Epidemiology of thalassemia among the hill tribe population in Thailand

Tawatchai Apidechkul; Fartima Yeemard; Chalitar Chomchoei; Panupong Upala; Ratipark Tamornpark

doi:10.1371/journal.pone.0246736

. 2021 Feb 11;16(2):e0246736. doi: 10.1371/journal.pone.0246736

Epidemiology of thalassemia among the hill tribe population in Thailand

Tawatchai Apidechkul ^1,^2,^*, Fartima Yeemard ¹, Chalitar Chomchoei ³, Panupong Upala ¹, Ratipark Tamornpark ¹

Editor: Michela Grosso⁴

PMCID: PMC7877639 PMID: 33571309

Abstract

Background

Thalassemia is a severe disease that occurs due to abnormalities in hemoglobin genes. Various genetic factors in different populations lead to different clinical manifestations of thalassemia disease, particularly among people who have a long history of migration and who have married among tribes, such as the hill tribe people in Thailand. This genetic epidemiological study aimed to estimate the prevalence of various forms of thalassemia among the six main hill tribe populations in Thailand.

Methods

A cross-sectional study was conducted to obtain information and blood specimens from school children belonging to one of the six main hill tribes in Thailand: Akha, Lau, Hmong, Yao, Karen, and Lisu. Hill tribe children who were attending grades 4–6 in 13 selected schools in Chiang Rai Province, Thailand, were invited to participate in the study. A validated questionnaire and 3 mL blood specimens were collected after obtaining information consent forms from both the children and their parents on a voluntary basis. A complete blood count (CBC) was performed, followed by osmotic fragility (OF) and dichlorophenol indophenol precipitation (DCIP) tests to screen for thalassemia. High-performance liquid chromatography (HPLC) and real-time quantitative polymerase chain reaction (qPCR) were used to identify hemoglobin type and α-thalassemia, respectively. A t-test, chi-square and logistic regression were used to detect the associations between variables at the significance level of α = 0.05.

Results

A total of 1,200 participants from 6 different tribes were recruited for the study; 50.0% were males, and 67.3% were aged 11–12 years. The overall prevalence of thalassemia carriers according to the screening tests was 9.8% (117 of 1,200). Among the cases, 83 were A2A (59 cases were α-thalassemia 1 carrier or α-thalassemia 2 carrier or homozygous α-thalassemia 2, and 24 cases were β-thalassemia trait with or without α-thalassemia); 1 case was EE (homozygous Hb E with or without α-thalassemia); 31 cases were EA (30 cases were the Hb E trait, and 1 case was Hb E trait with or without α-thalassemia); 1 case was A2A Bart’s H (Hb H disease α-thalassemia 1/α-thalassemia 2); and 1 case was A2A with abnormal Hb. The prevalence of the α-thalassemia 1 trait among the hill tribe population was 2.5%. The greatest prevalence of the α-thalassemia 1 trait was found in the Karen (3.0%) and Hmong (3.0%) tribes.

Conclusions

The prevalence of some forms of thalassemia in the hill tribe population is higher than that in the Thai and other populations. Effective and available thalassemia screening tests, including essential information to protect the next generation through the specific counseling clinic, are crucial, particularly due to increasing marriages within these populations.

Introduction

Thalassemia is a major inherited blood disorder caused by insufficient or nonfunctional hemoglobin [1]. Hemoglobin (Hb) is an important protein in red blood cells (RBCs) [2,3]. There are different forms of abnormal human red blood cells, which are indicated by the various levels of hemoglobin severity [3,4]. The Hb protein structure consists of 4 subunits, with each unit comprising heme and globin chains. There are two groups of globin chain: an α-like subunit, which consists of 141 amino acids (α-globin chain and ζ-chain), and a β-like subunit, which consists of 146 amino acids (β-globin chain, γ-globin chain, and δ-globin chain). Thalassemia is an abnormality in Hb in either quantitative or/qualitative aspects. Quantitative aspects result from aberrant expression of one of the globin chains: if expression of α-globin is reduced or absent, the disease is called “α-thalassemia”; if β-globin expression is reduced or absent, it is called “β-thalassemia”. Qualitative aspects lead to abnormal Hb, which mainly occurs from point mutations. There are two forms that are often found in the Thai population: Hb E, which is caused by missense mutation of the β-globin gene at codon 26 from GAG to AAG; and Hb Constant Spring (Hb CS), which is caused by mutation of the α-global gene (α^cs) at codon 142 from TAA (stop codon) to CAA [4].

Today, thalassemia is widely found with different variants in different populations [2]. In 2018, the World Health Organization (WHO) reported that at least 5.2% of individuals worldwide were thalassemia carriers, that approximately 1.1% of couples worldwide were at risk of having children with a hemoglobin disorder, and that 2.7/1,000 conceptions were affected [5]. Most children born with thalassemia in high-income countries survive with a chronic disorder, whereas children in developing countries die before 5 years of age [5]. In 2018, the Ministry of Public Health in Thailand reported that approximately 30–40% of Thai individuals (18–24 million people) were carriers of thalassemia or hemoglobin disorders, with more than 12,000 infants born with thalassemia every year [6]. Moreover, more than 600,000 thalassemia patients in Thailand are covered by the health system and require regular treatment, including blood transfusion [7]. These patients consume a large amount of medical resources for treatment and care. However, all medical expenses are covered by the government under the Thailand Universal Coverage Scheme (USC), which is required to cover approximately US $3,000 per year per person [8].

The United Nations reported that in early 2020, Thailand had a population of 69 million people [9]. In total, 4–5 million people were members of minority populations, such as hill tribes and other stateless populations [10]. The hill tribe people comprise a group having migrated from the south of China to settle along the highland and border areas of Thailand-Myanmar-Republic of Laos [11]. There are six main groups: Akha, Lahu, Hmong, Yao, Karen, and Lisu [11]. The hill tribe people have their own languages, cultures, and lifestyle, which are different from those of native Thai people. With globalization and economic constraints, the hill tribe people have opened their villages to allow for work, education, and other businesses [12]. Many individuals in newer generations have started to marry individuals from other tribes, including native Thai. However, there is little available information regarding thalassemia epidemiology in hill tribe populations.

This study aimed to estimate the prevalence of thalassemia carriers and demonstrate the variations in hemoglobin types and magnitudes of differences in thalassemia genes among hill tribe populations in Thailand.

Materials and methods

Study design

A cross-sectional study was performed to collect data and blood specimens from hill tribe children who were attending grades 4–6.

Study population

The target populations were hill tribe children who belonged to one of six main tribes: Akah, Lahu, Hmong, Yao, Karen, or Lisu.

Study sample

The study participants were recruited from 13 of the 162 primary schools [13] located in the hill tribe villages in Chiang Rai Province, Thailand. All children who were attending grades 4 to 6 in selected schools and able to identify themselves as members of one of the six main hill tribes were invited to participate in the study.

Sample size

The sample size was calculated to obtain the number required for estimating a prevalence with a specified level of confidence and precision. The outcome was the prevalence of various forms of thalassemia among the six main hill tribe populations in Thailand. The prevalence of thalassemia in a previous study was reported to be 13.0% [14]. The standard formula for a cross-sectional to estimate the sample size was applied, as follows [15]: n = [Z²_α/2 PQ]/e² (Z_α/2 = 1.96, P = 0.13 [15], Q = 0.87, and e = 0.05), where Z = value from the standard normal distribution corresponding to the desired confidence level (Z = 1.96 for 95% CI), P is the expected true proportion, and e is the desired precision; therefore, 173 participants from each tribe were needed. After adding an additional 10.0% for any error during the execution of the study, at least 192 participants from each tribe were required for analysis.

A validated questionnaire consisting of two parts was developed and used for data collection. In part one, 5 questions were used to collect personal information: age, sex, weight, height, and grade of school. In part two, 15 questions were used to ask questions regarding medical history and family history, such as the presence of medical conditions, history of blood transfusion, history of taking ferrous sulfate, and parents’ tribe. A 3-mL blood specimen was collected from each participant.

Data collection procedure

A total of 13 primary schools located in the hill tribe villages were randomly selected from 163 schools in Chiang Rai Province, Thailand [13]. Access to the schools for data collection was granted by the school directors. During a meeting with the school director, all essential information regarding the study was explained. Brief and essential information about the study was also provided to all children’s parents five days before data and blood specimen collection. All children were attending grades 4–6, and the children of parents who voluntarily signed the informed consent form were eligible and invited to join the study. On the day of data collection, all children were provided information about the study again and asked regarding their willingness to provide informed consent. The questionnaire was completed by the students within 10 minutes. Afterwards, 3 mL of blood was drawn and collected in ethylenediaminetetraacetic acid (EDTA) tubes. The blood specimens were transferred to the Mae Fah Luang Medical Laboratory Center for analysis on the same day.

Laboratory tests

A complete blood count (CBC) using the Sysmex/XN-550 was performed, followed by osmotic fragility (OF) [16] and dichlorophenol indophenol precipitation (DCIP) [17] screening tests. OF test was performed using KKU-OF reagent kit (Center for Research and Development of Medical Diagnosis Laboratory, Khon Kaen University, Khon Kaen Province, Thailand). A fresh EDTA-blood sample of 20 μL was used to add into the KKU-OF solution. Then, it was mixed by inventing, and placed the mixture at the room temperature for at least 15 minutes, before visual inspection. The interpretation was determined by observing the turbidity against the lines or letters; negative result was indicated by the clear solution, and positive result was indicated by turbidity. A positive quality control (mean corpuscular volume (MCV)>85fl, mean corpuscular hemoglobin (MCH) >28 pg, and Hb>13g/dL) and negative quality control (MCV<70 fl, MCH <25 pg, and Hb>10g/dL) were performed in parallel with unknown samples.

