MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY NGUYEN KIEU GIANG EPIDEMIOLOGICAL AND MUTATION GENE OF THALASSEMIA IN TAY WOMEN AND INTERVENTION SOLUTIONS AT COMMUNITIES, DINH HOA DISTRICT, THAI NGUYEN PROVINCE Specialization: Social Hygiene and Health Organization Code: 62 72 01 64 THAI NGUYEN - 2019 MINISTRY OF EDUCATION AND TRAINING THAI NGUYEN UNIVERSITY NGUYEN KIEU GIANG EPIDEMIOLOGICAL AND GENE OF THALASSEMIA IN TAY WOMEN AND INTERVENTION SOLUTIONS AT COMMUNITIES, DINH HOA DISTRICT, THAI NGUYEN PROVINCE Specialization: Social Hygiene and Health Organization Code: 62 72 01 64 Science instructor 1: Associate Professor Nguyen Tien Dung Science instructor 2: Professor Hoang Khai Lap THAI NGUYEN - 2019 INTRODUCTION TO DOCTORAL THESIS Necessity of the thesis Thalassemia is a haemolytic anemia that is inherited due to abnormalities in the synthesis of hemoglobin resulting in anemia at different levels According to the World Health Organization (WHO), about 7% of the world's population carries the Thalassemia gene, with between 300,000 and 400,000 newborns with severe hemolytic anemia In Vietnam, according to incomplete statistics, more than 12 million people are carrying the gene for thalassemia common in all provinces nationwide In particular, the proportion of people carrying the thalassemia gene in ethnic minority groups is higher than the Kinh majority With 53 ethnic groups with genotypes, the frequency of mutant genes may vary among ethnic groups, the study of the frequency of gene transfer in each ethnic group is essential for the development of the thalassemia prophylaxis program in Viet Nam Among ethnic minorities in Vietnam, the Tay ethnic group has a population of more than 1.6 million, topping the top 10 ethnic groups in the country The rate of 13.2% carrying the thalassemia gene in the Tay group migrated to the south was reported in 2010 However, the frequency of gene transfer, mutant genotype and frequency of alleles in the East group in East North has not been fully reported Thai Nguyen is a province in the midland and mountainous area of the North East, inhabited by a number of ethnic minority groups, of which the highest proportion is the Tay ethnic group However, there is no consistent data on the incidence of disease genes and mutant genotypes, as these are very useful information for thalassemia prevention in the region In addition, the development and testing of a preventive screening program - counseling at the grassroots level is rarely studied in Vietnam Objectives Identification of some epidemiological and moleculer epidemiological characteristics of thalassemia in Tay ethnic minority women in the reproductive age in Dinh Hoa district, Thai Nguyen province in 2015 Experiment and evaluate the effectiveness of thalassemia screening, monitoring and counseling in Dinh Hoa district primary health care Practical signification and new contributions of the thesis New contributions to science Application of molecular biology assay to analyze the rate of hemoglobin in determining the frequency of carrying thalassemia gene among Tay ethnic group in Thai Nguyen area The 27% carrying gene thalassemia has never been reported in previous studies in this population In particular, the frequency of carrying alpha thalassemia gene was reported in the Tay group at the site of study at 16.3% Successfully developed and applied screening, counseling and prenatal diagnosis in thalassemia prophylaxis at communi health station Screening and counseling provided directly by the health staff of communi The process of transporting and receiving blood samples closely, the implementation of disease diagnosis, genotypic diagnosis at the provincial level has solved the problem of access and availability of the health system Contribute to the reality The identification of mutant genotypes and the frequency of alleles in the Tay population at the study sites were significant in the prevention of thalassemia This information will be used to develop a thalassemia diagnostic panel for the Northeastern region in general, to estimate the incidence of new heavy organisms in the population and to contribute data to the development of the genetic map thalassemia in Vietnam The intervention model for thalassemia is feasible with a contingency cost estimate of ~ 1% for a new case compared to the cost of treating patients with new thalassemia major The study has transferred the OF screening technique to