The prevalence of beta thalassemia in students screening in the Lushnja district, Albania

1. The prevalence of beta thalassemia in students screening in the Lushnja district, Albania Etleva Refatllari,MD Laboratory Department UHC ”Mother Tereza”, Tirana, Albania XV BCLF Conference, Antalya 2007

2. Introduction • Thalassemia is among the most common genetic disorders worldwide (heterozygote advantage against malaria). • The β-thalassemias are widespread throughout the Mediterranean region, Africa, the Middle East, the Indian subcontinent and Far East.

3. Introduction • β-thalassemia is caused by any of more than 200 point mutations and, rarely by deletions. • Within each population at risk for β-thalassemia a small number of common mutations is found. • Today’s epidemiology of thalassemia is strikingly different from that of the past. ß-Thalassemia is now widespread in Europe, Americas and Australia. In 2002: • ~ 240 million healthy carriers worldwide. • ~ 200,000 ß-Thalmajor births per year (Cao et al, 2002). • The most affected children are born in countries with limited resources; as a result, these children do not receive the treatment they need, dying in childhood.

4. Introduction Providing resources for the treatment of existing patients is not enough if numbers increase each year with the birth of more affected children. In addition, with adequate treatment, the rate of patient survival increases and there is thus a cumulative increase in patient numbers to be provided for. Angastiniotis

5. Introduction • Prevention is essential • According to the WHO, the annual cost of a nationwide prevention programme in most countries is approximately equal to the cost of treating one annual birth cohort of patients for one year. The fall in thalassemia major birth rate in several national thalassemia prevention programes (Angastiniotis)

6. Thalassemia in Albania • ALBANIA is a relatively small country in the Mediterranean area, located on the Adriatic and Ionian seas. • Surface area: 28742 km2 • Population: 3.5million

7. Thalassemia in Albania • Until its eradication, Malaria has been the main medical and social cause for the decrease in the population of Albania. • The disease was endemic especially on the coastal provinces and lowland areas (Ashta, Adhami). • Malaria influenced Albania provoking high frequencies of thalassemic and sickle cell subjects as a memorial to its long and great presence in the past. • Arceological and paleopathological studies on sceletons from the first century B.C. have detected lesions in the bones that are typical for homozygous β-thalassemia. (Nemeskeri and Dhima 1988; Boletini1991)

8. Thalassemia in Albania • Albania is a country with a high endemicity of β-thalassemia. • According to the partial screening made during the ’70s the frequency of β-thalassemia carriers is about 7%. • The distribution of β-thalassemia carriers is quite heterogeneous; the frequency is higher in the western region (Boletini, Duka). • The thalassemia carrier rate was found as 4,9% among native Albanian immigrants in Greece (Manitsa 2002).

9. Thalassemia in Albania • During the ’90s, Boletini and Duka carried out molecular studies for different mutations associated with β-thalassemia. -eleven beta-globin mutations were identified - three β-thalassemia alleles (IVS-I-110, Codon 39, IVS-I-6) were present in nearly 83% of all β- thalassemia alleles. - their frequency was intermediate between those observed in the population of neighbor countries. - A few rare mutation are: IVS-I-1, codon 44, IVS-II-1, polyA, IVS-I-5, Codon 5, Codon 82-83 and IVS-II-745.

10. Difference in the frequencies of the allele IVS I-110

11. Difference in the frequencies of the allele Codon 39

12. Difference in the frequencies of the allele IVS I-6

13. Publications • D. Duka Studim epidemiologjik, molekular, hematologjik dhe biokimik i beta-talasemise ne Shqiperi. Punim doktorature mbrojtur ne 1996 (biblioteka e Fakultetit te Mjekesise) • E. Boletini, M. Svobodova, V. Divoky et al. Sickle cell anemia, sickle cell beta-thalassemia, and thalassemia major in Albania: characterization of mutations Human Genet 1994, 93: 182-187 • E. Boletini The haemoglobinopathies in Albania Meeting of the Mediterranean Blood Club, Milan, ITALY 1991 • M de Angioleti, G. Lacerra, E. Boletini et al. β- and α-globin genotypes in Albanian patients affected by β- globin gene disordes Haematologica 2002; 87

14. Publications • Refatllari E, Duka D, Boletini E, Bulo A, Spectrum of beta thalassemia mutations in Albania,14-th European Congress of Clinical Chemistry and Laboratory, Medicine 26 – 31 May , 2001,Prague , Czech Republic • Refatllari E, Duka D, Hani A, Bulo A Molecular characterization of beta thalasemia in Albania, 5-th Annual Conference of Pediatrics, 2-3 November 2001,Tirana ,Albania • Refatllari E, Bulo A, Refatllari A Performance evaluation of ferritin assay on the Brio-Sirio immunoenzymatic automated system. Ferritin levels in thalassemia major patients Laboratory Medicine 20-25 October, 2002, Kyoto, Japan • Refatllari E, Duka D, Boletini E, Hani A, Bulo A Spektri i hemoglobinopative dhe karakterizimi molekular i beta talasemise ne Shqiperi,Ditet Pediatrike Franko Shqiptare, 2-3 Maj, 2002

