| 1. Background1.1 The thalassemia is frequently in GuangdongThalassemia is the mostly spreaded and affected in population in the word, and China is one of the higher prevalence countries of thalassemia. In the southern provinces of Changjiang River of China, the prevalence of thalassemia is higher, and the top two highest prevalence of thalassemia of provinces is Guangxi and Guangdong. There were many thalassemia carriers in all cities of Guangdong, and the highest prevalence of thalassemia of city were Meizhou, Shaoguan, Qingyuan, Zhaoqing and Yunfu, and the East Guangdong is a lower prevalence of thalassemia. Some sampled surveys showed that there were 11% of individuals carried thalassemia in Guangdong, and in some higher prevalence area, there was one carried thalassemia of five persons in Guangdong. The α-and β-thalassemia were the popular thalassemia types in adults in China, and there were 23 genotypes, and 6 genotypes of α-thalassemia,17 genotypes of β-thalassemia.1.2 The thalassemia is a serious public health problem in GuangdongThalassemia major is huge hazard to public health in high prevalence area. Most a-thalassemia major fetuses would dead in perinatal period for serious fetal hydrops, and a few of them would dead in several hours after birth, and it also would increase the risk of complications of obstetrics. β-thalassemia major case would have no symptom in foetal period and in three monthes after birth, but it would appear progressing anaemia symptom after 3-6 monthed fater birth, and it would needed lifelong blood transfusion or hematopoietic stem cell transplant, the children affected β-thalassemia major would died in 5 years without therapy. For the limitation of treatment resources and medical care assurance level, the mean age of β-thalassemia major children was only 5 years for many of them couldn’t get the regularly and timely treatment. At the same time, the society and the family with β-thalassemia major children would face huge economic burden, for per β-thalassemia major case needed about 10,000 RMB every year, and that would be raised with grow-up. Base on the law of genetics, there were about 8000 a-thalassemia major birth and 1000 β-thalassemia major birth every year, and it would cause more than 1000 million a year. The symptom of α-and β-thalassemia intermediate was variable in different individual, and some individuals have no affect but some individuals neended blood tranfusion.The thalassemia is recessive genetic disease, the most thalassemia carriers couples have no any affects themsleves, but the same type thalassemia carrier couples could cause serious health harm to their off-spring. Base on the law of genetics, evary pregnant of the same type thalassemia carrier couples would have 1/4 probability of thalassemia major, and 1/2 probability of thalassemia gene carrier, and 1/4 probability of no any thalassemia. The globle experience showed that the most important strategy was to avoid the birth of thalassemia major, and the key measure was before marriage or progestation or prenatal thalassemia screening and diagnosis, and induced abortion for thalassemia major. Therefore, the govnerment of Guangdong province would launch the prenatal thalassemia prevention program by finance in 2012.There was about 30 years of thalassemia prevention in Guangdong, but that prevention didn’t become a public health program before 2012, and only a few hospitals afforded prenatal thalassemia screening. There were no enough data of the epidemiology on thalassemia of each city, and the prevention ability on thalassemia of health service institution, and the known degree of thalassemia prevention knowledge in population. The reported data were limitaion, and not enough for policy make. Therefore, it was urgent need to survey the epidemiology and prevention status on thalassemia in every city of Guangdong, it was very portant for finding key points and fund budget. This study was to get the base data to instruct the implement of the thalassemia prevention program of Guangdong, and it was very important for public health.2. Purposes and meaning2.1 PurposesTo survey the distribution of prevalence and genotype of thalassemia and the status of