While, DCIP test was performed using KKU-DCIP-Clear reagent (Center for Research and Development of Medical Diagnosis Laboratory, Khon Kaen University, Khon Kaen Province, Thailand). The DCIP test required three reagents; the DCIP reagent (a 2 mL dark-blue solution form), the cleaning reagent (a dry powder form), and the diluent. The procedure started from a 20 μL EDTA blood sample and added 2 mL of DCIP reagent into the tube. Then, mixing was operated by a vortex mixer or inventing. Afterwards, a 15-minute incubation at the temperature room (37° C) was required before a 20-μL of cleaning solution was added and mixed. A tube was placed a few seconds or until the blue color disappeared at room temperature before determining the result. A clear color solution was negative, while turbidity was a positive result. The OF and DCIP tests were found a high sensitivity, specificity, and accuracy to detect α-thalassemia, β-thalassemia, and Hb-E while using in parallel test [18].

If either or both tests presented a positive result, a high-performance liquid chromatography (HPLC) was performed using VARIANT-II-hemoglobin testing system (BIO-RAD^®, USA). Identifying β-thalassemia trait, we used the characteristics of Hb typing and also OF and DCIP tests according to the WHO guideline [19] and Thailand, Ministry of Public Health guideline [20]. The elution buffer 1 (Bio-Rad Laboratories, USA) was used to identify the hemoglobin type. Relative quantitative polymerase chain reaction (qPCR) was performed to identify α-thalassemia SEA and Thai deletion by using DMSc α-thal 1, which is produced by the Department of Medical Sciences, Ministry of Public Health, Thailand (Fig 1).

Statistical analysis

Data were doubled entered into an Excel sheet before being transferred into SPSS version 24 (SPSS, Chicago, IL) for analysis. The general characteristics of the participants are presented as the mean and standard deviation for continuous data; categorical data are described as percentages. A t-test and chi-square test were used for comparing means and proportions between groups, respectively. A logistic regression was used to detect the odds of having a positive for screening tests of thalassemia among the tribes.

Ethical considerations

All study protocols were approved by the Research Ethics Committee of Chiang Rai Public Health Office (No. CRPPHO 14/2562). Before starting the interview, all participants were provided with all relevant and essential information. An informed consent form was obtained on a voluntary basis from both parents and children before starting the interviews. Moreover, all questionnaires were destroyed properly after being coded and entered into the Excel sheet, such that the data could not be traced back to any individual.

Results

A total of 1,200 participants from 6 different tribes were recruited for the study; 50.0% were males, and 67.3% were aged 11–12 years. More than half of the participants were Christian (58.8%), all participants attended grades 4–6, and the participants were evenly distributed among the six tribes. The majority had 6 or fewer family members (Table 1).

Table 1. General characteristics of the participants.

Characteristics	n	%
Total	1,200	100.0
Sex
Male	600	50.0
Female	600	50.0
Age (years)
9–10	392	32.7
11–12	808	67.3
Mean = 11.03, Standard deviation (SD) = 0.84
Religion
Buddhist	495	41.2
Christian	705	58.8
Education
Grade 4	391	32.6
Grade 5	399	33.3
Grade 6	410	34.2
Number of family members
≤ 6	975	81.2
> 6	225	18.8
Number of siblings
None	184	15.3
1–2	667	55.6
≥ 3	349	29.1
Tribe
Akha	200	16.7
Lahu	200	16.7
Hmong	200	16.7
Yao	200	16.7
Karen	200	16.7
Lisu	200	16.7
Father’s tribe
Akha	202	16.8
Lahu	221	18.4
Hmong	194	16.2
Yao	194	16.2
Karen	195	16.3
Lisu	194	16.2
Mother’s tribe
Akha	481	40.1
Lahu	239	19.9
Hmong	91	7.6
Yao	181	15.1
Karen	161	13.4
Lisu	47	3.9
Paternal grandfather’s tribe
Akha	204	17.0
Lahu	188	15.7
Hmong	200	16.7
Yao	201	16.7
Karen	200	16.7
Lisu	207	17.3
Paternal grandmother’s tribe
Akha	135	11.3
Lahu	191	15.9
Hmong	281	23.4
Yao	202	16.8
Karen	190	15.8
Lisu	201	16.8
Maternal grandfather’s tribe
Akha	481	40.1
Lahu	239	19.9
Hmong	91	7.6
Yao	181	15.1
Karen	161	13.4
Lisu	47	3.9
Maternal grandmother’s tribe
Akha	234	19.5
Lahu	136	11.3
Hmong	292	24.3
Yao	153	12.8
Karen	104	8.7
Lisu	281	23.4

Open in a new tab

According to medical history, only 1.8% reported having a medical condition, 1.1% had anemia, and a large proportion had taken ferrous sulfate (98.7%) (Table 2).

Table 2. Medical history.

Medical history	n	%
Medical condition
No	1,179	98.3
Yes	21	1.8
Allergy	14	1.2
Asthma	2	0.2
Peptic ulcer	5	0.4
Having anemia
No	1,187	98.9
Yes	13	1.1
Receiving blood transfusion	0	0.00
Having a family member receiving blood transfusion for anemia
No	1,200	100.0
Yes	0	0.0
Having relatives receiving blood transfusion for anemia
No	1,200	100.0
Yes	0	0.0
Having taken ferrous sulfate
No	15	1.3
Yes	1,185	98.7

Open in a new tab

Comparisons of the mean corpuscular volume (MCV), mean corpuscular hemoglobin, hemoglobin (Hb), hematocrit (Hct), and red blood cells between those who were positive in either or both of the OF and DCIP tests and those who had negative results in both tests revealed significant differences (p-value = 0.010) (Table 3).

Table 3. Comparisons of mean MCV, MCH, Hb and Hct levels between thalassemia carriers and nonthalassemia carriers.

Biomarkers	Total (n = 1,200)	OF/DCIP positive (n = 117)	OF/DCIP negative (n = 1,083)	t-test	p-value
MCV	Mean = 80.86 SD = 6.89	Mean = 67.79 SD = 8.33	Mean = 82.27 SD = 4.97	18.10	<0.001^*
MCH	Mean = 26.47 SD = 2.40	Mean = 21.48 SD = 2.90	Mean = 27.01 SD = 1.59	19.75	<0.001^*
Hb	Mean = 13.02 SD = 0.96	Mean = 11.77 SD = 1.01	Mean = 13.16 SD = 0.85	14.25	<0.001^*
Hct	Mean = 39.81 SD = 2.92	Mean = 37.17 SD = 3.02	Mean = 40.09 SD = 2.76	10.39	<0.001^*
RBC	Mean = 4.94 SD = 0.43	Mean = 5.53 SD = 0.58	Mean = 4.88 SD = 0.36	-11.79	<0.001^*

Open in a new tab

* Significant level at α = 0.010.

The overall prevalence of thalassemia carriers according to the screening tests was 9.8%. The greatest prevalence of OF was found in Karen (13.0%), followed by Yao (9.0%) and Lahu (7.0%). Regarding the DCIP test, the highest prevalence was found in Hmong (6.0%), followed by Yao (4.0%). The proportions between positive and negative results of OF and DCIP tests were not statistically significant among the tribes. However, the differences in the proportions between sexes with regard to DCIP were significant (p-value = 0.002) (Table 4).

Table 4. Comparisons of OF and DCIP tests by tribe and sex.

Test	Positive		Negative		χ²	p-value
Test	n	%	n	%	χ²	p-value
Total of OF and DCIP	117	9.8	1,083	90.2	N/A	N/A
OF test	96	8.0	1,104	92.0	N/A	N/A
Tribe					10.19	0.070
Akha	11	5.5	189	94.5
Lahu	14	7.0	186	93.0
Hmong	15	6.5	185	92.5
Yao	18	9.0	182	91.0
Karen	26	13.0	174	87.0
Lisu	12	6.0	188	94.0
Sex					0.55	0.458
Male	45	7.5	555	92.5
Female	52	8.7	548	91.3
DCIP test	39	3.3	1,161	96.7	N/A	N/A
Tribe					8.82	0.116
Akha	2	1.0	198	99.0
Lahu	6	3.0	194	97.0
Hmong	12	6.0	188	94.0
Yao	8	4.0	192	96.0
Karen	5	2.5	195	97.5
Lisu	6	3.0	194	97.0
Sex					9.56	0.002^*
Male	10	1.7	590	98.3
Female	29	4.8	571	95.2

Open in a new tab

* Significance level at α = 0.05.

Regarding the proportions of OF- and DCIP-positive results in the one to two preceding generations, only maternal grandfathers and grandmothers presented significantly different results (p-value = 0.044) (Table 5).

Table 5. Proportions of OF and DCIP results in preceding generations.

