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Screening for Middle East respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts a prospective descriptive study Z A Memish 1 J A Al Tawfiq 2 3 H Q Makhdoom 4 A A Al Rabeeah 1 A Assiri 1 R F Alhakeem 1 F A AlRabiah 5 S Al Hajjar 5 A Albarrak 6 H Flemban 7 H Balkhy 8 M Barry 9 S Alhassan 10 S Alsubaie 11 and A Zumla 1 12 13 1 Global Centre for Mass Gatherings Medicine GCMGM Ministry of Health Riyadh 2 Saudi Aramco Medical Services Organization Saudi Aramco Dhahran Saudi Arabia 3 Indiana University School of Medicine Indianapolis IN USA 4 Jeddah Regional Laboratory Ministry of Health Jeddah 5 King Faisal Specialist Hospital 6 Prince Sultan Military Medical City 7 Alhada Military Hospital 8 National Guard Hospital 9 Adult infectious Diseases King Saud University 10 Security Forces Hospital Ministry of Interior 11 Paediatric Infectious Diseases King Saud University Riyadh Saudi Arabia 12 Division of Infection and Immunity University College London and 13 NIHR Biomedical Research Centre UCL Hospitals NHS Foundation Trust London UK Abstract The Saudi Arabian Ministry of Health implemented a pro active surveillance programme for Middle East respiratory syndrome MERS coronavirus MERS CoV We report MERS CoV data from 5065 Kingdom of Saudi Arabia individuals who were screened for MERS CoV over a 12 month period From 1 October 2012 to 30 September 2013 demographic and clinical data were prospectively collected from all laboratory forms received at the Saudi Arabian Virology reference laboratory Data were analysed by referral type age gender and MERS CoV real time PCR test results Five thousand and 65 individuals were screened for MER CoV hospitalized patients with suspected MERS CoV infection n 2908 57 4 healthcare worker HCW contacts n 1695 33 5 and family contacts of laboratory confirmed MERS cases n 462 9 1 Eleven per cent of persons tested were children 17 years of age There were 108 cases 99 adults and nine children of MERS CoV infection detected during the 12 month period 108 5065 2 case detection rate Of 108 cases 45 were females six children and 39 adults and 63 were males three children and 60 adults Of the 99 adults with MERS CoV infection 70 were hospitalized patients 19 were HCW contacts and ten were family contacts There were no significant increases in MERS CoV detection rates over the 12 month period 2 6 19 731 in July 2013 1 7 19 1100 in August 2013 and 1 69 21 1238 in September 2013 Male patients had a significantly higher MERS CoV infection rate 63 2318 2 7 than females 45 2747 1 6 p 0 013 MERS CoV rates remain at low levels with no significant increase over time Pro active surveillance for MERS CoV in newly diagnosed patients and their contacts will continue Keywords Clinical coronavirus demographic diagnosis MERS CoV Middle East real time PCR sample type SARS screening viral load Original Submission 29 December 2013 Accepted 17 January 2014 Editor M Drancourt Article published online 24 January 2014 Clin Microbiol Infect Corresponding author Z A Memish Collaborating Centre for Mass Gathering Medicine Ministry of Health Al Faisal University Riyadh 11176 Saudi Arabia E mail zmemish These authors contributed equally Introduction Understanding the natural history epidemiology and clinical presentation of new killer infectious diseases is dependent on and influenced by the WHO recommended surveillance strategies for case detection which largely focus on severe 2014 The Authors Clinical Microbiology and Infection 2014 European Society of Clinical Microbiology and Infectious Diseases ORIGINAL ARTICLE 10 1111 1469 0691 12562 illness and microbiological testing coupled with details from case studies Contact tracing activities allow for the detection of confirmed cases with a broader spectrum of illness For infectious diseases caused by viruses confirmed cases include only those with a positive PCR test result for viral genetic material in accordance with the laboratory guidelines Since the first case report of the novel Middle East respiratory syndrome MERS coronavirus MERS CoV in September 2012 1 the Kingdom of Saudi Arabia KSA Ministry of Health KSA MoH has been working closely with interna tional collaborators and the WHO to better understand and define the epidemiological demographic clinical and labora tory features of the new disease A molecular real time PCR diagnostic test was rapidly developed after the first case and was subsequently the method recommended by the WHO for detecting the presence of the MERS CoV infection 2 3 This test was used in a retrospective analysis on biobanked samples to confirm two cases of MERS from an earlier outbreak of respiratory infection in Jordan in April 2012 4 Important steps for the surveillance and control of MERS CoV infection are the early detection and isolation of patients with active MERS CoV disease and screening of their contacts Surveillance studies also help in defining and mon itoring transmission