Dinh Hoa district health center, transfer Transfer of thalassemia screening and counseling model for Dinh Hoa district Transfer of HbE screening technique to Thai Nguyen Blood Transfusion Center The structure of the thesis The thesis is presented in 121 pages, including: back ground (2 pages), overview (40 pages), subjects and research methods (17 pages), research results (23 pages), discussion (35 pages), conclusions (2 pages), recommendations (1 pages) The thesis consists of 37 table, 22 picture In the references 105, 80 documents in English, 25 documents in Vietnamese, 42 documents published in the last years (70 documents published in the last 10 years) The appendix includes the data collection tools, the sample list and the intervention images of the research team CHAPTER I LITERATURE OVERVIEW 1.1 The concept of thalassemia The term "thalassemia" or "haemolytic haemorrhage" refers to a group of pathological conditions characterized by a reduction in the synthesis of one of the two globin chains (α or β) that constitutes the normal human hemoglobin molecule (HbA, α2β2), resulting in a reduction in the hemoglobin synthesis in the endothelium, which is manifested externally by anemia 1.2 Genetic mechanism of thalassemia According to the inheritance characteristics of chromosomal recesses and the status of disease in the previous life, there are five cases of hereditary diseases to the next In cases where both parents have homozygous disease, 100% of cases are homozygous In case of a homozygous, a heterozygote: 50% of children with homozygotes, 50% of children with heterozygote In cases both perents have heterozygous: 50% of heterozygous children, 25% of normal children, 25% of homozygouts In case of an ordinary person, a heterozygote: 50% of children with heterozygosity, 50% of normal children In case of an ordinary person, a homosexual: 100% of children have heterozygous disease 1.3 Some epidemiological problems of thalassemia Characteristics of age groups: The different forms of disease manifested and affected in age groups are different Depending on age and other clinical characteristics, the physician may preliminarily diagnose the condition so that a proper diagnosis is identified for the diagnosis Age, gender is also one of the criteria to consider when developing a thalassemia screening program Ethnic characteristics: Many studies in the world claim that ethnic minority groups tend to carry genes much higher than that of majority ethnic groups This is explained by the epidemiological and cultural characteristics of ethnic minority groups Ethnic minorities also tend to communicate with the same locality and with the language group, which involves geographic proximity This is one of the factors that increase the rate of thalassemia gene in ethnic minority communities Gender: Gender is considered in the context of anemia and the complications of anemia caused by thalassemia Both male and female clinical conditions are affected by anemia and similar complications However, due to the specificity of pregnancy only in women, pregnant women with thalassemia have many health risks, risk during birth as well as postpartum complications Migration: Thalassemia is the first known disease in the Mediterranean, where it is described as prevalent in epidemic-endemic countries such as North Africa, the Middle East, India, South China and Southeast Asia, however, today the disease is reported in most countries and regions around the world Studies indicate that inter-territorial migration is the cause of widespread and diversified genomes worldwide Inbreeding: Inbreeding is the marriage between people who have blood relations, usually between cousins or sometimes uncles and nieces Inbreeding leads to inbreeding, which increases the likelihood of two people carrying the same gene to marry, and increases the likelihood of thalassemia homozygosity in the offspring Estimating the incidence of newborn infants in the population: Hardy-Weinberg's law is used to calculate the number of children born with thalassemia in 1,000 live births The frequency of genotypes, estimated by the Hardy-Weinberg formula, is as follows: The frequency of genes is constant over generations and allele frequencies in the latter are always proportional to p2: 2pq: q2 The condition of this argument is: (1) no mutation occurs resulting in new allele; (2) all individuals have the same opportunity in marriage and reproduction; (3) no