Pairs	Same-tribe		Different-tribe
Pairs	n	%	n	%
Father’s and mother’s tribes
OF
Positive	23	6.3	73	8.8
Negative	343	93.7	761	91.2
χ² = 2.10, p-value = 0.146
*DCIP*
Positive	9	2.5	30	3.6
Negative	357	97.3	804	96.4
χ² = 1.04, p-value = 0.306
*Both OF and DCIP positive (total = 19)*	4	N/A	15	N/A
Paternal grandfather’s and grandmother’s tribes
OF
Positive	87	8.1	9	6.9
Negative	983	91.9	121	93.1
χ² = 0.22, p-value = 0.631
*DCIP*
Positive	38	3.9	1	0.8
Negative	1,032	96.3	129	99.2
χ² = 2.85, p-value = 0.091
*Both OF and DCIP positive (total = 19)*	18	N/A	1	N/A
Maternal grandfather’s and grandmother’s tribes
OF
Positive	13	13.3	83	7.5
Negative	85	86.7	1,019	92.5
χ² = 4.01, p-value = 0.044^*
*DCIP*
Positive	4	4.1	35	3.2
Negative	94	95.9	1,067	96.8
χ² = 0.23, p-value = 0.628
*Both OF and DCIP positive (total = 19)*	2	N/A	17	N/A

Open in a new tab

* Significance level at α = 0.05.

Among either or both positives of OF and DCIP tests, 83 of 117 cases were A2A (32 cases had normal Hb typing, not rule out α-thalassemia, and 24 cases had the β-thalassemia trait with or without α-thalassemia). In addition, 1 case was EE (homozygous Hb E with or without α-thalassemia, 31 cases were EA (30 cases were Hb E trait, 1 case was Hb E trait with or without α-thalassemia), one case was A2A Bart’s H (Hb H disease α-thalassemia 1/α-thalassemia 2), and one case was A2A with abnormal Hb (MCV = 73.4lf, positive OF, positive DCIP, 46.0%HbAo, 3.0%HbA2/E, 0.5%HbF, A2A abnormal Hb, and negative for alpha-thalassemia (SEA and Thai deletion).

The prevalence of the α-thalassemia 1 trait (SEA deletion) among the hill tribes was 2.5% (2.3% in males and 2.7% in females) (Table 6).

Table 6. All laboratory results.

Type	n	Male n (%)	Female n (%)
Total	1,200 (100.0)	600 (50.0)	600 (50.0)
Screening test
Negative	1,083 (90.2)	552 (92.0)	531 (88.5)
Positive	117 (9.8)	48 (8.0)	69 (11.5)
OF positive	78 (66.7)	38 (79.1)	40 (57.9)
DCIP positive	20 (17.1)	3 (6.3)	17 (24.6)
Both OF and DCIP positive	19 (16.2)	7 (14.4)	12 (17.3)
HPLC (Hb typing)
A2A	83 (70.9)	39 (81.2)	44 (63.8)
Hb A2 ≤3.5% (Normal Hb typing, not rule out α-thalassemia)	59 (71.1)	25 (42.4)	34 (57.6)
Hb A2 3.6–8% (β-thalassemia trait with or without α-thalassemia)	24 (28.9)	14 (58.3)	10 (41.7)
EE	1 (0.9)	0 (0.0)	1 (1.4)
Hb E≥80.0% and Hb F ≤ 5.0% (Homozygous Hb E with or without α -thalassemia)	1 (100.0)	0 (0.0)	1 (100.0)
EA	31(26.4)	7(14.6)	24 (34.8)
Hb E >25.0% (Hb E trait)	30 (96.8)	7 (23.3)	23 (76.7)
Hb E< 25.0% (Hb E trait with or without α-thalassemia)	1 (3.2)	0 (0.0)	1 (100.0)
A2A Bart’s H	1 (0.9)	1(2.1)	0(0.0)
Hb H disease α-thalassemia 1/α-thalassemia 2	1(100.0)	1 (100.0)	0 (0.0)
A2A with abnormal Hb	1(0.9)	1(2.1)	0(0.0)
Suspected abnormal Hb	1(100.0)	1 (100.0)	0 (0.0)
PCR detects for α-thalassemia 1
Positive for α-thalassemia (SEA deletion)	30 (100.0)	14 (46.7)	16 (53.3)
α -thalassemia 1 trait	30 (100)	14 (46.7)	16 (53.3)

Open in a new tab

According to the PCR method to identify α-thalassemia 1, the greatest proportion of patients with the α-thalassemia 1 trait (SEA deletion) was found in Karen (3.0%) and Hmong (3.0%) tribes. Regarding β-thalassemia detection, the greatest proportions were found in Yao (4.5%) and Karen (3.5%) tribes, whereas the greatest proportions of the Hb E trait were found in Hmong (4.5%) and Lisu (3.0%) tribes. One case of A2A Bart’s H was found in a Lahu boy, one case of A2A with abnormal Hb was found in another Lahu boy, and one case of homozygous Hb E with or without α-thalassemia was found in a girl of Akha (Table 7).

Table 7. Proportions of Hb typing and thalassemia carriers by tribe.

Type	Total		Akha		Lahu		Hmong		Yao		Karen		Lisu
Type	n	%	n	%	n	%	n	%	n	%	n	%	n	%
Hb typing	117	100.0	12	10.3	18	15.4	23	19.7	21	17.9	27	23.1	16	13.7
A2A (Normal Hb typing, not rule out α -thalassemia)	59	50.4	9	4.5	10	5.0	10	5.0	7	3.5	15	7.5	8	4.0
A2A (β-thalassemia trait with or without α -thalassemia)	24	2.1	1	0.5	1	0.5	4	2.0	9	4.5	7	3.5	2	1.0
EE	1	0.9	1 (F)	0.5	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
EA	31	26.5	1	0.5	5	2.5	9	4.5	5	2.5	5	2.5	6	3.0
A2A Bart’s H	1	0.9	0	0.0	1 (M)	0.5	0	0.0	0	0.0	0	0.0	0	0.0
A2A with abnormal Hb	1	0.9	0	0.0	1 (M)	0.5	0	0.0	0	0.0	0	0.0	0	0.0
α-thalassemia 1
α-thalassemia 1 trait (SEA deletion)	30	25.6	4	2.0	5	2.5	6	3.0	5	2.5	6	3.0	4	2.0

Open in a new tab

* F: Female, M: Male.

In the logistic regression analysis to identify the associations between tribes and the OF and/or DCIP positives, it was found that Karen were more likely to have OF and/or DCIP positives than Akha (OR = 2.45, 95%CI = 1.20–4.98), in the overall model. While extracting into different sexes, Yao-males were more likely to have OF and/or DCIP positives than Akha-males (OR = 3.27, 95%CI = 1.02–10.53), and Karen-females were more likely to have OF and/or DCIP positives than Akha-males (OR = 5.25, 95%CI = 1.04–6.11). However, those who had same or different parents and grandparents were not found the association with OF and/or DCIP positives (Table 8).

Table 8. Logistic regression analysis in identifying the associations between the difference of tribes and OF and/or DCIP positives.

Tribe	Total n (%)	OF/DCIP		OR	95%CI	p-value
Tribe	Total n (%)	Positive n (%)	Negative n (%)	OR	95%CI	p-value
Total	1,200(100.0)	117 (9.8)	1,083(90.3)
Akha	200 (16.7)	12 (6.0)	188 (94.0)	1.00
Lahu	200 (16.7)	18 (9.0)	182 (91.0)	1.55	0.73–3.31	0.258
Lisu	200 (16.7)	16 (8.0)	184 (92.0)	1.36	0.63–2.96	0.435
Hmong	200 (16.7)	23 (11.5)	177 (88.5)	2.04	0.98–4.21	0.055
Yao	200 (16.7)	21 (10.5)	179 (89.5)	1.84	0.88–3.85	0.106
Karen	200 (16.7)	27 (13.5)	173 (86.5)	2.45	1.20–4.98	0.014^*
Male	600 (100.0)	48 (8.0)	552 (92.0)
Akha	100 (16.7)	4 (4.0)	96 (96.0)	1.00
Lahu	100 (16.7)	9 (9.0)	91 (91.0)	2.37	0.71–7.98	0.162
Lisu	100 (16.7)	6 (6.0)	94 (94.0)	1.53	0.42–5.60	0.519
Hmong	100 (16.7)	8 (8.0)	92 (92.0)	2.09	0.61–7.17	0.243
Yao	100 (16.7)	12 (12.0)	88 (88.0)	3.27	1.02–10.52	0.047^*
Karen	100 (16.7)	9 (9.0)	91 (91.0)	2.37	0.71–7.98	0.162
Female	600 (100.0)	69 (11.5)	531 (88.5)
Akha	100 (16.7)	8 (8.0)	92 (92.0)	1.00
Lahu	100 (16.7)	9 (9.0)	91 (91.0)	1.14	0.42–3.08	0.800
Lisu	100 (16.7)	10 (10.0)	90 (90.0)	1.28	0.48–3.38	0.622
Hmong	100 (16.7)	15 (15.0)	85 (85.0)	2.01	0.82–5.03	0.126
Yao	100 (16.7)	9 (9.0)	91 (91.0)	1.14	0.42–3.08	0.800
Karen	100 (16.7)	18 (18.0)	82 (82.0)	2.52	1.04–6.11	0.040^*
Father/ Mother’s tribe
Same	366 (30.5)	28 (7.7)	338 (92.3)	0.69	0.45–1.08	0.106
Different	834 (69.5)	89 (10.7)	745 (89.3)	1.00
Paternal grandfather/grandmother’ s tribe		s
Same	1,070 (89.2)	108(10.1)	962 (89.9)	1.51	0.75–3.06	0.253
Different	130 (10.8)	9 (6.9)	121 (93.1)	1.00
Maternal grandfather/grandmother’s tribe
Same	98 (8.2)	15 (15.3)	83 (84.7)	1.77	0.99–3.19	0.056
Different	1102 (91.8)	102 (9.3)	1,000(90.7)	1.00

Open in a new tab

* Significant level at α = 0.05.

