Epidemiological And Clinical Characteristics Of Hand, Foot And Mouth Disease In Guangdong Province During2009-2013

Many genotypes of enterovirus (EV), including EV71, coxasckievirus A (2,4,5,7,9,10,16) and coxasckievirus B (1,2,3,4,5) have been reported to be causative pathogen of hand, foot and mouth disease/herpangina (HFMD/HA), of which EV71and CVA16are the two most popular genotypes. Besides of the most commonly observed signs of fever and rash, respiratory infection, hepatic injury, myocardial injury, meningitis, encephalitis, acute flaccid paralysis, etc. are also observed in EV related HFMD/HA. Pathogen monitoring data shows that genotypes of EV that circulate in one region may vary along with periods. Constituents of EV genotype have changed in many countries in recent years, some newly introduced genotypes of EV claimed endemic outbreak of encephalitis or meningitis in some regions. We performed fluorescence real-time RT-PCR for testing EV, EV71, CVA16with rectal swab specimens from all suspected EV-related HFMD/HA patients admitted by Zhujiang Hospital during2009-2013, and genotypes of non-EV71/non-CVA16isolated from inpatients were determined by amplifying partial-lengths of VP1and/or5’-UTR. Collected information along with ascertained EV genotype was to throw light on dynamic change of EV genotype and clinical features of HFMD/HA in Guangdong province during2009-2013. Statistical analysis was performed by SPSS Version13.01(SPSS, Chicago, IL, USA). Categorical variables with minum expected number>5were analyzed by the Pearson chi-squared, those with minum expected number<5analyzed by the Fisher exact test, while continuous variables were analyzed by the independent-samples T-test, and two-sided P<0.05was deemed as statistically different. The research is divided into the following three-parts:I.Epidemiology of acute EV infection and circulated EV genotypes in Guangdong province during2009-2013A total of2484patients with EV related HFMD/HA were admitted by Zhujiang Hospital during2009-2013, of which1014cases were hospitalized patients. The genotypes of non-EV71/non-CVA16enterovirus from321inpatients admitted by Zhujiang hospital during2010-2013were successfully ascertained by sequencing partial lengths of VP1/5’-UTR.Statistical difference was observed patients admitted by Zhujiang hospital during2010-2013in constituents of infected EV71, CVA16and Other EVs (Z2=502.5; P=0.000), suggesting that constituents of EV genotype changed radically in Guangdong Province during2010-2013:percentages of EV71infected patients decreased from48.7%(2010) to7.3%(2013); percentages of CVA16infected patients decreased from21.8%(2010) to8.6%(2013); percentages of other EVs infected patients increased from29.8%(2010) to81.4%(2013). Similar changes were also observed in constituents of genotype of EV strains from HFMD/HA inpatients during2010-2013.A phenomenon of seasonal congregating distribution of EV infected patients was observed during2010-2013with most cases in May to August. Peak epidemic month slightly drifted in different years, and peak epidemic months of EV71, CVA16and other enteroviruses (EVs) were not always identical:peak epidemic months of EV71during2010-2013were May-June, June, May-July and May in order, while those of CVA16were May, no peak month, May and May; and those of other EVs were June, June-July, May and May-July.Most of EV infected patients were under5-year old: patient≤5-year old accounted for93.5%of total cases, patient<5-year old accounted for95.8%(2009),94.6%(2010),95.8%(2011),92.81%(2012) and81.6%(2013), respectively. Children of13m-36maccounted for more than half of the EV infected patients in each year of2009-2013with71.6%(2009),57.0%(2010),58.2%(2011),50.3%(2012) and53.0%(2013).321inpatients with non-EV71/non-CVA16infected HFMD/HA admitted during 2010-2013were successfully ascertained EV genotype, of which45,84,101and91inpatients were from2010,2011,2012,2013, respectively, and the genotypes were6,12,16and6correspondingly. Genotype