prenatal prevention of thalassemia in Guangdong.2.2 MeaningTo provide evidences for implementing public health programe of thalassemia prevention in Guangdong.3. Study contents3.1 The status on epidemiology of thalassemia. Through the detection of globin gene mutation, to know about the prevalence of genotypes of thalassemia in reproductive age population in all 21 cities of Guangdong province.3.2 To evaluate the effection of commonly used indicators and different combined prenatal csreening strategy of thalassemia. To compare the sensitivity, specificity, positive predictive value, negative predictive value, ROC curve and cost-effects of them.3.3 The status on thalassemian prevention service in Guangdong. Through questionaire to survey the service ability and problem of thalassemia prenatal screening, diagnosis, and intervention of different level hospitals in different cities. To survey the knowledge and participation and it’s influence factors of thalassemia prenatal prevention in targeted population.4. Study method and procedure4.1 Survey targets and site4.1.1 Sampled method. Used stratified and random cluster sampling. We randomly sampled one county or district in each city of all 21 cities of Guangdong, and selected 3-5 hospitals in each sampled county or distric which the living birth more than 1000 per year, and than surveyed 750 households laboring in each sampled county or district.4.1.2 Studied targets4.1.2.1 The evaluation of status and screening indications of thalassemia:The targets of this study were pregnant women and husbands and their offsprings laboring in the sampled hospital from June to December 2012. The sampled size of each county and district was calculated by formula based on the rate of thalassemia carriers of 11%. The number of sampled size of each hospital was distributed based on the percent of birth numbers in the county and district.The included condition:(1) The household regiter place of one of the couples was Guangdong province. (2) Birthed in the sampled hospitals.The excluded condition:The individuals who refused to be surveyed.4.1.2.2 The service ability of thalassemia prevention:About 80 relative managers and medical workers in the sampled hospitals in each county and district.4.1.2.3 The investigation of service participations:To invest about 200 paired of couples of the maternal and married medical exam participations in the sampled hospitals in each county and district.4.2 Laboratory test4.2.1 Sample. During the period of labor in hospital, under known and consent situation, the 2-3 ml periphery venous blood and umbilical core venous blood, anticoagulation by EDTA, placed in 4℃ refrigerator, and transported to genetic and metabolic disease of Guangdong maternal and children hospital in 3 days for hemoglobin electrophoresis analysis and globin mutations gene detection.4.2.2 MCV and MCH test. The MCV and MCH test was did in the laboring hospital in 30 minutes after the blood was sampled by the blood corpuscle autoanalyser.4.2.3 Hemoglobin electrophoresis was did in one week after blood was sampled by Capillarys2 Flex Piercing made in Sebia company of French.4.2.4 Globin gene mutations detection. Twenty-three mutations of a-and P-globin gene were identified by liquichip array system of Luminex 200. Three deletions a-globin genes were the Southeast Asian deletion (-SEA), the rightward deletion (-α37) and the leftward deletion (-α42); Three point mutations of α-globin gene were Hb Constant Spring, Hb Quong Sze and Hb Westmead; Seventeen point mutations of β-globin gene were codons 41/42 (-TCTT),654,-29 (A>G),-28 (A>G), codons 71/72 (+A), codon 17 (A>T), codon 43 (G>T), Hb E [β326(B8)Glu'Lys, GAG>AAG or codon 26 (G>A)], codons 27/28 (+C), codon 31 (-C),-32 (C>A),-30 (T>C), codons 14/15 (+G), IVS-I-1 (G>T), IVS-I-5 (G>T), Int and Cap.Genomic DNA was extracted from all the blood samples using an automation system Lab-Aid 820 of Zee San Biotech Company (Xiamen, Fujian, China). Twenty-three mutations of α-and β-globin gene were identified by liquichip array system of Luminex 200 [USA, R&D company], and the procedures of designing probes, multiplex PCR, attachment of probes to microspheres, hybridization and analysis were finished