Discussion

The overall prevalence of thalassemia carriers among the hill tribes according to the screening tests (OF and DCIP tests) was 9.8%; the greatest proportion of OF-positive results was found in Yao (9.0%), and the greatest proportion of DCIP-positive results was found in Hmong (6.0%). Females (4.8%) were significantly more likely to have a positive DCIP test than were males (1.7%). OF test positivity among children whose maternal grandfather and grandmother were in the same tribe was higher than among children whose maternal grandfather and grandmother were from different tribes. The major forms of Hb disorder was A2A (6.9%) and EA (2.6%). The prevalence of the α-thalassemia 1 trait among the hill tribe population was 2.5%.

A large study on the prevalence of thalassemia among 1,796 Thai women in northeastern Thailand reported that the highest prevalence of thalassemia in the country was 30.2%, with the highest prevalence of homozygous Hb E being 5.4%, the highest prevalence of the β-thalassemia trait being 0.6%, and the highest prevalence of the α-thalassemia 1 trait being 3.0% [21]. These results reflect that the prevalence of some types of thalassemia among the hill tribes is higher than that among Thai people, even those living in the areas with the highest prevalence of thalassemia.

The prevalence of some forms of thalassemia among the hill tribe people was lower than that in the Thai population, though the prevalence of other forms was higher than that in the Thai population. For instance, the prevalence of the Hb E trait among the hill tribe (2.5%) was lower than that among the Thai population. According to a study in northeastern Thailand, the most prevalent type of thalassemia is the Hb E trait (39.1%) [22]. Additionally, a study among people who did not present anemia or microcytosis in central Thailand did not detect the α-thalassemia 1 trait, but there was a 15.8% prevalence of the Hb E trait and a 0.6% prevalence of the β-thalassemia trait [23]. Another study among Thai blood donors found that the thalassemia trait prevalence was 21.1% [24]. In a study estimating the burden of α-thalassemia in Thailand using a comprehensive prevalence database of Southeast Asia, it was estimated that 3,595 (95% CI = 1,717–6,199) newborns will be born with severe α-thalassemia in Thailand in 2020 [25].

Several studies have sought to identify the prevalence of thalassemia among different non-Thai populations and have reported different prevalence rates for different types of thalassemia. For instance, a study among migrant workers in Thailand found the prevalence rates of the α-thalassemia 1 trait (1.8%) and the β-thalassemia trait (3.9%) to be highest among workers from Myanmar, whereas the prevalence of the Hb E trait was highest among workers from Laos [26]. A population-based study among Tai and Mon-Khmer ethnic groups in northern Thailand reported that 23.4% had the α-thalassemia trait and that 96.9% were heterozygous. The study also reported different prevalence of α-thalassemia traits among the tribes [27]. Apidechkul reported that several forms of Hb disorder were found among the Lahu women in Chiang Rai Province, Thailand; 4.3% were Hb E trait, 0.8% were β-thalassemia trait, and 0.8% were Hb E homozygous [28]. A study among the school children in northern Thailand, it was presented that the prevalence of β-thalassemia trait was 7.9% [29], which was greater than our study (2.0%).

The reported prevalence of thalassemia in different countries varies. A study in Pakistan found that early screening and detection of β-thalassemia could significantly reduce severe thalassemia in later generations in a population [30], and a study in Morocco presented a prevalence of α-thalassemia of 0.9% [31]. In Iraq, it was reported that the prevalence of thalassemia increased from 33.5/100,000 in 2010 to 37.1/100,000 in 2015, with β-thalassemia accounting for 73.9% of all cases [32]. Moreover, in the United States, the prevalence of thalassemia due to the immigration of people from different regions of the world has reportedly increased by 7.5% in recent decades [33]; 4.6% of Australian people were found to be carriers of α-thalassemia and β-thalassemia gene variants [34].

In our study, females were more likely to have thalassemia than males, and those whose maternal grandfather and grandmother were of the same tribe were significantly more likely to have thalassemia than those whose grandparents were of different tribes. Our study also found the α-thalassemia 1 trait to be presented at a higher rate in females than in males, with the opposite for the β-thalassemia trait. However, there are a few papers presenting these data.

A systematic review clearly demonstrated that thalassemia is associated with a large health care service burden in different countries [35]. Some forms of thalassemia require regular lifelong blood transfusions [36]. A study in northern Thailand showed that thalassemia was one of the significant risk factors for cardiac iron overload and cardiovascular complications [37]. Moreover, among nontransfusion-dependent thalassemia (NTDT) patients, major complications are cholelithiasis (35.0%), abnormal liver function (29.0%), and extramedullary hematopoiesis (EMH) (25.0%) [38], and a prospective study reported that nontransfusion-dependent Hb E/β-thalassemia and α-thalassemia (Hb H disease) result in the development of several severe diseases, particularly gallstones and serious infections [39].

Research projects related to genetic diseases among the hill tribe population in Thailand are very challenging due to several factors, including language and transportation barriers [40]. Therefore, a clear understanding of the research project among participants, particularly their parents, is required. Moreover, in disseminating the results of testing, all information should be provided carefully during the counseling process. One more point of the limitation of the study, it.

Conclusion

The hill tribe people in Thailand have with a high prevalence of thalassemia, particularly the α-thalassemia trait, the β-thalassemia trait, and the Hb E trait. Some tribes have a higher prevalence than others. A higher proportion the α-thalassemia trait and Hb E trait was found for females, with males exhibiting a higher incidence of the β-thalassemia trait. Moreover, those with grandfathers and grandmothers on either the father or mother’s side who were from the same tribe were at a greater risk of thalassemia than those whose grandparents’ were from different tribes; conversely, those whose parents were from different tribes were at a greater risk of thalassemia than those whose parents were from the same tribe. To reduce the prevalence of all types of thalassemia among the hill tribe people in Thailand, a screening program should be available at all health care centers located in hill tribe villages. Moreover, essential information on thalassemia, including prevention measures, should be provided to these populations in local hill tribe languages. A specific counseling clinic including prenatal diagnosis for hemoglobin disorders should be initiated in all levels of health institutes located in the hill tribe villages in Thailand.

Supporting information

S1 Appendix. Questionnaire used in the study (Thai).

(PDF)

Click here for additional data file.^{(81.9KB, pdf)}

S2 Appendix. Questionnaire used in the study (English).

(PDF)

Click here for additional data file.^{(73.6KB, pdf)}

S3 Appendix. Data file of the study.

(SAV)

Click here for additional data file.^{(172.9KB, sav)}

Acknowledgments

The authors thank all school directors and participants for providing all the essential information.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

TA was granted from The National Research Council of Thailand (NRCT) and Mae Fah Luang University to support grant(Grant No.23/2561), but they did not involve to the project.

