EV71 isolate TW/2086/98 was amplified and purified as an antigen for use in µ-capture ELISA. In comparison with standard method of cWith the conventional virus culture, as a standard method, the ssensitivity and specificity of thisfor µ-capture ELISA were 91.5% and 93.1%, respectively.20
Statistical Analysiss
The Ddata was were analyzed with the SAS Sstatistical Ppackage SAS system (Vversion 8.2, SAS Institute, Cary, North Carolina). We used the Sstudent’s t test for continuous variables; and used χ 2 tests for categorical data. Univariate analysis was done to screened for statistically significant variables. Then, aS step-wise multiple logistic regression analysis was performed to adjust confounders simultaneously and to calculate the multivariate-adjusted odds ratios for risk factors of EV71 infection and an unfavorable outcome. The α level of model selection was set to beat 0.15 for in-and-out models. A P p-value value will be considered significant if it was less than 0.05 was considered significant.
46
RESULTS
Demogr aphy and Iinfection Rrates
A total 173 families (including 343 children and 441 adults) were surveyed from April 2001 to August 2002; EV71 was isolated from 54% (94/173) families,Of these 173 families, 94 (54%) no viruses were isolated from 50/173families had positive EV71 isolation, 50 (29%), and did not have positive virus isolation, non-71 enteroviruses were isolated from 29/173 (17%) had positive non-71 enterovirus isolation. The 94 families with positive for EV71 isolation were further analyzed (Fig.
1).
Table 1 shows the demographic data and the rates for isolating EV71, detecting y, the positive EV71 isolation, positive EV71 IgM and demonstrating seropositive neutralizing antibodiesy seropositive rates among index cases, siblings, cousins, parents, grandparents, uncles/aunts and babysitters. The Ooverall infection rate was 52% (176/339) after EV71 was had been introduced into the family by the first cases.
Excluding the index cases, the infection rate in child household contacts was 84%
(89/106), and the infection rate in adult household contacts was 37% (87/233) (p<0.001). with χ2 test) The 60% (109/183) In addition, EV71 isolation rate (60%, 109/183) of from infected children was also significantly higher than the 11% (10/87) EV71isolation that rate (11%, 10/87) offrom infected adults (p<0.001) with χ2 test).
EV71 infection rates declined as age increased (Table 2). Furthermore, 100%
(71/71) of children younger than 2 years of age gotwere infected with EV71
infections. Among children, there was no significant differences of in the infection rate existed between siblings and cousins. Of Among adults, parents had a higher infection ratethe infected rate of parents (41%, 72/175) was higher than that other adults (26%, 15/58) of other adults (p=0.05). The Infectioninfected rate for mothers (43%, 40/92) of the mothers was similar to that thatrate for fathers (39%, 32/83) of the fathers (p=0.61). The EV71 seropositive rates of for all the family members were as high up asto 93% (401/429) during the convalescence.
2.Factor s Aassociated with EV71 Iinfection in Cchildren
Table 3 shows factors associated with EV71 infection in children. Male sex and age less than 6 years of age were associated with increased risk of EV71 infection.
Children attending kindergarten or school had a lower incidence of EV71 infection.
Larger family size and more children in the family did not significantly increase the risk of infection. Stepwise multiple logistic regression analysis indicated that the most significant factors associated with infection in children were age less than 6 years (adjusted OR=9.11, 95% confidence interval=2.90-28.65, p=0.0002) and male gender (adjusted OR=4.11, 95% confidence interval=1.19-14.15, p=0.025).
47
3.Sources of Ttr ansmission for to Pthe pr imar y Ccases in the family Source of transmission was identified inOnly 47% (44/94) of the primary primary infection cases in childrenchildren cases had clear contact history of identified source of EV71 infection, which is shown in(Fig. 1). Among the 44 families in which the source of the original EV71 infection was identified, 40%
(46/114) of adults became infected. Among these 46 infected adults, 20 became symptomatic and developed symptoms after their children had become ill. Children with primary infections transmitted EV71 to 79% (34/43) of the other children in their families.
