Abstract
Background
EV71 is associated with the fatal cases of brain stem encephalitis during large HFMD outbreaks from 1998 to 2008. EV71 may continuously shed from upper respiratory tracts and feces of HFMD patients for relatively long time after recovery. However, the persistence of viruses in the patients' secretions and excretions is not clear.
Methods
Serial throat swabs and feces of 34 definitely diagnosed patients, including 30 mild cases and 4 severe cases, were traced and collected with the interval of 2 to 4 days for up to 32 and 48 days, respectively, and tested by a nested RT-PCR.
Results
The EV-71 specific sequences were identified by a Nested RT-PCR in all specimens of 0-4 days, and 5-8 days. The positive rates of EV71 in throat swabs dropped markedly to 42.86% during 9-12 days, and maintained at 20-30% during 13-24 days, while that in feces reduced to 71.43% during 9-12 days, and maintained roughly 20% till 37-40 days. EV71 nucleotide of 36.36% cases disappeared simultaneously both in throats and feces, 39.39% cases showed longer persistence of EV71 nucleotides in feces, and 21.21% were longer in throats. The longest duration of shedding observed was 24 days for throat swabs and 42 days for fecal specimens.
Conclusions
EV71 shedding from respiratory tract may continue for nearly four weeks after onset, but its excretion through feces can persist more than five weeks.
Similar content being viewed by others
Background
Enterovirus 71 (EV71), which was firstly described in 1969 during an outbreak with central nervous system complications in California, causes a variety of neurological diseases, including aseptic meningitis, encephalitis, and poliomyelitis-like paralysis [1]. This virus is also one of only a few enterovirus serotypes most often associated with large outbreaks of hand, foot, and mouth disease (HFMD) [2, 3]. EV71 is associated with the fatal cases of brain stem encephalitis during large HFMD outbreaks in Malaysia (1997) [1) showed that the EV71 specific sequences were identified in all specimens of 0-4 days, and 5-8 days after onset. The positive rates of EV71 in throat samples dropped markedly to 42.86% during 9-12 days, and maintained at about 20-30% during 13 to 24 days after onset, while that in feces reduced to 71.43% during 9-12 days, and maintained roughly 20% afterwards till 37-40 days after onset. EV71 associated nucleotides were undetectable in respiratory samples fourth weeks after onset and almost undetectable in feces more than five weeks later. The longest EV71-postive time in throat swab was 24 days and that in feces was 42 days post-onset.
The positive rates of EV71 nucleotides in samples from the definitely diagnosed HFMD patients. The positive rates of EV71 related nucleosides identified in serially collected throats and feces samples from the definitely diagnosed HFMD patients. Square hole represents RT-PCR positive rates from feces sample. Black square represents RT-PCR positive rates from throat swabs.
To evaluate the negative conversion rate in clinical samples of EV71 nucleotides after disease, the time period of EV71 negative closest to that of EV71 positive was taken as the time of EV71 nucleotide negative conversion. Figure 2 showed that none of the tested cases converted to EV71 negative in throats or in feces in the first four days. In the time period of 5-8 days, only one feces showed EV71 negative conversion. EV71 negative conversion happened mostly in the time periods of 9-12 days and 13-16 days after onset. 48.48% throat swabs and 17.65% feces samples in the period of 9-12 days, as well as 12.12% throat swabs and 38.24% feces samples in the period of 13-16 days were EV71 negative, respectively. Afterwards, the EV71 negative conversion rates increased gradually in throats and feces, but EV71 nucleotides in feces samples lasted clearly longer than that in throats. No significant difference in persistence of EV71 nucleotides were analyzed between mild and severe cases, between genders and among the onset ages.
To figure out the possible different profiles of EV71 disappearance, persistent states of EV71 nucleotides in throats compared to feces of individual patient were analyzed. 36.36% (12/33) cases revealed almost same time period of EV71 nucleotide disappearance both in throats and feces, 39.39% (13/33) cases showed longer persistence of EV71 nucleotides in feces, and 21.21% (7/33) were longer in throats (Figure 3). Furthermore, the identifications of EV71 nucleotides in each time period of individual case were counted. Only two out thirty-three cases showed EV71 positive in the subsequent samples after previous negative in their serial throat samples, however, 6 cases were EV71 positive reversion in the serial feces samples.