References

1.Centers for Disease Control and Prevention (CDC). Thalassemia. Available from. https://www.cdc.gov/ncbddd/thalassemia/facts.html Accessed 11 January 2020.
2.Angastiniotis M, Lobitz. Thalassemia: an overview. International Journal of Neonatal Screening. 2019; 5(16): 10.3390/ijns5010016 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Tari K, Ardalan PV, Abbaszadehdibavar M, Atashi A, Jalili A, Gheidishahran M. Thalassemia an update: molecular basis, clinical features and treatment. International Journal of Biomedicine and Public Health. 2018; 1(1): 48–58. [Google Scholar]
4.Taher A, Weatherall DJ, Cappellini MD. Thalassemia. The Lancet. 2018; 391(10116): 156–67. [DOI] [PubMed] [Google Scholar]
5.World Health Organization (WHO). Global epidemiology of hemolobin disorders and derived service indicators. Available from: https://www.who.int/bulletin/volumes/86/6/06-036673/en/. Accessed 11 January 2020.
6.Ministry of Public Health Thailand. Thalassemia in Thailand: 2018. Available from: https://pr.moph.go.th/?url=pr/detail/all/02/116500 Accessed 17 January 2020.
7.Ministry of Public Health Thailand. Thalassemia in Thailand: 2018. Available from: https://pr.moph.go.th/?url=pr/detail/all/02/116500. Accessed 17 January 2020.
8.National Health Security Office (NHSO). Thalassemia care and treatment for Thai population. Available from: https://www.nhso.go.th/FrontEnd/NewsInformationDetail.aspx?newsid=MTQyOQ. Accessed 17 January 2020.
9.United Nations. Country profile: Thailand 2020. Available from: http://data.un.org/CountryProfile.aspx/_Images/CountryProfile.aspx?crName=Thailand. Accessed 22 January 2020.
10.United Nations. Country profile: Thailand 2020. Available from: http://data.un.org/CountryProfile.aspx/_Images/CountryProfile.aspx?crName=Thailand. Accessed 22 January 2020.
11.Princess Maha Chakri Siridhorn Anthropology center. Hill tribe. 2018. Available from: http://www.sac.or.th/main/index.php Accessed 22 January 2020.
12.Apidechkul T. Sexual behaviors and seroprevalence of HIV, HBV, and HCV among hill tribe youths of northern Thailand. BMC Public Health. 2019; 19(1101). 10.1186/s12889-019-7459-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Ministry of Education, Thailand. Education management information system: Chiang Rai Province, Thailand. Available from: https://data.bopp-obec.info/emis/school.php?Area_CODE=5703 Accessed 5 February 2020.
14.Wongprachum K, Sanchaisuriya K, Vidamaly V, Sorparisth S, Dethvongphanh M, Norcharoen B, et al. Pilot screening program for thalassemia in a country with limited resources: a collaboration model between close neighboring countries. Southeast Asian J Trop Med Public Health. 2016; 47(5): 1040–7. [PubMed] [Google Scholar]
15.Charan J, Biswas T. How to calculate sample size for different study design in medical research? Indian J Psycho Med. 2013; 35(2): 121–6. 10.4103/0253-7176.116232 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Fucharoen G, Fucharoen S. Thalassemia screening in Thailand. Jounral of Medical Technology and Physical Therapy. 2008; 20(3): 165–77. [Google Scholar]
17.Fucharoen G, Sanchaisuriya K, Sae-ung N, Dangwibul S, Fucharoen S. A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia. Bull World Health Organ. 2004; 82: 364–72. [PMC free article] [PubMed] [Google Scholar]
18.Sangkitporn S, Sankitporn S, Sangnoi A, Supagwiput O, Tanphaichitr VS. Validatio of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E. Southeast Asian J Trop Med Public Health. 2005; 36(6): 1538–42. [PubMed] [Google Scholar]
19.World Health organization (WHO). Management of haemoglobin disorders. Available from: https://www.who.int/genomics/WHO-TIF_genetics_final.pdf.
20.Ministry of Public Health, Thailand. Thailand national guideline of thalassemia and abnormal hemoglobin detection and diagnosis. Available from: http://e-library.dmsc.moph.go.th/ebooks/files/Guideline1.pdf.
21.Trisakul N, Siripulsak P, Chuesupalobol W. Prevalence of thalassemia, hemoglobinopathy, at-risk couples and incidence of thalassemia from the screening program prenatal and postnatal diagnosis at Yasothorn hopistal. J Hematol Transfus Med. 2008; 19: 285–92. [Google Scholar]
22.Chaibunruang A, Somkayasit K, Chewasateanchai M, Sanugul P, Fucharoen G, Fucharoen S. Prevalence of thalassemia among newborns: a revisited after 20 years of a prevention and control program in northeast Thailand. Mediterr J Hematol Infect Dis. 2018; 10(1): e2018054 10.4084/MJHID.2018.054 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Insiripong S, Yingsitsiri W, Boondumrongsagul J, Noiwatanakul J. prevalence of thalassemia traits in people without anemia or microcytosis. J Hematol Transfus Med. 2015; 24: 25–9. [Google Scholar]
24.Kittisares K, Palasuwan D, Noulsri E, Palasuwan A. Thalassemia trait and G6PD deficiency in Thai blood donors. Transfusion and Apheresis Science. 2019; 58(2): 201–6. 10.1016/j.transci.2019.03.009 [DOI] [PubMed] [Google Scholar]
25.Hockham C, Ekwattanakit S, Bhatt S, penman BS, Gupta S, Viprakasit V, et al. eLife. 2019; 8: e40580 10.7554/eLife.40580 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Buakhao J, Khumpun P, Winichahoon P, Fucharoen S, Paiboonsukwong K. Prevalence and characterization of thalassemia among migrant workers from Cambodia, Lao PDR and Myanmar in Thailand. Southeast Asian J Trop Med Public Health. 2017; 48(4): 846–74. [Google Scholar]
27.Lithanatudom P, Khampan P, Smith DS, Svasit S, Fucharoen S, Kanwanpong D, et al. The prevalence of alpha-thalassemia amongst Tai and Mon-Khmer ethnic groups residing in northern Thailand: a population-based study. Journal of Hematology. 2016; 21(8): 480–5. 10.1080/10245332.2016.1148374 [DOI] [PubMed] [Google Scholar]
28.Apidechkul T. Prevalence of thalassemia carriers among the Lahu hill tribe population, Chiang Rai, Thailand. Asian Biomedicine. 2015; 9(4): 527–33. [Google Scholar]
29.Yanola J, Kongpan C, Pornprasert S. Prevalence of anemia, iron deficiency, thalassemia and glucose-6-phospahte dehydrogenase deficiency among hill tribe school children in Omkoi District, Chiang Mai Province, Thailand. Southeast Asian J Trop Med Public Health. 2014; 45(4): 920–5. [PubMed] [Google Scholar]
30.Raza S, Farooqi S, Mubeen H, Shoaib MW, Jabeen S. Beta thalassemia: prevalence, risk and challenges. International Journal of Medicine and Health Research. 2016; 2(1): 5–7. [Google Scholar]
31.Laghmich A, Ismaili FZ, Barakat A, Nourouti NG, Khattab M, Mechita MB. Alpha-thalassemia in north Morocco: prevalence and molecular spectrum. BioMed Research International. 2019; 10.1155/2019/2080352 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Kadhim KA, Baldawi KH, Lami FH. Prevalence, incidence, trend, and complications of thalassemia in Iraq. Hemoglobin. 2017; 41(3): 164–8. 10.1080/03630269.2017.1354877 [DOI] [PubMed] [Google Scholar]
33.Sayani FA, Kwiatkowski JL. Increasing prevalence of thalassemia in America: implications for primary care. Journal of Annuals of Medicine. 2015; 47(7): 592–604. 10.3109/07853890.2015.1091942 [DOI] [PubMed] [Google Scholar]
34.Crighton G, Wood E, Scarborough R, Ho PJ, Bowden D. Hemoglobin disorders in Australia: where are now and where will we be in the future? International Medicine. 2016; 770–9. 10.1111/imj.13084 [DOI] [PubMed] [Google Scholar]
35.Musallam KM, Revella S, Vichinsky E, Rachmilewitz EA. Nontransfusion-dependent thalassemias. Hematological. 2013; 98(6): 833–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Muncie HL, Campbell JS. Alpha and beta thalassemia. American Family Physician. 2009; 80(4): 339–44. [PubMed] [Google Scholar]
37.Tantiworawit A, Tapanya S, Phrommintikul A, Saekho S, Rattarittamrong E, Norasetthada L, et al. Prevalence and risk factors for cardiac iron overload and cardiovascular complications among patients with Thalassemia in northern Thailand. Southeast Asian J Trop Med Public Health. 2016; 47(6): 1335–42. [PubMed] [Google Scholar]
38.Winichakorn P, Tantiworawit A, Rattanathammethee T, Hantrakool S, Chai-Adisaksopha C, Rattarittamrong E, et al. Anemia. 2015; 18: 10.1155/2015/793025 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Ekwattanakit S, Siritanaratkul N, Viprakasit V. A prospective analysis for prevalence of complications in Thai nontransfusion-dependent Hb E/β-thalassemia and α-thalassemia (Hb H disease). Am J Hematol. 2018; 93: 623–29. 10.1002/ajh.25046 [DOI] [PubMed] [Google Scholar]
40.Apidechkul T, Laingoen O, Suwannaporn S. Inequity in accessing health care service in Thailand in 2015: a case study of the hill tribe people in Mae Fah Luang district, Chiang Rai, Thailand. J Health Res. 2016; 30(1): 67–71. 10.14456/jhr.2016.10 [DOI] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0246736.r001

Decision Letter 0

Michela Grosso

Transfer Alert

This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present.

23 Jul 2020

PONE-D-20-14674

Epi-genetics of thalassemia among the hill tribe population in Thailand

PLOS ONE

Dear Dr. Apidechkul,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses all the the points raised during the review process, as reported below.

Please submit your revised manuscript by Sep 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Michela Grosso, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include additional information regarding the questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

3. Please state in your methods section the participant recruitment date.

4. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 7 in your text; if accepted, production will need this reference to link the reader to the Table.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Tawatchai Apidechkul et al. “Epi-genetics of thalassemia among the hill tribe population in Thailand” presents results of a genetic screening, aiming to estimate the prevalence of various forms of thalassemia among the six main hill tribe (minority) populations in Thailand. The issue of management of hemoglobinopathies patients is still apparent despite attempts to be controlled with prenatal testing and it is even nowadays one of the most common genetic diseases. The prevalence of all forms of thalassemia is high in countries with diverse ethnicity, high immigrant rate and in minority societies where consanguineous marriages are widely in practice. The early onset of the disease (few months after birth) and the application of expensive therapeutic protocols, including blood transfusions and chelating agents, adds an unendurable financial burden to healthcare systems.

This study appears to have been carefully performed and conclusions are relevant to the main scope of the study, the mandatory establishment of genetic counseling and prenatal diagnosis for hemoglobin disorders, especially in minority societies such as the hill tribe population in Thailand.

However, there are a number of major issues in the manuscript that need to be addressed before resubmission of revised version:

1) The term “epigenetics” in the title is irrelevant to the subject of this study. Epigenetics refers to biochemical alterations of the histone proteins or genome (DNA) or non-coding RNAs that differentially regulate gene expression levels.

2) The manuscript needs an extensive grammatical and syntactical editing. In its present form does not meet the criteria for publishing.

3) The authors need to be as precise as possible about how they did the genetic analysis:

a)The equation in the paragraph “sample size”, although its reference is given, its terms are not (even briefly) described.

b) The whole section of “materials and methods” is insufficient. The lab tests must be described and results obtained by each method performed, must be stated. Differences detected between lab methods and presented in the discussion section, must be explained and substantiated.

4) A figure presenting α-type and β-type globin genes with specific mutations highlighted from the study should accompany the manuscript to support the text and offer to the audience a better understanding of the data presented.

5) The discussion section is confusing. Authors display results of the study but significant and non-significant data are not distinguished, hereditary risk factors for the next generations are not clearly defined, the prognostic value of their results is not supported.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Argyro Sgourou

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Feb 11;16(2):e0246736. doi: 10.1371/journal.pone.0246736.r002

Author response to Decision Letter 0

7 Aug 2020

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

: Thank you, I have checked to ensure that the journal requirements are met.

