Abstract

Introduction

As of March 8, 2015, Middle East respiratory syndrome coronavirus (MERS-CoV) has caused at least 1082 mostly severe cases of respiratory infection, 439 of these fatal, since its discovery in 2012.1 Apart from its geographical focus in countries in and around the Arabian Peninsula and laboratory evidence of widespread infection of dromedary camels, little is known about the actual epidemiology of the disease in human beings.1, 2, 3, 4, 5 Few primary infections were acquired through direct camel exposure, but the relevance of this infection pattern is unclear in the absence of systematic determinations of the proportion of infections in individuals who have had contact with camels.5, 6, 7 Moreover, most reported patients with primary MERS-CoV infection have no history of camel exposure, suggesting the existence of other, as-yet-unknown sources of infection. Camel milk as a food-borne source seems unlikely.8

For secondary infections acquired through human-to-human contact, mathematical projections predicted that transmission chains in the population cannot be sustained.9, 10, 11 The apparent transmission rate in household settings is low, with fewer than 50% of index patients transmitting the infection to contacts who subsequently had no pronounced clinical symptoms.12 However, a highly fatal outbreak of MERS-CoV infection centred in Jeddah, Saudi Arabia, in March–April, 2014, was apparently caused by human-to-human transmission in several nosocomial settings, without any evidence that the causative virus differed from other MERS-CoV strains in people, in terms of replication in cell culture, immune escape, or excretion level.13 Undiscovered MERS-CoV infections might thus exist in the human population.

Serology is key to the understanding of infection statistics at a population level. However, only a few serological studies of MERS-CoV have been done.14, 15 Although none of those preliminary studies showed any evidence of previous infection with MERS-CoV in the population of Saudi Arabia, the examined cohorts have been small and methods applied in those studies have not been validated for sensitivity and specificity. We previously presented the first validated recombinant ELISA (rELISA) for MERS-CoV and combined this test with recombinant immunofluorescence assay (rIFA) and a plaque reduction virus neutralisation test (PRNT) into a staged strategy for MERS-CoV serology that was shown to identify even subclinical infections on detection of specific antibodies.12 Here, we apply the established testing algorithm on a nationwide serum collection. Additional serum samples were obtained from individuals occupationally exposed to dromedary camels.

Methods

Results

Between April 1, and June 30, 2013, we obtained 5765 serum samples from local primary health-care clinics for individuals selected to participate in a survey of the national burden of disease,16, 17 and a further 4244 samples between Dec 1, 2012, and Dec 1, 2013 from primary health-care centres. Data for patient demographic characteristics (appendix) were available for 8719 (87%) of 10 009 stored serum samples.

Additionally, we obtained serum samples from 87 camel shepherds who participated in a MERS-CoV surveillance study from Feb 1, to May 31, 2014, and 140 expatriate slaughterhouse workers in Makkah during August, 2013. The camel shepherds were aged 21–84 years (mean 37·9 years, SD 13·02) and resided in six different provinces (Al-Jouf, Asir, Eastern, Madinah, Makkah, and Riyadh). The slaughterhouse workers were aged 15–62 years (mean 37·13, SD 8·64).

The mean sample size per province was 770 (range 321 to 2343 [SD 585]), representing between 19·7 and 97·1 samples per 100 000 individuals per province (mean 52·9, SD 26·7). The age distribution of samples with available age information matched that of the population of Saudi Arabia (figure 1 ). Of the 8719 individuals with available demographic information, 4341 (50%) were male.

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Figure 1

Age distribution of the study sample compared with the total population

Census for the population of Saudi Arabia in 2013 was retrieved from the Saudi Arabia Central Department of Statistics and Information (detailed numbers in the appendix).

All serum samples from the general population were first screened for MERS-CoV-specific IgG by rELISA, yielding reactive results in 152 (1·52%, 95% CI 1·29–1·78) of 10 009 samples. Subsequent testing by rIFA and PRNT₅₀ confirmed less than 0·2% to be stage 1 and stage 2 seropositive (table 1 ; appendix). Because repeat samples were missing, the timepoint of seroconversion was established by IgM detection. Coronavirus IgM had been previously shown to be maintained for a maximum of 180 days after infection.22 Only one of 15 seropositive individuals had a reciprocal MERS-CoV IgM titre of at least 100, suggesting recent seroconversion (appendix). In accordance with our previous study,12 seropositivity with rELISA was ten times higher than with rIFA. Seroprevalence did not differ between the two subcohorts (seven [0·16%] of 4244 patients in the primary health-care study group vs eight [0·14%] of 5765 in the burden of disease study cohort group, p=0·94; appendix). Men had a higher proportion of infections (11 [0·25%] of 4341) than did women (two [0·05%] of 4378; p=0·025). Cases were identified in four of six age groups without significant age preference (appendix).