Discussion
HFMD has been an important public health concern worldwide, especially in the Asia-Pacific region. Up to now, more than 900,000 HFMD cases have been reported in mainland of China. Enteroviruses can be isolated from both the lower and upper alimentary tract and can be transmitted by both fecal-oral and respiratory routes. Fecal-oral transmission may predominate in areas with poor sanitary conditions, whereas respiratory transmission may be important in more developed areas. The relative importance of the different modes of transmission probably varies with the particular EV and environmental setting [1]. Like other infections of enteroviruses, e.g. Coxsackie's viruses and polioviruses, EV71-caused HFMD often occurred sporadically or epidemically, sometimes without clear transmission chain [2, 15]. One of the reasons is believed to be the wide and continuous circulation of EV71 among humans as well as environment [16, 17]. On the other hand, long time of virus persistence and shedding in the patients' secretions and excretions makes infectious sources be more abundant. Additionally, relatively stronger resistance of viruses in environment let the disease more easily transmit [18].
With the established nested RT-PCR, we prove a long persistence of EV71 nucleotides in the throats and feces of the patients with HFMD. Only about half of the patients show EV71 negative in their respiratory secretions and stools two weeks after onset. The traditional techniques for detecting and characterizing enteroviruses rely on the time-consuming and labor-intensive procedures of viral isolation in cell culture and neutralization by reference antisera [1, 19]. Virus persistent time in the throat in this study seems to be longer than that of previous data, mostly obtained from virus culture, which showed EV71 shedding up to 2 weeks [13]. Isolation of enteroviruses from specimens need appropriate cultured cell lines and suitable specimen. The best specimens for isolation of virus are, in order of preference, stool specimens or rectal swabs, throat swabs or washings, and cerebrospinal fluid. Throat swabs or washings and CSF are most likely to yield virus isolates if they are obtained early in the acute phase of the illness [19, 20]. Overall, the positive rate of virus isolation for enteroviruses from throat swabs in acute period is less than 50%. Even combined throat plus vesicle swabs enables the identification of virus increase, but still less than 70% [9, 13]. It is obvious that measurement of EV71 shedding merely based on virus culture will result in a portion of false negative. Certainly, identification of positive EV71 nucleotides by PCR in specimen does not indicate the presence of 100% live virus. Therefore, combination of the results from those two techniques may be more helpful for evaluating EV71 shedding. Additionally, detection of virus in a sample does not equal to being able to set up an efficient infection or transmission, which may be influenced by numerous factors, e.g virus load, exposing time and pathway, environmental and host situations.
In line with the concept that the EV shedding time from gastrointestinal tract usually longer than from respiratory tract [13], our study also illustrates a similar pattern that more than 20% cases maintain EV71 positive in the stool samples after clearance of EV71 nucleotides in their throats. It highlights a special requirement of decontamination for feces after recovery.
Conclusions
Excretion of EV71 may persist for months after infection, but most cases become negative 2 weeks after onset. Thus, the patients during the first 2 weeks should be at high risk to spread the pathogens.
References
Knipe DM, Howley PM: Enteroviruses: polioviruses, coxsackie-viruses, echoviruses, and newer enteroviruses. Fields virology. 2007, Lippincott/The Williams & Wilkins Co., Philadelphia, PA, 840-892. 5
McMinn PC: An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol. 2002, 26: 91-107. 10.1111/j.1574-6976.2002.tb00601.x.
Shimizu H: Molecular epidemiology of enterovirus 71 infection in the Western Pacific Region. Pediatr In. 2004, 46: 231-235.
AbuBakar S, Chee HY, Al-Kobaisi MF, **aoshan J, Chua KB, Lam SK: Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res. 1999, 61: 1-9. 10.1016/S0168-1702(99)00019-2.
Chen KT, Chang HL, Wang ST, Cheng YT, Yang JY: Epidemiologic features of hand-foot-mouth disease and herpangina caused by enterovirus 71 in Taiwan, 1998-2005. Pediatrics. 2007, 120: 244-252. 10.1542/peds.2006-3331.
Chen SC, Chang HL, Yan TR, Cheng YT, Chen KT: An eight-year study of epidemiologic features of enterovirus 71 infection in Taiwan. Am J Trop Med Hyg. 2007, 77: 188-191.
Wang JR, Tuan YC, Tsai HP, Yan JJ, Liu CC, Su IJ: Change of major genotype of enterovirus 71 in outbreaks of hand-foot-and-mouth disease in Taiwan between 1998 and 2000. J Clin Microbiol. 2002, 40: 10-15. 10.1128/JCM.40.1.10-15.2002.
