Anemia prevalence, severity and determinant factors among children aged 6-71 months in rural Hunan Province, China: a community-based cross-sectional study CURRENT STATUS: UNDER REVIEW

In recent ten years, with the rapid development of children nutrition improvement projects, the previous epidemiological data cannot reflect the actual level of anemia among children in China, especially in rural areas. Therefore, this study analyzed the prevalence, severity and associated determinant factors of anemia among children aged 6-71 months in rural Hunan Province. A community-based cross-sectional study was conducted. Through multistage stratified cluster sampling, 5229 children aged 6 to 71 months and their caregivers were randomly selected from 72 villages across 24 towns in 12 counties from rural Hunan. The demographic characteristics of children and their caregivers, feeding practice, caregivers’ anemia-related feeding knowledge, and gestational conditions of mothers were acquired by using a unified questionnaire. Peripheral blood from the left-hand middle fingertip was sampled from each child, and hemoglobin concentration was measured using a HemoCue301 portable hemoglobin analyzer (Sweden). Associated determinant factors analyses involving overall anemia and anemic severity were conducted on multivariate logistic regression models.


Abstract Background
In recent ten years, with the rapid socioeconomic development and the extensive implementation of children nutrition improvement projects, the previous epidemiological data cannot reflect the actual level of anemia among children in China, especially in rural areas. Therefore, this study analyzed the prevalence, severity and associated determinant factors of anemia among children aged 6-71 months in rural Hunan Province.

Methods
A community-based cross-sectional study was conducted. Through multistage stratified cluster sampling, 5229 children aged 6 to 71 months and their caregivers were randomly selected from 72 villages across 24 towns in 12 counties from rural Hunan. The demographic characteristics of children and their caregivers, feeding practice, caregivers' anemia-related feeding knowledge, and gestational conditions of mothers were acquired by using a unified questionnaire. Peripheral blood from the lefthand middle fingertip was sampled from each child, and hemoglobin concentration was measured using a HemoCue301 portable hemoglobin analyzer (Sweden). Associated determinant factors analyses involving overall anemia and anemic severity were conducted on multivariate logistic regression models.

Results
The overall anemia prevalence was 9.7% (506/5229), and the prevalence of mild, moderate and severe anemia was 6.7% (349/5229), 2.9% (151/5229) and 0.1% (6/5229), respectively. Children age groups of 6-11 months, 12-23 months and 36-47 months, exclusive breast-feeding within 6 months after birth, and maternal moderate/severe anemia were significantly associated with an increased risk of overall anemia in children. Children age groups of 6-11 months and 12-23 months, maternal moderate/severe anemia were significantly associated with an increased risk of mild anemia in children. Children age groups of 6-11 months, 12-23 months and 36-47 months, lower caregivers' anemia-related feeding knowledge level, and maternal moderate/severe anemia were significantly associated with an increased risk of moderate/severe anemia in children. Children who underwent regular physical examination were less likely to have moderate/severe anemia. The common protective factor for overall, mild and moderate/severe anemia in children was high family income.

Conclusions
The anemia status of preschool children in rural Hunan Province was a mild public health problem and associated with children age group, feeding practice, regular physical examination, family income, caregivers' anemia-related feeding knowledge level, and maternal moderate/severe anemia.