Among the 53% (50/94) 50 families in which the original source of EV71
transmission was not identified, adults in 28% (26/94) with EV71-infected children of unknown source of infection, at least 26 (28%) families had adults with had
asymptomatic EV71 infections. in their families. At least two individuals became ill on the same day in 19% (18/94) families. In these cases, an asymptomatic
EV71-infected adult family member could have been the source of transmission to the children. In addition, there were 18 families with at least two cases who became ill on the same day, so it indicated presence of asymptomatic EV71-infected family member to transmit the virus.
Among the 44 families whose first EV71-infected children had known source of transmission, 40% (46/114) adults in the families also got EV71 infection. Among these 46 infected adults, 20 had symptoms and all their disease developed later than their first EV71-infected children’s illness. The first EV71-infected children also transmitted EV71 to 79% (34/43) of the other children in their families.
Therefore, theH household secondary attack infection rates was were estimated to be 79% (34/43) infor the children and 40% (46/114) infor the adults (Fig. 1).
TheI intervals between primary case and secondary cases in the family ranged from 0 to 15 days., the Mmedian interval was 3 days and the mean interval was 3.7 (SD 2.6) days.
There were 18 families with at least two cases who became ill on the same day, and all these 18 families did not have the identified source of EV71 infection.
4.Clinical Ssyndromes and Ooutcomes of EV71 Infected children and adults.
The Cclinical syndromes and outcomes of for all the infected children and adults are shown in Table 4. EV71-infectedC children had significantly higher rates than adults of for complications, long-term sequelae and fatalitiesy than adults.
Of 183 infections ined children, 6% (11/183) were asymptomatic, and 73%
(133/183) were uncomplicated. Complications such as meningitis, encephalitis, polio-like syndrome and cardiopulmonary failure occurred inand 21% (39/183)had complications such as meningitis, encephalitis, polio-like syndrome or
48
cardiopulmonary failure;. 7% (Thirteen of these13/183) had suffered from long-term sequelae of limbthat included muscular weakness/atrophy, dys-swallowing
dysfunction, cranial nerve palsiesy or central hypoventilation. and 5%Five percent ( (10/183) died.
Table 5 shows factors associated with an unfavorable outcome in infected children based on univariate analysis. Age was the most significant factor. Secondary cases, contact history with HFMD/HA, larger family size and more children in the family were not associated with a significantly higher rate of an unfavorable outcome.
Children in kindergarten and school had lower rates of an unfavorable outcome.
Stepwise multiple logistic regression analysis indicated that the most significant factor associated with an unfavorable outcome in infected children was age less than 3 years (adjusted OR=6.19, 95% confidence interval=1.77-21.6, p=0.0044).
Of the 87 EV71-infected adults, 53% (46/87) were asymptomatic., All symptomatic adults recovered completely from and the other had uncomplicated illnesses that included such as HFMD/HA, herpangina, febrile illnessfever, upper respiratory tract infection or and viral exanthema.
;
all recovered.
Table 5 shows factors associated with unfavorable outcome in infected children in univariate analysis. Age was the most significant factor associated with clinical outcome of children. The secondary children cases in the family, contact history with HFMD/HA, larger family size and more children in the family did not have significantly higher rate of unfavorable outcome than the primary children cases.
Kindergarten or school children had lower rate of unfavorable outcome possibly due to older age.
After stepwise multiple logistic regression analysis, the most significant factors associated with unfavorable outcome in infected children was younger than 3 years of age (adjusted OR=6.19, 95% confidence interval=1.77-21.6, p=0.0044).
49
COMMENT
New York Virus Watch data showed that secondary coxsackievirus infections were more frequent in mothers (78%) than in fathers (47%).16However, we observed similar incidence of EV71 infection between mothers (43%, 40/92) and fathers (39%, 40/92). The EV71 infection rates of parents (41%, 72/175) were higher than other adults (26%, 15/58), and this indicated that closer contact or longer contact made higher incidence of EV71 transmission.
