Clinical Nutrition 31 (2012) 543e548

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Clinical Nutrition
journal homepage: http://www.elsevier.com/locate/clnu

Original article

Influence of vitamin A status on the antiviral immunity of children with hand,

foot and mouth disease
Siyuan Chen a, d, Yi Yang c, d, Xiufeng Yan b, Jiande Chen c, Hui Yu b, Weiping Wang a, *
a
Department of Pediatric Healthcare, Children’s Hospital of Fudan University, Shanghai, PR China
b
Department of Infectious Diseases, Children’s Hospital of Fudan University, Shanghai, PR China
c
Pediatrics Institute, Children’s Hospital of Fudan University, Shanghai, PR China

a r t i c l e i n f o s u m m a r y

Article history: Background & aims: Vitamin A (VA) deficiency has been shown to affect antiviral immunity and thus may
Received 21 September 2011 be related to the progress and outcome of hand, foot and mouth disease (HFMD) in young children. Our
Accepted 9 December 2011 objective was to determine whether children with HFMD associated with VA insufficiency displayed
a decline in antiviral immunity.
Keywords: Methods: 450 children with HFMD and 113 non-infected children were included in this study. Dietary
Vitamin A
investigations were performed using a 24-h dietary questionnaire. The serum concentrations of VA were
Hand
measured by high-performance liquid chromatography. The serum levels of interferon-a (IFN-a) and
Foot and mouth disease
Antiviral immunity
enterovirus 71 (EV71) IgM antibodies were detected using an enzyme-linked immunosorbent assay
Children (ELISA).
Results: The mean serum VA concentration for all patients was 0.73
0.26 mmol/L, and 237 (52.7%) of
them presented low concentrations (0.7 mmol/L). Both serum concentrations of VA and IFN-a in the
patients with complications were significantly lower than in patients without complications (P < 0.01).
The decreased concentrations of IFN-a and EV71-IgM were positively related to lower VA levels
(correlation coefficient ¼ 0.58 and 0.41, respectively, P < 0.001).
Conclusions: Most of the children with HFMD presented VA insufficiency, which was associated with their
reduced immunity and more severe illness.
Ó 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction maintenance of mucous membrane health and adequate immune

Vitamin A (VA) deficiency has already been recognized as a risk
factor for some viral infectious diseases, such as measles, in young
children. Since 1987, the WHO have recommended VA treatment of
children with measles.1 Hand, foot, and mouth disease (HFMD) is
a common viral infection that mostly affects children under four
years of age, with typical pathological damage in the skin and
mucous membranes.2 Notably, VA plays important roles in the
Abbreviations: DCs, dendritic cells; DRIs, dietary reference intakes; EP Tube,
Eppendorf microetest tube; EV71, enterovirus 71; HFMD, hand, foot and mouth
disease; IFN-a, interferonea; RAE, retinol activity equivalents; VA, vitamin A.
* Corresponding author. Department of Pediatric Healthcare, Children’s Hospital
of Fudan University, 399 Wanyuan Road, Shanghai 201102, PR China. Tel./fax: þ86
21 64931883.
E-mail addresses: jijiyuanyuan@163.com (S. Chen), yyang@shmu.edu.cn
(Y. Yang), vivia_yan@hotmail.com (X. Yan), 09211240001@fudan.edu.cn (J. Chen),
yuhui20@yahoo.com (H. Yu), wpwang@fudan.edu.cn (W. Wang).
d
These two authors contributed equally to this work.
function, and VA insufficiency is most commonly found in young
children from developing countries, including China.3e5 Thus,
whether the VA status affects the susceptibility and progress of
HFMD in these young children is worth studying.
Several reports, including our previous studies, have revealed
the widespread influence of VA and its metabolites on the devel-
opment and function of the immune system in children,6 including
its effects on T- and B-cells,7,8 antigen-presenting cells,9 dendritic
cells (DCs),10 and other immune system components or structures.
However, few studies have examined the effect of VA on the anti-
viral immunity of HFMD infection in children. Therefore, in this
study, the dietary intake and serum concentrations of VA from
children with HFMD were evaluated. The serum level of interferon-
alpha (an important innate antiviral cytokine) and the production
of the antiviral antibody for Entervirus 71 (a main pathogen that
causes HFMD) were measured to determine whether the VA status
of HFMD subjects was associated with poor immune function and/
or illness severity.