constituents of non-EV71/non-CVA16from acute EV infected inpatients admitted by Zhujiang Hospital during2010-2013presented the following characteristics:CVA6was the dominant genotype, accounting for68.9%(2010),58.3%(2011),54.5%(2012) and81.1%(2013); the overwhelming majority of cases were coxsackievirus infected cases, which accounted for97.8%(2010),97.6%(2011),86.1%(2012) and100%(2013), respectively; echovirus infection was rare with sporadic cases; the number of echovirus infected inpatients and detected genotypes of echovirus increased significantly in2012with8genotypes and14inpatients, while only1-2genotypes and1-2inpatients found in other years.To clarify changes of complication constituent of EV related HFMD/HA inpatients during2010-2013, data of882HFMD/HA inpatients were summarized and analyzed without incorporating inpatients with congenital heart disease, previous neurological diseases or obvious bacterial infections in illness. The cases each year were as the following:214(2010)>326(2011),204(2012) and138(2013). Severe central neural system involvement (SCNSI), acute respiratory infection (ARI), liver injury, myocardial injury and diarrhea were seen in HFMD/HA inpatient, and a significant change of complication constituent was observed during2010-2013. A decreasing tendency of MCNSI (mild central neural system involvement, CNSI) and SCNSI occurred since2011inEV infected inpatients as follows:the percentages of MCNSI were14.5%(2010),27.6%(2011),14.7%(2012) and10.1%(2013); percentages of SCNSI were16.8%(2010),7.1%(2011),12.7%(2012) and2.2%(2013). A rebound of SCNSI appeared in2012(12.7%of2012vs7.1%). A trend of increasing percentage of MARI (mild acute respiratory tract infection) was seen in inpatients with EV related HFMD/HA during2010-2013:21.0%(2010),16.6%(2011),22.5%(2012) and37.7%(2013). A portion of inpatients suffering SARI (severe acute respiratory tract infection) were seen in each year of2010-2013:16.4%(2010),11.0%(2011),19.6%(2012)and13.8%(2013).During2011-2013, increasing trends of liver injury and myocardial injury were observed in HFMD/HA inpatients since2011,II. Clinical characteristics of HFMD/HA inpatients caused by different EV genotypesTo clarify complications of HFMD/HA caused by different EV genotypes, data of HFMD/HA inpatients were summarized and analyzed without incorporating inpatients with congenital heart disease, previous neurological diseases, obvious bacterial infections in illness and dual EV genotypes co-infection. The inpatients included409cases infected with EV71,169cases with CVA16,207cases with CVA6,19cases with CVA4,41cases with CVA10,17cases with Echo and36with other coxsackievirus. The infection with EV of different genotypes shared the following common clinical features,in which fever and rash were the most common signs, and vesicle was the most frequently observed rash type; ARI were common complications, ranking only second to rash and fever in different genotypes infection except for EV71infection; a small portion of cases presented elevated enzymes associating with myocardial injury or hepatic injury in illness.We compared the occurrence of complications in HFMD/HA inpatients infected by EV of different genotypes. The comparison revealed that the mortality of inpatients infected by EV71or Echo was11.8%and3.2%, respectively, while no death occurred in inpatients with CA (CA4,6,10and16) infections. These results indicated the possible roles of EV71and Echo as the causative genotypes of death. CNSI occurred in inpatients with different genotypes of EV infection. In addition, the occurrences of CNSI varied a lot in different EV genotypes:50.6%(EV71),29.4%(Echo),26.3%(CVA4),12.2%(CVA10),10.1%(CVA6) and2.4%(CVA16). Moreover, the occurrence of SCNSI in inpatients infected by EV71and Echo was high, being20.0%and23.5%, respectively, while its occurrence in those infected by CVA4, CVA10, CVA16or CVA6was low, being5.3%,2.4%,0.6%and0.0%, respectively. Notably, in the total28cases with complicating paralysis,27cases