In this prospective family cohort study, we found that EV71E infections in young children are associated with serious diseases; we also found the high ratio of asymptomatic adults and high household transmission rate, which make controlling transmission of EV71 infections difficult. Long periods of viral shedding may account for widespread transmission of enteroviral diseases. This is certainly the case for polio and coxsackievirus infections.The greater spread of polio and coxsackievirus may derive from longer periods of viral shedding.14 However, the period of EV71 viral shedding has not been well defined. In a previous Our previous study, we found that EV71 cases might shed the viruswas present in the the stool of infected patients for up to 5 weeks.21The other, Previous research has demonstrated a higherwhich could not be stopped by hand washing only. Our previous study showed that higher rate of EV71 isolation rate from throat swabs than the rate from rectal swabs or or stool: 90%
vs. 32%, respectively.22 We speculated that aerosol transmission would explain the high secondary infection rate in Taiwan, despite hand washing precautions in practice since 1998.23Consequently, Based on the results of the high EV71 isolation rate from throat swab and the high household secondary attack rate, we speculate that main transmission route during the acute EV71 infection may be aerosol transmission and isolation of EV71 patients may be mandatory necessary to prevent the spreadaerosol transmission.
New York Virus Watch data indicate that secondary coxsackievirus infections are more frequent in mothers (78%) than fathers (47%).15 However, we found similar secondary EV71 infection rates in both mothers and fathers: 43% and 39%,
respectively (Table 1). EV71 infection rates for parents (41%, 72/175) were higher than for other adults (26%, 15/58), suggesting that close or longer contact facilitated EV71 transmission.
As Figure 1 shows, it is likely that sequence of transmission was not only child-to-adult, but also adult-to-child. Because EV71 seropositive rates for all family members were as high as 93% during convalescence (Table 1), it is likely that almost all the susceptible family members were infected once EV71 had been introduced.
The high infectivity of EV71 is similar to that of poliovirus.14The aerosol
transmission may also explain the reason why EV71 still causes lots of cases after
50
hand washing has been highly advocated since 1998 enterovirus epidemic in Taiwan.23
Among children, Intrafamilial transmission produced a higher rate of clinical symptoms (93%) compared to extra-familial transmission (29%) in our previous EV71 seroepidemiological studyin children cause significantly higher rate of clinical symptoms (93%) in comparison to social transmission (29%) in our previous study.11 The difference in illness development may be due to Vhigher viralus load, of
intrafamilial infection, host genetic factors or virus virulence may account for this difference. Because the rate of asymptomatic infection with EV71 after social contact Since the asymptomatic ratio is high (about 71%),11 it was difficult to identify the source of primary infections. We were successful in only 47% of the cases. after social contact with EV71, the infection source of the first symptomatic case in the family is difficult to find and we could only identify 47% source of infection in this study. Such observation also indicated that asymptomatic EV71 cases may also transmit the virus to other people without caution and this also makes prevention of EV71 infection more difficult.
EV71 infections in adults were less serious than those in children. Adults could be infected by EV71 and their symptoms and clinical outcomes were significantly different from those of children. Although there were only two reported fatal adult EV71 cases in the literature,2,14Although EV71 infection is a differential is still a consideration for in cases of adult encephalitis, unexplained pulmonary edema or cardiopulmonary failure and has been reported in the deaths of two adults,2,17 most infected adults are asymptomatic or suffer from symptoms of a mild upper respiratory tract infection. However, most of infected adults did not have typical syndrome, hand, foot, and mouth disease. Consequently, EV71 transmission by infected adults who are asymptomatic or mildly symptomatic Therefore, infected adults may transmit the virus into the community without warning sign.is the likely source of many infections.
In those cases (50/94) in which the primary source of infection could not be identified, we estimate that up to 28% (26/94) of EV71 infections were introduced into the
family by an asymptomatic adult (Fig. 1).
Although Tthe first symptomatic cases in a family in the family almost werewas usually in a child, and mchildren and most of the symptomatic parents developed symptoms later than their offspringchildren. In these cases, it is likely that EV71 was These observations suggested that EV71 might be introduced into the family by children and secondarily transmitted to adults. Therefore, either way transmission is possible.