: We added that both Thai and English versions of the questionnaire that we developed for this particular study were used.

3. Please state in your methods section the participant recruitment date.

: The information and method of selection is described in the “Data collection procedure” section. In brief, a simple random method was used to select the schools, and all students graded 4-6 in selected schools were eligible and invited to participate in the study after obtaining consent from their parents.

: Thank you, this has been clarified on page 6.

Comments to the Author

However, there are a number of major issues in the manuscript that need to be addressed before resubmission of revised version:

: Thank you for the comment. As my training background is in “epidemiology”, which is the study and analysis of the distribution, patterns and determinants of health and disease conditions in defined populations, and our work focuses on the genetics of thalassemia by using PCR and HPLC, we used the term of “Epi-genetics”.

However, the reviewer did not provide alternate phrasing, and we have changed the title to “Epidemiology of thalassemia among the hill tribe population in Thailand: a cross sectional study”, which seems to well reflect the content and STROBE guideline.

2) The manuscript needs an extensive grammatical and syntactical editing. In its present form does not meet the criteria for publishing.

: The manuscript has been revised twice by the American Journal Experts with the code 07A3-ECE2-5B78-98D6-6F9F .

we hope that the language has been improved.

3) The authors need to be as precise as possible about how they did the genetic analysis

: Thank you for the comment. We have added detail to the methods section.

a)The equation in the paragraph “sample size”, although its reference is given, its terms are not (even briefly) described.

: Thank you for the comment; this has been clarified.

: The details of the laboratory methods have been added to the “Laboratory test” section. The results in each step or method are clearly presented in Tables 4-7, including elaboration of the main findings in each section.

: In the discussion, we added to present the overall results and comparisons with other studies and populations, with possible reasons for differences. We also added information per your comments.

: Relevant information has been added to the introduction section (first paragraph)

: Thank you for the comment. However, the main aim of the study was to estimate the prevalence and the major form of thalassemia in each specific group of population, including sex and tribe. Therefore, for the main result of the study, we used the laboratory findings to present the situation of thalassemia among the hill tribe, which has never been reported. This is basic and pioneering scientific information regarding thalassemia in these populations. Detecting the risk factors for the next generation was not the main goal and is not to common because thalassemia is genetic-related disease.

We use the term of ‘epidemiology’ to refer to description (Descriptive epidemiology) the characteristics of the person (sex and tribe) and the proportion of different forms of thalassemia, which is a descriptive epidemiology focus.

However, in Tables 4 and 5, the chi-square test is presented to detect differences in the proportion on the OF and DCIP results among patients with different characteristics. The results indicate that females are more likely to have thalassemia than males and that those whose grandfather (father of mother) and grandmother (mother of mother) are of the same tribe were more likely to have thalassemia than those whose grandfather (father of mother) and grandmother (mother of mother) are from different tribes.

Moreover, α-thalassemia 1 was found at a higher rate in females than males, though β-thalassemia was higher among males than females.

We have heavily reviewed all the literature of different sources of medical papers, but unfortunately there is no articles regarding these points.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Kind regards,

Assist Prof. Dr. Tawatchai Apidechkul, MSc (Infectious Epidemiology), PhD (Epidemiology)

Deputy Dean, School of Health Science, MFU

Director, Center of Excellence of Hill Tribe Health Research, WHO-CC

Former Hubert H Humphrey Fellow (2013-2014), Emory University

Global Health Delivery Intensive (Harvard School of Public Health)

Attachment

Submitted filename: Journal_Requirements.docx

Click here for additional data file.^{(20.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0246736.r003

Decision Letter 1

Michela Grosso

2 Dec 2020

PONE-D-20-14674R1

Epidemiology of thalassemia among the hill tribe population in Thailand

PLOS ONE

Dear Dr. Apidechkul,

More in details, we encourage you to address all comments raised by reviewers #1, #3 and #4, particularly those regarding the Material and Methods Section.

Please submit your revised manuscript by Jan 16 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Michela Grosso, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #3: Partly

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #1: • The title of the study absolutely suits the text.

• The manuscript has been edited by an expert as authors claim, however, the text flow is tedious, specific points are still difficult for native speakers to follow, the analysis of the results are greatly extended, but with little necessity. The above mentioned issues reduce the readership for the manuscript and does not meet the criteria for publishing in PLOS ONE journal. Nevertheless, genetic analysis of existing minorities worldwide should gain attention due to high frequencies of pathological alleles observed among them.

• Carriers of pathological thalassemia alleles, should be identified as heterozygotes or double heterozygotes in case of the co-existence of two distinct alleles for thalassemia traits.

• In materials and methods the research outline should be presented clearly. The questionnaires cannot be considered as ”research instruments”.

• A list of abbreviations is needed.

• It would be more interesting to reduce the text in terms of appearing as a “letter to the editor” (than original article), with sharp analysis of results and clear definition of the consequent needs for constant genetic counseling and prenatal diagnosis for hemoglobin disorders in these minorities (hill tribe population of Thailand).

Reviewer #3: 1-I agree with all first reviewer comments, additionally, I have the following comments:

2-Materials and methods still need additions and clarifications ,starting from the manufacturer of all kits used[ KKU-OF reagent KKU-DCIP-Clear reagent kit ] instruments as well (e.g what was the brand name of qPCR used and manufacturers, HPLC machine( Variant II ??) .

3-OF and DCIP tests needs more clarifications ( as supplement to the paper probably ). These tests are not widely spread and the method of action needs to be mentioned. How the authors considered each of these tests results as positive or negative ? any references ? these are manual kits? ,Spectrometer used ? which wavelength ? Please write concise details about each tests to be more >

4- Since the above tests were the screening tests , we need to know the implication of each test . DCIP is mentioned by the authors to measure unstable Hbs, while no such Hb was mentioned in the results .I think HbE ais detected by this kit too.

5-Screening was performed using both tests as mentioned but figure -1- states OF or DCIP. Please clarify .

6- Did the authors followed previous guidelines regarding hemoglobinopathies screening and have chosen to organised their plan accordingly ? I am asking because I cant understand why after double positive tests, HPLC test was performed ? Why not after single positive tests for example ?

7- Abbreviations in Fig 1 need standardization . What did the author mean with A2A?

8-How could the authors identify HbBarts ?

9-What were the types of beta genes mutations reported in this study ? The authors have mentioned beta 0 and beta + mutation??

10-What was the abnormal Hb type detected (mentioned in Fig 1 ) please ?HPLC usually identify Hb structural variants like HbD, HbE ,HbO ,HbS,.......

11-What was the distribution of alfa-thalassemia SEA and Thai deletion among alfa thal minor cases detected ?

12-In Fig -1- the authors mentioned that qPCR was used to detect alfa thal mutations, while they have reported beta thal mutation and HbE as well ( beta structural variant ) .There are errors in the results of qPCR, some of them are mentioned in HPLC results too. Please re-arrange the whole Fig 1 .

13-i wished to see a comparison of alfa and beta thal mutations reported with surrounded districts or populations , or even the tribes included in the current study.

In conclusion ,

Material and methods needs a good revision, and results accordingly.

Reviewer #4: Comments for the author

This paper demonstrated the epidemiology of thalassemia among the hill tribe population in Thailand. The study showed differences in the prevalence of thalassemia and hemoglobinopathies among different tribes. The author also reported the relation between positive OF tests and the same-tribe grandparents. These are interesting findings reflecting the association between consanguineous marriage and thalassemia disease. I have the following comments on this paper:

1.The authors should clarify the remaining 32 patients who have positive OF tests with normal hemoglobin typing by excluding other conditions that may cause false-positive OF tests. It is interesting if the author can further test for alpha-thalassemia-2 (if not, please add this issue in the limitation part).

2.The author should demonstrate the “magnitude of the relation between thalassemia and the same-tribe grandparents” by using other statistical methods, for example, the logistic regression to show odds ratio (OR) and 95% CI.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Argyro Sgourou, SST HOU

PLoS One. 2021 Feb 11;16(2):e0246736. doi: 10.1371/journal.pone.0246736.r004

Author response to Decision Letter 1

5 Jan 2021

Response to reviewers’ comments

Dear editor and reviewers,

In this revised version we have revised and confirmed in whole section of laboratory results with consulting experts in the field. We also made more analysis according to the comment of reviewer no.4. We did improvement in telling the story in whole text particularly in first paragraph, and many paragraphs in the method and results sections. It’s also improved by the native speakers to make sure that it could be good enough for the journal.

I can confirm that all the points suggested from revisers have been carefully improved.

I do very hope very hope that you happy in this version.

Reviewer #1: • The title of the study absolutely suits the text.

: Thank you.

: The whole paper has been revised and improved by both authors, and the native speakers.

: We thank you for your comment here. Even it is very difficult for us to demonstrate an excellent English, but after carful revisions many times, we do very hope that you will happy in this version.

• Carriers of pathological thalassemia alleles, should be identified as heterozygotes or double heterozygotes in case of the co-existence of two distinct alleles for thalassemia traits.

: Thank you for the great comment. We have revised all terminologies and presented only essential forms in the whole text. However, if I did not make clear to response your comment, please clarify more. Thank you so much.

• In materials and methods the research outline should be presented clearly. The questionnaires cannot be considered as “research instruments”.

: Thank you, we agree with and it’s deleted. We have added laboratory procedures.

• A list of abbreviations is needed.

