Haemoglobin variants and Plasmodium falciparum malaria in children under five years of age living in a high and seasonal malaria transmission area of Burkina Faso

Abstract

Background
Genetic factors play a key role in determining resistance/susceptibility to infectious disease. Susceptibility of the human host to malaria infection has been reported to be influenced by genetic factors, which could be confounders if not taken into account in the assessment of the efficacy of interventions against malaria. This study aimed to assess the relationship between haemoglobin genotypes and malaria in children under five years in a site being characterized for future malaria vaccine trials.

Methods
The study population consisted of 452 children living in four rural villages. Hb genotype was determined at enrolment. Clinical malaria incidence was evaluated over a one-year period using combined active and passive surveillance. Prevalence of infection was evaluated via bi-annual cross-sectional surveys. At each follow-up visit, children received a brief clinical examination and thick and thin blood films were prepared for malaria diagnosis. A clinical malaria was defined as Plasmodium falciparum parasitaemia >2,500 parasites/μl and axillary temperature ≥37.5°C or reported fever over the previous 24 hours.

Results
Frequencies of Hb genotypes were 73.2% AA; 15.0% AC; 8.2% AS; 2.2% CC; 1.1% CS and 0.2% SS. Prevalence of infection at enrolment ranged from 61.9%-54.1% among AA, AC and AS children. After one year follow-up, clinical malaria incidence (95% CI) (episodes per person-year) was 1.9 (1.7-2.0) in AA, 1.6 (1.4-2.1) in AC, and 1.7 (1.4-2.0) in AS children. AC genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 1–2 years [rate ratio (95% CI) 0.66 (0.42-1.05)] and 2–3 years [rate ratio (95% CI) 0.37 (0.18-0.75)]; an association of opposite direction was however apparent among children aged 3–4 years. AS genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 2–3 years [rate ratio (95% CI) 0.63 (0.40-1.01)].

Conclusions
In this cohort of children, AC or AS genotype was associated with lower risk of clinical malaria relative to AA genotype only among children aged one to three years. It would be advisable for clinical studies of malaria in endemic regions to consider haemoglobin gene differences as a potentially important confounder, particularly among younger children.

Full text

R E S E A R C H Open Access
Haemoglobin variants and Plasmodium falciparum
malaria in children under five years of age living
in a high and seasonal malaria transmission area
of Burkina Faso
Edith C Bougouma
1
, Alfred B Tiono
1
, Alphonse Ouédraogo
1
, Issiaka Soulama
1
, Amidou Diarra
1
, Jean-Baptiste Yaro
1
,
Espérance Ouédraogo
1
, Souleymane Sanon
1
, Amadou T Konaté
1
, Issa Nébié
1
, Nora L Watson
3
, Megan Sanza
3
,
Tina JT Dube
3
and Sodiomon B Sirima
1,2*
Abstract
Background: Genetic factors play a key role in determining resistance/susceptibility to infectious disease.
Susceptibility of the human host to malaria infection has been reported to be influenced by genetic factors, which
could be confounders if not taken into account in the assessment of the efficacy of interventions against malaria.
This study aimed to assess the relationship between haemoglobin genotypes and malaria in children under five
years in a site being characterized for future malaria vaccine trials.
Methods: The study population consisted of 452 children living in four rural villages. Hb genotype was determined
at enrolment. Clinical malaria incidence was evaluated over a one-year period using combined active and passive
surveillance. Prevalence of infection was evaluated via bi-annual cross-sectional surveys. At each follow-up visit,
children received a brief clinical examination and thick and thin blood films were prepared for malaria diagnosis. A
clinical malaria was defined as Plasmodium falciparum parasitaemia >2,500 parasites/μl and axillary temperature
≥37.5°C or reported fever over the previous 24 hours.
Results: Frequencies of Hb genotypes were 73.2% AA; 15.0% AC; 8.2% AS; 2.2% CC; 1.1% CS and 0.2% SS.
Prevalence of infection at enrolment ranged from 61.9%-54.1% among AA, AC and AS children. After one year
follow-up, clinical malaria incidence (95% CI) (episodes per person-year) was 1.9 (1.7-2.0) in AA, 1.6 (1.4-2.1) in AC, and
1.7 (1.4-2.0) in AS children. AC genotype was associated with lower incidence of clinical malaria relative to AA genotype
among children aged 1–2 years [rate ratio (95% CI) 0.66 (0.42-1.05)] and 2–3 years [rate ratio (95% CI) 0.37 (0.18-0.75)];
an association of opposite direction was however apparent among children aged 3–4years.ASgenotypewas
associated with lower incidence of clinical malaria relative to AA genotype among children aged 2–3 years [rate ratio
(95% CI) 0.63 (0.40-1.01)].