rates case load and epidemic risk assessment and assist in instituting infection control measures with new diagnostic methods and treatments Although MERS CoV case detection is critically dependent on the degree of awareness of the attending physician accurate laboratory testing is also essential in making a diagnosis Soon after the detection of the first case of MERS in Jeddah in September 2012 1 the KSA MoH put in place a proactive surveillance and screening programme for inpatients admitted with respiratory illness suspected of being caused by MERS CoV It also included active screening of contacts of confirmed MERS cases KSA MoH recommendations for MERS CoV screening are based on the WHO guidelines on case definition detection and contact investigations 5 7 This led to an increase in the numbers of requests for MERS CoV screening from hospitals throughout the KSA We report these laboratory data on the use of real time PCR tests on clinical samples received from 5065 individuals screened for MERS CoV during a 12 month period commencing from the first case detection in September 2012 Methods Selection of individuals for MERS CoV screening The KSA MoH has implemented a pro active early case detection and surveillance system for MERS CoV It recom mends sending respiratory tract samples from critically ill patients admitted to hospitals with fever and lower respiratory tract infection symptoms Screening for MERS CoV is also recommended for family and healthcare worker HCW contacts of proven cases of MERS CoV infection All samples are transported to and are processed by the KSA MoH virology laboratory in Jeddah which is accredited and regu lated by the Central Board for Accreditation of Health Care Institute Quality assurance and control for all diagnostic tests is monitored through Internal Policy Procedures and by external quality assurance schemes Collection of clinical specimens Respiratory specimens collected from patients and contacts were sputum samples nose and throat N T swabs nasopharyngeal NP swabs and tracheal aspirate samples Sputum was collected directly into a sterile leak proof screw capped sputum collection sterile container NP swabs and N T swabs were collected with sterile synthetic tip Dacron flocked swabs For NP specimens the swabs were inserted through the nostril parallel to the palate into the nasopharynx Swabs were left in place for a few seconds to absorb secretions For N T swabs both nostrils and the throat were swabbed with separate swabs All swabs were placed immediately into sterile tubes containing 2 3mLof viral transport medium For inpatients lower respiratory tract samples 2 3 mL of bronchoalveolar lavage fluid and tracheal aspirate were obtained and placed into sterile leak proof screw capped sterile dry containers Labelling storage and transportation of specimens When there were short periods of transportation 48 h of specimens to the laboratory specimens were held in a refrig erator at 2 8 C rather than frozen for periods exceeding 48 h specimenswereshippedondryiceatC070 Cassoonaspossible after collection Each specimen container was labelled with the patient s ID number the specimen type and the date on which the sample was collected All specimens were pre packed to preventbreakage and spillage Specimen containers were sealed with parafilm and placed in zip lock bags Absorbent material to absorb the entire contents of the secondary container con taining the primary container was placed to separate the primary containers containing specimen to prevent breakage RNA extraction Extraction of RNA was performed with Roche MagNa Pure LC RNA Viral isolation Kit Sputum samples were pretreated with 2 9 lysis buffer for 30 min in a shaking incubator Swabs were placed in lysis buffer Two hundred microlitres of each sample was added to the MagNA Pure LC plate which contains 2014 The Authors Clinical Microbiology and Infection 2014 European Society of Clinical Microbiology and Infectious Diseases CMI 2 Clinical Microbiology and Infection CMI 96 wells Reaction reagents were then loaded and checked before the samples were run according to the manufacturer s instructions for nucleic acid extraction in the specimen area MERS CoV screening test A biosafety level 2 facility equipped with microbiological safety cabinets was used for the handling of clinical specimens and for extracting RNA for PCR PCR is the recommended method for detecting the virus At least three sites in the genome of MERS CoV have been identified as suitable targets for the diagnostic test the upstream E protein gene upE open reading frame ORF 1A and ORF 1B and sequences of the specific primers have been published Positive controls for the upE screening and the ORF 1A confirmation assays are available Clinical samples were screened by real time PCR as previously described 6 with amplification targeting both upE and ORF 1A for confirmation these are standard assay used in the KSA for MERS CoV testing The test result was