Background
Anemia is defined as the below-normal red blood cell count or hemoglobin level per unit volume in peripheral blood. Children anemia is a public health problem worldwide. Data from World Health Organization (WHO) showed that the anemia prevalence among children aged 6-59 months was 42.6% globally in 2011, and maximized in Africa (62.3%) and Southeast Asia (53.8%) [1]. The anemia prevalence of children under 5 years old in China was 12.6% and differed geographically, as it was higher in rural areas (13.3%) than in urban areas (10.3%) [2].
The causes of anemia are multifactorial, including the shortage of hematopoietic materials (e.g. iron, folic acid, vitamin A or B12), infectious diseases (e.g. malaria) and inherited hemoglobin diseases [3].
Anemia epidemiological investigation among children under 5 years old was already conducted in 2010 in China to clarify the anemia prevalence among children at different age groups and in different regions [2]. However, the nutrition status of children has been significantly improved along with the rapid socioeconomic development in China and the extensive implementation of children nutrition improvement projects in rural China in recent ten years [15,16]. Hence, the data from the 2010 epidemiological investigation already cannot reflect the actual level of anemia among children under 5 years old in China, especially in rural areas. Recently, some children anemia status investigations have been conducted in rural China, but the investigated age ranges are narrow and concentrated on children under 2 years old, in particular, relevant data on anemic severity are insufficient [5,7,[17][18][19].
Childhood anemia will adversely affect the health of children, including physical development, and may cause irreversible damages to motion, cognitional and behavioral development [3,20,21]. The age from 0 to 6 years old is the critical period for children's growth and development, and it is also a high age group for anemia. Owing to limitations in socioeconomic conditions and the feeding skills of caregivers, rural children are more susceptible to anemia. Hence, systematical research about the determinant factors on anemia in the whole age range of 0-6 years is critical for the prevention and treatment of children anemia in rural areas. In this study, a community-based cross-sectional study was conducted to clarify the status, severity and associated determinant factors of anemia among children aged 6-71 months in rural Hunan Province, and to propose some appropriate intervention measures for government to further improve the anemia status of rural children.

Materials And Methods Subjects
The subjects were children under 6 years old (aged 6-71 months) and their caregivers from rural Hunan Province between August and November 2019. The sample size was determined according to relevant equations for cross-sectional studies [22]. Since the anemia prevalence among children in rural China was estimated to be 13.3% [2], the size of a test α was 0.05, permissible error d was 0.10, the design effect of complex samples was 2 and the non-response rate was 10%, the final sample size was calculated to be 5500 (= 2500 × 2 × 1.1).
Subjects were selected by multistage stratified cluster sampling. The 14 cities in Hunan were divided by the economic condition into three levels: high, moderate and low. Then 2 cities from each economic level, 2 counties from each selected city, 2 towns from each selected county, and 3 villages from each selected town were randomly selected. From each village, all eligible children and their caregivers were included into our study. Totally, 5229 children from 72 villages across 24 towns in 12 counties were involved. The inclusion criteria for children were as follows: (1) residence in the study area for at least 6 months; (2) no acute or chronic diseases 1 month before the investigation; (3) no drug treatment 1 month before the investigation.

Data Collection
This study consisted of a questionnaire survey and a peripheral hemoglobin detection. The questionnaire covered 3 aspects: (1) demographic characteristics of children (gender, age, ethnicity, birth weight, preterm birth, left behind child), feeding practice (feeding practice with 6 months after birth, nutrient supplements), passive smoking, and regular physical examination; (2) demographic characteristics of caregivers (type, education level, occupation) and knowledge of anemia-related feeding; (3) gestational conditions of the mothers (moderate/severe anemia, pregnancy comorbidity, folate supplementation, iron supplementation). Peripheral blood hemoglobin concentration was detected using a HemoCue301 portable hemoglobin analyzer (Sweden).