Conclusions: In this cohort of children, AC or AS genotype was associated with lower risk of clinical malaria relative to
AA genotype only among children aged one to three years. It would be advisable for clinical studies of malaria in
endemic regions to consider haemoglobin gene differences as a potentially important confounder, particularly among
younger children.
Keywords: Plasmodium falciparum, Malaria, Haemoglobin abnormalities, Children, Epidemiology, Burkina Faso
* Correspondence: s.sirima.cnlp@fasonet.bf
1
Centre National de Recherche et de Formation sur le Paludisme,
Ouagadougou, Burkina Faso
2
Groupe de Recherche et Action en Santé, Ouagadougou, Burkina Faso
Full list of author information is available at the end of the article
© 2012 Bougouma et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Bougouma et al. Malaria Journal 2012, 11:154
http://www.malariajournal.com/content/11/1/154

Background
Malaria remains one of the most important causes of
morbidity and mortality in endemic areas, primarily
affecting children under five years of age [1]. The highest
death burden occurs in young children who have not yet
developed protective immune mechanisms against the
parasite. A minority of children appear to have a natural
biological advantage thought to partially impede parasite
growth [2].
Malaria is a complex disease that depends on many
host genetic factors [3]. Indeed, resistance to Plasmo-
dium falciparum is an important adaptive trait of human
populations living in endemic areas [4]. Haemoglobin S
(HbS) has become a stable polymorphism within malaria-
endemic regions, associated with a limited life expectancy
among homozygous individuals who suffer from sickle cell
disease, and an extended life expectancy of heterozygous
individuals who are more likely to evade malaria [5-7].
HbAS is widely known to confer significant protection
from severe and uncomplicated malaria [6-12] although
underlying mechanisms not precisely defined. Similar
protection afforded by haemoglobin C (HbC) was more
recently demonstrated although findings are less conclu-
sive. Clinical studies performed in Nigeria and Mali has
found no protection [13-15], while other Malian study
and Burkina study indicated an association between
HbAC and clinical malaria [16,17].
Several innate or immune mechanisms have been
hypothesized to explain malaria-protective effects of
HbS or HbC [2,18-20] Erythrocytes containing HbS or
HbC may impede parasite growth and replication rela-
tive to normal red cells when subject to low oxygen ten-
sions [18]. Protein targets of specific antibodies may be
more rapidly exposed in HbS-containing red blood cells
[21] resulting in an enhanced immune response to infec-
tion [18,22]. It is also possible that unknown innate pro-
tective processes may up-regulate the malaria-specific
immune response [23] or enhance nonspecific immunity
to malaria [24].
The comparison of malaria indicators among popula-
tions with different genetic backgrounds that are uni-
formly exposed to the same parasite strains is one
approach to the study of human heterogeneities in re-
sponse to the infection [9,17]. To characterize malaria
risk in the population residing in the malaria vaccine
trial site in Saponé, Burkina Faso, a haemoglobin geno-
typing study was conducted in children under five years
of age living in this malaria endemic region. The study
aimed to describe the relationship between abnormal
haemoglobin genotypes and malaria in children under
five years in a site being characterized for future malaria
vaccine trials. Age-specific patterns of association were
hypothesized to reflect the development of acquired im-
munity throughout early childhood.
Methods
Study area and period
The study was conducted in four villages (Dawelgue,
Tanghin, Kounda, and Watenga) in the Saponé Health
District, located 50 km south-west of Ouagadougou, the
capital city of Burkina Faso, in the Bazega province
(Figure 1). The region is populated almost exclusively
by the Mossi ethnic group, and farming is the main sub-
sistence activity. The area has a rainy season lasting from
June to October, corresponding to the high malaria trans-
mission season, and a long dry season from November to
May. The main malaria vectors are Anopheles gambiae,
Anopheles arabiensis,andAnopheles funestus. The ento-
mological inoculation rate (EIR) in 2001 was estimated at
0.3 and 44.4 infective/bites/person/month during the dry
and rainy seasons, respectively in study area [25]. A demo-
graphic surveillance system (DSS) for monitoring vital
events has been operating in the villages since 2002.