by the system.4.3 Questionnaire investigation. The relative managers and medical personnels of the sampled hospitals were invested by self-fill questionnaire, and the service participations were invested by face to face ask.4.4 Statistical methodThe data were input by Epidata 3.0 and analysised by R 3.0.1 software (May 16, 2013, copyright 2013, R Foundation for Statistical Computing, Vienna, Austria). We used the packages of R 3.0.1 included "vcd", "car", "psych", "genetics", "pROC", "multcomp", "gplots", "MASS", "nnet" and "reshape". For the quantity data, we used mean, standard error, scatterplot, linear chart to describe their character, and to do the statistic test by t test, F test, correlate or linear regression, and the P value less than 0.05 was the significant statistic level. For the quality data, we used percentage, rate or ratio, pie chart, and bar chart to describe their character, and to do the statistic test by chi square test, rank sum test or Logistic regression analysis. The screening indicators were evaluated by ROC curve.The 95% confidence intervals (95% CI) of prevalence of gene were calculated by Bootstrap, and adjusted by the population data. Calculated the sensitivity, specificity, positive predictive value and negative predictive value to compare different methoded. Used Hardy-Weinberg-Equilibrium to test the frequency of allele gene.5. Quality control5.1 The questionaires and blood samples were unified encoded and printed.5.2 All the participants of survey must be unified trained.5.3 Each county or district had a chargeman to deal with the survey matters and check the questionaires. And 10% of questionaires were sampled to be checked again and the mistakes or missed items less than 35.5.4 Used the same sample tube and transported box, and recorded the samples send and reception information, and all blood samples were transported in 3 days and tested in 7 days.5.5 The blood routine test must be accepted the clinic laboratory test external quality control.5.6 The unqualified blood sample needed to be resampled or replaced by a similar targets’ blood sample in the same hospital.5.7 The data of questionaires were double inputed unified.6. Results6.1 There was a high prevalence of thalassemia carriers in pregnant couples and their offsprings in Guangdong, and the thalassemia prevention was serious.There was a high prevalence of thalassemia carriers in pregnant couples and their offsprings in Guangdong, and the prevalences of thalassemia carriers were different in different area and different household register population. Compared with Guangdong household register couples, the non-Guangdong household register couples had a lower prevalence of thalassemia carrier, but they also needed public service of thalassemia prevention. Our results shown that in the pregnant women and husbands and their offsprings, the prevalence of a-thalassemia carrier was 12.76% (1816/14230),11.31% (1611/14249) and 11.61% (1655/14254) respectively, the prevalence of β-thalassemia carrier was 3.91% (557/14230),3.68% (524/14249) and 3.74% (533/14254) respectively, the prevalence of α-with β-thalassemia carrier was 0.75%(107/14230) respectively,0.50% (71/14249) and 0.61% (259/14254) respectively; the prevalence of α-thalassemia intermediate was 0.28% (40/14230), 0.13%(18/14249) and 0.18% (25/14254) respectively, the prevalence of β-thalassemia intermediate was 0.01% (1/14230),0.02%(3/14249) and 0.03% (4/14254). There were no α-and β-thalassemia major in pregnant women and husbands, the prevalence of a-thalassemia major was 0.04% (5/14254) and the prevalence of β-thalassemia major was 0.01% (1/14254) in fetus and newborns. In 13284 pairs of couples,1.66% (221/13284) of them both carried a-thalassemia and 0.20% (27/13284) of them both carried p-thalassemia.In all detected 42733 individuals, the top five genotype of a-thalassemia was αα/-SEA6.33% (2707), αα/α3.7α3.29% (1407), αα/α42αl.l5% (493), αα/αwsa0.90% (386), and αα/αcsa0.31%(134) respectively, they accounted 95.99 (5127/5341) of all a-thalassemia; the top five genotype of β-thalassemia was βA/β41-421.74% (744), βA/P6541.12%(479), βA/β-280.61% (262), βA/β170.36%(154), βA/βE 0.11% (46) respectively, they accounted 