A Study On Enterovirus-associated Hand-foot-mouth Disease And Lower Respiratory Infection Disease In Children

Objective To analyse the enterovirus(EV) infection in the children suffering hand-foot-and-mouth disease or lower respiratory tract infection, and to investigate the usability of real-time PCR for clinical diagnosis and treatment especially in the diseases related to EV infection.Methods Children diagnosed of hand-foot-and-mouth disease in the outpatient infection during April to September,2009 were enrolled as the first part of the subjects, and throat swab, herpes fluids and serum of these children were collected. EV genome were detected with real-time PCR using primers such as EV universal, coxsackievirus A16 and EV 71. Anti-Cox A16 and anti-EV71 IgMs in the serum were detected with ELISA. The results were analyzed with a comparison between the two methods. Some positive specimen were selected following with a PCR amplification of VP 1 section on the genome. The products were sequenced, blast aligned and analyzed of sequential evolution after identification with electrophoresis.Children with lower respiratory tract infection in the respiratory care ward during June,2008 to September,2009 were selected as the subjects of the second part of the thesis. Serum and respiratory specimen of these cases were collected. IgM in the serum and EV genome in the respiratory specimen were detected using ELISA or real-time PCR respectively. Both the results were analyzed with some other respiratory virus infection in the cases. Specimen containing EV RNA identified by RT-PCR were amplified according to VP4 region or 5'non-coding region, and the products were sequenced and blast aligned for determination of the serotype of the virus.Results In the 174 cases of hand-foot-mouth disease, EV RNA were detected in 167 cases, in which 112 cases(67.1%) were CA16 positive and 46 cases (27.5%)were EV71 positive, and CA16:EV71 was 2.43:1. In 131 cases with double sera collected, there were 51 cases(38.9%) with CA16 IgM positive and 25 cases(19.1%) with EV71 IgM positive in the early collected sera, and in the later samples,98 cases(74.8%) with CA16 IgM positive and 32 cases(24.4%) with EV71 IgM positive. When compared the results of the early samples with that of the RT-PCR, the consistence is not good with Kappa values of 0.353(CA16) and 0.735(EV71), while according to the results of the later samples there were good consistence with Kappa values of 0.83 and 0.898. The sequencing results showed that the genome extract from the CA16 positive specimen belong to coxsakie B with a homology of 88.7%-98.5%, that from the EV71 positive specimen belong to C4 subtype, with a homology of 96.5%-98.2%.In 402 cases with sera collected, there were 85 cases(21.1%) with EV IgM positive. There were 74 Coxsackievirus-IgM positive and 56 Echo-IgM positive, in which 45 cases were double positive. The EV IgM positive rates were 11.1%-30.3% during the research period. The highest infection occurred in July,2008 and the lowest in Decemberm,2008. And according to ages, there was a higher infection in the school children. In the 85 EV-positive cases,62 cases coinfected with MP, and 10 cases coinfected with common respiratory virus. In the 402 respiratory specimen cases,15 cases were identified positive of EV RNA using RT-PCR, and the positive rate was 3.7%. According to the sequencing results, EV serotype were identified, including 3 cases of EV68,3 cases of ECHO30,2 cases of CVB2, and 1 cases of EV71, CVA3, CVA9, CVB1 and CVB3, in which, EV71, CVA3, CVA9, ECHO30, EV68 and CVB1 were related to pneumonia, and ECHO30, CVB2 and CVB3 were related to bronchitis.Conclusion The mainly pathogen causing hand-foot-and-mouth disease in children in the summer,2009 were CA16 and EV71. EV71 infection, mainly C4 subtype, was highly elevated according to the earlier reported. As to the early diagnosis of hand-foot-mouth disease, Real-time PCR is more appropriate than the serological test. EV-IgM was detected during the whole year and the serotypes that relates to lower respiratory tract infection were coxsackie virus, EV68, EV71 and ECHO30, etc. in the recent years. The diagnosis of EV infection in the respiratory disease needs further study.