Definition of variables
Ethnicity was divided into Han and minority. The birth weight < 2500 g, 2500-3999 g, and ≥ 4000 g were considered as low birth weight, normal birthweight, and macrosomia, respectively. Birth < 37 gestational weeks was regarded as premature birth. Left behind children referred to those children whose mothers or/and fathers left home for work and not live with them.. The feeding practice within 6 months after birth was divided into exclusive breast-feeding, predominant breast-feeding, mixed feeding, and formula feeding. Exclusive breast-feeding meant the infant only fed on breast milk and did not eat any other food or beverage or even water, but can be fed with drugs, vitamins or minerals under medical indications. Predominant breast-feeding meant the infant mainly fed on breast milk, but also took other liquids, including water, juice, dropping liquid and syrup. Mixed feeding meant the infant was fed with both breast milk and other milk and milk replacements. Formula feeding meant the infant was not fed with breast milk, but only with other milk and milk replacements. Children's nutrient supplements included vitamin A, vitamin D, calcium, iron, zinc and DHA, and we investigated whether the child was supplemented one or more of the above nutrient agents in the past one week.
Passive smoking meant a nonsmoker inhaled at least 15 minutes every day the smoke exhaled by smokers for at least 1 day within 1 week. Caregivers were those who took care of the diets, living and personal security for children and were divided into two types: parents, and grandparents/others. The caregivers' education level was classified into primary school or below, junior high school, senior high school, college or above. The occupation of caregivers was divided into housework, government agencies staff, business service staff, farmer, others. The knowledge of anemia-related feeding was acquired according to the following 4 questions concerning anemia prevention in children, including (1) which nutrient deficiency is related to anemia; (2) what is the most suitable food for iron supplement for children; (3) what is the most suitable supplementary food to be first added for infants; (4) whether eating more vegetables and fruits can promote iron absorption. According to their answers to the questions of anemia prevention, the anemia-related feeding knowledge level of caregivers was divided into 3 groups: a high-level group (with all 4 answers correct); a moderate-level group (with 3 answers correct); a low-level group (with 2 or less answers correct).
Maternal moderate/severe anemia was defined as a hemoglobin level < 100 g/L [23]. The maternal hemoglobin concentration in this study was the concentration in the third trimester of pregnancy, which was obtained based on the participants' recall for their hemoglobin detection during pregnancy.
The maternal pregnancy comorbidities included gestational diabetes mellitus, hypertensive disorders complicating pregnancy, cardiac diseases complicating pregnancy, liver diseases complicating pregnancy, and thyroid dysfunction.

Detection of Peripheral blood hemoglobin in children
Peripheral blood was collected from the left-hand middle fingertip of each child by using exclusive reagents (blood spots), and hemoglobin concentration was detected using a HemoCue301 portable hemoglobin analyzer (Hemocue, Inc, Ängelholm, Sweden).

Anemia diagnostic criteria in children
According to WHO criteria, for children aged 6-59 months, anemia is defined at Hb < 110 g/L (100-109, 70-99, and < 70 g/L correspond to mild, moderate and severe anemia, respectively). For children aged 5-11 years old, anemia is defined as Hb < 115 g/L, in which 110-114, 80-109, and < 80 g/L correspond to mild, moderate and severe anemia, respectively [23]. Hemoglobin (Hb) concentrations of children living at different altitudes were calibrated, except at altitude < 1000 m.

Quality Control
The questionnaire survey was completed by child health care doctors, who conducted a face-to-face inquiry survey for the caregivers. Hb concentration was measured by technicians. Prior to the survey, the whole staffs involved were trained unifiedly, and only the qualified ones were allowed to take part in on-site investigations. During the survey, all copies of the questionnaire were checked by quality control personnel: each copy should be filled in in a complete and standard way. Any unlogical or missed response should be corrected in time. Data were double-inputted on Epidata 3.1(Jens M.Lauritsen, Michael Bruus and Mark Myatt, Odense, Denmark) and tested in terms of consistency.
For any inconsistent data, the original copy should be checked to ensure the high accuracy of any inputted data.

Statistical analysis
Statistical analyses were conducted on SPSS 19.0 (IBM, Chicago, IL, USA). The overall anemia prevalence among children with different characteristics was compared using chi-square test. The anemia severities among children with different characteristics were compared via nonparametric rank sum test. The significant variables identified from univariate analyses were involved into multivariate logistic regression models of overall anemia and anemia severities. The independent variables were selected by stepwise regression (forward: LR). The strength of association between determinant factors and anemia was evaluated by using odds ratios (ORs) with 95% confidence interval (CI). All statistical tests were two-tailed, and the significant level was P < 0.05.

Ethics Approval And Consent To Participate
The study protocol was approved by the Ethics Committee of Hunan Provincial Maternal and Child Health Care Hospital (No.2019-S036). The study was conducted in accordance with the Declaration of Helsinki. Written informed consents were obtained from all the caregivers of children involved in this study.

Characteristics of children
A total of 5500 children and their caregivers were investigated. After excluding 271 children for incomplete core information (e.g. sex, age, hemoglobin concentration), and the final sample size for analysis was 5229. As shown in Table 1, of the 5229 children investigated, 50.5% were boys and 49.5% were girls. The major age groups of children were 36-47 and 48-59 months, which accounted for 21.3% and 22.0% respectively. Most of the children were in Han ethnic group (89.2%). The proportions of low birth weight, preterm birth, and left-behind children were 3.6%, 4.8% and 56.8% respectively. The proportion of exclusive breast-feeding within 6 months after birth was 53.5%; and the proportion of nutrient supplements in the past one week was 47.8%. 44.8% of the children were exposed to passive smoking, and 88.7% of the children underwent regular physical examinations. The proportion of family annual income ≤ 30000 RMB was 61.5%.

Characteristics Of Caregivers
As shown in Table 2, the caregivers were mostly parents (65.6%). Most of the caregivers had junior middle school education (38.5%), but the proportion of primary school or below was as high as 21.0%.

Prevalence And Severity Of Anemia In Children
The overall anemia prevalence was 9.7% (506/5229), and the prevalence of mild, moderate and severe anemia was 6.7% (349/5229), 2.9% (151/5229) and 0.1% (6/5229), respectively. The overall anemia prevalence in age group of 6-11 months was highest among all age groups (24.5%), and the prevalence of mild and moderate/severe anemia was 16.6% and 7.9%, respectively. Of the children whose mothers suffered from moderate/severe anemia during pregnancy, the anemia prevalence was 15.3%, and the prevalence of mild and moderate/severe anemia was 9.6% and 5.6%, respectively.
Univariate analyses showed that the overall anemia was associated with 8 factors, including children age group, feeding method within 6 months after birth, nutrient supplementation, family income, education level of caregivers, caregivers' anemia-related feeding knowledge level, maternal moderate/severe anemia during pregnancy, and pregnancy comorbidities ( Table 4). The anemia severities of children were associated with 8 factors, including children age group, ethnicity, nutrient supplements, regular physical examination, family income, education level of caregivers, caregivers' anemia-related feeding knowledge level, and maternal moderate/severe during pregnancy. Determinant factors of overall anemia and anemia severities in children Table 5 shows the determinant factors of overall anemia and anemia severities among children in multivariate logistic regression analyses. Children age groups of 6-11 months, 12-23 months and 36-47 months, exclusive breast-feeding within 6 months after birth, and maternal moderate/severe anemia were significantly associated with an increased risk of overall anemia in children. Children age groups of 6-11 months and 12-23 months, maternal moderate/severe anemia were significantly associated with an increased risk of mild anemia in children. Children age groups of 6-11 months, 12-23 months and 36-47 months, lower caregivers' anemia-related feeding knowledge level, and maternal moderate/severe anemia were significantly associated with an increased risk of moderate/severe anemia in children. Children who underwent regular physical examination were less likely to have moderate/severe anemia. The common protective factor for overall, mild and moderate/severe anemia in children was high family income.

Discussion
Anemia status of children The finding of this study showed that the overall anemia prevalence among children aged 6-71 months in rural Hunan Province was 9.7%, which was significantly lower than the preschool children in Yingyangbao contributed to a low risk for childhood anemia [28,29].
In our study, the anemia severity was mainly mild, followed by moderate, which was in agreement with similar studies conducted in Ethiopia [30] and Haiti [31]. There were two possible reasons for the fact. On the one hand, children with mild anemia were mostly asymptomatic and thus did not attract enough attention from their caregivers, so the caregivers might not seek medical intervention and did not give the children prompt and effective treatment [30]. On the other hand, childhood anemia was nutritional anemia in most cases and was dominated by mild iron-deficiency anemia.

Determinant Factors On Anemia
Age of children Many of the published observational studies have confirmed that child age is a key determinant factor on anemia [11,13,25,30,32,33]. An epidemiological investigation in Iran reported that the older age of children was a protective factor for childhood anemia, and the risk of anemia decreased by 12% with the rise of 1 month of age [32]. A cross-sectional study conducted by Endidaye in Ethiopia found that the risks of anemia in age groups of 6-11 months and 12-23 months were 5.67 and 5.80 times of the age group of 48-59 months, respectively [33]. The association of child age with anemia in our study was consistent with the above researches, found that as the child getting older, the overall anemia prevalence decreased from 24.5% (in the age group of 6-11 months) to 6.2% (in the age group of 60-71 months) which meant that children at younger age were the most vulnerable group for anemia. The likelihood of being anemic for the children in the age group of 6-11, 12-23 and 36-47 months was 5.11, 1.92 and 1.64 times of the age group of 60-71 months. Like the overall anemia prevalence, age group was also associated with the anemia severity, as younger age was a risk factor of both mild anemia and moderate/severe anemia. Since the age group of 6-23 months was the key period for complementary food introduction and eating habits nurturance, inappropriate complementary food introduction or poor eating habits in this age group would easily cause insufficient intake of iron nutrient and led to iron-deficiency anemia [7,14,30]. Moreover, in China, when children reached 36 months of age, they could go to kindergartens for collective life, but some kindergartens provided low-quality nutritious diets that did not meet the demands of children for normal growth and development and consequently, the risks of anemia at the age group of 36-47 months was increased [34].

Feeding Practice
As reported, feeding practice was closely associated with childhood anemia, and breast-feeding was at increased risk of childhood anemia compared to formula feeding [10,17,35,36]. An observational study conducted in China showed that compared with formula feeding, both exclusive breast-feeding and mixed feeding increased the risks of iron deficiency and iron-deficiency anemia among infants aged 9 months [35]. A cross-sectional study conducted in South Korea also found that infants aged 8-15 months fed only with or mainly with breast milk might be more prone to iron deficiency and irondeficient anemia [36]. Our study showed that exclusive breast-feeding within 6 months after birth increased the risk of anemia by 40% compared with formula feeding, but we did not find any association between predominant breast-feeding or mixed feeding with anemia in children. The possible explanation was that the iron stored in infants' body would be depleted within 4 months after birth, and the iron concentration in breast milk was low, and despite the high bioavailability, the iron in breast milk did not meet the demand for growth and development by infants. A cohort study involving 270 infants (followed up from birth to 6-8 months after birth) from Bolivia confirmed this viewpoint that the hemoglobin and serum ferritin concentrations significantly dropped with the prolonged time of exclusive breast-feeding [10].
Exclusive breast-feeding as the recommended feeding practice for infants within 6 months after birth by WHO was indisputably beneficial to infants and young children, and was outstanding with complete nutrition composition and immunodominance, that was incomparable by other ways of feeding.

Hence, 2012 Children's Nutritional Disease Management Technical Specification issued by China
Ministry of Health definitely provided that term infants depending on exclusive or predominant breastfeeding should be supplemented with iron since the 4th month after birth, and the daily intake should be 1 mg/kg elemental iron, which would prevent the occurrence of iron-deficient anemia.

Maternal Anemia
Many of the published papers have confirmed that maternal anemia can induce inadequate iron store in infants and is more likely to cause iron-deficient anemia in infants after birth [9,33,37]. A cohort study from India found the hemoglobin and serum ferritin levels in infants born to anemic mothers were significantly lower than those born to non-anemic mothers both at birth and 14 weeks after birth [37]. An observational study in Southern Africa reported that maternal anemia raised the risk of anemia in children aged 6-59 months by 52%-71% [9]. In the present study, maternal moderate/severe anemia was an independent risk factor of childhood anemia and was significantly associated with the anemia severity. Compared with children born to mothers without moderate/severe anemia, the risks of mild anemia and moderate/severe anemia in children born to mothers with moderate/severe anemia were increased by 56% and 117%, respectively, and the overall anemia risk rose by 76%. Hence, prenatal care and gestational nutrition of mothers and prompt addressing of maternal anemia are significant for anemic prevention in children.

Preterm Birth And Low Birth Weight
Like maternal anemia during pregnancy, preterm birth and low birth weight also can lead to inadequate fetal iron store, which makes preterm infants and low-birth-weight infants more susceptible to anemia. A cross-sectional study involving 1127 infants aged 6 months showed the anemia prevalence of preterm infants was significantly higher than that of term infants (38.5% vs. 10.2%) [5]. An observational study conducted in Switzerland reported that low birth weight raised the risk of anemia in infants aged 6-23 months by 16% [8]. Our study found no association between preterm birth or low birth weight with childhood anemia. This might be due the fact that the preterm infants or low-birth-weight infants in China were routinely supplemented with iron (2 mg/kg/day) since the 4-th week after birth until 1 year old, which efficiently compensated for the inadequate iron store due to preterm birth or low birth weight and thereby prevented iron-deficient anemia in preterm infants and low-birth-weight infants.

Regular Physical Examination And Caregivers' Anemia-related Feeding Knowledge
This study found out that regular physical examination was a protective factor for moderate/severe anemia in children, but found no association between regular physical examination and mild anemia in children. The possible reason for this association might be that children with moderate/severe anemia had obvious clinical symptoms and were more likely to pursue medical interventions and thus would be effectively treated in time. In addition, our study found that the lower caregivers' anemiarelated feeding knowledge level was a risk factor of moderate/severe anemia in children, which was consistent with a study conducted in Indonesia [38]. As reported, maternal knowledge of anemia promoted the development of some health behaviors related to anemia reduction (e.g. rational diets, regular physical examination, preventive iron agent supplementation), which would decrease the occurrence of anemia in children [38]. In the present study, the caregivers' anemia-related feeding knowledge level largely decided the diet quality of children, while the diet quality was closely related with anemia in children. Two observational studies from Ethiopia also showed that poor diet would greatly increase the risk of childhood anemia [11,39].

Family Income
High family income was also identified as a protective factor for childhood anemia, which was supported by previous studies [7,11,13]. Families with high incomes have the ability to purchase and provide good nutritious foods, so the dietary patterns for their children are more reasonable, which decreases the occurrence of anemia.

Limitations
This study has several limitations. First, owing to the inherent characteristics of cross-sectional studies, the relationship between investigated factors and anemia identified in our study was statistical association, rather than causality. Second, the data collection about maternal anemia status and feeding practices within 6 months after birth were both based on the recalling of past event by the caregivers, which unavoidably led to recall bias. Third, anemia diagnosis was based on hemoglobin levels alone, but no etiological diagnosis, which limited the classification of subtypes of anemia. Finally, certain important factors were unavailable in our study, such as complementary food introduction, dietary patterns, parasitic infections, and genetic hemoglobin disorders. Therefore, these factors were not included in the final analysis. Nevertheless, this large-size epidemiological survey involved 5229 children from 72 villages across 24 towns in 12 counties of Hunan Province, and the investigated ages covered the whole preschool age range. Hence, our findings reflected the current anemia status and determinant factors of preschool children in rural Hunan and would help health administrative departments to take measures to reduce the anemia-induced burdens in rural areas.

Conclusions
The anemia status of preschool children in rural Hunan Province was a mild public health problem and associated with children age group, feeding practice, regular physical examination, family income, caregivers' anemia-related feeding knowledge level, and maternal moderate/severe anemia. The key population for childhood anemia prevention and control in rural areas is children aged 6-23 months.
Exclusive breast-feeding should be encouraged for infants within 6 months after birth, but attention should be paid to preventive iron agent supplementation. While strengthen the health education for caregivers' scientific feeding knowledge, the prevention and treatment of maternal anemia during pregnancy needs to be further improved.
Abbreviations WHO: World Health Organization; Hb: Hemoglobin; OR: Odds Ratio; CI: Confidence Interval.

Declarations
Ethics approval and consent to participate