89.96%(1873/1873) all β-thalassemia.The prevalence of α-, β-, and α-with β-thalassemia carriers, that was 7.75% (603/7781),1.89%(147/7781) and 0.19%(15/7781) respectively in East Guangdong, that was 13.99% (6439/6439),4.75% (306/6439) and 0.87% (56/6439)in West Guangdong, that was 14.46%(1441/9964),4.60% (458/9964) and 0.99% (99/9964)in Mountain Area of Guangdong, that was 11.52% (2137/18549),3.79% (703/18549) and 0.48% (2929/18549) in Pearl River Area of Guangdong. As to the prevalvence of α-and β-thalassemia carrier in every sampled area, Yangjiang 23.73%, Yunfu 23.34, Meizhou 20.41% , Heyuan 19.75% , Qingyuan 19.49%, Zhaoqing 19.47%, Maoming 18.14%, Jiangmen 17.66%, Shaoguan 17.04%, Zhuhai 16.81%, Huizhou 16.70%, Zhanjiang 16.50%, Foshan 15.82%, Shenzhen 15.51%, Dongguan 14.41%, Guangzhou 13.09%, Zhongshan 12.49%, Shanwei 10.80%, Chaozhou 10.20%, Jieyang 10.16%and Shantou 8.47%. In couples of household register in local city, and in other cities of Guangdong, and not in Guangdong, the prevalence of thalassemia carrier was 17.16%(3491/20349),16.04%(906/5647) and 11.64% (289/2483).6.2 The effect evaluation on different thalassemia screening indicators and combined screening strategy.6.2.1 There were good effect of MCV/MCH or Hb A2 for thalassemia screening, but still were a high risk of missed diagnosis.MCV and MCH were effective for thalassemia screening of pregnant couples, but the effect of different type of thalassemia was different, pregnant women and husbands should use different cutoff value of MCV/MCH for better effect. As to the area under ROC curve of MCV/MCH screening thalassemia carrier, a-thalassemia carrier, p-thalassemia carrier, a-thalassemia intermediate, a-thalassemia minor and intermediate, and p-thalassemia intermediate, that was 0.87/0.88、0.83/0.84、 0.91/0.91、0.92/0.97、0.92/0.93 and 0.94/0.98 in pregnant women respectively, that was 0.88/0.92,0.83/0.88,0.93/0.93,0.98/0.93,0.94/0.95 and 0.96/0.97 in husbands respectively. The best cutoff value of MCV/MCH for thalassemia screening of pregnant women was 79.45fL/25.15pg, and that of husbands was 83.75fL/28.05pg, they were different from the cutoff value in force of 82fL/27pg. There was a bad effect of MCV/MCH for thalassemia screening of newborns, and it’s area under ROC curve was 0.79/0.84, and the cutoff value was 101.95fL/25.65pg.The effects of Hb A2 screening different type thalassemia of couples were different, and the effects for p-thalassemia and a-thalassemia intermediate were better. The screening thalassemia of newborns needed combined usage of Hb A, Hb A2, Hb Bar’s and Hb F, the effect of screening P-thalassemia was better. As to the area under ROC curve of Hb A2 screening thalassemia carrier, a-thalassemia carrier, β-thalassemia carrier, a-thalassemia intermediate, a-thalassemia minor and intermediate, and p-thalassemia intermediate, that was 0.72,0.98,0.86,0.74 and 1.00 in pregnant women respectively, and that was 0.82,0.97,0.94,0.91 and 0.97 respectively. The cutoff of Hb A2 screening a-thalassemia, it was less than 2.45 or more than 3.75 in pregnant women, it was less than 2.55 and more than 3.45 in husbands; The cutoff of Hb A2 screening β-thalassemia, it was more than 3.25 in pregnant women and more than 3.45 in husband. As to the area under ROC curve of Hb A, Hb A2 and Hb Bar’s screening a-thalassemia was 0.35、0.54 and 0.35 respectively, as to the area under ROC curve of Hb A, Hb A2 and Hb F screening β-thalassemia was 0.91,0.52 and 0.10.6.2.2 There were better effects of parallel screening strategy or both couples screening, but the costs were higher.The result shown that there was limitation of single indicator for thalassemia screening, and the combined screening strategy should be used to thalassemia prenatal prevention. There were three strategy of prenatal thalassemia screening commonly used, they were MCV/MCH and Hb A2 serried or parallel screening based on pregnant women (SSPW or PSPW), and serried screening based on couples (SSC), the three strategy all were good effects for prenatal thalassemia screening. SSPW had the lowest cost but highest missed diagnosis risk, PSPW and SSC had better effect but higher cost, it was prefer to PSPW and SSC as the public strategy