0261-5614/$ e see front matter Ó 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
doi:10.1016/j.clnu.2011.12.005

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544 S. Chen et al. / Clinical Nutrition 31 (2012) 543e548
2. Materials & methods
This cross-sectional, observational study was conducted at the
Children’s Hospital of Fudan University between October 2010 and
June 2011. In total, 450 patients with median (interquartile range)
age of 25 (17e37) months were included in the study. All patients
were diagnosed with HFMD according to the standardized clinical
case definition published by the Ministry of Health of People’s
Republic of China.11 A case was defined as a patient who had clinical
symptoms of HFMD, typically vesicles on the hand or foot and oral
lesions, with or without fever. Patients with HFMD should be
hospitalized if there is evidence of central nervous system
involvement (i.e., dispirited, drowsiness, headache, vomiting, irri-
tability, convulsion and so on). The severity of the condition of
patients with HFMD was classified according to the associated
complications, including encephalitis, meningitis, meningoen-
cephalitis, and so on. For comparison, a gender- and age- matched
control group (n ¼ 113) was recruited from among non-infected
children. This study was approved by the Ethics Committee of the
Children’s Hospital of Fudan University. Parents were informed
about the study, and written informed consent was obtained.
2.1. Sample collection and handling
Blood samples were obtained from the patients on admission
and from non-infected children during healthcare visits to the
Children’s Hospital of Fudan University. Venous blood samples
were taken by a registered nursing using a vacutainer needle and
delivered into aluminum foil-wrapped tubes. To separate the
serum, the blood sample was centrifuged at 3000 rpm for 3 min.
Then, the serum was aliquoted in marked Eppendorf test tubes and
frozen at 70  C until assays to determine the VA and IFN-a levels
were performed. Stool specimens were collected from patients at
the time of hospitalization. Clinical data were collected by
reviewing hospital records.
2.2. Dietary investigation
Daily food intake was investigated using a 24-h dietary ques-
tionnaire, and the VA intake was enumerated in retinol activity
equivalents per day (mg RAE/day). The VA intake of patients with
HFMD was compared with Chinese Dietary Reference Intake (DRI),
which is an age-based measure.12 Dietary intake and food
consumption data were analyzed by a dietician using the Nutrient
Elements Calculator V1.6, based on the China food composition
tables and developed by the Institute of Food Safety and Nutrition
at China’s Center for Disease Control and Prevention.
2.3. Serum vitamin A levels
VA levels of the serum samples were analyzed by high-
performance liquid chromatography according to the method
proposed by Driskell13 with some modifications, using a Waters
2487 dual lambda absorbance detector. VA was detected at 315 nm.
Serum was pipetted into an Eppendorf Micro Test Tube (EP Tube).
Ethanol was added to the serum, and then the tubes were vortexed
for 60 s. Hexane was added, and the mixture was vortexed for 120 s
(the tube was bounced as it was being vortexed to ensure thorough
mixing of the two layers). Then the tubes were centrifuged for
10 min. A 500-mL aliquot of the top layer fluid was transferred to
another EP tube. These tubes were placed in a room-temperature
water bath, and the hexane was evaporated with a stream of
nitrogen. The residue was redissolved in 100 mL of methanol and
vortexed for 10 s. Then, 20 mL of the solution was injected into the
chromatography system.
