Epidemiologic Study Of Hand,Foot And Mouth Disease Caused By CA16Infection In Guangzhou From2009to2011and The Analysis Of The Kinetics Of CA16-IgM Antibodies

Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus. It was noticed that coxsackievirus A16(CA16) and enterovirus71(EV71) were two major etiological agents of hand, foot and mouth disease in children. Both viruses belong to the Enterovirus genus of the Picornaviridae family and possess a single-stranded positive-sense RNA genome.The diameter of the virus is about24-30nm.The genome is translated as a single large polyprotein that is composed of four capsid proteins, VP1to VP4. We pay more attention to EV71infection, because it leads to serious complications and even to death. The causes of death are mainly for severe neuropathy, such as brain stem encephalitis and aseptic meningitis. EV71infection is prone to occur in children below3years. Due to EV71and CA16turn to become dominant strain to cause epidemic and be coinfected in outbreak of HFMD, CA16has become increasingly concerned about.Large outbreaks of the disease have occurred frequently in the Asia-Pacific region in recent years, HFMD is gradually becoming a potential danger to global public health disease. Rapid and effective diagnosis is very important to prevent the spread of the disease and reduce the death. Methods of diagnosis of enterovirus infection include virus isolation, nucleic acid detection and serological testing. This study is to analysis the clinical sample if children infected with HFMD and the HFMD caused by CA16infection in Zhujiang hospital during the years2009to2011. The methods of the detection include fluorescence PCR, virus isolation and neutralization test. We analyzed the epidemic trends and gene evolution of CA16by constructing of phylogenetic tree based on the CA16VP1fragments. According to the clinical information of patients with hand, foot and mouth in the years from2009to2011, we analyzed the myocardial enzyme spectrum by different enterovirus infection; In addition, we analyzed the kenetic of IgM antibodies in CA16infection and cross-reactivity of CA16-IgM ELISA by detection the serum of HFMD patient with IgM capture ELISA. The research is to reveal epidemic trend and gene evolution of CA16through etiology and serology data, hoping to benefit monitoring CA16prevalence status and contributing to controlling its outbreak.This study is divided into two chapters:First chapter Epidemiologic study of hand, foot and mouth disease caused by CA16infection in Guangzhou from2009to2011Clinical date and clinical specimens were collected from1239patients diagnosed as HFMD by Clinical diagnosis in Zhujiang hospital from2009to2011, including525outpatients and714inpatients. There were813male and426female. The mean age was about35months.Clinical date includes age, gender, data of onset, clinical symptoms, cardiac marker, time of specimens colletction. All the specimens included1239rectal swabs and497serums.All the rectal swabs were detected by Fluorescent PCR, some of which were detected by isolation and then were serotyped by Fluorescent PCR.13CA16isolations and clinical swab specimens for VP1gene amplification, sequencing, were followed by analysis of the phylogenetic tree. All the497acute and convalescent sera were carried out a panel of myocardial enzyme spectrum test, including AST, CK, CK-MB, LDH and HBDH.The age of the HFMD patients diagnosed by clinical from2009to2011were between1month and35years old. The results suggest that various age groups were at risk of infection of hand-foot-mouth disease. Most of the patients with HFMD were under5years old. The peak of the onset age was from1to3years, and is less than1year. Below1year old children with maternal antibodies existing, so they are not easy to be infected. Because of the immature immune system and free of the maternal antibodies, children under5years old become the high-risk population of hand foot and mouth disease.From2009to2011, the gender ration of male to female in HFMD patient were2.34:1,1.87:1and1.88:1respectively. The gender ration of male to female in HFMD patient are2.34:1,1.87:1and1.88:1respectively, of which the gender ration in CA16infection are2.75:1,1.49:land2.57:1respectively. The result above showed that infection rate of male is higher than female. Maybe male is more activer than female so increasing the chance to contact with the goods of enterovirus infection.The positive rate detected by real-time