significant immigration from other fully populated populations 1.4 Studies on the epidemiology and molecularities of thalssemia 1.4.1 In the World According to the World Health Organization (WHO), about 7% of the world's population carries the Thalassemia gene, with between 300,000 and 400,000 newborns with severe hemolytic anemia Figure 1 Map of alpha and beta thalassemia mutations in the world 1.4.2 In Viet Nam Tablet 1 The contribution carriers thalassemia gene in Viet Nam Local -Ethnic Sampl es Rat e(%) Author Kinh (Hue) 1100 5,6 Phan Le Minh Tuan (2014) Kinh (An Giang) 1572 19, Pham Ngoc Dung (2011) 19, Phan Thi Thuy Hoa (2010) 7,0 12, O'Riordan, S (2010) Kinh (Quang Binh) 130 Kinh (Khanh Hoa) Tay, Dao (Thai Nguyen) Muong (Hoa Biinh) C’Tu, Ta Oi (Hue) 452 712 Vu Thi Bich Van (2010) 22, Duong Ba Truc (2010) 54 42, Nguyen Van Hoa (2013) 15, Phan Le Minh Tuan Phan Le Minh Tuan Ta Oi (Hue) 1100 C’Tu (Hue) 1100 (2014) 11, S’Tieng E de M’Nong 3917 266 551 (2014) 63, 32, O'Riordan, S (2010) (Khanh Hoa, Binh Phuoc) 24, 1.5 The screening and diagnostic tests of thalassemia 1.5.1 The screening tests of thalassemia Screening of thalassemia by OF test Screening of HbE by DCIP Screening of thalassemia by CBC test – MCV, MCH 1.5.2 The diagnostic tests of thalassemia Diagnostic the types of thalassemia by analysis the rate of hemoglobin typing Diagnostic the thalassemia mutation gene by moleculer analysis 1.6 Prevention of thalassemia Currently, there are three cases of thalassemia that are listed as preventive measures: • homozygous beta thalassemia, or Cooley disease • Thalassemia / HbE (beta thalassemia - HbE) • Homozygous alpha thalassemia, or Bart's Hydrops Fetalis 1.6.1 Approach to develop a thalassemia prophylaxis program Thalassemia affects three groups of people in the community: common people, people with thalassemia, people with thalassemia Prophylactic treatment with thalassemia should have a comprehensive impact on these groups to prevent the risk in these groups at the time of marriage After marriage and pregnancy, the diagnosis of fetal disease (CTS, pre-embryo transfer diagnosis ) is the last resort to be prevented The approach to prevention for each target group is different With the community in mind, when it is not yet known whether individual carriers carry genes, the first step is to test the gene carrying the gene There are many screening stages: screening through cord blood storage Film screening at school age, college students Humanitarian Blood Screening Clinic Periodic health screening Check the subject of marriage registration Screening pregnant women in the early months 1.6.2 Thalassemia Prevention Programs in the World Screening program for pregnant women Pre-Marriage Screening Program Other thalassemia prophylaxis programs 1.6.3 Research on screening and prenatal screening in Vietnam Vietnam has no national thalassemia prophylaxis program, thalassemia interventions are mainly focused on screening models, TSH A small number of authors propose interventions based on CT scans in community hospitals or community-based IEC (A)Figure Thalassemia screening procedures (B)Screening, counseling, prenatal diagnosis at at An Giang Hospital Tu Du Hospital, Ho Chi Minh City CHAPTER II SUBJECT AND METHODODOLOGY 2.1 Research subjects 2.1.1 Descriptive study Tay woman from 15-49 years old, marrieriedried and have child; Communes health workers intervene 2.1.2 Interventional studies (1) Health workers in 24 communes and Cho Chu town, Dinh Hoa district, Thai Nguyen province (2) Pregnant women under 12 weeks of age registered for management of pregnancy at the commune health stations (3) Husbands of pregnant women under 12 weeks of age who are registered for management of pregnancy at the commune health stations who are positive for OF screening 2.1.3 Location and time of study 2.1.3.1 Study location: Dinh Hoa district, Thai Nguyen province 2.1.3.2Time of the study: June 2015 to June 2017 and divided into two phases (1) Cross-sectional (2) Interventions (Figure 2.2) Figure Time of the study 2.1.4 Methodology 2.1.4.1 Research design Design of the cross sectional (Phase I) Design of Interventional studies (Phase 2) 2.1.4.2 Calculation of sample size, sample selection Use the method of calculating sample size for population and random sampling for crosssectional studies 300 Tay women were selected for descriptive study With intervention research on basic health staff, sample size and method of