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2.4. Serum IFN-a concentration
The expression of IFN-a protein was determined using a human
IFN-a ELISA kit (eBioscience, USA) according to the manufacturer’s
instructions. This involved adding 60 mL of dilution buffer and 40 mL
of serum from each sample to microplate wells. Next, 50 mL of
horseradish peroxidase-conjugated antibodies were added to all
wells. Microplate strips were then covered and incubated for 2 h at
room temperature (18e25  C). After washing 3 times with washing
buffer,100 mL of tetramethylbenzidine substrate solution were added
to all wells, which were then incubated at room temperature for
10 min. Then, 100 mL of stop solution was added to each well. The
optical density was detected at a wavelength of 450 nm on a micro-
plate reader (Wellscan MK3, Labsystems, Finland). A standard curve
ranging from 7.8 to 500 pg/ml was constructed, using serial dilutions
of a human IFN-a standard provided with the kit. Samples with
values greater than the negative control but <7.8 pg/ml were
assigned values based on extrapolation of the standard curve.
2.5. Detection of human enterovirus 71
The diagnosis of human enterovirus 71 was performed by
fluorescence-based quantitative real-time PCR (qRT-PCR). Viral RNA
was extracted from the stool specimens using an RNA Extraction Kit
(DAAN Gene Co., Ltd., China), according to the manufacturer’s
instructions. The LightCycler RNA amplification hybridization probe
kit (DAAN Gene) was used in this study. The test kit allows for one-
step qRT-PCR to be performed using the LightCycler instrument (MJ
Research, USA). The reaction mix in the hybridization probe kit
contains a mixture of hybridization probe, primer and dNTP mix.
Each reaction was performed in a reaction capillary by mixing the
reagents, followed by spinning the mixture down briefly with the
help of a centrifuge (Eppendorf, Germany). Each reaction contained
5 mL of RNA, 15 mL of reaction mix, 2 mL of reverse transcriptase and
3 mL of Taq enzyme. After reverse transcription at 40  C (25 min) and
initial denaturation at 94  C (3 min), amplification was performed in
40 cycles at 93  C for 15 s and 55  C for 45 s.
2.6. Detection of IgM anti-EV71
EV71-IgM antibodies were detected using a human enterovirus
71-type IgM antibody ELISA kit (IBL, Germany). Briefly, 40 mL of
dilution buffer and 10 mL of serum samples from each sample were
added to microplate wells, and then 50 mL of horseradish
peroxidase-conjugated antibodies was added and mixed gently,
then incubated at 37  C for 60 min. After washing 5 times with
washing buffer, 50 mL of chromogen solution A and chromogen
solution B, respectively, were added to each well. Samples were
then incubated at 37  C for 15 min. Stop solution (50 mL) was then
added to each well, and optical density was measured at a wave-
length of 450 nm within 15 min. A standard curve ranging from 0 to
80 ng/ml was constructed.
2.7. Statistical analysis
Statistical operations were performed with the SPSS Statistical
Package, version 13.0. Descriptive statistics were presented as
mean
standard deviation (SD) and a median (interquartile range)
for normally and non-normally distributed data, respectively.
Student’s t-test and ANOVA were used for continuous variables
with normal distributions, when appropriate. The ManneWhitney
U test was used for continuous variables without a normal distri-
bution, whereas the Chi-squared test was used for the analysis of
categorical data. Pearson correlation coefficients or Spearman rank
correlations were performed to test for significant associations
 S. Chen et al. / Clinical Nutrition 31 (2012) 543e548 545

