we screened serum samples collected from inpatient children in cambodia through routine national dengue surveillance . the study set was extracted from the institut pasteur in cambodia biobank of strictly anonymized samples collected from 9,408 febrile inpatients during 20002011 . ethics clearance was obtained from the cambodian national ethics committee for human research before testing commenced . after exclusion of data entry errors , outliers in terms of year of participation , and insufficient data or samples , the database included 7,823 children 215 years of age for whom age , sex , and province of residence were documented . to avoid any influence from maternal antibodies , we excluded children < 2 years of age from the study . provinces were allocated to geographic quadrants and mapped by using arcgis 10 ( esri co. , redlands , ca , usa ) ( technical appendix figure 1 ) . random sampling was applied by using stata 11 ( statacorp lp , college station , tx , usa ) with a representation of samples for each year . a total of 1,707 anonymized samples ( 1 sample / child ) all available samples from the sparsely populated northeastern quadrant ( 4% of the dataset ) were included . because the southeastern quadrant ( bordering vietnam ) is the most populated quadrant , samples were selected in approximate proportion to population ( 46% of the dataset ) . samples from the southwestern ( 18% of dataset ) and northwestern ( 31% of the dataset ) quadrants , each of which borders thailand , were selected proportionally to represent a total of 48% from the quadrants bordering thailand ( table ) . * all - age population in 2008 ( census nearest to retrospective study midpoint ) . data from the national institute of statistics , directorate general for health , cambodia demographic icf macro , 2011 ; general population census of cambodia , 2008 ; and ministry of planning , 2009 . the 1,707 serum samples were screened by use of a microneutralization assay to detect neutralizing antibodies against an ev71 strain ( genotype c4a ) isolated from an infected child during the 2012 outbreak in cambodia . the assay was conducted on vero e6 cells by mixing 2-fold serial dilutions ( 1:8 to 1:8,192 ) of heat - inactivated human serum samples with 100 l ( 2,000 50% tissue culture infective doses / ml ) of the ev71 strain . all serum samples were tested in duplicate , and positive control serum was added to each reaction plate for quality control purposes . the lowest dilution at which cytopathic effect was observed in > 50% of wells was considered the antibody titer of the serum sample . a titer of > 1:16 was considered the cutoff for a positive antibody response and was a more stringent cutoff than that used in previously published ev71 seroprevalence studies , in which the cutoff was usually > 1:8 ( 36 ) . to reconstruct the historical annual probability of infection this reconstruction assumed that after infection , detectable antibody titers are long lasting ; this method has been used to estimate the historical force of infection for other diseases , such as dengue ( 7 ) . we estimated a separate annual probability of infection for each year from 1994 through 2011 . because no patients in our dataset had been born before 1994 , we could not estimate the force of infection before this time ( online technical appendix ) . because samples included in this study were from children with a denguelike illness , rates of ev71 infection among study participants might not accurately reflect rates among all children in cambodia . however , because an average of 87.8% of patients recruited by the national dengue surveillance program had a laboratory - confirmed dengue infection ( 8) , the febrile episode that triggered the hospitalization could be only slightly associated with ev71 infection and thus would have negligible influence on the ev71 neutralizing titers of the patient population . among children in this study , the overall seroprevalence of ev71 neutralizing antibodies was 88.8% : 1,300 ( 94.8% ) of 1,371 ( 95% ci 93.5%95.9% ) among children 215 years of age sampled during 20062011 and 216 ( 64.3% ) of 336 ( 95% ci 58.9%69.4% ) among those 27 years of age sampled during 20002005 ( figure 1 ) . this profile across age groups remained unchanged in more stringent analyses with higher cutoff values ( technical appendix figure 2 ) in which , despite levels of seropositivity decreasing with higher cutoff titers , the reduction was consistent across all age groups . seroprevalence of ev71 relative to sex did not differ significantly ( 89.8% among girls vs. 87.7% among boys ; p = 0.18 ) . age - associated seroprevalence of enterovirus 71 ( ev71 ) infection in cambodia , estimated by detection of ev71 seroneutralizing antibodies in inpatient children 215 years of age , 20002011 . epidemic curves derived from the seroprevalence data show the dynamics of infection for the whole country ( figure 2 ) and across the 4 quadrants ( technical appendix figure 3 ) . the reconstructed curves were coherent , showing large - scale , countrywide circulation of the virus since 2002 . seroprevalence peaks every 23 years indicate a cyclical pattern of ev71 outbreaks . this pattern has been reported from other asia - pacific countries ( 911 ) and probably represents the time needed for establishment of a new cohort of immunologically naive patients . in countries with a larger population , such as china , infection might peak annually ( 12 ) . annual probability of enterovirus 71 infection ( ev71 ) in cambodia during 19942011 , estimated by detection of ev71 seroneutralizing antibodies in inpatient children 215 years of age . serum samples were collected from routine national dengue surveillance in cambodia . despite our use of a more stringent cutoff value , the seroprevalence detected in our study was considerably higher than that reported from previous studies in the region , during which a cutoff of 1:8 was invariably used ( 36 ) . if we had used a neutralization titer of 1:8 , seroprevalence would have been 93.1% ( n = 1,590 positive samples ) . intense circulation of ev71 was therefore occurring in cambodia long before the 2012 outbreak . in cambodia and other asia - pacific countries , some of these strains , such as coxsackieviruses a6 and a16 , have also been associated with severe neurologic illnesses in children . previous studies have established that cross - neutralization occurs among different ev71 strains and genogroups ( 13,14 ) . however , there is no evidence of cross - neutralization between ev71 and other enteroviruses ( 15 ) . cross - neutralization at high dilutions would probably not have generated a consistent profile of seropositivity across children of different ages ( technical appendix figure 2 ) . thus , the high level of seropositivity observed in this study is probably specific for ev71 . our data support the widespread circulation of ev71 at least a decade before its reported emergence in 2012 . furthermore , reconstructed epidemic curves suggest that ev71 outbreaks occurred in a cyclical pattern in cambodia and that the virus infected large proportions of immunologically naive children every 23 years . before 2012 , this circulation remained undetected , highlighting the need to further reinforce the surveillance systems in developing countries . also needed is enhanced medical education for better identification of infectious diseases such as hand , foot and mouth disease , which , despite its association with relatively specific clinical signs , requires careful physical examination of patients . it is still unknown why so many severe cases were detected during the 2012 ev71 outbreak in cambodia . however , seroepidemiologic studies in other settings have also confirmed widespread circulation before outbreaks ( 5,9 ) . for combatting this pathogen , developments in vaccines and antiviral drugs are urgently needed . distribution of provinces into quadrants , age - associated seroprevalence , estimated annual probability of infection by geographic quadrant during 19942011 , and statistical model used to estimate annual probability .
enterovirus 71 is reported to have emerged in cambodia in 2012 ; at least 54 children with severe encephalitis died during that outbreak . 
 we used serum samples collected during 20002011 to show that the virus had been widespread in the country for at least a decade before the 2012 outbreak .