In conclusion, EV71 infections in children, especially those less
51
than 3 years old, are associated with serious complications, long-term sequelae and death. HThe EV71 seropositive rates of all the family members were high up to 93% during the convalescence, and it indicated that almost all the susceptible in the family were infected once EV71 was introduced into the family. The striking high infectivity of EV71 was just similar to that of poliovirus.15 igh infectivity, the absence of effective antiviral drugs and the high ratio of Regarding the high asymptomatic adults in theratio of general population and high household transmission rate, make it will be very difficult to controlling transmission of the EV71 infection infections difficult. At this time, developing a v by using antiviral drugs or hand-washing measurement, not to mention there is no effective antiviral drug against EV71. Vaccine development may be the onlyappears to be the most effective way toapproach to control the transmission of the EV71 infections.
52
Author Contr ibutions: Study concept and design: Chang, Lin.
Acquisition of data: Chang, Tsao, Hsia, Shih, CG Huang, Fang, YC Huang.
Analysis and interpretation of data: Chang, Tsao, Hsia, Lin Drafting of the manuscript: Chang, Lin.
Critical revision of the manuscript for important intellectual content: Chang, Tsao, Hsia, Shih, CG Huang, Fang, YC Huang, Lin.
Statistical expertise: Chang.
Obtained funding: Chang, Lin.
Administrative, technical, or material support: Tsao, Shih, Huang, Lin Study supervision: Lin.
Funding/Suppor t: In conclusion, we found high household transmission rate of EV71 and there was either child-to-adult or adult-to-child transmission. About one half of EV71-infected adults did not have symptoms and infected children tended to have severe disease than adults. This study was supported by grants from the Chang Gung Memorial Hospital (CMRP1089) and National Science Council (NSC 90-2314-B-002-463 and NSC 91-3112-B-002-029).
53
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2. Shindarov LM, Chumakov MP, Voroshilova MK, et al. Epidemiological, clinical and pathomorphological characteristics of epidemic poliomyelitis-like disease caused by enterovirus 71. J Hyg Epidemiol Microbiol Immunol 1979;23:284-95.
3. Nagy G, Takatsy S, Kukan E, Mihaly I, Domok I. Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS diseases in Hungary in 1978. Arch Virol 1982;71:217-27.
4. Chan LG, Parashar UD, Lye MS, et al. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: Clinical and pathological characteristics of the disease. Clin Infect Dis 2000;31:678-83.
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natural immunity to poliomyelitis in Louisiana. II. Description and analysis of episodes of infection observed in study group households. Am J Hyg
1957;65:367-85.
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continuing surveillance of viral infections in metropolitan New York families. VII.
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1969;89:51-61.
16. Morens DM, Pallansch MA, Moore M. Polioviruses and other enteroviruses. In:
Belshe RB, ed. Textbook of human virology, 2nd ed. St. Louis, MO: Mosby Yearbook; 1991:427-97.
17. Chan KP, Goh KT, Chong CY, Teo ES, Lau G, Ling AE. Epidemic Hand, Foot and Mouth Disease Caused by Human Enterovirus 71, Singapore. Emerg Infec Dis 2003;9:78-85.
18. Grandien M, Fosgren M, Ehrnst A. Enterovirus. In: Lennette EH, Lennette DA, Lennette ET, eds. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 7th ed. Washington, DC: American Public Health Association;
1995:279-98.