Table 1 the serum VA concentration was lower in the group of 1w<4 years
The vitamin A intake of HFMD patients in different age groups. of age than in any other group, but the difference did not reach
Number of Dietary reference Vitamin A intake DRIsa % statistical significance. Moreover, the subjects with complications
patients intakes (DRIs) (mg RAE/day) had a lower serum concentration of VA than that presented in
<1 year 21 400 290.5
114.5 72.6% patients without complications (Table 2).
1 w <4 year 229 500 297.1
126.1 59.4%
4 w <7 year 48 600 375.8
172.5 62.6%
7 year 2 700 349.4
65.0 49.9% 3.4. Serum concentrations of IFN-a
a
Vitamin A intake/Dietary reference intakes  100%.
Of the 450 children with HFMD, no measurements of the
concentration of IFN-a were possible in 34 due to insufficient
between variables as appropriate. A probability value less than 0.05 serum samples. Therefore, a total of 416 (283 male and 133 female;
was considered to be significant. median age, 25 months) patients were included in the analysis of
both VA and IFN-a. The patients were divided in two groups
according to the level of serum VA: VA  0.7 mmol/L (n ¼ 218) and
3. Results
VA gt; 0.7 mmol/L (n ¼ 198); 113 non-infected children (74 male and
39 female, median age was 24 months) served as the control group.
3.1. Subject characteristics
Fig. 1 illustrates the serum concentration of IFN-a among these
three groups. The results show that the production of IFN-a in the
Of the 450 patients in the present analysis, 303 (67.3%) were
patients with HFMD was significantly increased compared with the
male, and 147 (32.7%) were female. The median patient age at
non-infected children. Furthermore, the subjects who were diag-
disease onset was 25 (17e37) months; 375 (83.3%) patients were
nosed with HFMD and had VA gt; 0.7 mmol/L presented higher
younger than 4 years of age at the time of this study. All 450
median serum concentrations of IFN-a than the other two groups.
patients were hospitalized in the infectious disease ward of the
Furthermore, the IFN-a concentration was lower in the patients
Children’s Hospital of Fudan University. The body weights of these
with complications [67.1 (46.6e109.0) pg/ml] than in the patients
HFMD patients were within the normal range, and none of them
without complications [87.7 (52.3e130.7) pg/ml, P ¼ 0.004]. More
had apparent malnutrition. The mean
SD serum VA concentra-
importantly, as shown in Fig. 2A, the serum concentration of IFN-
tion for all patients was 0.73
0.26 mmol/L, and 237 (52.7%) of them
a was positively associated with the VA concentration (r ¼ 0.58,
presented serum VA concentrations  0.7 mmol/L. No patients had
n ¼ 416, P < 0.001).
xerophthalmia. None of the patients had received VA therapy. The
mean duration of hospitalization for patients with VA  0.7 mmol/L
was longer than that for patients with VA gt; 0.7 mmol/L (3.51
1.33 3.5. Serum concentrations of EV71-IgM antibodies
days vs. 3.23
1.18 days, P ¼ 0.019). In total, we found 226 (50.2%)
stool specimens that yielded positive EV71 test results and 170 In total, we found 226 (50.2%) patients with EV71 antibody
(37.8%) patients who had complications; none had died at the time expression. Levels of serum EV71-IgM antibodies were significantly
of the study. lower in the patients with VA insufficiency (28.1
9.4 ng/ml;
n ¼ 154) than in the patients without insufficiency (32.9
11.8 ng/
ml; n ¼ 72), P ¼ 0.004. Similar to the serum concentration of IFN-a,
3.2. Dietary vitamin A intake
the EV71-IgM antibodies were positively associated with VA
concentration (r ¼ 0.41, n ¼ 226, P < 0.001) (Fig. 2B).
Of the 450 patients, 300 (206 male and 94 female, median age
was 25.5 months) were able to complete the dietary question-
3.6. The incidence rate of complications
naires. The mean
SD intake of VA from food (n ¼ 300) was
309.6
136.2 mg RAE/day, whereas the median (interquartile
According to the level of serum VA, the 450 HFMD patients were
range) intake was 285.0 (220.3e388.2) mg RAE/day. There was no
divided into two groups: VA  0.7 mmol/L (n ¼ 237) and VA gt;
significant difference in the intake of VA from food between boys
0.7 mmol/L (n ¼ 213). As an indicator of illness severity, the inci-
and girls (P ¼ 0.89), but intake was greater in the group of patients
dence of complications was compared between the two groups
with VA gt; 0.7 mmol/L [307.4 (244.4e411.3) mg RAE/day, n ¼ 122]
using a Chi-squared test. In total, we found 170 patients who had
than it was in the patients with VA  0.7 mmol/L [274.1
(197.0e366.4) mg RAE/day, n ¼ 178], P ¼ 0.001. These 300 patients
were divided to 4 groups by age, and the VA intakes are presented Table 2
in Table 1. The 24-h recall analyses showed deficient intakes in all 4 The serum vitamin A concentrations in patients with HFMD.
groups when compared with the DRIs; almost 92% (277/300) of n Vitamin A concentrations Pa
these patients failed to meet 100% of the DRIs for VA. Another (mmol/L)
observation in this questionnaire was that the deficient intake Gender
seems to be more serious in the 1w<4-year age group. Further- Male 303 0.73
0.26 0.79
more, dietary VA intake was positively related to the serum Female 147 0.74
0.26
concentration of VA (correlation coefficient ¼ 0.27, P < 0.001). Age
<1 year 29 0.80
0.33 0.1
1w<4 year 346 0.72
0.26
3.3. Serum concentrations of vitamin A 4w<7 year 70 0.77
0.26
7 year 5 0.90
0.11
Duration of hospitalization
The mean
SD serum VA concentration for all patients
3 day 274 0.76
0.26 0.006
(n ¼ 450) was 0.73
0.26 mmol/L. In total, 237 (52.7%) of all patients >3 day 176 0.69
0.26
had values indicative of VA insufficiency (0.7 mmol/L). The VA Complications
concentrations did not differ significantly by either gender Yes 170 0.66
0.23 < 0.001
(P ¼ 0.79) or age (P ¼ 0.1). No association was found between serum No 280 0.78
0.27