of thalassemia prenatal screening in the enough resource area. As to the missed diagnosis rate, sensitivity, specificity, positive predictive value and negative predictive value, the SSPW was 50.68%,49.32%,98.77%,40.37% and 99.14% respectively, PSPW was 11.76%,88.24%,85.13%,9.12% and 99.77% respectively, SSC was 11.31%,88.69%, 59.86%,3.60% and 99.68%. As to screening the couples who both carried α0-thalassemia or haematoglobin H disease, the missed diagnosis rate, sensitivity, specificity, positive predictive value and negative predictive value of SSPW was 17.46%,82.54%,98.35%,19.26% and 99.92% respectively, that of PSPW was 4.76%, 95.24%,88.18%,3.70% and 99.97%, that of SSC was 3.17%,96.83%,59.31%, 1.12% and 9.99%; And the missed diagnosis rate, the differ was significant between SSPW and PSPW (χ2=3.938, P=0.0472< 0.05), between SSPW and SSC (χ2=5.489, P=0.0191<0.05), the differ was not significant between PSPW and SSC (χ2=0.0, P=1.0>0.05). The mean fees for detecting one pair of couple who both carried α0-thalassemia or haematoglobin H disease, SSPW was 37049.23 RMB, PSPW was 50836.00 RMB, SSC was 40321.64 RMB.As to screening the couples who both carried β-thalassemia, the missed diagnosis rate, sensitivity, specificity, positive predictive value and negative predictive value of SSPW was 11.11%,88.89%,100.0%,100.00% and 99.98%, that of PSPW was 0.00%, 100.00%,95.55%,4.38% and 100.00%, that of SSC was 0.00%,100.00%,91.69%, 2.39% and 100.00%. The mean fees for detecting one pair of couple who both carried β-thalassemia, SSPW was 27166.40 RMB, PSPW was 49837.86 RMB, SSC was 45429.29 RMB.4.3 The status on the prevention of thalassemia in Guangdong6.3 There were low service ability and low availability of thalassemia prevention in Guangdong.6.3.1 There was insufficient prevention service of thalassemia in primary hospitals of Guangdong, the status and ability were significant different in different developed level area. In the surveyed hospital of Pearl River Area and non-Pearl River Area,51.92% and 28.85% of them offered genetic counseling,69.23% and 57.69% of them offered hemoglobin electrophoresis analysis,53.85% and 44.23% of them offered globin mutation gene detection. The number of health technical personnel and outpatient of gynecology and obstetrics were the key factors on whether the hospital offered genetic counseling or not. The number of outpatient of gynecology and obstetrics were the key factors on whether the hospital offered thalassemia screening or not.6.3.2 The training on thalassemia prevention for primary health personnel was not enough, some primary health personnel had a few mistake recognition of thalassemia prevention. In the 1833 surveyed health personnel,31.71% of gynaecology and obstetrics doctors,26.33% of pediatrics doctors,16.19% of laboratory technician, and 12.50% of ultrasound technician accepted the training of thalassemia prevention in last three year. In the interviewees, only 51.94% knew MCV/MCH could screening thalassemia,45.50% knew hemoglobin electrophoresis could screening thalassemia,55.65% knew the fetus of couples who both carried same type of thalassemia,6.71% thought that couldn’t avoid the birth of thalassemia major,10.09% didn’t know whether ould avoid the birth of thalassemia major or not, 92.80% thought that the key strategy was to aviod the birth of thalassemia major, 54.12% didn’t know the prevalence of thalassemia carrier in Guangodng. In 557 obstetrician, only 37.88% of them knew take chorionic villi for biopsy in 11-14 gestation week,37.88% of them knew take amniotic fluid for prenatal diagnosis after 16 gestation week,35.73% knew take umbilical vein blood for prenatal diagosis after 23 gestation week.6.3.3 In the participations of married and prenatal medical test, there was a low percent of knew correct knowledge of thalassemia prevention, that affected their attitude and compliance of thalassemia prevention. In the 5790 surveyed individuals, 27.69% didn’t know thalassemia. In the surveyed individuals who knew the thalassemia, only 75.93% thought thalassemia was genetic disease,33.25% thought thalassemia could be cured,67.44% thought the thalassemia could be avoided to birth (but 14.37% of them thought