1. Alpha+ thalassaemia and the severity of Plasmodium falciparum infection in microcytic children from the Kumasi metropolis of Ghana
Clement Opoku-Okrah1,2; Caroline Smith2; Mike Gordge2; Martin Parry2
Claire Robertson2; Tsiri Abgenyega1; Emmanuel Nakua1
Kwame Nkrumah University of Science and Technology, Ghana1
University of Westminster, Biosciences, London, UK2
KNUST

2. Malaria
More than 90% of global morbidity and mortality of malaria is suffered by Africa
Mostly affect children less than 5 years old. (Mockenhaupt et al., 2004)

3. Malaria Situation in Ghana
Malaria is hyper-endemic
Over 900,000 cases of suspected malaria involving children <5years are reported each year
Malaria accounts for 22% of under-five mortality (Roll back malaria, Ghana, 2005).

4. Protective effects of inherited red cell disorders
It is well recognised that protection against malaria is provided by a variety of inherited red cell disorders
G6PD deficiency
Southeast Asian ovalocytosis
Duffy antigen negative status
Structural haemoglobinopathies
Thalassaemia

5. Alpha+-thalassaemia
In people of African descent the -α3.7 deletional α+- thalassaemia is predominant
In the Northern and Ashanti regions of Ghana, prevalence of α+-thalassaemia, is estimated at 26-33% (Mockenhaupt et al., 2001; 2004a)
The disease is often mistaken for iron deficiency anaemia and treated inappropriately
Evidence suggests that a+-thalassaemia ameliorates the severity of malaria, but does not reduce initial infection rates

6. OBJECTIVES
Determine prevalence of malaria parasitaemia among children attending outpatient clinic in Kumasi, Ghana
Determine the prevalence of α+-thalassaemia among those children with malaria infection
Determine whether the presence of an  + thalassaemia genotype influences the density of malaria parasitaemia

7. Screened Subjects (n=1672) Mps-ve and Mps +ve, confounders (n=940)
Enrolled Subjects Mps +ve and non- confounders (n=732)
Screened Subjects (n=1672)
Mps+ve (904);
Mps +ve (n=904)
Mps –ve (n=768)
Mps-ve and Mps +ve, confounders (n=940)
Hb AS (n=123)
Mps-ve (79)
Mps +ve (44)
HbAC (n=147)
Mps –ve (68)
Mps +ve (79)
G6PD deficiency n=102) Mps –ve (40) Mps +ve (62)
No Mps (n=768). Some already included in Hb AS, Hb AC and G6PD deficiency
Mean Hb RBC MCV MCH GMPD
Normal Genotype (αα/αα)
(n=358)
Alpha+ thalassaemia (n=175)
Homozygous (-α/-α) (n=49)
Heterozygous (-α/αα) (n=126)
PCR
Microcytic (MCV  76 fL) Hypochromic (MCH  25 pg) (n= 238)
Normocytic (MCV > 76 fL) Normochromic (MCH > 25 pg) (n=114)
Microcytic (MCV  76 fL) Normochromic (MCH > 25 pg) (n= 351)
Normocytic (MCV > 76 fL) Hypochromic (MCH  25 pg) (n= 29)
No Amplification (n=56).
Study Population

8. Parasitological Studies
Ring form of Plasmodium falciparum
Parasitological Studies
Thick and thin blood films were stained with 10% Giemsa and read for malaria parasites
Parasitaemia was graded as:
Low (1-999/μL)
Moderate ( /μL)
High ( /μL).
Severe malaria was defined as Mean Parasite Density (MPD) ≥100,000/μL
Gametocyte

9. PCR for –α3.7 deletion W W M H W N L Primer sequence
2000bp
1800bp
Name 5’-3’ Sequence GenBank ID: Nucleotide
α2/3.7-F CCCCTCGCCAAGTCCACCC HUMHB A4:
3.7/20.5-R AAAGCACTCTAGGGTCCAGCG HUMHB A4:
α2-R AGACCAGGAAGGGCCGGTG HUMHB A4:
Ethidium bromide-stained gels showing results from multiplex PCR for the 3.7 thalassaemia. Sizes of the amplified fragments are given in base pair (bp). Lane L is the 100 bp DNA; M is -a/-a homozygote (2000 bp); H is -a/aa heterozygote (1800 bp and 2000 bp); W is αα/ααWildtype (1800 bp);N is negative (Chong et al., 2000)

10. RESULTS
The frequency of P. falciparum malaria in the studied population was 54.1%
Frequencies for α+-thalassaemia genotypes were:
heterozygous (-α/αα) %
homozygous (-α/-α) %
total carriage rate (α/αα & -α/-α) 29.3%

11. Results (2)
Among microcytic patients, mean parasite density was lower in the presence of a+-thalassaemia genotype, compared with normal genotype
Severe malaria (defined as parasite density ≥100,000/μL) was less prevalent in microcytic patients with a+- thalassaemia genotype than in those with normal genotype

12. P<0.001 Parasite density/μL
Homozygous(-α/-α) Heterozygous (-α/αα) Normal genotype(αα/αα)

13. Percentage of patients
The effect of alpha+ thalassaemia genotypes on Plasmodium falciparum(n=533), Error bars:95% confidence interval (p<0.001)

14. CONCLUSIONS
There is a prevalence of 29.3% a+-thalassaemia among malaria-infected children in this population
Microcytic hypochromic anaemia should not, therefore be assumed to be due to iron deficiency
The presence of an a+-thalassaemia genotype (both -α/αα and α/-α) reduces the severity of P. falciparum parasitaemia, compared to normal genotype.
The mechanism of this protection is still not clear

15. THANK YOU