VA and age (P ¼ 0.22). Similar to the trend line of dietary VA intake, a

Student’s t test or ANOVA.

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546 S. Chen et al. / Clinical Nutrition 31 (2012) 543e548

Table 3
The clinical complications of HFMD children in different VA levels.

Type of clinical Number of VA  0.7 mmol/L VA > 0.7 mmol/L Pc

complications patients (n ¼ 237) n (%) (n ¼ 213) n (%)
Encephalitis 10 6 (2.5) 4 (1.9) 0.88
Meningitis 71 50 (21.1) 21 (9.9) 0.001
Meningoencephalitis 73 43 (18.1) 30 (14.1) 0.24
Encephalomyelitis 1 0 (0.0) 1 (0.5) e
Pulmonary edema 6 4 (1.7) 2 (0.9) 0.78
Liver damagea 2 2 (0.8) 0 (0.0) e
b
Cardiac damage 7 4 (1.7) 3 (1.4) 1.0
Total 170 109 (46.0) 61 (28.6) <
0.001
a
A case who had serum alanine transarninase (ALT) level > 40 IU/L was regard as
liver damage.
b
A case who had serum CK-MB level >25 IU/L and/or serum cardiac troponin I
(cTnI) level >1 ng/ml was regard as cardiac damage.
c
Comparison of the prevalence between VA  0.7 mmol/L and VA > 0.7 mmol/L
groups (chi-square test).

average level of normal children aged 0w5 years in China was

Fig. 1. The serum concentrations of IFN-a in different groups. Box plots show the
median (horizontal line in the center of each box) and 25th and 75th percentiles
(bottom and top of each box). The HFMD VA gt; 0.7 mmol/L group had a higher median
serum concentration of IFN-a [112.3 (78.4e155.5) pg/ml, n ¼ 198] than the other two
groups (P < 0.01). The median serum IFN-a concentration was higher in the HFMD
VA  0.7 mmol/L group [57.3 (39.9e85.4) pg/ml, n ¼ 218] than in the control group
[35.7 (26.0e42.8) pg/ml, n ¼ 113] (P < 0.01). Data were analyzed by ManneWhitney U
test.
clinical complications. The type and frequency of complications
were shown in Table 3. In the VA  0.7 mmol/L group, there were
109 patients who had complications, and in the VA gt; 0.7 mmol/L
group, there were 61 patients who had complications. These data
indicate that the patients with low levels of VA were more likely to
have complications (46% vs. 28.6%, P < 0.001).
4. Discussion
VA is an essential micronutrient with established roles in
embryogenesis, growth, reproduction, maintenance of epithelial
integrity, and optimal function of the immune system.14 The last
two functions are of particular concern when considering the
potential impact of VA deficiency superimposed with HFMD
infection. It is generally agreed that a serum VA concentration less
than 0.7 mmol/L is considered as VA deficiency.15 A 2006 study
showed that the incidence of VA deficiency in a cohort of Chinese
children aged 0e6 years was 12.2%.16 Another study found the
1.06
0.33 mmol/L.17 In this investigation, the prevalence of VA
insufficiency was 52.7% in the 450 patients with HFMD, which is
remarkably high. Furthermore, the mean serum VA concentration
was below the value reported by the literature.
The etiology of the depressed serum VA concentrations in the
HFMD patient has not been identified. Data from some studies
suggest that serum VA concentrations decrease transiently during
the acute-phase response to infection.18e21 The mechanisms are
unclear, but low VA levels may be due to decreased intake,
increased consumption, or increased catabolism. Reduced hepatic
synthesis, secretion of a retinoleretinol binding protein complex
and increased urinary excretion of VA have also been reported.22 In
the current study, the result of dietary investigation shows that 92%
of the patients with HFMD failed to meet 100% of the DRIs for VA.
There is a positive correlation between the intake of VA and the
serum VA, which suggests that the low intake of VA could be
a contributor to VA insufficiency in the HFMD patients. However,
because the children with HFMD were sick and probably for several
days prior to the questionnaire their intake was limited due to
illness, the 24-h dietary questionnaire may not provide a reliable
estimate of these children’s usual intake. Therefore, the etiology of
the depressed serum VA concentrations during HFMD is unclear
based on the present study. Further studies are needed to establish
the significant of this phenomenon.
HFMD is a common illness in young children that is caused by
viruses belonging to the enterovirus genus of the picornavirus

Fig. 2. The correlation between serum vitamin A and other variables for children with HFMD. The concentrations of both IFN-a and EV71-IgM were positively associated with VA
(correlation coefficient ¼ 0.58 and 0.41, respectively, P < 0.001). Data were analyzed by Spearman rank coefficients (A) or Pearson correlation (B).