target selection, convenience 12 health workers in communes of Dinh Hoa district were allocated to the intervention group 36 health workers in the remaining 18 communes were allocated to the control group 2.1.4.3 Research indicators Group of epidemiological indicators: age, sex, occupation, near-geographical marriage, inblood marriage, economic Hematological indexes: RBC, Hb, HCT, MCV, MCH, Group screening tests: OF, DCIP Group of indicators K.A.P: point and classify knowledge, attitude, practice Indicators of communication: Number of consultations, number of consultations, number of counseling materials distributed, number of couples agreeing with counseling 2.1.4.4 Research content Descriptive study: To determine the proportion of thalassemia carriers gene, genotype, and frequency of alleles Tay minority ethnic women aged 15-49 in Dinh Hoa, Thai Nguyen were selected for the study Subjects were interviewed using a questionnaire that was available to collect 20 MCV&/or MCH (-) 137 90,13 Total 12 152 100 Comment: The compatibility between OF (+) screening test with MCV / MCH (+) screening is 84.15% Between OF (-) and MCV / MCH (-) is 90.13% OF screening can omit 9.87% compared to automatic blood formula testing machine, whereas OF can detect 15.85% more suspicious cases of thalassemia gene that MCV / MCH missed Table 30 Hb electrophoresis results of 13 couples at risk of having fetus with thalassemia Couples at risk Screening by Hb typing α CBC Risk of fetus β thalassemia thalassemia H Positive couple usband W s Positive ife A2A (HbA2>3,5%) A2A H Positive A2A W Positive A2A H Positive (HbA2>3,5%) A2A couple usband Positive ife H Positive Not heterozygous 50% β thal heterozygous 50% HbE heterozygous (HbE=25,8%) A2A α thal Not risk DHT** W Positive ife A Bart’s H-CS H Positive A2A couple usband not excluded homozygous Not Not risk excluded W s homozygous, EA couple usband Not excluded * W 25% β thal 50% β thal excluded ife excluded (HbA2>3,5%) couple usband s Not Positive A2A ife * The fetus with the alpha thalassemia gene has not been excluded ** If the husband does not carry the alpha gene, the child of this pair is 100% carrying the gene of which 50% α0 thal heterozygous, 50% HbCS heterozygous Comment: In 13 couples, they were tested for Hb electrophoresis, couples had abnormal results: pairs of both husband and wife carrying heterozygous beta thalassemia gene; couples of wives heterozygous beta thalassemia; pair of wives heterozygous HbE; pair of wives HbH-CS; The remaining pairs have normal results on Hb electrophoresis 12/13 pairs cannot rule out the risk of having a baby with alpha thalassemia 21 Figure Results of prenatal diagnosis of alpha and beta thalassemia mutations in fetuses at risk Table 32 Number of subjects consulted by health workers at screening stages Subjects consulted Consulting content Pregnant women C) in this case falsifies the end of set so that the alpha globin sequence is synthesized to be 31 a.a This structure is not stable and most of the formed HbCS decomposes, the rate in peripheral blood 0.05) when comparing the rate of knowledge acquisition between the control group (30.56%) and the intervention group (33.33) %) But after year of intervention, the knowledge point had a significant change between the control groups and the intervention, the rate achieved in the intervention group was 83.33% while in the control group was 36.11% The difference was statistically significant with p 0.05) This is consistent with the general situation in thalassemia prevention practice at the grassroots level Practical activities of grassroots health workers such as medical examination and treatment or communication organizations on diseases often associated with the targets of the higher levels entrusted to them, accompanied by funding allocated for activities However, with thalassemia, there has not been a written decision to determine which targets are assigned at the grassroots level Practicing thalassemia prophylaxis at the grassroots level of health care has absolutely no written guidance or guidance from higher levels The practice of health workers is only recorded in some stations with thalassemia patients Regarding the method of assessing the effectiveness of K.A.P change interventions, comparing knowledge points at the time before and after on the same object used The score K.A.P will be specified into a scale with opposing levels to perform a test of change according to