15. Publications • Refatllari E, Duka D, Boletini A, Hani A, Bulo A Karakterizimi molekular dhe diagnoza laboratorike e beta-talasemive ne Shqiperi Konferenca e Pare Kombetare e Mjekesise Laboratorike Shqiptare, Tirane, 31 Maj-1Qershor 2004. • E. Boletini, D. Duka, E. Refatllari, A. Hani, A. Bulo Report diagnostics condition of thalassaemia in AlbaniaBalkan Journal of Clinical Laboratory Federation, Vol 12:2005 • E. Refatllari, A. Bulo-Kasneci,R. Zaganjori, N. Heta, I. Korita, E. Kolici Sebia automated system electrophoresis in haemoglobinopathies diadnosis, AlbaniaBalkan Journal of Clinical Laboratory Federation, Vol 13:2006 • E. Refatllari, A. Bulo-Kasneci,R. Zaganjori, A. Barbullushi, P. Daja, N. Heta, I. Korita, E. Kolici, M. Haruni Glucose-6-phosphate dehydrogenase deficiency in albanian students, AlbaniaBalkan Journal of Clinical Laboratory Federation, Vol 13:2006

16. Thalassemia in Albania • Untill now, we don’t have any prevention strategy or national program • Each year there are 15-20 new cases of thalassemia major • Actually we have ~500 patients with thalassemia major. The improvement in the clinical management of the disease over the last 15 years led to an impressive increase in the lifespan of patients. The quality of life is directly linked to quality and quantity of treatment • For the health care system in Albania it is difficult to perform an adequate treatment for the increasing numbers of patients

17. Thalassemia in Lushnja • Lushnja is located about 80 km from Tirana, the capital of Albania • Lushnja is actually a strategic point of communication between the north and the south of Albania

18. Thalassemia in Lushnja • Thalassemia is one of the major concerns for the health care system in this district • This area is the epicenter of incidence of thalassemia in Albania • A screening activity in this area, is considered very cost effective • The effectiveness of the screening process is much higher in the fertile age population in this area

19. Aim of the study • To evaluate the prevalence of the thalassemia carrier status in high school students of Lushnja • To educate the participants and medical care givers about thalassemia, its inheritance and the significance of this screening project • To increase the awareness of the population in the area about the importance of routine screening • Preventing a certain number of potential new cases in the future

20. Main preparatory activities • Definition and approval of the methodology and respective indicators • Training of the staff (technical laboratory and nurse staff) • Educational meeting with children and teachers in the selected schools • Distribution of the informative leaflets. This leaflet is very important for 2 main reasons: a) it will explain to students and parents what is thalassemia and why testing is very important for all of them b) it will be filled with important personal data and it includes parental consent, too.

21. Main preparatory activities • The screening program was carried out on a volontary basis • We collaborate with national and local health and educational authorities • The screening study was performed by ASoLaM • Financial suport was provided by USAID – Albania Our program consisted of educating, screening and consueling senior high school students about beta thalassemia

22. Methods • The target population was made of healthy high school adolescent students • The group selected to participate in the project is appropriate because: - the expected prevalence of the carrier status in this area is very high - very soon they will start their sexual life and will, eventually, be parents - the group is easily accessible - the educational and cultural level is relatively high

23. The widespread of study • We prepared the results using the micromapping method to obtain a more complete picture of the situation. • Samples number was propotional as populations number in all areas

24. Material • From May till December 2006 we performed our study in 8 high schools of the Lushnja district • 1820 students agreed to undergo the screening program • The age of the students varied from 14-19 years old. The average age was 17 years old • 61.6% of the participants was female, while 38.4% male

25. Methods • Venous blood was taken into EDTA tube • Complete blood count and red blood cell indices were measured by a Mythic 22 automated cell counter on the same day of collection • Hemoglobin electrophoresis was done on Hydrasis – Sebia automated system

26. Methods • A primary screening of all the samples to determine red cell indices • A secondary screening involving haemoglobin analysis in subject with reduced MCV and/or MCH • A secondary screening involving haemoglobin analysis in subject with familial history • Samples with MCV <79 (microcytosis) and MCH <27 (hypocromia) were selected for electrophoresis • Samples with HbA2≥3,5% were diagnosed as thalassaemia minor

27. Results • 30% of the 1820 samples were observed to have non normal RBC numbers and indices. • 7,58% were beta-thalassemia carriers • 1,48% were carriers of Hb S

28. Results • Electrophoresis samples

29. Distribution of β-thalassemia trait • The prevalence of carriers results greater in lower seaside regions(10-11%) then in the city (4,38%) • The lowest prevalence is found in the northern region (Cerme – 2.86%)

30. Distribution of β-thalassemia trait

31. Distribution of HbS trait Prevalence of HbS carriers was greater in the northern parts of the region (8,57%).

32. Distribution of HbS trait

33. Selective distribution of thalassemia and HbS There is a selective distribution of thalassemia and drepanocytosis

34. Comparison of the data of the two studies There isn’t any difference between our results and those of the 1975 study in specific area.

35. ResultsRBC indices

36. The hemoglobin values in different groups

37. RBC numbers in different groups

38. MCV and MCH values in different groups • Microcytosis and hypochromia are present in two groups; Thalassemia minor and Iron deficiency anemia. • No differencies were found in MCV and MCH values between normal subjects and HbS carriers.

39. RBC,HB and MCV in IDA and MT

40. RDW values in thalassemia minor and Iron deficient subject The difference between two groups is not significant (p=0.004)

41. Conclusions • Our data confirmed the high prevalence of β-thalassemia carriers in Lushnja (7.58%) • The prevalence of HbS carriers was grater in the northern parts of the region (8.57%) • Total blood count is very useful to suspect the thalassemia, but not HbS trait • Sebia automated Hb electrophoresis has a high sensibility and specificity for β-thalassemia diagnosis and for identification of Hb variants

42. Conclusions • Our data suggests long-term application of educational and screening programs • Lushnja screening project will serve as an example of national screening and education program in Albania • The high prevalence of HbS trait in the area justified complete screening based on determining red cell indices and haemoglobin pattern analysis

43. Monastery of Ardenica -Lushnja

44. Karavasta lagoon – Lushnja

45. Divjaka beach-Lushnja

46. Apolonia Thank you