were found in inpatients infected by EV71and the other one in inpatients infected by CVA16. No complication of paralysis was observed in other EV infected inpatients. These results indicated EV71was the dominant genotype of causing paralysis in enterovirus infection in Guangdong province.Occurrence of ARI was observed to be high in inpatients with infections of EV71, Echo, CVA4, CVA10, CVA6and CVA16, with the highest occurrence of63.2%in inpatients with CVA4infection and the lowest occurrence of30.1%in inpatients with EV71infection. The highest percentile of myocardial injury was Echo infection (23.5%), while the lowest was CVA16infection (5.9%). The highest percentile of diarrhea was Echo infection (11.8%) followed by CVA6(3.9%) and EV71(2.4%) infection, no diarrhea was found in CVA4or CVA10infection. Liver injury in EV71, Echo, CVA4, CVA10, CVA6and CVA16infection were only1.8%-6.7%in hospitalized patients, and Echo infection was the highest with6.7%, while CVA16infection was the lowest with1.8%.The characteristics of the27EV71infected cases with paralysis were summarized as following:EV71infection could cause various degrees of paralysis (0-4grade); different segments of spinal cord and cranial nerve could be involved in EV71infection; motor nerve of spinal cord are the major attacked neuron, and sensory neuron could also be inflicted; different location of CNS could be involved in one case; nearly60%cases suffered different degrees of paralysis sequela.Cardiopulmonary failure was the most common cause for13deaths in EV71infection. Neurogenic pulmonary edema, severe pulmonary infection and pneumothorax were precipitating factors for cardiopulmonary failure. MODS induced by sepsis led to two deaths.Additionally, cladogram constructed with full-length of VP1of98EV71strains from patients shows that all EV71strains circulated in Guangdong province in2009-2012are C4a subtype, and amino acid sequence of VP1demonstrates that no significant difference exists between strains from severe/dead cases of HFMD/HA and strains from mild cases, no amino acid substitution of particular position of VP1is found to be related with illness severity. Cladogram constructed with full-length of VP1of35CVA16strains shows that two subtypes (Bla and B1b) co-circulated in Guangdong in2009-2012, and the two subtypes of CVA16circulated in each year of2009-2012in Guangdong province. Ⅲ. Study on special cases of EV related HFMD/HA◆Clinical features of EV related HFMD/HA with acute gastroenteritis. Thirty cases of acute gastroenteritis (AGE) were found out of804EV infected inpatients during2009-2012, accounting for3.73%. of the30cases,26cases were successfully identified infected genotype., of which EV71infections were ten (38.5%) were, CVA6infections seven (26.9%), CVA10infection three (11.5%), CVA (CVA4,9,16,21) and Echo (Echo2,14) each accounted for one infection. Most of the30cases were mild in gastrointestinal related symptom and mild AGE accounted for86.7%. The30AGE cases were characterized by high percentile of diarrhea (93.3%) and short duration of diarrhea (1-4d accounted for78.6%), low percentiles of abdominal pain (6.7%) and severe dehydration (10.0%). Frequent and long-duration of vomiting was not common (vomiting≤2days accounted for83.3%, and vomiting (24h)<4times/day accounted for75%). The HFMD/HA cases with AGE were significantly higher than those without AGE in multiple organ dysfunction syndrome (MODS)(10.0%vs0.40%)(P=0.001), hepatic injury (13.3%vs.3.2%)(P=0.013), myocardial injury (23.3%vs6.1%)(P=0.003) and convulsion (23.3%vs7.1)(P=0.006), but no significant difference was observed between cases with AGE and those without AGE in mortality (6.7%vs1.6%)(P=0.093), altered consciousness (13.3%compared with8.4%)(P=0.316), SCNSI (16.7%over11.4%)(P=0.913), MCNSI (23.3%over20.8%)(x2=0.112, P-0.738), SARI (23.3%vs2.8%)(P=0.000) and MARI (43.3%vs24.7%)(x2=5.974, P=0.015),◆Comparisons of clinical features between cases with exanthema and those without exanthema in acute EV