good directions In this study, Mc Nermar test is recommended for use when comparing pre- and post-intervention rates on the same subject The difference evaluated above changes from not good / not well-achieved / achieved and vice versa, good / unsatisfactory / not yet good If the transition from good to good is higher than the opposite after intervention, and the difference is statistically significant, there is evidence to conclude that the intervention is effective 2.4.2 Effectiveness of output impact on the intervention object 29 2.4.2.1 Select the object Pregnant women less than 12 weeks were selected as the target audience (the target for the incidence of new births with thalassemia) of the intervention Selecting this group will help assess the effectiveness of prevention in the shortest time If you choose other subjects such as children, couples to register for marriage, then they must follow up until they get married and become pregnant to assess the new situation In addition, pregnant women are also those who have a high demand for health care for themselves and their babies Communication and counseling for this group will be more effective than children, adolescents or marriage registration groups and other age groups, which are the target groups for the thalassemia screening program Husbands of pregnant women who agree to screen for screening are also recruited into the study to ensure the determination of the genetic risk for the fetus is accurate 2.4.2.2 Develop prenatal screening and diagnosis program In this study, we developed and tested a closed screening intervention program based on recommendations from the Ministry of Health and successful models in other countries At the same time, there are some points to be adapted to the Vietnamese health system and some specific criteria at the study site According to our survey in Dinh Hoa, only the district hospital laboratory has sufficient facilities for thalassemia screening tests Meanwhile, the management of pregnancy is largely at the commune level Moreover, if screening (+), the subjects will have to go to the provincial health center to the electrophoresis test and the central level to genetic testing as well as CDTS This can lead to people who have to travel a lot, are expensive, have to take blood several times and wait for test results at each level This will certainly affect the accessibility and payment of people If the screening is a woman who is 3.5% is used to diagnose cases of carrying thalassemia gene β thalasemia The couple's HbA2 ratio in this study was greater than 3.5% so the couple was carrying the thalassemia beta gene Their children definitely have a 25% risk of getting beta thalassemia Results of genetic testing of couples and husbands with CD17 mutation (β0 heterozygous) and wife with mutation CD-28 (β + heterozygous) combined with mutation SEA (α0 heterozygous) Thus, the test indicators show that the couple is forced to prenatal diagnosis to make sure the status of the thalassemia gene is present in the fetus Genetic testing results again show that relying solely on Hb electrophoresis will miss the type of a0 thalassemia heterozygous in the wife, and may lead to inaccurate results in prenatal diagnosis Fortunately, in this case the husband does not carry the α0 thalassmia gene, the couple's child only needs to perform prenatal diagnosis for β thalassemia Due to the diagnosis of a serious fetal risk due to the β-28 / β17 phenotype, the Minisequencing technique was applied to sequence the condon position -28 and codon 17 Also to increase the Strict part for test results, GAP PCR technique for separate SEA mutations was performed for the fetus, although the fetus is completely incapable of homozygous α0 thalassemia Prenatal diagnosis results for the fetus show that the child does not inherit a mutation in the beta globin gene, but inherits the α0 thalassemia mutation from the mother (type SEA) The result of the combination of fetal genes is (β / β) | (- SEA / αα), so the fetus does not suffer from thalassemia major, this couple is completely assured of giving birth this time Previously, the first-born child of the couple suffered from thalassemia β thalassemia major, their child still had to receive iron daily blood transfusions With the result of the fetus carrying heterozygous - SEA will be useful information for couples in the prevention of thalassemia for their grandchildren, later on by pre-marital screening