McMinn P, Lindsay K, Perera D, Chan HM, Chan KP, Cardosa MJ: Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J Virol. 2001, 75: 7732-7738. 10.1128/JVI.75.16.7732-7738.2001.
Singh S, Vincent TK, Chow MC, Chan PKP, Poh CL: Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers. J Clin Microbiol. 2002, 40: 2823-2827. 10.1128/JCM.40.8.2823-2827.2002.
Yang F, Ren LL, **ong ZH, Li JG, **ao Y, Zhao R, He YQ, Bu G, Zhou SL, Wang JW, ** Q: Enterovirus 71 outbreak in the People's Republic of China in 2008. J Clin Microbiol. 2009, 47: 2351-2352. 10.1128/JCM.00563-09.
Bible JM, Iturriza-Gomara M, Megson B, Brown D, Pantelidis P, Earl P, Bendig J, Tong CYW: Molecular epidemiology of human enterovirus Kingdom from 1998 to 2006. J Clin Microbiol. 2008, 46: 3192-3200. 10.1128/JCM.00628-08.
Hamaguchi T, Fujisawa H, Sakai K, Okino S, Kurosaki N, Nishimura Y, Shimizu H, Yamada M: Acute encephalitis caused by intrafamilial transmission of enterovirus 71 in adult. Emerg Infect Dis. 2008, 14: 828-830. 10.3201/eid1405.071121.
Chung PW, Huang YC, Chang LY, Lin TY, Ning HC: Duration of enterovirus shedding in stool. J Microbiol Immunol Infect. 2001, 34: 167-170.
Zhang Y, Tan XJ, Wang HY, Yan DM, Zhu SL, Wang DY, Ji F, Wang XJ, Gao YJ, Chen L, An HQ, Li DX, Wang SW, Xu AQ, Wang ZJ, Xu WB: An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong. China J Clin Virol. 2009, 44: 262-267. 10.1016/j.jcv.2009.02.002.
Chang LY, Tsao KC, Hsia SH, Shih SR, Huang CG, Chan WK, Hsu KH, Fang TY, Huang YC, Lin TY: Transmission and clinical features of enterovirus 71 infections in household contacts in Taiwan. JAMA. 2004, 291: 222-227. 10.1001/jama.291.2.222.
Herrero LJ, Lee CS, Hurrelbrink RJ, Chua BH, Chua KB, McMinn PC: Molecular epidemiology of enterovirus 71 in peninsular Malaysia, 1997-2000. Arch Virol. 2003, 148: 1369-1385. 10.1007/s00705-003-0100-2.
Brown BA, Oberste MS, Alexander JP, Kennett ML, Pallansch MA: Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol. 1999, 73: 9969-9975.
Abad FX, Pintó RM, Bosch A: Survival of enteric viruses on environmental fomites. Appl Environ Microbiol. 1994, 60: 3704-3710.
Schmidt NJ, Emmons RW: Enteroviruses and reoviruses. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 1989, Washington, DC: American Public Health Association, 513-569. 6
Yin-Murphy M: Acute hemorrhagic conjunctivitis. Prog Med Virol. 1984, 29: 23-24.
Pre-publication history
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/178/prepub
Acknowledgements
We thank the staff of the department of epidemiology and infectious disease, Fuyang municipal CDC for collecting human specimens. This work was supported by China Mega-Project for Infectious Disease (2009ZX10004-101, 2008ZX10004-002), National Basic Research Program of China (973 Program) (2007CB310505) and the SKLID development Grant (2008SKLID102).
Author information
Authors and Affiliations
Corresponding author
Additional information
Competing interests
We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our works; there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
Authors' contributions
JH and XJM collected and analyzed data. JFW participated in epidemiology investigation and blood sampling. YHL, YLH, CC, CT, CG and MW carried out PCR tests. XPD designed and coordinated the study, analyzed data and drafted the manuscript. All authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
Rights and permissions
Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
About this article
Cite this article
Han, J., Ma, XJ., Wan, JF. et al. Long persistence of EV71 specific nucleotides in respiratory and feces samples of the patients with Hand-Foot-Mouth Disease after recovery. BMC Infect Dis 10, 178 (2010). https://doi.org/10.1186/1471-2334-10-178
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/1471-2334-10-178