Enrolment was initiated on 23 January, 2007 and the
final study visit for the last subject was completed on 29
February, 2008.
Study participants
The study population consisted of children under five
years of age identified from the above four villages
whose parents were also resident in the study area. The
sample was identified from a census list generated from
the DSS census list of all children under five years living
in the study region. Children were recruited approxi-
mately equally among age groups as follows: nought to
11 months, 12–23 months, 24–35 months, 35–47 months
and 48–49 months.
Study design and sample collection
Exclusion criteria from the cohort included major con-
genital defects, any chronic disease, or anaemia defined
as measured haemoglobin <6 g/dl. Inclusion criteria
were as follows: i) written/thumb-printed informed con-
sent obtained from the parent or legal guardian of the
child, ii) permanent resident in the study area and
expected to remain so for at least one year following
enrolment, iii) age between nought and five years.
Prevalence of infection and malariometric indices were
evaluated during bi-annual cross-sectional surveys
within the low and high transmission seasons. The inci-
dence of clinical malaria was documented through active
and passive surveillance from the first survey through
one year follow-up.
Cross-sectional surveys
Each cross-sectional survey consisted of a brief clinical
examination and collection of blood by finger prick. The
sampled blood was used for thick and thin blood film prep-
aration for malaria microscopic examination. Haemoglobin
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measurements were done using a HemoCue machine. A
venous blood sample of 5 ml was additionally collected at
the first survey for haemoglobin genotyping.
Active case detection
During the 12-month period of longitudinal surveillance,
each child was visited twice per week by nurses living in
the villages. The visit consisted of a brief history and
clinical examination. Among children with an axillary
temperature ≥37.5°C or reported history of fever in the
past 24 hours, finger-prick blood samples were collected
for haemoglobin measurement and malaria microscopic
examination. Subjects with fever and positive rapid diag-
nostic test (RDT, OptiMAL) were recommended to visit
the medical centre for treatment with artemether-
lumefantrine.
Passive case detection
Parents of the children were encouraged to report to the
nearest community clinic or hospital any time their child
showed a sign of sickness in between home visits. Each
child in attendance at either health facility received a
brief clinical examination; any child with an axillary tem-
perature ≥37.5°C or reported history of fever in the past
24 hours was tested for malaria and treated accordingly.
Parasitological diagnosis
Blood slides from either cross-sectional or longitudinal
surveys were stained with Giemsa for microscopic iden-
tification of the Plasmodium species and determination
of parasite density. Thick and thin blood films were air-
dried; thin blood films were fixed with methanol, and
both were stained with 3% Giemsa. One hundred high
power fields were examined and the number of malaria
parasites of each species and stage were recorded. The
number of parasites per microlitre of blood was calcu-
lated by assuming that there were 20 white blood cells
per high-power field and a fixed cell count of 8,000 per
μl. Each film was read twice by two experienced techni-
cians and a third reading was undertaken if discrepan-
cies exceeding 30% occurred between the two readers.
Haemoglobin typing
DNA was extracted using a QIAGEN kit (QIAamp DNA
blood mini kit) and the haemoglobin was typed by poly-
merase chain reaction-restriction fragment length poly-
morphism (PCR-RFLP). Briefly, DNA samples were
amplified using a 5’-AGG AGC AGG GAG GGC AGG
A-3’forward primer and a 5’-TCC AAG GGT AGA
CCA CCA GC-3’reverse primer. The 358 base pair bp
fragment obtained was digested with MnlI for discrimin-
ation between HbAA (173 pb, 109 pb, and 60pb), HbSS/
Figure 1 Study site in Burkina Faso.
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HbCC andHbSC (173 pb, 109 pb, and 76 pb), HbAS/
HbAC (173 pb, 109 pb, 76 pb and 60). A second diges-
tion with DdeI allowed for further discrimination for am-
biguous results between HbSS (331 pb), HbCC (201 pb
and 130 pb), HbSC (130 pb, 201 pb and 331 pb), HbAS
(130 pb, 201 pb and 331 pb) and HbAC (201 pb and 130
pb). All digestion was carried out for three hours at 37°C
and the products were run on a 3% agarose gel [17].
Malariometric indices
Malariometric indices were evaluated during the low
and high transmission seasons using measures obtained
from the biannual surveys. A clinical P. falciparum mal-
aria episode was defined as an axillary temperature of
≥37.5°C or history of fever in the past 24 hours and P. fal-
ciparum trophozoite count >2,500 parasites/μl. Spleno-
megaly of any size was identified by Hackett’s
classification [26]. Haemoglobin was measured using a
HemoCue machine and reported as g/dl. Child age was
defined as age (years) at enrolment.
