Burden of Anemia in Relation to Thalassemia and Iron Deficiency among Vietnamese Pregnant Women

Anemia is a serious public health problem, especially in developing countries. A number of factors might contribute to the condition but generally it is assumed that iron deficiency (ID) is the major cause [1,2]. However, ID in the absence of anemia can exist if a negative iron balance does not persist long enough. The more severe stage of ID-associated anemia is called ID anemia (IDA). In pregnant women, ID may result in a number of adverse outcomes for both mothers and infants, including an increased risk of maternal morbidity, perinatal mortality, and a low birth weight of the infant [2].

Other factors that might affect the synthesis of red blood cells or hemoglobin include deficiencies of micronutrients such as vitamin A, vitamin B₁₂, and folate, as well as infectious diseases, especially malaria and hookworm infestation [1]. Other causes of anemia are linked to genetic diseases, such as thalassemia and hemoglobinopathies, and are highly endemic in certain areas of Southeast Asia. Individuals who carry thalassemia genes may appear anemic as a result of a reduction in globin chain synthesis; this is particularly true for the clinically significant thalassemias, namely α⁰-thalassemia (α⁰-thal), β-thalassemia (β-thal), and hemoglobin E (Hb E) [3]. These three forms are prevalent in many parts of the world including Southeast Asia where ID is also prevalent [3,4,5].

The Socialist Republic of Vietnam is one of the low middle income countries in Southeast Asia in which anemia is highly prevalent, especially in rural areas. The prevalence of anemia ranges from 25 to 55.6%, depending on the study area and population [6,7,8,9,10,11,12,13]. In pregnant women, a prevalence of anemia of 40-50% has been reported [7,9]. An ID prevalence of 20-30% among nonpregnant women has been reported in some regions in Northern Vietnam [12,14]. Many efforts have been made to reduce the prevalence of anemia through iron supplementation programs [13,15,16,17,18,19]. However, the programs have not covered the whole country because of limited resources, and this has led to the existing burden of anemia in many regions.

A recent study among Vietnamese women of reproductive age and young children showed a prevalence of 9.1-11.6% for anemia, 12.9-13.7% for ID, and 3.2-5.4% for IDA [20]. Among pregnant women in Thua Thien Hue, the prevalence of anemia, ID, and IDA is limited. In addition, none of the previous studies has paid attention to thalassemia as a cause of anemia. It is still unknown whether thalassemia or ID is the main culprit in anemia. It is not clear to what extent thalassemia traits exist predominantly in the area and are causing anemia. So far, only one study has reported thalassemia prevalence among the Vietnamese (Kinh ethnicity) in South Vietnam, with prevalences of 3.4% for α⁰-thal, 1.6% for β-thal, and 3.4% for Hb E [21].

The main objective of this study was to determine the prevalence of anemia, ID, and IDA, as well as to investigate the burden of anemia in relation to thalassemia and ID among a representative group of Vietnamese pregnant women living in Thua Thien Hue, Central Vietnam.

The survey was conducted in Thua Thien Hue. The province is located on the north central coast of Vietnam. The province is divided into 9 administrative units, 1 provincial city, Hue, and 8 districts. Six districts are located in the plain area and the other two are in mountainous areas. The distance from the city to the mountainous Nam Dong district is 60 km and to the mountainous Aluoi district it is 100 km. The majority of the population (96%) is Vietnamese (Kinh ethnicity). Ethnic minority groups consist of the Katu, Ta Oi, and Bru-Van Kieu [22]. Local groups include the Pacoh and Pahy. The average GDP per capita in 2010 was USD 1,150 [23].

The province has 152 communes. Each commune has one community health center (CHC). Samples were obtained via a clustered sampling method. Primary sampling units were CHCs in each district. Two to 5 CHCs from each district were selected on the basis of probability-proportional-to-size random sampling. Thirty out of the 152 communes and an additional 10 communes in Hue City were recruited. The catchment areas are shown in figure 1. Pregnant women who attended the antenatal care service at the selected health centers were recruited consecutively from September 2011 to May 2012. Based on the main objective of determining the prevalence of anemia, the sample size was calculated using the expected anemia prevalence of 0.45 (average value from previous studies) with an allowable error of 0.1 and a design effect of 2. Therefore, at least 191 women were required.

A total of 425 pregnant women volunteered for this study. All participants were healthy according to standard antenatal care testing. The standard antenatal care for first-visit pregnant women included measurements of weight, height, blood pressure, and heart rate, as well as interviews regarding the individuals' health status and demographic information. Only women pregnant at the time of their first visit to the CHC and with a gestational age <12 weeks were recruited. Women with a chronic illness (including asthma, diabetic disease, hypertension, and heart disease) or taking iron tablets were excluded. Demographic data and blood specimens were collected by well-trained health officials. Informed consent was obtained from all participating women via either their signature or fingerprint. Ethical approval for the study protocol was obtained from the Scientific Committee of the Hue College of Medicine and Pharmacy and the Institutional Review Board of Khon Kaen University.