considered to be positive if both assays gave positive results In cases of discordance between the first and second assays or if the result was considered to be a doubtful positive result another clinical sample was requested and analysed Data collection Data from laboratory forms accompanying clinical samples received at the KSA MoH virology laboratory specifically requesting MERS CoV testing during the period 1 October 2012 to 30 September 2013 were collected and analysed Demographic and laboratory PCR test data and MERS CoV viral load data were recorded Where data were missing from the records or where clarification was required data were obtained through direct communication with attending physi cians and other healthcare providers Serological testing No serological testing data were available because there are no validated accurate serological diagnostic tests for MERS CoV available to date Statistical analyses Demographic clinical and laboratory descriptive data were tabulated Univariate analysis was performed with binary logistic regression analysis A p value of 0 05 was considered to indicate statistical significance Results A total of 5065 individuals 625 children and 4440 adults were screened for MERS CoV during the 12 month period hospitalized patients with suspected MERS CoV infection n 2908 57 4 HCW contacts n 1695 33 5 and family contacts of laboratory confirmed MERS cases n 462 9 1 Fig 1 There were 108 cases 99 adults and nine children of MERS CoV infection detected during the 12 month period 108 5065 2 case detection rate Table 1 Of 108 MERS cases 45 were females six children and 39 adults and 63 were males three children and 60 adults Of the 99 adults with MERS CoV infection 70 were hospitalized patients 19 were HCW contacts and ten were family contacts Of the nine children with MERS CoV infection two were hospitalized patients and seven were family contacts A significant increase in the number of screened and tested specimens was evident over the study period Fig 2a but there were no significant increases in MERS CoV detection rates over the 12 month period Fig 2b The monthly case detection rates of MERS CoV were 2 6 19 731 in July 2013 1 7 19 1100 in August 2013 and 1 69 21 1238 in September 2013 Fig 2b Male patients had a significantly higher MERS CoV infection rate 63 2318 2 7 than females 45 2747 1 6 p 0 013 Table 2 Children 17 years n 9 HP 2 HCW n a FC 7 PATIENTS AND CONTACTS SCREENED Total n 5065 Children 17 years n 625 Adults 17 years n 4440 MERS CoV RT Real time PCR positive n 108 MERS CoV Real time PCR negative n 4957 Adults 17 years n 99 HP 70 HCW 19 FC 10 Children 17 years n 616 HP 459 HCW n a FC 157 Adults 17 years n 4341 HP 2377 HCW 1676 FC 288 FIG 1 Flow chart screening of hospitalized patients and contacts FC family contacts HCW healthcare workers and their contacts HP hospital patients NA not applicable 2014 The Authors Clinical Microbiology and Infection 2014 European Society of Clinical Microbiology and Infectious Diseases CMI CMI Memish et al Screening for Middle East respiratory syndrome 3 Discussion Since the first KSA case report in September 2012 the KSA MoH has recommended mandatory testing for MERS CoV of all cases of respiratory illness requiring intensive care admission This is the largest study describing the incidence rates of MERS CoV in the KSA over a period of 12 months The real time PCR MERS CoV diagnostic test has been in use for detecting new MERS cases in hospitalized patients and for screening of HCW and family contacts of confirmed MERS cases 7 11 Six months after MERS CoV was discovered at the end of March 2013 there were only 17 MERS CoV cases reported globally nine of which were from the KSA 12 In light of the infrequent but continuing detection of sporadic MERS CoV cases in the community and the hospital outbreak at Al Hasa 10 the WHO constituted an Emergency Com mittee under the International Health Regulations to advise the Director General on the status of the MERS CoV situation 13 The important issue at that time was whether MERS CoV was going to progress to cause a major pandemic as did the SARS epidemic in the early 2000s which was also caused by a novel coronavirus SARS CoV 14 Our results show that pro active surveillance during the months after the Al Hasa outbreak which occurred in April May 2013 showed no significant increase in the MERS case detection rates in the ensuing 8 months The monthly positive rates of MERS CoV were 2 7 in July 2013 and then 1 7 in AugustandSeptember2013 TheinitialincreaseinJuly2013was related tothe intensification of the surveillance of MERS CoV in the KSA following the healthcare related outbreak at Al Hasa 10 From our data three areas of transmission can be focused on for active surveillance and screening The first pattern is the occurrence of sporadic cases in communities The true incidence of the disease in the community is not known and remains to be defined through case control serological surveys when accurate rapid sensi tive and specific serological tests become available In the community the asymptomatic cases or those with minimal symptoms are difficult to identify and are usually missed Those who become acutely ill in the community present to emergency rooms where real time PCR testing of respiratory samples is performed The second pattern of transmission is transmission within families 8 The rate of intrafamilial transmission is not known Our results provide an estimate of a rate of 3 6 for acquisition of MERS CoV from close family contacts How ever this finding is not conclusive as no serological assays were utilized for screening mild or subclinical cases How ever the finding is in agreement with previous observations of low secondary attack rates among family members or contacts of patients in the KSA and European countries 8 15 22 TABLE 1 Middle East respiratory syndrome coronavirus MERS CoV screening by referral type and age group Patients and contacts Children aged 17 years Adults aged 17 years All MERS CoV real time PCR Positive Total Positive a Positive Total Positive a Positive a Total Positive a Hospital patients 2 461 0 43 70 2441 2 86 72 2908 2 51 HCW contacts NA NA NA 19 1695 1 12 19 1695 1 12 Family contacts 7 164 4 2 10 298 3 36 17 462 3 6 Total 9 625 1 4 99 4440 2 19 108 5065 2 1 HCW healthcare worker NA not applicable For children p value for positive PCR in hospitalized patients vs family contacts 0 0021 For adults p value for positive PCR in hospitalized patients vs HCWs 0 0029 p value for hospitalized patients vs family contacts 0 46 p value for HCWs vs family contacts 0 025 a Percentage within each age group 0 200 400 600 800 1000 1200 1400 Total number tested Total number tested Total Total Positive Positive 3 2 5 1 5 0 5 0 2 1 FIG 2 Kingdom of Saudi Arabia Middle East respiratory syndrome coronavirus case screening a and detection rates b over time 2014 The Authors Clinical Microbiology and Infection 2014 European Society of Clinical Microbiology and Infectious Diseases CMI 4 Clinical Microbiology and Infection CMI The third transmission pattern is nosocomial transmission to HCWs In one study seven HCWs with MERS CoV infection were reported Two of them were asymptomatic and five had mild upper respiratory tract symptoms 11 The current study sheds more light on the transmission of MERS CoV in healthcare setting The positivity rate of MERS CoV by PCR was only 1 12 of all tested HCWs Transmission within healthcare settings was retrospectively reported from Jordan 4 and then subsequently reported in France the KSA the UK the United Arab Emirates and Qatar and included HCWs treating MERS CoV patients 23 27 These data suggest that the current risk of transmission within healthcare facilities remains small and that the recommended infection control measures are adequate 6 9 13 20 Studies of family and hospital case clusters of MERS CoV infections in the KSA and other European countries that have reported MERS cases indicate that a spectrum of clinical illness occurs 7 9 15 16 Reports from Tunisia and the UAE of MERS CoV infections occurring in siblings whose father s illness was a probable MERS CoV infection case and the case from the UK 8 24 25 show that the siblings who are not immunocompromised only manifest mild respiratory illnesses and do not require hospitalization In the UK family cluster among 33 close contacts 20 household and 13 non house hold there were only two cases 6 attack rate of confirmed MERS CoV infection one with mild illness and one with severe illness 15 There were no cases of MERS CoV infection among 59 HCWs who were in contact with the index case without wearing full personal protective equipment 15 The fact that we have subsequently identified milder or asymptomatic cases of MERS in HCWs children and family members of contacts of MERS cases indicates that the severe cases represent only the tip of the iceberg and there is a spectrum of milder clinical disease that requires definition Our data indicate that MERS CoV affects both genders and although a few cases of MERS CoV in children have been detected it remains mainly a disease of adults across all age groups To date there is still no evidence of sustained community transmission Despite extensive investigation and testing of thousands of contacts by the KSA MoH only a few instances of transmission to HCWs or family contacts have been identified Almost all patients who died or who had been hospitalized had severe disease or other comorbidities 9 10 The mortality rate and severity of disease are exaggerated to some degree by the detection of such cases The case fatality rate has fallen in recent months owing to the detection of milder and asymptomatic cases 23 27 The most critical characteristic of pandemic MERS CoV strains would be progression to efficient human to human transmis sion The number of sporadic MERS cases being reported has been small and indicates that the virus appears to be not readily capable of