: We do not sure that the style of Ploseone allows us to present a list of abbreviation separately. However, we have double-checked all abbreviations are placed properly in whole text.

: Thank you for great comment. We have made improvement in whole text particularly in the first paragraph of introduction.

Reviewer #3:

1-I agree with all first reviewer comments, additionally, I have the following comments:

: We have carefully revised and improved all points on the first reviewers’ comments

2-Materials and methods still need additions and clarifications, starting from the manufacturer of all kits used [ KKU-OF reagent KKU-DCIP-Clear reagent kit] instruments as well (e.g what was the brand name of qPCR used and manufacturers, HPLC machine ( Variant II ??) .

: Thank you, all essential information have been placed. Page No.5,

: Variant-II was used for HPLC. Page 5

3-OF and DCIP tests needs more clarifications (as supplement to the paper probably). These tests are not widely spread and the method of action needs to be mentioned. How the authors considered each of these tests results as positive or negative ? any references ? these are manual kits? ,Spectrometer used ? which wavelength ? Please write concise details about each tests to be more.

: Thank you so much for the great comment. We have put extended information relevant to these methods.

: We have added detail of these procedures at page 5 with references.

4- Since the above tests were the screening tests, we need to know the implication of each test. DCIP is mentioned by the authors to measure unstable Hbs, while no such Hb was mentioned in the results. I think HbE ais detected by this kit too.

: Thank you for the comment. At the stage of using either OF or DCIP, we did not intend to make interpretation since there were screening tests.

: Bascially, OF test intends to screen for alpha-thalassemia-1 and beta-thalssemia, while DCIP is screened for Hb E. Then, in our study, after having a positive of either one or both were confirmed by HPLC and PCR before making interpretation.

:If OF+ only, it’s suspected to be alpha-thalassemia and/ or betha-thalssemia, while having DCIP+ only, it’s suspected to be Hb E trait. If having positive in both tests, it could be suspected Hb E with or without alpha-thalassemia, and/or b-thalassemia.

5-Screening was performed using both tests as mentioned but figure -1- states OF or DCIP. Please clarify.

: Sorry for the mistake, it’s correct. Specimens were tested by both tests.

: Positive to either OF or DCIP, or both OF and DCIP were tested by HPL and PCR.

7- Abbreviations in Fig 1 need standardization. What did the author mean with A2A?

: Thank you, there have been made clarification all abbreviations.

: HbA which is adult hemoglobin, hemoglobin A1 or �2�2.

: HbA is the most common adult form of hemoglobin and exist as tetramer containing two alpha subunits and two beta subunits (�2�2)

: HbA2 is a less common adult form of hemoglobin and is composed of two alpha and two delta.

: A2A is a normal variant of hemoglobin A that consists of two alpha and two delta chains (�2�2) and is found at low levels in normal human blood.

8-How could the authors identify HbBarts ?

: By doing Hb typing in the HPLC method, those who have presented the low Hb A2 level due to having Hb H (�4) or Hb Bart’s (�4) which is few of �-globin then the �-globin and �-globin remain a large portion and catching each other to obtain 4 lines. Then while we doing HPLC, the band of Hb Barts is presented.

9-What were the types of beta genes mutations reported in this study ? The authors have mentioned beta 0 and beta + mutation??

: It could be (β0/β) or (β+/β)

: However, in our current method, we could not identify in specific genes mustation. Sorry.

10-What was the abnormal Hb type detected (mentioned in Fig 1 ) please ?HPLC usually identify Hb structural variants like HbD, HbE ,HbO ,HbS,.......

: Thank you for the notice.

: A case of abnormal Hb type was detected by HPLC with detail; MCV=73.4lf, positive OF, positive DCIP, 46.0%HbAo, 3.0%HbA2/E, 0.5%HbF, A2A Hb typing with abnormal Hb, and negative for alpha-thalassemia (SEA and Thai deletion).

11-What was the distribution of alfa-thalassemia SEA and Thai deletion among alfa thal minor cases detected?

: Only alpha-thalassemia 1 trait (SEA deletion) was found, data presented in table 6 and 7.

12-In Fig -1- the authors mentioned that qPCR was used to detect alfa thal mutations, while they have reported beta thal mutation and HbE as well (beta structural variant) .There are errors in the results of qPCR, some of them are mentioned in HPLC results too. Please re-arrange the whole Fig 1 .

: Thank you for great comment, we have made revised in whole structure in figure no. 1, and also in the context.

13-i wished to see a comparison of alfa and beta thal mutations reported with surrounded districts or populations, or even the tribes included in the current study.

: It’s reported in the reference no. 21 and 28. There was very few publications on the hill tribe people in Thailand. One more study reported on beta thalassemia among the hill tribe school children (Ref. No. 29).

In conclusion ,

Material and methods needs a good revision, and results accordingly.

: Thank you very much for the great comments. We have revised improved a large scale particularly in extending the content of OF and DCIP including other information as comments.

Reviewer #4: Comments for the author

: I have careful looked into your point, unfortunately I do not see the particular point. From Table 6, 117 of 1,200 cases have OF+/DCIP+/ or OF+ and DCIP+.

: In total, 97 cases of OF+; 79 case A2A, 1 case EE, 15 cases EA, 1 case A2A Barts H, and 1 case A2A with abnormal Hb.

: another 4 cases of A2A have OF- but DCIP+.

: Therefore, all OF positives have presented some form of Hb disdorder.

: 30 cases of alpha thalassemia 1 (SEA deletion), 29 cases have A2A (Hb A2<3.5%), and one case has A2A Bart’s H.

: However, if I missed the point please kindly let me know.

: Thank you, we have used logistic regression to see the magnitude of having OF+ or DCIP+ or positive both OF and DCIP in different models. We have added the information into the result (Page 18-19) and discussion sections.

Thank you,

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(21.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0246736.r005

Decision Letter 2

Michela Grosso

26 Jan 2021

Epidemiology of thalassemia among the hill tribe population in Thailand

PONE-D-20-14674R2

Dear Dr. Apidechkul,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Michela Grosso, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: I appreciate the authors efforts in correcting the whole work.One minor comment please; the laboratory section in MM needs language editing please .

Thank you

Reviewer #4: This is a large epidemiologic study of thalassemia among the hill tribe population. The author showed differences in the prevalence of thalassemia and hemoglobinopathies among different tribes. The author demonstrated the relation between positive OF tests and the same-tribe grandparents. These findings reflected the association between consanguineous marriage and thalassemia disease. The author has addressed all comments. I have no further comment.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #3: No

Reviewer #4: No

Attachment

Submitted filename: Reviewer attachment.docx

Click here for additional data file.^{(11.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0246736.r006

Acceptance letter

Michela Grosso

29 Jan 2021

PONE-D-20-14674R2

Epidemiology of thalassemia among the hill tribe population in Thailand

Dear Dr. Apidechkul:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Michela Grosso

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Appendix. Questionnaire used in the study (Thai).

(PDF)

Click here for additional data file.^{(81.9KB, pdf)}

S2 Appendix. Questionnaire used in the study (English).

(PDF)

Click here for additional data file.^{(73.6KB, pdf)}

S3 Appendix. Data file of the study.

(SAV)

Click here for additional data file.^{(172.9KB, sav)}

Attachment

Submitted filename: Journal_Requirements.docx

Click here for additional data file.^{(20.9KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(21.7KB, docx)}

Attachment

Submitted filename: Reviewer attachment.docx

Click here for additional data file.^{(11.5KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0246736.ref001] 1.Centers for Disease Control and Prevention (CDC). Thalassemia. Available from. https://www.cdc.gov/ncbddd/thalassemia/facts.html Accessed 11 January 2020.

[pone.0246736.ref002] 2.Angastiniotis M, Lobitz. Thalassemia: an overview. International Journal of Neonatal Screening. 2019; 5(16): 10.3390/ijns5010016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref003] 3.Tari K, Ardalan PV, Abbaszadehdibavar M, Atashi A, Jalili A, Gheidishahran M. Thalassemia an update: molecular basis, clinical features and treatment. International Journal of Biomedicine and Public Health. 2018; 1(1): 48–58. [Google Scholar]

[pone.0246736.ref004] 4.Taher A, Weatherall DJ, Cappellini MD. Thalassemia. The Lancet. 2018; 391(10116): 156–67. [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref005] 5.World Health Organization (WHO). Global epidemiology of hemolobin disorders and derived service indicators. Available from: https://www.who.int/bulletin/volumes/86/6/06-036673/en/. Accessed 11 January 2020.

[pone.0246736.ref006] 6.Ministry of Public Health Thailand. Thalassemia in Thailand: 2018. Available from: https://pr.moph.go.th/?url=pr/detail/all/02/116500 Accessed 17 January 2020.

[pone.0246736.ref007] 7.Ministry of Public Health Thailand. Thalassemia in Thailand: 2018. Available from: https://pr.moph.go.th/?url=pr/detail/all/02/116500. Accessed 17 January 2020.

[pone.0246736.ref008] 8.National Health Security Office (NHSO). Thalassemia care and treatment for Thai population. Available from: https://www.nhso.go.th/FrontEnd/NewsInformationDetail.aspx?newsid=MTQyOQ. Accessed 17 January 2020.

[pone.0246736.ref009] 9.United Nations. Country profile: Thailand 2020. Available from: http://data.un.org/CountryProfile.aspx/_Images/CountryProfile.aspx?crName=Thailand. Accessed 22 January 2020.

[pone.0246736.ref010] 10.United Nations. Country profile: Thailand 2020. Available from: http://data.un.org/CountryProfile.aspx/_Images/CountryProfile.aspx?crName=Thailand. Accessed 22 January 2020.