After informed consent had been obtained, a 3-ml venous blood sample was taken. None of the participants refused blood sampling. All samples were stored at 4°C and transferred to Hue Central Hospital for determination of hematological parameters. This investigation was performed within 6 h of blood collection. Parts of blood and plasma samples were sent on ice to the Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Khon Kaen University, Thailand. All specimens were processed for laboratory investigations within 12 h of arriving at the CMDL. Due to an insufficient blood volume for some samples, only 399 (94%) out of 425 samples could be used for further analyses.

Hemoglobin concentration and other hematological parameters were measured using a Sysmex KX-21 automated blood cell counter (Sysmex Corp., Kobe, Japan). Plasma ferritin was determined using a chemiluminescent immunoassay (Beckman Coulter, USA). Hemoglobin profiles were analyzed using automated capillary zone electrophoresis (Capillarys 2; Sebia Co. Ltd., Lisses, France). All automated analyses were validated against the commercial quality control samples provided by the companies from which the reagents had been purchased.

The thalassemias investigated included α⁰-thal, β-thal, and Hb E. α⁰-thal, i.e. SEA and THAI deletion, was identified using PCR-based techniques [24]. The diagnosis of β-thal was made in cases with Hb A₂ >4% accompanied by MCV values <80 fl [25]. Hb E was diagnosed based on the presence of a peak on the electropherogram at zone 4, as indicated by the manufacturer.

Anemia was diagnosed using an Hb level <11.0 g/dl as the cutoff value [1]. ID was diagnosed in cases with plasma ferritin <15 ng/ml [2]. IDA was defined in cases with Hb <11.0 g/dl and plasma ferritin <15 ng/ml. All anemic cases were investigated further for α⁺-thalassemia (α⁺-thal), the 3.7- and 4.2-kb deletion, hemoglobin Constant Spring (Hb CS), and Hb Pakse as described previously [26,27]. In order to identify anemic individuals with a falsely normal ferritin because of inflammation, C-reactive protein (CRP) levels were determined using the particle immunoassay methodology (CRPH reagent kit Synchron System; Beckman Coulter). This investigation followed standard instructions provided by the manufacturer.

Data analysis was performed using the Minitab statistical software program (Minitab Inc., USA) version 12. The primary outcome, i.e. the prevalence of anemia, ID, and IDA, is given as percentages with 95% CI. To test the difference between proportions, i.e. the difference in general characteristics between anemic and nonanemic groups as well as the proportional difference between ID and thalassemia, either the Z test or the χ² test was applied. The differences in hematological parameters between two independent groups were tested using the Student t test. Nonparametric statistics, medians, and interquartile ranges were calculated to describe the ferritin values. The Mann-Whitney U test was applied to test significant differences in ferritin levels between two independent groups. p < 0.05 was considered statistically significant.

None off the 425 first-visit pregnant women who participated had either taken iron supplementation or were suffering from chronic illnesses. Three hundred ninety-nine samples were eligible for laboratory investigations. General characteristics of the study population are shown in table 1. The average age of the women was 28.4 years with a range of 18-46, and the mean body mass index (BMI) 20.5. More than 85% of participants belonged to the predominant ethnic group in Vietnam, i.e. the Kinh group. The majority of women (60.7%) were from rural areas. Only 7% of the participants had a poor economic background, as classified by the local government using monthly income as an indicator. Forty-four percent of the participants were primipara, 36% had 1 child, and 20% had 2 children or more.

The prevalences of anemia, ID, IDA, and thalassemia are given in table 2. Seventy-seven females were anemic (19.3%, 95% CI 15.5-23.5). The prevalence of ID was 20.1% (95% CI 16.2-24.3) and that of IDA was 6.0% (95% CI 3.9-8.8). The overall prevalence of the three clinically significant thalassemia traits was 7.3% (95% CI 4.9-10.3). Of the 77 anemic women, 24 (31.2%) had ID and 20 (26.0%) had thalassemia genes. The rest (42.9%) were anemic due to unknown causes (table 3). Analysis of CRP among anemic individuals with unknown causes revealed normal CRP levels, ranging from <0.2 mg/l to 6.85 mg/l (reference value: >7.48 mg/l). Comparison of the general characteristics of the females including age, BMI, ethnicity, income, and number of children and the proportion of thalassemia and ID between the anemic and nonanemic groups revealed no significant difference for any of the variables mentioned. The results of these evaluations are not displayed.

Table 4 provides hematological parameters for various conditions. The group of anemic individuals with thalassemia had significantly higher mean Rbc and RDW values, but significantly lower mean Hb, MCV, and MCH levels, compared to the group of anemic females with unknown reasons for their anemic status (Student's t test, p < 0.01). Ferritin levels among ID individuals were significantly lower than in those with thalassemia and an unknown cause (Mann-Whitney U test, p < 0.01).