Statistical analysis
Demographic characteristics (age, sex and health dis-
trict) and season-specific malariometric indices were
compared among normal (AA) and abnormal Hb geno-
types using Kruskal-Wallis tests for continuous and Chi-
square or Fischer’s exact tests for categorical measures.
Clinical malaria incidence was calculated within strata
defined by commonly occurring (AA, AC, and AS) geno-
type and season of follow-up as the total number of clin-
ical malaria episodes observed by active and passive
surveillance divided by the total number of person-years
at risk. Children were excluded from incidence analysis
for 28 days after recording an episode, to ensure that the
infection causing the episode was only recorded once.
The study period ended 365 days after the first visit or
on the date of death/emigration from the study locale
(N = 2).
Age-specific risks associated with Hb genotype were
evaluated using a Poisson regression model of haemo-
globin genotype as a predictor of rate of malaria epi-
sodes observed over one year follow-up. The model was
used to estimate rate ratios associated with AC, AS or
either genotype relative to AA within age strata after ad-
justment for health district. Analyses were repeated
among the full cohort to formally test for interactions of
AC and AS genotype with age group. Cumulative inci-
dence of clinical malaria within genotype strata was esti-
mated using the Kaplan-Meier method. Malaria-free
survival curves were compared among genotypes using
the log-rank test.
All data were double entered in Epi Info Version 6.04
(CDC, Atlanta, USA) and statistical analyses were per-
formed in SAS Version 9.2.
Ethical processes
The study protocol was explained to the local population
prior to study initiation. Written informed consent was
obtained from the parents or guardians of all participat-
ing children before enrolment. The protocol of this
study was approved by the Burkina Faso Health Re-
search Ethics Review Committee.
Results
Baseline characteristics of the study cohort
The study cohort was divided into five predefined age
groups: nought to one year (n= 75), one to two years
(n=93), two to three years (n=97), three to four years
(n = 102), and four to five years (n = 85). Mean age of
the cohort was 32 months; 47.8% were female (Table 1).
Frequencies of each haemoglobin genotype were 73.2%
AA; 15.0% AC; 8.2% AS; 2.2% CC; 1.1% CS and 0.2% SS.
Age and sex distributions were similar among genotype
groups.
Malariometric indices
Prevalence of P. falciparum infection decreased from
59.7% to 50.4% from the low to high season; geometric
Table 1 Demographic characteristics by Age, sex and
haemoglobin genotype
Haemoglobin
type
Gender All
Male Female
AA N 175 156 331
Age (months), mean 33.2 32.0 32.6
Age (months), SD 17.1 15.9 16.5
AC N 33 35 68
Age (months), mean 29.0 29.5 29.3
Age (months), SD 18.0 17.2 17.5
AS N 18 19 37
Age (months), mean 31.3 32.7 32.0
Age (months), SD 11.7 16.5 14.2
CC N 6 4 10
Age (months), mean 19.1 17.1 18.3
Age (months), SD 17.9 17.2 16.6
SC N 4 1 5
Age (months), mean 41.7 1.7 33.7
Age (months), SD 15.4 0 22.3
SS N 1 0 1
Age (months), mean 43.6 0 43.6
Age (months), SD 0 0 0
Non-AA N 62 59 121
Age (months), mean 29.8 29.2 29.5
Age (months), SD 16.4 17.3 16.8
Note: Age and sex distribution were similar among genotype.
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mean parasite density increased from 1,579 (1,315–
1,896) to 2,748 (2,104–3,589) trophozoites/μl. Prevalence
of clinical malaria varied minimally (11.3% to 12.9%)
(Tables 2 and 3). Clinical malaria prevalence and mean
haemoglobin did not vary by Hb genotype within either
season; however P. falciparum infection was less preva-
lent among children with CC relative to AA genotype at
each survey (p <0.05 for each).
Incidence of clinical malaria
Incidence of clinical malaria (95% CI) among the full co-
hort was 1.8 (1.7-1.9) episodes per person-year after one
year follow-up. The season-specific incidence rate was
3.1 times higher in the high relative to the low transmis-
sion season (Table 4). Within each season incidence
rates were higher among children with AA genotype
relative to abnormal genotypes, although differences
were not statistically significant.