infection. The ninety one cases without rash in illness were found out of804EV infected inpatients during2009-2012, accounting for11.7%, and713cases presented rashes in illness. Cases without rash were significantly higher than those with rashes in death (6.6%VS1.1%)(P=0.001), SCNSI (18.7%vs10.7%)(P=0.024), SCNSI+cardiopulmonary failure (7.7%vs2.8%)(P=0.015), altered consciousness (15.4%vs8.4%)(x2=7.808, P=0.005) and convulsion (14.3%vs6.9%)(x2=6.232, P=0.020). Percentiles of no exanthema differed a lot among different genotypes of EV that circulated in Guangdong province during2009-2012. The percentiles of inpatients without exanthema in different genotypes of EV ranking from high to low were37.5%(Echoviruses),28.0%(other CVs group),13.1%(CVA16group),12.8%(EV71group),6.3%(CVA4group),3.2%(CVA10group) and2.3%(CVA6group).◆CVA16related nephrotic syndrome. An HFMD inpatient infected with CVA16suffering nephrotic syndrome found. This patient presented rash, eyelid edema, hypertension (118/78mmHg), hypoproteinemia (serum albumin11.3g/L) oliguria, proteinuria, hyperlipidemia (TC7.48mmol/L, TG2.76mmol/L, HDLC1.65mmol/L, LDLC5.38mmol/L, LPa2407mg/l, ApoAl2.11g/L, ApoB1.93g/L). Other related examinations showed as HBsAg-C0IU/ml (0-0.05), HBsAb-C324.0IU/ml (0-10), both anti-nuclear protein antibody negative, anti-double DNA antibody negative、 anti-streptolysin negative and rheumatoid factor negative. The all above results are summarized as the follows.●Constituent of EV genotype inducing HFMD/HA changed drastically in Guangdong province during2010-2013,in which EV71and CVA16shifted gradually from dominant genotypes to subordinate genotypes, and correspondingly, CVA6ascended from the subordinate genotype to dominant genotype. A variety of EV genotypes co-circulated in Guangdong province during2010-2013, specifically,8,14,18and8kinds of EV genotypes were identified from HFMD/HA inpatients in2010,2011,2012and2013, respectively.●All EV71strains in Guangdong province during2009-2012are C4a subtype, while two subtypes of CVA16(B1a and B1b) co-circulated in Guangdong province during that period. No significance was observed in sequence of VP1between severe EV71infected cases and mild cases, and no amino acid substitution of VP1was found related with illness severity of HFMD/HA caused by EV71.●Constituent of complications of EV infected HFMD/HA patients changed drastically during2009-2013with CNSI and SCNSI decreasing and ARI increasing since2010, leading to weakening threats of EV related HFMD/HA since2010.●EV71was the major genotype related with SCNSI, death and paralysis in HFMD/HA during2009-2013.●Cardiopulmonary failure was the direct causes leading to death in EV71infection, while severe pulmonary infection or MODS induced by sepsis also attributed to death in EV71infection. EV71infection can cause0-4grades of paralysis, different segments of spinal cord and cranial nerves could be inflicted in infection. Motor nerve is the major inflicted CNS in paralysis, however, sensory nerve can also be involved; most patients with paralysis during illness suffered various degrees of neurological sequela.●Illness of most coxsackievirus-related HFMD/HA are mild with low percentile of CNSI, and respiratory infection dominates complications.●Echovirus related HFMD/HA is characterized by high percentiles of death, SCNSI, myocardial injury and no rash.●Most of eneterovirus related AGE are mild, HFMD/HA cases with AGE are higher than those without AGE in occurrence of MODS, hepatic injury, myocardial injury and convulsion, however, the former are not high in incidences of death, altered consciousness, respiratory tract infection and central nervous system involvement.●The patients without rash could manifest as serious symptoms, even more severe than patient with rashes in mortality, SCNSI+cardiopulmonary failure, altered consciousness and convulsion, which suggests that the EV infected patients without rash should not be ignored.●ACVA16related HFMD with nephrotic syndrome was observed.