This is the first study to report the burden of anemia in relation to thalassemia and ID among Vietnamese pregnant women. The overall prevalence of anemia, ID, and IDA was significantly lower than in previous reports. Improvements in health care, better living standards due to enhancement of the economy, and changes in food habits could possibly explain the decline in anemia, ID and IDA.

Based on a national survey conducted in 1995, the estimated prevalence of anemia among Vietnamese pregnant women was 52.5% [8]. More recent studies conducted in 2001 and 2003 resulted in an anemia prevalence of 43-53% [7,9]. The anemia prevalence of 19% reported here is therefore unexpectedly low. This lower prevalence might be due to the fact that in this study only women with a gestational age <12 weeks were included. Hence, their hemoglobin levels had not yet been affected by hemodilution, as occurs in the more advanced stages of pregnancy [28].

In low and middle income countries, the prevalence of IDA is usually estimated on the basis of the prevalence of anemia, since generally ID accounts for the majority of anemia cases [1,2]. Likewise, in Vietnam it is thought that the prevalence of IDA might be high. The low IDA prevalence of 6% therefore is unexpected. A recent study conducted in Vietnamese children and nonpregnant women revealed results similar to those of this study, with markedly reduced prevalences of anemia, ID, and IDA [20]. Similar findings were also obtained in many studies conducted in Thailand in that the prevalence of IDA was not high [29,30,31,32]. The outcomes of the studies indicate that the burden of anemia due to ID in the region has improved.

Considering that Southeast Asia is an endemic area for thalassemia [3], the clinically significant thalassemia traits for α⁰-thal, β-thal, and Hb E might be causally linked to anemia as well. Several studies conducted in Northeast Thailand have demonstrated that anemia in the region is associated with thalassemia and hemoglobinopathies rather than ID [29,30,31,32]. This seems not to be the case in Vietnam since the prevalence of the 3 clinically significant thalassemias is relatively lower. Similar to the results of this study, an overall prevalence of 8.4% for the clinically significant thalassemias has been reported among Kinh ethnicity in South Vietnam [21], indicating that thalassemia may not be the main burden in anemia among the Vietnamese.

Instead, frank anemia due to ID seems to be a more significant problem for pregnant women. This conclusion is based on the observation that for various forms of thalassemia including α⁺-thal and Hb CS, as demonstrated in table 3, 26.0% (20/77) of cases of anemic pregnant women are attributed to thalassemia, but 31.2% (24/77) can be explained by ID, leaving the reasons for almost 43% of anemia cases unexplained. In addition to ID and thalassemia, other micronutrient deficiencies, parasitic infestation, and chronic diseases may cause anemia. Anemia due to chronic disease among the participants may be rejected as a possible reason for anemia since all of the women had been healthy based on standard antenatal care testing and self-reports. There is controversy regarding whether parasitic infestations are associated with anemia [6,7,8,12,33]. Theoretically, anemia due to parasites, especially hookworm, is a consequence of chronic blood loss, resulting in ID and subsequently IDA [34], but hookworm infestation is not reported to be a problem in the population investigated here [12]. Based on data from the Department of Parasitology and Malaria Control, Thua Thien Hue Health Service [pers. commun.], it appears that the occurrence of hookworm and malaria infestations is rather low, with an average prevalence of 6.4% for hookworm infestation and <0.1% for malaria infestation. Most importantly, none of the cases observed belonged to the group of pregnant women. The results of normal CRP levels among anemic women with unexplained reasons for anemia would exclude the possibility of falsely normal ferritin values because of inflammation [2]. Instead, the consumption of green leafy vegetables is high and is the main source of iron, which is then poorly absorbed [34]. Besides inhibition of iron absorption, vitamin B₁₂ deficiency might contribute to anemia, but more in depth investigations are needed to establish these factors. Perhaps other micronutrient deficiencies are also responsible for anemia among anemic pregnant women, but this also requires further investigation.

Significant differences in hematological parameters among the Vietnamese pregnant women with different causes of anemia could be observed. All anemic women had mild degrees of anemia (i.e. Hb >10.0 g/dl). It is obvious that the low MCV and MCH values are indicative of thalassemia among the Vietnamese (table 4). Most individuals with ID had normal MCV and MCH values, which is not generally observed since usually MCV and MCH are reduced. In theory, the degree of hematological changes among individuals with ID depends on the duration of the deficiency as well as the severity of the anemia [34]. The data reported here suggest that approximately one fifth of Vietnamese pregnant women are at high risk for IDA. This emphasizes the need for campaigning to persuade pregnant women to attend antenatal care clinics early and take iron tablets to prevent adverse outcomes for both the mother and the fetus. For nonanemic pregnant women, intermittent iron supplementation might be the method of choice in order to avoid side effects which are commonly reported for daily supplements, resulting in unsatisfactory compliance rates. The recommendation applies to countries where the prevalence of anemia is <20% [35].

This study reflects the situation in a poorly served rural part of Vietnam and might represent the situation in other underserved areas in Vietnam. However, the situation in more conveniently accessible rural and urban areas might be different.

This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHeP-GMS), Khon Kaen University.