In age-stratified analyses adjusted for health district,
AC genotype was associated with lower incidence of
clinical malaria relative to AA among children aged 1–
2 years [rate ratio (95% CI) = 0.66 (0.42, 1.04); p = 0.07]
and 2–3 years [rate ratio (95% CI) = 0.37 (0.18, 0.75);
p = 0.01] (Table 5). An association of opposite dire ction
was however apparent among children aged 3–4 years:
rate ratio (95% CI) = 1.61 (1.08, 2.41); p = 0.02. An associ-
ation of AS genotype with lower incidence of clinical
malaria relative to AA approached significance among
children aged 2–3 years: rate ratio (95% CI) = 0.63 (0.40,
1.101); p = 0.06.
In analyses inclusive of all children aged 1–3 years and
adjusted for health district, presence of either AS or AC
genotype was associated with reduced risk of similar magni-
tude: rate ratio (95% CI) = 0.63 (0.49, 0.83); p = <0.001.
Among the full cohort, risk associated with AC genotype
varied significantly among children younger versus older
than three years after adjustment for health district (p for
interaction <0.001).
Time to first malaria episode did not significantly dif-
fer by AA, AC or AS genotype among the full cohort
(p = 0.21) (Figure 2). Among children aged 1–3 years,
however, delayed first malaria was apparent among chil-
dren with AC (241 days) or AS (201 days) genotype rela-
tive to AA (198 days) (p = 0.04) (Figure 3). Associations
of genotype with time to first malaria were not apparent
among other age groups.
Discussion
In this cohort of children under five years of age living in a
high malaria-transmission region of Burkina Faso, AC or AS
Hb genotype was associated with lower risk of clinical mal-
aria relative to the AA genotype among children aged one
to three years. This association was attenuated though
approached significance among the full cohort. These data
suggest that Hb genotype should be considered a potentially
important confounder, particularly among younger children,
in evaluations of clinical malaria risk in endemic regions.
The haemoglobin S and C genes occurred in the co-
hort with similar frequencies to those previously pub-
lished [17,27,28]; 26% were carriers of either HbS or
Table 2 Malariometric indices by haemoglobin genotype
Infection characteristic AA
(N = 331)
AC
(N = 68)
AS
(N = 37)
CC
(N = 10)
SC
(N = 5)
SS
(N = 1)
All
(N = 452)
Symptomatic malaria
(fever and >0 parasites/μl), N (%)
39
(11.8%)
7
(10.3%)
4
(10.8%)
1
(10.0%)
0
(0.0%)
0
(0.0%)
51
(11.3%)
P. falciparum infection
(>0 parasites/μl), N (%)
205
(61.9%)
39
(57.4%)
20
(54.1%)
2
(20.0%)*
4
(80.0%)
0
(0.0%)
270
(59.7%)
Symptomatic malaria
(fever and >2500 parasites/μl)
22
(6.6%)
3
(4.4%)
0
(0.0%)
0
(0.0%)
0
(0.0%)
0
(0.0%)
25
(5.5%)
P. falciparum infection
(>2500 parasites/μl), N (%)
82
(24.8%)
15
(22.1%)
6
(16.2%)
1
(10.0%)
1
(20.0%)
0
(0.0%)
105
(23.2%)
Gametocyte carriage, N (%) 101
(30.5%)
21
(30.9%)
10
(27.0%)
3
(30.0%)
3
(60.0%)
0
(0.0%)
138
(30.5%)
Splenomegaly, N (%) 67
(20.2%)
9
(13.2%)
4
(11.1%)
1
(10.0%)
0
(0.0%)
0
(0.0%)
81
(18.0%)
Haemoglobin (g/dl), mean (SD) 9.7
(1.5)
9.5
(1.6)
9.5
(1.5)
9.4
(1.5)
11.1
(0.8)
10.1 9.6
(1.5)
P. falciparum density
(per/μl), GM (95% CI)
1546
(1259,1898)
1904
(1159, 3128)
1305
(527, 3231)
3088
(51, 185428)
1418
(104,19382)
—1579
(1315, 1896)
Gametocyte density
(per/μl), GM (95% CI)
51
(41, 64)
48
(30, 75)
95
(54, 166)
81
(2, 3110)
22
(0, 62183)
—52
(43, 64)
Low transmission season.
*P for difference vs AA <0.05.