[pone.0246736.ref011] 11.Princess Maha Chakri Siridhorn Anthropology center. Hill tribe. 2018. Available from: http://www.sac.or.th/main/index.php Accessed 22 January 2020.

[pone.0246736.ref012] 12.Apidechkul T. Sexual behaviors and seroprevalence of HIV, HBV, and HCV among hill tribe youths of northern Thailand. BMC Public Health. 2019; 19(1101). 10.1186/s12889-019-7459-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref013] 13.Ministry of Education, Thailand. Education management information system: Chiang Rai Province, Thailand. Available from: https://data.bopp-obec.info/emis/school.php?Area_CODE=5703 Accessed 5 February 2020.

[pone.0246736.ref014] 14.Wongprachum K, Sanchaisuriya K, Vidamaly V, Sorparisth S, Dethvongphanh M, Norcharoen B, et al. Pilot screening program for thalassemia in a country with limited resources: a collaboration model between close neighboring countries. Southeast Asian J Trop Med Public Health. 2016; 47(5): 1040–7. [PubMed] [Google Scholar]

[pone.0246736.ref015] 15.Charan J, Biswas T. How to calculate sample size for different study design in medical research? Indian J Psycho Med. 2013; 35(2): 121–6. 10.4103/0253-7176.116232 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref016] 16.Fucharoen G, Fucharoen S. Thalassemia screening in Thailand. Jounral of Medical Technology and Physical Therapy. 2008; 20(3): 165–77. [Google Scholar]

[pone.0246736.ref017] 17.Fucharoen G, Sanchaisuriya K, Sae-ung N, Dangwibul S, Fucharoen S. A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia. Bull World Health Organ. 2004; 82: 364–72. [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref018] 18.Sangkitporn S, Sankitporn S, Sangnoi A, Supagwiput O, Tanphaichitr VS. Validatio of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E. Southeast Asian J Trop Med Public Health. 2005; 36(6): 1538–42. [PubMed] [Google Scholar]

[pone.0246736.ref019] 19.World Health organization (WHO). Management of haemoglobin disorders. Available from: https://www.who.int/genomics/WHO-TIF_genetics_final.pdf.

[pone.0246736.ref020] 20.Ministry of Public Health, Thailand. Thailand national guideline of thalassemia and abnormal hemoglobin detection and diagnosis. Available from: http://e-library.dmsc.moph.go.th/ebooks/files/Guideline1.pdf.

[pone.0246736.ref021] 21.Trisakul N, Siripulsak P, Chuesupalobol W. Prevalence of thalassemia, hemoglobinopathy, at-risk couples and incidence of thalassemia from the screening program prenatal and postnatal diagnosis at Yasothorn hopistal. J Hematol Transfus Med. 2008; 19: 285–92. [Google Scholar]

[pone.0246736.ref022] 22.Chaibunruang A, Somkayasit K, Chewasateanchai M, Sanugul P, Fucharoen G, Fucharoen S. Prevalence of thalassemia among newborns: a revisited after 20 years of a prevention and control program in northeast Thailand. Mediterr J Hematol Infect Dis. 2018; 10(1): e2018054 10.4084/MJHID.2018.054 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref023] 23.Insiripong S, Yingsitsiri W, Boondumrongsagul J, Noiwatanakul J. prevalence of thalassemia traits in people without anemia or microcytosis. J Hematol Transfus Med. 2015; 24: 25–9. [Google Scholar]

[pone.0246736.ref024] 24.Kittisares K, Palasuwan D, Noulsri E, Palasuwan A. Thalassemia trait and G6PD deficiency in Thai blood donors. Transfusion and Apheresis Science. 2019; 58(2): 201–6. 10.1016/j.transci.2019.03.009 [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref025] 25.Hockham C, Ekwattanakit S, Bhatt S, penman BS, Gupta S, Viprakasit V, et al. eLife. 2019; 8: e40580 10.7554/eLife.40580 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref026] 26.Buakhao J, Khumpun P, Winichahoon P, Fucharoen S, Paiboonsukwong K. Prevalence and characterization of thalassemia among migrant workers from Cambodia, Lao PDR and Myanmar in Thailand. Southeast Asian J Trop Med Public Health. 2017; 48(4): 846–74. [Google Scholar]

[pone.0246736.ref027] 27.Lithanatudom P, Khampan P, Smith DS, Svasit S, Fucharoen S, Kanwanpong D, et al. The prevalence of alpha-thalassemia amongst Tai and Mon-Khmer ethnic groups residing in northern Thailand: a population-based study. Journal of Hematology. 2016; 21(8): 480–5. 10.1080/10245332.2016.1148374 [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref028] 28.Apidechkul T. Prevalence of thalassemia carriers among the Lahu hill tribe population, Chiang Rai, Thailand. Asian Biomedicine. 2015; 9(4): 527–33. [Google Scholar]

[pone.0246736.ref029] 29.Yanola J, Kongpan C, Pornprasert S. Prevalence of anemia, iron deficiency, thalassemia and glucose-6-phospahte dehydrogenase deficiency among hill tribe school children in Omkoi District, Chiang Mai Province, Thailand. Southeast Asian J Trop Med Public Health. 2014; 45(4): 920–5. [PubMed] [Google Scholar]

[pone.0246736.ref030] 30.Raza S, Farooqi S, Mubeen H, Shoaib MW, Jabeen S. Beta thalassemia: prevalence, risk and challenges. International Journal of Medicine and Health Research. 2016; 2(1): 5–7. [Google Scholar]

[pone.0246736.ref031] 31.Laghmich A, Ismaili FZ, Barakat A, Nourouti NG, Khattab M, Mechita MB. Alpha-thalassemia in north Morocco: prevalence and molecular spectrum. BioMed Research International. 2019; 10.1155/2019/2080352 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref032] 32.Kadhim KA, Baldawi KH, Lami FH. Prevalence, incidence, trend, and complications of thalassemia in Iraq. Hemoglobin. 2017; 41(3): 164–8. 10.1080/03630269.2017.1354877 [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref033] 33.Sayani FA, Kwiatkowski JL. Increasing prevalence of thalassemia in America: implications for primary care. Journal of Annuals of Medicine. 2015; 47(7): 592–604. 10.3109/07853890.2015.1091942 [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref034] 34.Crighton G, Wood E, Scarborough R, Ho PJ, Bowden D. Hemoglobin disorders in Australia: where are now and where will we be in the future? International Medicine. 2016; 770–9. 10.1111/imj.13084 [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref035] 35.Musallam KM, Revella S, Vichinsky E, Rachmilewitz EA. Nontransfusion-dependent thalassemias. Hematological. 2013; 98(6): 833–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref036] 36.Muncie HL, Campbell JS. Alpha and beta thalassemia. American Family Physician. 2009; 80(4): 339–44. [PubMed] [Google Scholar]

[pone.0246736.ref037] 37.Tantiworawit A, Tapanya S, Phrommintikul A, Saekho S, Rattarittamrong E, Norasetthada L, et al. Prevalence and risk factors for cardiac iron overload and cardiovascular complications among patients with Thalassemia in northern Thailand. Southeast Asian J Trop Med Public Health. 2016; 47(6): 1335–42. [PubMed] [Google Scholar]

[pone.0246736.ref038] 38.Winichakorn P, Tantiworawit A, Rattanathammethee T, Hantrakool S, Chai-Adisaksopha C, Rattarittamrong E, et al. Anemia. 2015; 18: 10.1155/2015/793025 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246736.ref039] 39.Ekwattanakit S, Siritanaratkul N, Viprakasit V. A prospective analysis for prevalence of complications in Thai nontransfusion-dependent Hb E/β-thalassemia and α-thalassemia (Hb H disease). Am J Hematol. 2018; 93: 623–29. 10.1002/ajh.25046 [DOI] [PubMed] [Google Scholar]

[pone.0246736.ref040] 40.Apidechkul T, Laingoen O, Suwannaporn S. Inequity in accessing health care service in Thailand in 2015: a case study of the hill tribe people in Mae Fah Luang district, Chiang Rai, Thailand. J Health Res. 2016; 30(1): 67–71. 10.14456/jhr.2016.10 [DOI] [Google Scholar]

PERMALINK

Epidemiology of thalassemia among the hill tribe population in Thailand

Tawatchai Apidechkul

Fartima Yeemard

Chalitar Chomchoei

Panupong Upala

Ratipark Tamornpark

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Materials and methods

Study design

Study population

Study sample

Sample size

Data collection procedure

Laboratory tests

Fig 1. Steps of data collection and laboratory analysis.

Statistical analysis

Ethical considerations

Results

Table 1. General characteristics of the participants.

Table 2. Medical history.

Table 3. Comparisons of mean MCV, MCH, Hb and Hct levels between thalassemia carriers and nonthalassemia carriers.

Table 4. Comparisons of OF and DCIP tests by tribe and sex.

Table 5. Proportions of OF and DCIP results in preceding generations.

Table 6. All laboratory results.

Table 7. Proportions of Hb typing and thalassemia carriers by tribe.

Table 8. Logistic regression analysis in identifying the associations between the difference of tribes and OF and/or DCIP positives.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Michela Grosso

Roles

Transfer Alert

Author response to Decision Letter 0

Decision Letter 1

Michela Grosso

Roles

Author response to Decision Letter 1

Decision Letter 2

Michela Grosso

Roles

Acceptance letter

Michela Grosso

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases