EV71 as one kind of enterovirus, shedding from respiratory tract after onset may continue for nearly four weeks17, that is the reason why EV71 can persist in throat secretions for a period of time18, and it can survive for a long period outside the host with suitable environment19. National Centre for Immunization and Respiratory Diseases (NCIRD) mentioned that some populations, especially adults, might not show any clinical symptoms, but they can still spread the virus to others20. Therefore, in this study children’s caregiver samples were collected by nasal and throat swabs and tested for the presence of enterovirus, EV71, CVA16 and CVA6. Overall, seven children’s caregiver samples (7.14%) had been detected positively of enterovirus. Such proportion of children’s caregivers with enterovirus detected involved in this study was higher than in previous reports (enterovirus positive rate of 1.7% on aged above 16-year-old adults from the data of Deng’s study)21. In Deng’s study, stool specimens were collected randomly and tested by qPCR. We assume that higher positive rate of enterovirus in our study may due to viruses exist in nasal and throat secretions longer than in stool, although Teng et al.22 observed that the duration of enterovirus (EV71 and CVA16) last for a long time in patients’ stool. Estimated 7.14% of children’s caregivers have positive enterovirus, means they would be huge reservoir of HFMD viruses that could transmit the viruses to children. In this study we did not collected the nasal and throat swabs from corresponding children of each parent or grandparent simultaneously, the source of infection between children and their caregivers could not be confirmed as there might be a cross propagation occurred between them, but children’s caregivers could be considered as potential reservoir of HFMD infection.

From the results of environmental samples in Table 1, totally seven wiping samples collected from playgrounds (7.61%) had positive results of enterovirus. Playground is one of main transmission places for children and their caregivers. Previous study of Xie had the results that adjusted odds ratios (OR) of hospital HFMD cases to community controls for exposure to public playgrounds were 6.03 (95% CI: 2.84, 12.80), as well as the attributable fractions of this risk factor (57.2%)15 was the highest among five risk factors they studied. Such result was also found in reported previous exposures, considered playgrounds as an important risk factor of HFMD23. Based on the findings of these previous studies, we collected a large number of wiping samples in public playgrounds in order to estimate the virus level around such kind of area. Just by wiping some points of facilities’ surface, enterovirus rate could be as high as 7.61%. If children continuously wipe or touch the surface more than just some points of it, they will have greater chance of contacting with enterovirus. Our study suggested that enterovirus could be transferred and spread among children by wiping the surface of playground facilities and regular disinfection of surface is essential.

In our study, only one air sample (1.49% of 67 air samples) had positive result. Air samples were collected for 20 minutes long as the minimum collecting time was not well testified, because there was lack of guidelines about air samples of HFMD. Only in sampling plan of swine respiratory pathogens, air samples were collected for detection. Updated methodology used in Corzo’s study24 was that collector ran for 30 minutes on air sampling, which was 10 minutes more than the time lasted in our study. Short time period of air samples collection might give rise to the possibilities of virus levels being below the detection limit. If time of air collection increases, more positive results of enterovirus might be detected. As the only one tested positive out of eight samples collected in the same indoor market place, this air sample’s cycle threshold (Ct) value was relatively high (40.93 on average). Such results had not enough evidences to explain air environment of these indoor market places as the reservoir of large number of infectious cases caused by primary virus agents. Besides, high population density of public places might be essential factor to increase the frequency of HFMD transmission through the air, which should be considered carefully in the further studies.

Neither children’s caregiver samples nor environmental samples had been detected positively of predominant virus such as EV71, CVA6 or CVA1625. However, other coxsackievirus, such as CVA4, CVA5, CVA10 and CVB2 to B5 might be potential agents of HFMD as well3,4,5. In 2014 in Guangzhou Xintang area, enterovirus was the major pathogen of HFMD among diagnosed cases of HFMD (enterovirus positive rate of 55.76%, CVA16 positive rate of 20.73% and EV71 positive rate of 23.51%)26. From this study, other species of enterovirus beside CVA16 and EV71 could be the pathogens of HFMD. Positive detection of enterovirus in our study means the possibility of EV71, CVA6, CVA16 or other coxsackievirus still existing in nasal secretions, throat secretions or surface of facilities in playgrounds, which cannot deny that there is risk of HFMD virus transmission from children’s caregivers or playgrounds. And the reason why no positive detection of EV71, CVA6 or CVA16 could be small sample size that should be increased in further studies.

If children’s caregivers and public playgrounds can serve as the reservoir of the agent of HFMD, it is crucial to the control of HFMD spread. The range of activities for adults is generally far greater than five-years-old children, and there is a greater chance that these caregivers are able to contact with enterovirus. Considering the infection of adults is mostly asymptomatic infection27, it makes them difficult to be identified, as preventing and controlling the spread of enterovirus-related diseases. The difficulty can also apply to the public playgrounds. Playgrounds are often fully packed, even during weekdays and especially during the weekends and the holidays. Thus, it would be a challenge to ensure the surface of these recreational facilities pathogen-free and identify the contaminated surface. Overall, 4 out of 10 playgrounds in Guangzhou were detected EV positive as the ratio was 40%. The question remained was whether this result relevant to disinfection measures, visitors’ average daily amount, indoor or outdoor as well as the types of entertainment facilities in these playgrounds.

In order to implement the measurements to prevent and control HFMD among children, disinfection management on public entertainment facilities should be improved. Currently the automatic alert and response system were confirmed that they had good sensitivity on the detection of HFMD infection during the outbreaks28, but it is not enough for prevention. Sampling from environment with potential infections and monitoring on general population to identify recessive infection as regular basis should be into the HFMD surveillance system. Future study should be carried on with sampling from children at the same places as their caregivers and tracing back to the HFMD individual cases, because is essential to establish a cohort research and identify the source of HFMD transmission between children and their caregivers. Considering of children’s caregivers, target samples should locate on specific groups such as household members, nurses or tutors. Larger sample size under the calculation of 5% statistical power, and focus on associations between each specific facility and HFMD cases around the same public area, such as children slide and parallel bars. Besides, in this study, enterovirus was detected positively by qPCR, but whether it has infectivity and the infectivity level need cell culture experiments and animal trials to be confirmed.