fluorescent PCR from2009to2011were87.85%(94/107),91.32%(505/553) and88.95%(515/579) respectively. The percentage of HFMD caused by CA16in total HFMD caused by enterovirus are43.69%(45/94),19.80%(122/505) and20.00%(107/515) respectively. The results showed that CA16was one of the dominant pathogenic serotype during the HFMD epidemic in Guangzhou, especially in the year2009. It had a downward trend in recent2years.April, May and June were the high incidence period of HFMD in Guangzhou2009. The HFMD epidemic in2010began in March, the high incidence of which was in the month April to July and the peak in November. The months from May to August were the high incidence period of HFMD and the peak period was in June and November2011. The prevalence of HFMD caused by CA16is similar to the total HFMD epidemic in the years2009,2010. The HFMD caused by CA16in2011began in May, the high incidence of which was in the month April to July and the peak in November. In general the trend of CA16epidemic is dropped somewhat during the3years. There were49severe cases in2010, among which2patient infected with CA16, accounting for4.08%. Among48severe cases in2011,2patient infected with CA16, accounting for2.08%. The result suggested that the symptom of HFMD caused by CA16was mild generally, rarely server.58clinical samples from CA16-positive patient detected by real-time fluorescent PCR in2009and2010were used to culture and isolate virus. The results showed that the virus isolation rate of CA16infected patients is44.83%. By3generations of blind pass, the supernatant of the cell which had cytopathic effect was detected by real-time fluorescent PCR. The results were consistent with the detection of real-time fluorescent PCR before isolation. The clinical specimens were further confirmed as CA16. We could find that the sensitivity of virus isolation is low.All the497serums were detected serum myocardial enzyme spectrum. We divided the serums into two parts:acute serum (0-7days), early convalescent serum (7-14days). And also, the enterovirus was divided into three parts:EV71infected group, CA16infected group and the other enterovirus infected group. The abnormal rate of this three groups was26.60%,44.19%and45.54%respectively。After statistical analysis, we discovered that the abnormal rate of acute serums was different in those three groups, and the rate of the CA16infected group was higher than the EV71infected group. CA16may have a higher tropism to myocardial cells than EV71and may cause myocardial damage more easily. In contrast with this phenomenon, EV71infection is more likely to result in severe central nervous system disease. The cardiac index CK and CK-MB of CA16infection were all different in acute and early recovery stage by statistical analysis, while EV71infection not. The results showed myocardial injury caused by CA16infection could be recovery somewhat after a week. CA16infection is prone to damage cardiac cells while the recovery of patient infected CA16was faster than EV71.All the13CA16strains in Guangdong2010were B1genotype divided into two different lineages on the basis of phylogenetic analysis. Four isolates belonged to subgenogroup B1b and other nine strains grouped to B1a, which suggested that these isolates may be originated from different ancestral source although they were isolated from the same region. Additionally, these isolates had high homology with isolates from Shenzhen in2009, but lower homology with strains before2005. This phenomenon suggested that the newly isolated strains after2009in Guangdong region may had the same origin, which was different from the strains from Shenzhen before2005. The isolates had lower homology with the strains in Taiwan. It suggested that they may have the different origin.Second chapter kinetics of IgM antibodies in CA16infectionHFMD patient diagnosis by laboratory in Southern Medical University Zhujiang Hospital from March2009to December2010, were studied, of which156were male,74were female. Age of the230patient was from4months to169months and the median age was26months. All patients had been laboratory diagnosis as being infected with HEV71, CA16and other enterovirus infections by detected rectal swabs with real-time PCR or combination of virus isolation, some patients confirmed by neutralization test. A total of434acute-and convalescent-phase serum specimens were collected between days1and158after the onset of symptoms from these230HFMD patients. In addition, we