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Table 1. Demographicy Data and Rates forIsolating the positive rates ofEV71 , Detecting EV71 EV71 isolation,IgM, Demonstrating Seropositivity for Neutralizing Antibodies yand Infectionamong family members
Subjects Number Age Sex
(Male/ Female)
Positive EV71 Iisolation Rrate
Positive EV71 IgM Rrate
EV71 Nneutralizing Sseropositive Rrate during Convalescence*
Infectioned Rrate†
Children
Index Ccases 94 2.5±2.0 61/33 87% (82/94) 95% (88/93) 98% (91/93) 100% (94/94)
Siblings 81 5.1±3.5 39/42 21% (17/81) 77% (59/77) 96% (76/79) 84% (68/81)
Cousins 25 4.8±6.6 16/9 40% (10/25) 71% (17/24) 92% (22/24) 84% (21/25)
Adults
Parents 175 31.5±5.4 83/92 5% (9/175) 17% (29/175) 91% (159/175) 41% (72/175)
Grandparents 36 59.3±10.4 14/22 0% (0/36) 15% (5/34) 94% (34/36) 28% (10/36)
Uncles/Aunts
19 27.4±8.7 10/9 5% (1/19) 16% (3/19) 84% (16/19) 26% (5/39)
Babysitters 3 36.6±7.7 0/3 0% (0/3) 0% (0/3) 100% (3/3) 0% (0/3)
* EV71 neutralizing seropositive rate during the convalescence was defined as an EV71 neutralizing antibody serotiter equal or over 8 four weeks after illness onset. † EV71 infection was defined as either positve positive EV71 isolation, a four-fold changes change inof EV71 neutralizing antibody serotiters, or positive presence of EV71 IgM.
56
Table 2. EV71 Infection Ratesby in Different Age Groups
Age Ggroup (Yin ears)
EV71 Iinfection Rrates Adjusted Oodds Rratio*
95% CI PValue
Age≦6 96% (159/165) 1.0 --
--6<age≦18 72% (26/36) 0.10 0.033-0.30 <0.001
18<age≦40 39% (71/181) 0.025 0.010-0.059 <0.001
Age>40 27% (14/51) 0.014 0.005-0.040 <0.001
*Adjusted odds ratio was calculated using the age <=6 group as the reference group and was adjusted by sex.
CI denotes confidence interval.
57
Table 3. Factors Associated with EV71 Infection in Children
Factor Infected
(N=183)
Notn I-infected (N=17)
P Value
Male/Female Rratio 1.57 (112/71) 0.31 (4/13) 0.003
Mean Aage 3.3±2.4 10.0±7.6 0.002
Age≦6 Years 86% (158/183) 35% (6/17) 0.001
Family Ssize≧6 52% (95/183) 53% (9/17) 0.94
Number of Children number≧3
45% (82/183) 59% (10/17) 0.27
Kindergarten or Sschool Aattendance
34% (62/183) 71% (12/17) 0.006
58
Table 4. Clinical Syndromes and Outcomes of 183 EV71-infected Children and 87 EV71-infected Adults
Syndromes / Outcomes Number of Children
No. (%) (N=183)
Number of Adults No.
(%) (N=87)
P Value
Syndr omes <0.001*
Asymptomatic 11 (6%) 46 (53%)
Uncomplicated Ccases 133 (73%) 41 (47%)
HFMD 90 (49%) 7 (8%) Herpangina 19 (10%) 8 (9%) Nonspecific Ffebrile Iillness 4 (2%) 1 (1%) Upper Rrespiratory Ttract Iinfection 16 (9%) 18 (21%) Enteritis 2 (1%) 2 (2%) Viral Eexanthema 2 (1%) 5 (6%)
Complicated Ccases 39 (21%) 0
HFMD plus Mmeningitis 9 (5%) 0 HFMD plus Eencephalitis 11 (6%) 0 HFMD plus Ppolio-Llike Ssyndrome 5 (3%) 0 HFMD plus Eencephalomyelitis and
Ccardiopulmonary Ffailure
14 (8%) 0
Outcomes 0.003†
Complete Recovery 160 (87%) 87 (100%)
With Ssequelae 13 (7%) 0
Death 10 (5%) 0
HFMD denotes hand, foot, and mouth disease.
59
* p-V value was measured with χ2 test in to compareison of the percentages of asymptomatic cases, uncomplicated cases and complicated cases between between infected children and infected adults.
†p-V value was measured with χ2 test in to compareison of the percentages of recovery, sequelae and death between
†p-V value was measured with χ2 test in to compareison of the percentages of recovery, sequelae and death between