, patient; rRT-PCR, real-time reverse transcription PCR. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 755 RESEARCH Table 1. Characteristics of MERS-CoV–infected patients, by clinical severity outcome, Saudi Arabia, August 1, 2015–August 31, 2016* No. (%) patients p value† Group 1, Group 2, Group 3, Mortality, Ventilation support, Characteristic n = 13 n = 7 n = 12 G1 and G 2 vs. G3 G1 vs . G2 Demographics Sex M 7 (54) 6 (86) 7 (58) 0.724 0.329 F 6 (46) 1 (14) 5 (42) Nationality Saudi 5 (38) 6 (86) 10 (83) 0.139 0.070 Non-Saudi 8 (62) 1 (14) 2 (17) Age group, y 25–44 10 (77) 4 (57) 0 <0.001 0.548 45–64 3 (23) 2 (29) 7 (58) >65 0 1 (14) 5 (42) Median age (range), y 33 (2 6–62) 41 (30–73 ) 62 (55–78 ) <0.0 01 0.0 47 Underlying conditions None reported 10 (77) 0 0 0.004 0.003 Any reported underlying condition 3 (23) 7 (100) 11 (92) Unknown underlying condition 0 0 1 (8) DM‡ 3 (23) 4 (80) 11 (100) 0.001 0.047 DM and possible DM§ 3 (23) 6 (86) 12 (100) 0.002 0.047 Hypertension 1 (8) 2 (29) 11 (100) <0.001 0.270 Cardiac disease¶ 0 0 6 (55) 0.001 NA Pulmonary disease# 0 2 (29) 2 (18) 0.602 0.111 On oxygen at home** 0 1 (14) 1 (9) 1.000 0.350 Renal disease†† 0 0 3 (27) 0.001 NA Cardiac, pulmonary, or renal disease 0 2 (29) 9 (82) <0.001 0.111 History of cerebrovascular accident 1 (8) 0 3 (27) 0.318 1.000 Cancer in previous 12 mo 0 0 1 (9) 0.3 55 N A Possible preillness exposure Secondary,‡‡ healthcare personnel 5 (38) 0 0 NA NA Secondary, household contact 4 (31) 1 (14) 1 (8) NA NA Secondary, hospital visitor 2 (15) 1 (14) 2 (17) NA NA Secondary, hospital inpatient 0 0 3 (25) NA NA Any secondary exposure 11 (85) 2 (29) 6 (50) 0.249 0.022 Direct camel contact 0 0 2 (17) Multiple possible exposures 0 1 (14) 0 No recognized risks§§ 2 ( 15) 4 (57) 4 (33) Primary vs. secondary exposure¶¶ Primary## 2 (15) 4 (67) 6 (50) 0.452 0.046 Secondary 11 (85) 2 (33) 6 (50) Symptoms before admission Absence of symptoms 4 (31) 0 0 NA NA Any reported symptom 9 (69) 7 (100) 12 (100) NA NA Fever 8 (62) 6 (86) 11 (92) 0.212 0.354 Cough 7 (54) 6 (86) 10 (83) 0.422 0.329 Dyspnea 1 (8) 7 (100) 11 (92) 0.008 <0.001 Vomiting 4 (31) 2 (29) 2 (17) 0.676 1.000 Diarrhea 2 (15) 1 (14) 4 (33) 0.379 1.000 Sore throat 2 (15) 1 (14) 1 (8) 1.000 1.000 Rhinorrhea 1 (8) 1 (14) 1 (8) 1.000 1.000 *Group 1, on room air; group 2, ventilated but survived; group 3, died. CoV, coronavirus; DM, diabetes mellitus; MERS, Middle East respiratory syndrome; NA, not applicable. †p values are for Fisher exact or Kruskall–Wallis tests comparing long-term and short-term. ‡Based on documented medical history of DM; comparison excludes 3 patients with possible DM status; group 1, n = 13; group 2, n = 5; group 3, n = 11. §Includes 18 patients with a documented history of DM and 3 patients with possible DM status who exhibited multiple periods of hyperglycemia (random glucose readings >395 mg/dL) but who had no documented history of DM. ¶Cardiac disease includes congestive heart failure, coronary artery disease, a history of coronary artery bypass, or a history of myocardial infarction. Reports of isolated hypertension were not included. #Pulmonary disease includes chronic obstructive pulmonary disease, asthma or reactive airway disease, or use of supplemental oxygen at home. **Patients who were on supplemental oxygen at home (both patients were on bilevel positive airway pressure) required mechanical ventilation when hospitalized. ††Renal disease includes reports of chronic kidney disease. ‡‡Secondary exposure defined as reported contact with a known MERS case-patient. §§No recognized risks defined as no reported contact with a known MERS case-patient or camel (direct or indirect contact). ¶¶Comparison excludes 1 patient with multiple exposures; group 2, n = 6. ##Primary exposure defined as no reported contact with a known MERS case-patient; includes direct camel contact and patients with no recognized risks. 756 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 MERS-CoV, Saudi Arabia Table 2. Demographic and exposure characteristics of survivors with prolonged MERS-CoV detection, Saudi Arabia, August 1, 2015– August 31, 2016* Days to negativity Characteristic Total, N = 19 <11 d, n = 11 >11 d, n = 8 p value† Demographics Sex M 13/19 (68) 6/11 (55) 7/8 (88) 0.177 F 6/19 (32) 5/11 (45) 1/8 (12) Nationality Saudi 10/19 (53) 5/11 (45) 5/8 (63) 0.650 Non-Saudi 9/19 (47) 6/11 (55) 3/8 (37) Age group, y 25–44 14/19 (74) 9/11 (82) 5/8 (63) 0.262 45–64 4/19 (21) 1/11 (9) 3/8 (38) >65 1/19 (5) 1/11 (9) 0/8 Median age, y (range) 36 (26 –73) 30 (26 –73) 40 (27 –62) 0.0 83 Underlying conditions None reported 10/19 (53) 8/11 (73) 2/8 (25) 0.070 Any reported underlying condition 9/19 (47) 3/11 (27) 6/8 (75) DM‡ 7/18 (39) 2/11 (20) 5/7 (71) 0.049 DM and possible DM§ 8/19 (42) 2/11 (20) 6/8 (75) 0.024 Hypertension 3/19 (16) 1/11 (9) 2/8 (25) 0.546 Cardiac disease¶ 1/19 (5) 1/11 (9) 0/8 1.000 Pulmonary disease# 1/19 (5) 1/11 (9) 0/8 1.000 On oxygen at home** 1/19 (5) 1/11 (9) 0/ 8 1.0 00 Possible preillness exposure Secondary,†† healthcare personnel 5/19 (26) 4/11 (36) 1/8 (13) NA Secondary, household contact 5/19 (26) 3/11 (27) 2/8 (25) NA Secondary, hospital visitor 3/19 (16) 0/11 3/8 (38) NA Secondary, hospital inpatient 0/19 0/11 0/8 NA Any secondary exposure 13/19 (68) 7/11 (64) 6/8 (75) 0.796 Direct camel contact 0/19 0/11 0/8 Multiple possible exposures 2/19 (11) 1/11 (9) 1/8 (13) No recognized risks‡‡ 4/19 (21) 3/11 (27) 1/8 ( 13) Primary vs. secondary exposure§§ Primary¶¶ 4/17 (24) 3/10 (30) 1/7 (14) 0.603 Secondary 13/17 (78) 7/10 (70) 6/7 (86) *Values are no. (%) patients except as indicated. CoV, coronavirus; DM, diabetes mellitus; MERS, Middle East respiratory syndrome; NA, not applicable. †p values are for Fisher exact or Kruskall–Wallis tests comparing long-term and short-term. ‡Based on documented medical history of DM; comparison excludes 1 patient with possible DM status; total, N = 18; prolonged shedding >11 days, n = 7. §Includes 7 patients with a documented history of DM and 1 patient (of possible DM status) who had no documented history of DM but exhibited multiple periods of hyperglycemia (>200 mg/dL) during hospitalization, with a maximum random glucose reading of 404 mg/dL. ¶Cardiac disease includes congestive heart failure, coronary artery disease, a history of coronary artery bypass, or a history of myocardial infarction. Reports of isolated hypertension were not included. #Pulmonary disease includes chronic obstructive pulmonary disease, asthma or reactive airway disease, or use of supplemental oxygen at home. **Patient who was on supplemental oxygen (bilevel positive airway pressure) at home required mechanical ventilation when hospitalized. ††Secondary exposure defined as reported contact with a known MERS case-patient. ‡‡No recognized risks defined as no reported contact with a known MERS case-patient or camel (direct or indirect contact). §§Comparison excludes 2 patients with multiple exposures; total, N = 17; shed <11 days, n = 10; shed >11 days, n = 7. ¶¶Primary exposure defined as no reported contact with a known MERS case-patient; includes direct camel contact and patients with no recognized risks. in a subset of patients. For specimens submitted to CDC, we We used Fisher exact, Kruskall–Wallis, or log rank tests to estimated viral load on the basis of the upE C value (or N2 compare groups and exact logistic regression for multivari- t C value if upE testing was negative and N3 was positive). able analysis. We compared antibody titers with estimated t viral load in different specimen types by using the Spear- Antibody Responses man test for correlation. All data were analyzed using Mi- We compared the proportion of serum specimens with de- crosoft Excel 2016 (https://products.office.com) and SAS tectable antibody responses between survivors and patients version 9.4 (https://www.sas.com). who died. We assessed specimens collected <14, <21, and <28 days after illness onset; during 28–56 days after onset; Results and then at 1 year. Cohort Description Statistical Analyses During August 1, 2015–August 31, 2016, a total of 33 We summarized patient characteristics by illness severity MERS-CoV–infected patients were enrolled. Among and, among survivors, by time to MERS-CoV negativity. these, 4 were classified as asymptomatic on admission, and Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 757 RESEARCH Table 3. Clinical features of survivors with prolonged MERS-CoV detection, Saudi Arabia, August 1, 2015–August 31, 2016* Days to negativity Clinical feature Total, N = 19 <11 d, n = 11 >11 d, n = 8 p val ue† Symptoms before admission‡ No symptoms 4/18 (21) 3/10 (30) 1/8 (13) 0.603 Fever 13/18 (72) 7/10 (70) 6/8 (75) 1.000 Cough 11/18 (61) 5/10 (50) 6/8 (75) 0.367 Dyspnea 6/18 (33) 2/10 (20) 4/8 (50) 0.321 Vomiting 5/18 (28) 2/10 (20) 3/8 (38) 0.608 Diarrhea 3/18 (17) 0/1 0 3/8 ( 38) 0.0 69 Clinical course‡ Room air 13/18 (72) 9/10 (90) 4/8 (50) 0.118 Ventilator support§ 5/18 (28) 1/10 (10) 4/8 (50) Abnormal chest radiograph 9/18 (50) 4/10 (40) 5/8 ( 63) 0.6 37 Medications¶ Ribavirin plus peg-IFNα 2/19 (11) 0/11 2/8 (25) 0.164 Oseltamivir 12/19 (63) 6/11 (55) 6/8 (75) 0.633 Antibiotics 15/19 (79) 8/11 (73) 7/8 (88) 0.603 Parenteral steroids 3/19 (16) 0/11 3/8 (38) 0.058 Group 2 only# 3/5 (60) 0/1 3/4 (75) 0.400 Inhaled steroids 2/19 (11) 0/11 2/8 (25) 0.164 Group 2 only# 2/5 (40) 0/1 2/4 (50) 1.000 Bronchodilators 5/19 (26) 2/11 (18) 3/8 (38) 0.603 Antipyretics 9/19 (47) 6/11 (55) 3/8 (38) 0.650 *Values are no. (%) patients except as indicated. Group 2, ventilated but survived; MERS-CoV, Middle East respiratory syndrome coronavirus. †p values are for Fisher exact or Kruskall–Wallis tests comparing long-term and short-term. ‡Excludes patient no. 30, who was admitted and intubated before onset because of injuries sustained in a road traffic accident (N = 18). §Includes mechanical or nonmechanical (i.e., bilevel positive airway pressure) ventilation. ¶Medication given during MERS-CoV detection period (on the basis of diagnostic testing in respiratory specimens). #Assessing steroid use among G2 patients only. 9 reported symptoms but remained on room air during hos- to date of illness onset (Figure 1; Appendix 2 Table 2). pitalization (Figure 1; Appendix 1 Figure 1; Appendix 2 Time to admission (median 4 days) did not differ between Table 1, https://wwwnc.cdc.gov/EID/article/25/4/18-1595- groups. Time to MERS-CoV negativity among survivors App1.xlsx); 10 of these 13 patients were identified through ranged from day 1 to day 44 after illness onset and was contact tracing (5 were healthcare personnel) and were typically longer among G2 than G1 patients. Twelve of 13 hospitalized to ensure isolation. Twenty patients required patients in G1 were discharged by day 21 after onset; the ventilator support (1 bilevel positive airway pressure [Bi- mildly ill patient who was in the hospital until day 40 af- PAP] and 19 mechanical ventilation), 12 of whom died. We ter onset (Pt 23) has been described previously (36). Dura- grouped 13 patients into G1, 7 into G2, and 12 into G3; 1 tion of hospitalization for G2 patients was 19–70 days, and patient (patient [Pt] 30) was initially hospitalized and intu- duration of intubation was 14–31 days. G3 patients died bated after a road traffic accident, before MERS onset, and 10–73 days after onset. was excluded from analyses regarding severity and clinical course resulting from MERS-CoV infection. Daily Symptoms Patient ages ranged from 26 to 78 years, and 63% were Common symptoms before admission were fever (78%), male (Appendix 2 Table 1). Twenty-three (70%) patients cough (72%), and dyspnea (59%) (Table 1). Dyspnea be- had >1 underlying medical condition, 19 of whom had doc- fore admission was associated with a more severe outcome umented DM; an additional 3 patients were considered of (p<0.001). Among the 4 patients who reported no symp- possible DM status because they exhibited multiple periods toms on admission, 2 were mildly symptomatic during hos- of hyperglycemia (random glucose readings >395 mg/dL) pitalization (Appendix 2 Table 3). but had no documented history of DM. Death was associat- Among G1 patients, fever and cough were com- ed with older age (p<0.001), DM (p = 0.001), hypertension monly reported, and the proportion of patients with (p<0.001), cardiac disease (p = 0.001), or renal disease (p = either symptom appeared to align with the proportion 0.001) (Table 1). Among survivors, ventilator support was who concurrently had detectable MERS-CoV in clini- associated with DM (p = 0.047), older age (p = 0.047), or cal respiratory specimens (Figure 2, panel A). Cough preillness primary exposure (p = 0.046) (Table 1). Among persisted in 5 G1 patients for <4 days after MERS-CoV the 12 patients with a primary exposure, 8 had DM (Ap- negativity (Figure 2, panel B). Chest radiographs of 4 pendix 2 Table 1). G1 patients were described as abnormal, typically with Clinical course and time to MERS-CoV negativity unilateral findings (Appendix 2 Table 4). Oxygen satura- (in clinical respiratory specimens) is depicted according tion remained >92% in G1 patients. Among G2 patients, 758 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 MERS-CoV, Saudi Arabia the proportion of patients mechanically ventilated ap- MERS-CoV RNA in Respiratory Specimens peared to align with the proportion who had detectable MERS-CoV upE C values from clinical diagnostic reports are t MERS-CoV in the LRT (Figure 2, panel C); only 1 G2 depicted in Figure 3. MERS-CoV RNA levels in the URT of patient (Pt 13, who had underlying pulmonary disease) most G1 patients peaked in the first week after onset (Figure was MERS-CoV–positive for a short period but required 3, panel D). Among patients who died, RNA levels peaked in extended mechanical ventilation. Among the 12 G3 pa- the LRT during weeks 2 and 3 (Figure 3, panel E), after which tients, 11 were MERS-CoV RNA–positive until death RNA levels typically began to decrease (Figure 3, panel C); 4 (Figure 2, panel D). patients died with negative or probable rRT-PCR results. Figure 2. Symptom progression and MERS-CoV detection during hospitalization at a MERS referral hospital, Saudi Arabia, August 1, 2015–August 31, 2016. Each panel depicts the number of patients hospitalized on a given day for each category shown; MERS-CoV detection is based on the clinical diagnostic reports in the upper or lower respiratory tract. A, B) Number of group 1 patients with fever (measured oral temperature >38.0°C or measured axillary temperature >37.5°C) and reported cough during (A) and after (B) the MERS-CoV detection period. C, D) Number of patients intubated (dashed lines) and the number of patients who were positive for MERS-CoV on a given day for group 2 (C) and group 3 (D). MERS, Middle East respiratory syndrome; MERS-CoV, Middle East respiratory syndrome coronavirus. Group 1, on room air; group 2, ventilated but survived; group 3, died. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 759 RESEARCH We next assessed characteristics of survivors with pro- 2 Table 5). Evidence for this association was stronger when longed MERS-CoV detection periods (on the basis of clinical patients with DM and possible DM were combined (Tables diagnostic reports of URT specimens) (Tables 2, 3; Appendix 2, 3; Appendix 2 Table 5). No other underlying medical con- 2 Tables 5, 6). Patients who reached negativity >11 days after ditions were associated with prolonged detection. Survivors onset were more likely to have DM than patients who cleared with prolonged detection (>14 or >21 days) were also more the virus earlier (p = 0.049; when adjusting for severity group, likely to require ventilator support (Appendix 2 Table 5), but p = 0.061) (Tables 2, 3). This association was also observed in this was not significant when adjusting for DM. patients who reached negativity >14 days after onset (p = 0.013) Based on respiratory specimens submitted to CDC and when adjusting for severity group (p = 0.030) (Appendix (Appendix 1 Figures 2, 3), full-genome sequences from 13 Figure 3. MERS-CoV RNA detection in the respiratory tract, based on clinical diagnostic reports, among MERS-CoV patients, Saudi Arabia, August 1, 2015–August 31, 2016. A–C) UpE real-time reverse transcription PCR C values of t group 1 (A), 2 (B), and 3 (C) patients, by days since illness onset (day 0). Panel A depicts URT specimens, and panels B and C depict LRT specimens collected during MV; Pt 29 (a G2 patient who received BiPAP ventilation) is depicted in panel A because only URT specimens were collected for this patient. The dashed line represents the limit of detection, above which specimens were considered MERS-CoV–negative or not detected. Probable results, meaning that only 1 of 2 real- time reverse transcription PCR assays were positive, are depicted on the dashed line for graphing purposes. Patients with limited C values t or unknown specimen types are not depicted. Patients 11 and 32 did not report any symptoms throughout their illness. Pt 30 is depicted alongside G2 patients. Pt 23 reached negativity 37 days after illness onset, as described previously (36). *Indicates patients with a documented history of diabetes mellitus. D, E) Minimum C values reported, t which was determined for a subset of patients with sufficient data. Panel D depicts URT specimen results among group 1 patients and Pt 29; panel E depicts LRT specimen results in group 2 and 3 patients, collected from the LRT during MV. Group 1, on room air; group 2, ventilated but survived; group 3, died. BiPAP, bilevel positive airway pressure; C, cycle threshold; CoV, coronavirus; LRT, t lower respiratory tract; MERS, Middle East respiratory syndrome; min, minimum; MV, mechanical ventilation; Pt, patient; URT, upper respiratory tract; upE, upstream of the envelope. 760 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 MERS-CoV, Saudi Arabia patients belonged to the NRC-2015 (39) clade (or lineage 5 onset; 7 patients (21 specimens, all MERS-CoV–negative) [40]) (GenBank accession nos. MG520075 and MG757593– were excluded because specimens were only collected after MG757605). Viable MERS-CoV was isolated from 3 of 37 the virus had been cleared from the respiratory tract. Four- URT specimens; 2 specimens were from a mildly ill patient teen of 24 patients had MERS-CoV RNA detected in whole (Pt 23) collected on days 13 and 15 after onset (described blood, 9/20 in serum, 5/10 in stool, and 3/16 in urine (Fig- previously [36]), and 1 specimen was collected on day 13 ure 4; Appendix 2 Table 2). In G1, MERS-CoV RNA was from a patient who subsequently died (Pt 8). detected in the whole blood or serum of 4/8 patients (<2.3 × 103 copies/mL) for <13 days and in the stool of 3/5 patients MERS-CoV RNA in Nonrespiratory Specimens (<7.5 × 103 copies/mL) for <15 days; only 1 patient with CDC received 252 nonrespiratory specimens for MERS- RNA-positive stool had concurrent gastrointestinal symp- CoV testing, collected from 31 patients <3 months after toms. Specimens were limited in G2, but MERS-CoV RNA Figure 4. Estimated viral loads in non–respiratory tract specimens collected from hospitalized MERS-CoV patients, Saudi Arabia, August 1, 2015–August 31, 2016, and submitted to the US Centers for Disease Control and Prevention. Specimen types are shown by severity group. Estimated viral loads are based on upstream of the envelope (upE) real-time reverse transcription PCR cycle threshold values, or N2 cycle threshold values if the upe real-time reverse transcription PCR was negative. The dashed line represents the limit of detection, below which specimens were considered MERS-CoV–negative or not detected. Round data points represent specimens collected during the MERS-CoV detection period (defined by clinical results from respiratory specimens). Diamond data points represent specimens collected after the MERS-CoV detection period (defined by clinical results from respiratory specimens); no specimens were positive for MERS-CoV after the detection period. *Patients with a documented history of diabetes mellitus. MERS-CoV, Middle East respiratory syndrome coronavirus; Pt, patient. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019 761 RESEARCH was detected in the blood of 2/4 patients (<1.3 × 103 copies/ Serum Antibody Responses mL) and the stool of 1/2 (3.5 × 105 copies/mL). In G3, high CDC tested 74 serum specimens collected <56 days after viral loads were detected in the whole blood or serum of 9 onset; 41 specimens were from 16 survivors, and 33 speci- patients, reaching as high as 2.1 × 106 copies/mL. All 3 pa- mens were from 11 case-patients who died. Time between tients with MERS-CoV RNA detected in urine died (<6.0 × onset and collection did not differ between patient groups 104 copies/mL); 1 patient had chronic kidney disease. We (survived, median 15 days, range 1–50 days; died, me- attempted but were unable to isolate live MERS-CoV from dian 17 days, range 5–45 days). Four specimens from 4 5 stool specimens and 3 urine specimens with elevated survivors were collected ≈1 year after illness onset. Time MERS-CoV RNA levels. courses of antibody responses are shown by patient in Fig- ure 5 and Appendix 1 Figures 4–6. Among case-patients who died, 5/6 had detectable neutralizing responses during the first 2 weeks of illness; by week 3, all of these case-patients with available specimens had detectable antibodies by MN (Table 4). Notably, 1 of these case-patients exhibited a 9-day delay in the devel- opment of a detectable response by VSV-MERS-S pseu- doparticle assay compared with MN (Appendix 1 Figure 7); the detectable S-specific response by ELISA was also delayed for this patient. Overall, the 2 neutralizing assays were better correlated in specimens from survivors than from patients who died (Appendix 1 Figure 8). Survivors and case-patients who died had responses by N- and S-ELISA (Table 4), although detectable N- specific responses preceded S-specific responses in 3 pa- tients. By 1 year, N-specific responses had waned in 2 of the 4 patients tested. Co-detection of Antibodies and Viral RNA We next compared neutralizing antibody titers to estimated viral load in the same serum specimen and to estimated vi- ral load in respiratory specimens collected on the same day (Figure 6; Appendix 1 Figure 9). Among specimens with detectable antibodies by MN (Figure 6), viral RNA was of- ten co-detected in serum, URT, and LRT specimens, even beyond 21 days after onset, when antibody titers were typi- cally higher. Co-detection in serum (p = 0.032) and URT (p = 0.003) specimens was observed more frequently among patients who died than among those who survived. Discussion We characterized MERS-CoV infection dynamics by pa- tient demographics, underlying medical conditions, expo- sure route, and symptom progression and antibody respons- Figure 5. MN antibody titers of serum collected from MERS-CoV es by clinical outcome. Our findings demonstrate a possible patients, by patient, severity group, and days since illness onset association between DM and prolonged MERS-CoV RNA (day 0), Saudi Arabia, August 1, 2015–August 31, 2016. A) Group detection in survivors, when adjusting for severity. The 1 patients; B) group 2 patients; C) group 3 patients. The dashed line represents the limit of detection, below which specimens were prevalence of DM is high in Saudi Arabia; estimated na- considered not to have detectable antibodies. Pt 11 did not report tional prevalence ranges from 14.4% (41) to 18.6% (42). any symptoms throughout their illness. Pt 30 was hospitalized DM is frequently reported among MERS case-patients and mechanically ventilated before MERS onset because of (7,43), is a risk factor for illness among those with primary a road traffic accident. *Patients with documented history of exposures (13), and has been associated with increased se- diabetes mellitus. MERS-CoV, Middle East respiratory syndrome coronavirus; MN, microneutralization assay; ND, antibodies not verity (44) and mortality (26,44), as was observed in our detected; Pt, patient. study. DM was less frequently reported in MERS patients 762 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 25, No. 4, April 2019