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 S. Chen et al. / Clinical Nutrition 31 (2012) 543e548
family. In the current study of 450 patients in total, 226 (50.2%)
patients provided stool specimens that were positive for Entero-
virus 71 (EV71), which demonstrated that the main pathogen of
HFMD was EV71. EV71 is also the most common etiological agent
isolated from HFMD patients with neurological complications. As
vitamin A deficiency has been known to increase susceptibility to
infection-especially by damaging the mucosa-and to raise the
incidence of infectious respiratory and alimentary tract diseases,23
it becomes much more interesting to explore the relationship
between VA stature and antiviral immune factors.
The human antiviral immune response comprises two branches:
innate and acquired immunity. The innate immune system is the
first line of host defense against pathogens and is the major
contributor to acute inflammation induced by microbial infection.
Acquired immunity is involved in the elimination of pathogens in
the late phase of infection.24 Recent studies have suggested that
EV71 infection is detected by cellular foreign nucleic acid sensors
that initiate innate antiviral responses, including the activation of
type I interferon (IFN) and proinflammatory cytokines.25 Type I IFN
(mainly IFN-a and IFN-b) plays a critical role in the first-line defense
against viral infection. The early induction and action of type I IFN
result in cellular resistance to viral infection as well as the inhibition
of viral replication and viral dissemination.26 Notably, the findings
of this study indicate that the serum IFN-a level was markedly
reduced and positively related with the lack of VA in patients with
HFMD. The result suggests that low VA level plays an important role
in the insufficient production of IFN-a in patients with HFMD.
Our previous study demonstrated that VA can enhance immu-
noglobulin production and promote the humoral immune
response.7,8,10 The results of the current study suggest that the
production of EV71-specific IgM antibody was significantly
decreased in the HFMD patients with VA insufficiency as compared
with those without VA insufficiency, and the serum EV71-specific
IgM antibodies were positively associated with the serum
concentrations of VA. Therefore, the VA deficiency could compro-
mise not only innate immunity but also adaptive immunity in
young patients with HFMD.
Previous studies have demonstrated that immature or impaired
immunity in individuals infected with EV71 may be associated with
increased morbidity and mortality.27,28 In this study, the incidence
of complications was higher and the median duration of hospital-
ization was longer in patients with VA  0.7 mmol/L. In addition, the
patients with complications had lower serum concentrations of
both VA and IFN-a than those without complications. Therefore, we
hypothesize that HFMD might lower serum VA concentrations,
which in turn exacerbates the pathogenetic condition, possibly by
depressing antiviral immune responses. However, the exact
mechanism requires further elucidation.
Certain limitations should be noted when interpreting the
results of this study. First, the results are based on cross-sectional
data, and the sample size is relatively small; therefore, its explan-
atory power is limited. Furthermore, clinical trials of vitamin A
therapy in children with HFMD could determine whether patients
would benefit from such an intervention.
In conclusion, our results showed that VA status is associated
with the antiviral immunity and pathogenetic condition of HFMD
in young children. The children with HFMD mostly presented low
VA concentrations and simultaneously had lower serum IFN-
a levels, decreased immune antibody production and more severe
illness. To our knowledge, this is the first study to investigate the
influence of VA status on antiviral immunity in young patients with
HFMD. These findings encourage further work to evaluate the
potential benefits of VA supplementation in the prevention and
treatment of HFMD. In addition, further studies are clearly needed
to determine the reasons for VA insufficiency in HFMD patients so
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547
that appropriate interventions can be implemented to improve the
VA status of individuals with HFMD.
Conflict of interest
The first two authors contributed equally to this work. All
authors have no financial and personal relationships with other
people or organizations or any other conflicts of interest that could
inappropriately influence this study.
Statement of authorship
Y.Y., and W.W. designed the research; S.C., J.C., X.Y., and H.Y.
conducted the research; S.C. analyzed the data and wrote the
manuscript; Y.Y. and W.W. revised the manuscript. W.W. had
primary responsibility for final content. All authors read and
approved the final manuscript.
Acknowledgments
This study was not sponsored by any outside source.
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