for adult children For grassroots health workers, the test information will help to monitor the cases of 31 carrying genes in the management area 2.4.2.5 Results of advisory communication In this study, communication behavioral change interventions, especially face-to-face counseling, are applied in the stages of the screening model Communication activities on diseases and preventive measures are carried out when women come to register for pregnancy management The objects will be communicated with basic knowledge about diseases, consequences and prevention Volunteers participating in the study will be screened and consulted with test results soon after The consultation is divided into stages depending on the results of couples at each stage During the 12 months of intervention, a total of 177 pregnant women under 12 weeks of pregnancy came to manage pregnancy All pregnant women voluntarily join the program after being communicated by health staffs Of these 68 pregnant women were positive for OF, the husbands of these women continued to be consulted for screening tests There are 11 cases of positive women with screening tests but the husband has not been screened simultaneously In which cases of husbands going to work away from home left cases of refusing to tests (Table 3.32) CONCLUSION Epidemiological characteristics, characteristics of thalassemia genes of the study subjects and the value of screening tests in the community Subjects of the study are Tay ethnic women aged 15-49 years, of which the age group of 25-34 accounts for the highest proportion (60.4%) The inbreeding rate is 3%; married within lifetime is 0.3% Most married close to geography, the marriage rate in the same district is 88% of which the same commune is (50%) Marriage rate in other districts is 12% The prevalence of anemia in the community is high (27.3%), of which minor anemia is 16.3%; Moderate anemia is 10.7% and major anemia is 0.3% Microcytic and hypochromic anemia with average MCV of 72.8 ± 10.58 fl, average MCH is 22.7 ± 3.95 pg The rate of sharing thalassemia gene among the Tay ethnic group in Dinh Hoa and Thai Nguyen was 27% In which alpha thalassemia accounted for the highest rate, 16.3%, beta thalassemia was 7.3%, HbE was 1.7% and alpha-beta thalassemia combination was 1.7% Diverse phenotypes and genotypes, phenotypic groups with 14 types of gene mutations have been identified including: SEA / αα; THAI / αα, αα / -α3.7, αα / αCSα; SEA / -α3.7, SEA / αCSα, β / β41 / 42, β / β17, β / β71 / 72, β / β-28, β / β26 HBE, SEA / αα; β / β17 SEA / αα; β / β41 / 42, αα / -α3.7; β / β17 types of single allele mutations were identified, including: - SEA (47.3%); THAI (1.1%); -α3.7 (9.9%); -αCSα (6.6%); Cd 41/42 (9.9%), CD 17 (4.4%), Cd71 / 72 (2.2%); Cd-28 (1.1%), Cd26 (5.5%) The burden of disease caused by thalassemia is estimated to be ~ 32/5000 babies living with major disease Married married couples in the same district of the same commune have a higher rate of carrying thalassemia genes (34.2%) than couples of other districts (22.2%) or in the same district of other communes (22 , 7%) The proportion of people with thalassemia gene in the anemia group was 67.1%, 11.9% in the anemic group The OF test has a good screening value for people with thalassemia gene well, sensitivity is 77.8%, specificity is 91.3%, predictive value (+) is 76.8%, predictive value ( -) is 91.7%, AUC is 0.846 If combined with DCIP for HbE screening, the combined thalassemia screening value of OF / DCIP increases markedly with sensitivity of 85.19%; specificity is 87.76%; (+) predictive value is 71.88%; the predictive value (-) is 94.12%; The AUC is 0.864 Effectiveness of thalassemia prevention intervention in the community 32 2.1 The K.A.P change result of commune health workers in the prevention of thalassemia disease 48 health workers in 24 communes / towns in Dinh Hoa district were intervened to change K.A.P to be able to implement thalassemia prevention program These officials have an average working time of 20 years Of which, 70.83% of health workers are women, 81.25% are ethnic minorities and over 80% are doctors or medicos After year of intervention, positive changes in K.A.P of health workers are very clear and effective Changing knowledge: Before the intervention, the rate of health workers with knowledge reached 30.3%, after the intervention, the rate increased to 80.3% The change from not yet reached is statistically significant (p