collected105serums from75patients with acute respiratory infections as controls. All these patients had been laboratory-confirmed previously as being infected respiratory virus. All the serums were detected by IgM-capture ELISA.To measure the kinetics of CA16-IgM, the serums of CA16infected patients were tested by CA16-IgM ELISA. The IgM positive rate reached25%in the serum collected within the first day after onset, increased continuously to37%,67%and86%on day two, three and five.The100%positive rate reached at day8. Twenty weeks after onset, IgM detection rate decreased rapidly. Only one of four CA16sera taken after90days of disease was weakly positive for CA16-IgM (S/Co=1.09).To measure the specificity of CA16-IgM capture ELISA, serum from EV71infected patients, other enterovirus infected patients and respiratory virus infected patients were tested. CA16-IgM was apparently positive in58of211EV71,16of48other enterovirus and3of105respiratory virus infected serum.For further analyse the cross-reactivity of the assay, a total of119sera from66CA16infected patients were tested for both HEV71-and CA16-IgM simultaneously. For the CA16patients, CA16-IgM was detected in only83of119samples while cross-reactivity towards EV71-IgM was seen in30.3%. While of the36EV71-IgM positive sera, the ratio of OD450value for CA16-IgM divided by that for EV71-IgM greater than1.0can successfully identify33(91.7%) CA16infection.We collected date of the serums and swabs which was taken on the same day to compare the correlation between real-time PCR and CA16-IgM capture ELISA. For CA16, the difference between ELISA and real-time PCR was not statistically, while the measure agreement was low (Kappa value=0.276, P=0.000).Generally, the CA16-IgM capture ELISA for detection of foot and mouth disease caused by CA16infection is an alternative method with its advantage of convenience and low cost.Summarization:According to the results of two chapters above, we summarize as follows:1. The percentage of CA16infection in total enterovirus from2009to2011were43.69%(45/94),19.80%(122/505) and20.00%(107/515) respectively. The results showed that HFMD caused by CA16epidemic in Guangzhou has a downward trend in recent2years.2. The prevalence of HFMD caused by CA16is similar to the total HFMD epidemic in the years2009and2010. The HFMD caused by CA16in2011began in May, the high incidence of which was in the month July to December and the peak in November. The number of the male is more than the female in both the HFMD patients and the HFMD patients caused by CA16during the3years. Most of the patients with HFMD were under5years old. Patients below5years accounted for92.11%and96.35%in all patients with HFMD and HFMD caused by CA16respectively.3. Level of ardiac marker of CA16infected patients was higher than that of EV71infected patients. CK and CK-MB of CA16infected patients with acute phase (0-7days) and early convalescent phase (8-14days) were all different. This result showed CA16could have a higher tropism to cardiac cells than EV71while the recovery of patient infected CA16was faster than EV71. 4.13CA16virus gene were amplified and sequenced based on the CA16VP1fragments. Phylogenetic tree results suggest the genotype was B1. We inferred the2010Guangzhou CA16epidemic was caused by genotype B1.5. The CA16-IgM antibody can be detected within the first day after onset, lasted several weeks. The100%positive rate reached at day8. Twenty weeks after onset, IgM detection rate decreased rapidly. Only one of four CA16sera taken after90days of disease was weakly positive for CA16-IgM (S/Co=1.09).6. As genome and amino acid homology between enterovirus, there was cross-reactivity of CA16-IgM ELISA. To measure the specificity of CA16-IgM capture ELISA, serum from EV71infected patients, other enterovirus infected patients and respiratory virus infected patients were tested. The positive rate was27.48%(58/211),33.33%(16/48) and2.86%(3/105) respectively. The ratio of OD450value for CA16-IgM divided by that for EV71-IgM greater than1.0can successfully identify91.7%(33/36) CA16infection.7. We detected the serums and swabs which were taken on the same day to compare the correlation between real-time PCR and CA16-IgM capture ELISA. For CA16, the difference between ELISA and real-time PCR was not statistically, while the measure agreement was low (Kappa value=0.300). In short, CA16-IgM capture ELISA can be used in large epidemic of HFMD and is suitable to primary hospital.