the way of avoid birth was take medicine, and 8.54% of them thought that was vaccination), only 31.23% thought the fetus of couples who both carried the same type of thalassemia needed prenatal diagnosis,18.15% didn’t knew the best time to do thalassemia prevention, only 26.70% got the thalassemia knowledge from doctors. In the interviewees,21.00% thought it wasn’t necessary to do thalassemia prenatal screening, only 68.44% accepted the advice of thalassemia prenatal screening during the married medical test,28.01% didn’t will to accept free thalassemia screening, only 68.64% thought the thalassemia major needed induced abortion,15.53% thought thalassemia major fetus shoud be born and reared by government and society. In who knew the thalassemia and didn’t knew the thalassemia, 13.06% and 41.73% thought it wasn’t necessary to do thalassemia screening,70.72% and 58.64% accepted the advice of doctor of thalassemia screening. In the interviewees from Pearl River Area, East Guangdong, West Guangdong, Mountain Area, the percent of knew the thalassemia was 82.62%,51.02%,71.63% and 66.78%. In addition, the cognitive dissonance of thalassemia of paired maled and female interviewees could affect the compliance of thalassemia prenatal screening.7. Conclusions7.1 The thalassemia was a major public health problem in Guangdong, and there was a high prevalence of thalassemia carriers and inherited down to the next generation, the thalassemia prevention needed long-term strategy and budget of goverment.7.2 The effects of MCV/MCH or Hb A2 for thalassemia screening were good, and the parrallel screening or both couples screening for prenatal thalassemia screening had better effects, the latter two strategies should be prefered for thalassemia prevention of Guangdong.7.3 There were low service ability of thalassemia prevention in primary health instiutions and medical workers in Guangdong. Strengthened the laboratory construction and personnels training were the technical support for thalassemia prevention in Guangdong.7.4 There were low knowledge and availability level of thalassemia prevention in Guangdong. The widely scoity education of thalassemia prevention was the basic measure for thalassemia prevention.8. Prevention strategyEstimation based on the prevalence of thalassemia carrier, genetic law of thalassemia, and birth number in Guangdong, there was over 1 billion illness burden caused by thalassemia. The status of thalassemia prevention was serious, but the ability and input of thalassemia prevention were limited. We referred the following intergrated strategy of thalassemia prevention in Guangdong:8.1 Input of 20 to 30 million yuan of government budget per year for free thalassemia screening, prenatal diagnosis and intervention in premarital, progestation, and prenatal population. MCV/MCH and Hb A2 serried screening for both couples was referred. And to register the thalassemia carrier electronic health record for newborns and reproductive couples.8.2 Establish multi-department cooperation for extensive society education on thalassemia prevention, and improve the awareness and selection of thalassemia prevention.8.3 Training of thalassemia prevention for entire medical staffs in Guangdong, and include into public compulsory course.8.4 Help to set up classified clinic laboratory system in poor area in Guangdong by government fund.8.5 Establish referral network of thalassemia prenatal diagnosis covered whole Guangdong.9. The advantages of this study9.1 The data could show the prevalence of thalassemia of whole province and also could thow that of each city.9.2 Could system evaluate the cost-effect of different thalassemia prenatal screening strategy of MCV/MCH combined hemoglobin ingredients.9.3 To system evaluate the status of thalassemia prevention of Guangdong based on service suppliers and service receptors.10. The shortcoming of this study10.1 The feasibility and effect of preferred prenatal screening strategy of thalassemia by our study needed to further study.10.2 This study was only focused on the status survey and evaluation, but evaluation of the globin gene mutations test method needed to further study. |
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