Confirmed cases of MERS-CoV infection were identified in six of the 13 administrative provinces of Saudi Arabia, with higher numbers in central provinces that generally are more rural (figure 2 ). The 13 provinces were classified into seven central and six coastal provinces, on the basis of whether they had a shoreline. Significantly more confirmed infections were in central provinces (14 [0·26%] of 5479 samples) than in coastal provinces (one [0·02%] of 4529; p=0·003).

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Figure 2

Serologically confirmed cases of Middle East respiratory syndrome coronavirus (MERS-CoV) in the general population of Saudi Arabia from Dec 1, 2012, to Dec 1, 2013

Red areas represent provinces that contain individuals with stage 2 seropositive MERS-CoV. Percentages show the number of stage 2 positive serum samples per number tested in each province (detailed numbers and full serological results per province in appendix).

The proportion of both camel shepherds and slaughterhouse workers who were seropositive was significantly higher than in the general population (table 1). Seropositive individuals were identified in five of six age groups with even distribution (appendix).

Both registered patients (ie, patients with confirmed MERS)21 and index patients from the household study were significantly older than the serologically confirmed individuals in our present study (table 2 ; detailed laboratory results in the appendix).

Discussion

In our study, we identified antibodies against MERS-CoV in 0·15% of samples from the general Saudi Arabian population. The true number of infections is difficult to estimate because our sample consisted of only 10 009 (0·03%) of the whole population of 29 994 300 people. However, by combining two cross-sectional subcohorts we could cover all 13 provinces and achieved an age distribution of our sample that largely matched that of the general population (panel ). All geographical regions of the country were sampled with a reasonably low variation in the proportions of population that were studied per province. To have optimum sample coverage per province, we included samples from an earlier study (unpublished report) for which detailed information about sex and age was not available for 26% of samples. Nevertheless, samples from both subcohorts should be similar in age and sex composition because both were previously obtained according to calculations by the National Census Bureau to represent the composition of the Saudi Arabian population. This similarity was lent support by the highly similar seroprevalence when we analysed both subcohorts separately. Even if we had only used data from the more thoroughly documented burden-of-disease study subcohort for which we had sex and age information for 97% of the samples, the overall seroprevalence would have been maintained (0·15% in the overall sample vs 0·14% in the subcohort).

To obtain an estimate of the total number of seropositive individuals in Saudi Arabia we did a straightforward extrapolation based on the proportion of stage-2-positive serum samples in both population subcohorts (excluding shepherd and slaughterhouse worker samples). Extrapolation by simple multiplication suggests that 44 951 seropositive individuals (95% CI 26 971–71 922) could exist in a total population of 29 994 300. Our serological assays might have missed some infections because the high specificity of rIFA and PRNT could encompass a reduction in sensitivity of the overall serological algorithm. However, even a projection based on rELISA results only would raise the estimated infection rate by about ten times, which is several orders of magnitude lower than figures expected for a respiratory disease that continuously transmits between human beings. In accordance with our previous study,12 seropositivity with rELISA was ten times higher than that with rIFA.12 This rise can be attributed to the higher sensitivity of rELISA and by putative crossreactivity of low-affinity IgG, which is raised in individuals with recent infections from the common-cold coronavirus.23

Despite low detection rates, antibodies were identified widely across Saudi Arabia, including in provinces where outbreaks in people have not been reported. Widespread acquisition of infections without subsequent causation of outbreaks corresponds to the concept of a predominantly zoonotic disease that does not readily spread between human beings. The raised prevalence in rural regions and the significantly increased proportion of infection in groups that are occupationally exposed to camels emphasises the importance of zoonotic acquisitions by dromedary camels.5, 6, 7 However, even though the analysed shepherd samples were collected in six different regions during the first months of 2014, the sampling was not randomised and samples were not strictly taken at the same time as those from the general population. Similar limitations apply for the slaughterhouse worker samples. Direct comparison of seroprevalences between these groups and the general population cohort might thus be biased. Matched case-control studies could be used to substantiate the raised seroprevalence in exposed groups.

Direct animal contact has been reported for only a few primary cases in Saudi Arabia and elsewhere. The younger age of serologically confirmed patients in our study compared with people with laboratory confirmed infection underlines the possibility that MERS-CoV might cause unrecognised infections that cause no or mild symptoms in healthy, younger (aged 15–44 years), and socially active populations. These subclinical infections could act as an unknown source for index patients who cannot recall contact with camels. Importantly, the predominance of men even within serologically confirmed cases emphasises a potential association with animal exposure because, typically, more men than women in Saudi Arabia associate with camels during leisure activities. Cases of MERS in younger people (aged <45 years) might seem mild or even subclinical, similar to cases in our study of household contact clusters,12 in which contacts were younger than index patients and mostly asymptomatic. MERS might constitute a zoonotic disease that causes enough short-transmission chains in the young male population of Saudi Arabia, for which older and predisposed individuals constitute a sentinel population.

Acknowledgments

Contributors

Declaration of interests

References