**P for difference vs AA <0.01.
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Table 4 Incidence of malaria episodes (fever and parasitemia >2500/μl) by haemoglobin genotype and season
Season Haemoglobin
genotype
Number of
children
Number of
episodes
Mean number
of episodes
per child
Total
person-
years
Incidence rate
(95% CI)
(per person-year)
Low AA 331 184 0.6 177.0 1.0 (0.9, 1.2)
AC 68 31 0.5 37.1 0.8 (0.6, 1.2)
AS 37 15 0.4 20.4 0.7 (0.4, 1.2)
CC 10 1 0.1 5.8 0.2 (0.0, 1.0)
SC 5 1 0.2 2.8 0.4 (0.0, 2.0)
SS 1 0 0.0 0.6 0.0 (0.0, 6.4)
Total 452 232 0.5 243.6 1.0 (0.8, 1.1)
High AA 305 363 1.1 112.4 3.2 (2.9, 3.6)
AC 64 74 1.1 23.0 3.2 (2.5, 4.0)
AS 35 35 0.9 13.1 2.7 (1.9, 3.7)
CC 9 8 0.8 3.7 2.2 (0.9, 4.3)
SC 5 5 1.0 1.8 2.9 (0.9, 6.7)
SS 1 0 0.0 0.4 0.0 (0.0, 8.8)
Total 419 485 1.1 154.4 3.1 (2.9, 3.4)
Low and High AA 331 542 1.6 288.0 1.9 (1.7, 2.0)
AC 68 104 1.5 59.8 1.7 (1.4, 2.1)
AS 37 50 1.4 33.1 1.5 (1.1, 2.0)
CC 10 9 0.9 9.3 1.0 (0.4, 1.8)
SC 5 6 1.2 4.5 1.3 (0.5, 2.9)
SS 1 0 0.0 1.0 0.0 (0.0, 3.7)
Total 452 711 1.6 395.7 1.8 (1.7, 1.9)
Note: Incidence of malaria episodes was calculated as the number of malaria cases.
divided by total person-years (p-yrs) at risk).
Table 3 Malariometric indices by haemoglobin genotype
Infection characteristic AA
(N = 305)
AC
(N = 64)
AS
(N = 35)
CC
(N = 9)
SC
(N = 5)
SS
(N = 1)
All
(N = 419)
Symptomatic malaria
(fever and >0 parasites/μl), N (%)
37
(12.1%)
13
(20.3%)
2
(5.7%)
0
(0.0%)
2
(40.0%)
0
(0.0%)
54
(12.9%)
P. falciparum infection
(>0 parasites/μl), N (%)
152
(49.8%)
34
(53.1%)
19
(54.3%)
1
(11.1%)*
4
(80.0%)
1
(100.0%)
211
(50.4%)
Symptomatic malaria
(fever and >2500 parasites/μl)
27
(8.9%)
9
(14.1%)
1
(20.0%)
0
(0.0%)
0
(0.0%)
0
(0.0%)
37
(8.8%)
P. falciparum infection
(>2500 parasites/μl), N (%)
84
(27.5%)
22
(34.4%)
4
(11.4%)*
0
(0.0%)
2
(40.0%)
1
(100.0%)
113
(27.0%)
Gametocyte carriage, N (%) 60
(19.7%)
11
(17.2%)
3
(8.6%)
2
(22.2%)
0
(0.0%)
0
(0.0%)
76
(18.1%)
Splenomegaly, N (%) 26
(8.5%)
7
(10.9%)
2
(5.7%)
0
(0.0%)
0
(0.0%)
0
(0.0%)
35
(8.4%)
Haemoglobin
(g/dl), mean (SD)
10.2
(1.5)
9.9
(1.4)
9.9
(1.5)
10.0
(1.5)
11.1
(1.1)
10.5 10.1
(1.5)
P. falciparum density
(per/μl), GM (95% CI)
2690
(1978, 3659)
5823
(3117, 10878)*
986
(383, 2541)*
364 1632
(7, 375567)
9740 2748
(2104, 3589)
Gametocyte density
(per/μl), GM (95% CI)
41
(31, 53)
20
(11, 37)*
23
(3, 173)
34
(4, 263)
(NA) (NA) 36
(28, 45)
High transmission season.
*P for difference vs AA <0.05.
**P for difference vs AA <0.01.
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HbC. The mechanism by which HbS protects against
malaria has been the subject of speculation for more
than 50 years. While protection may largely be conferred
by the physical characteristics of HbS erythrocytes, a
number of studies suggest that HbAS may also enhance
the acquisition of naturally acquired immunity [19,29].
Associations between the HbC trait and malaria risk,
however, have not been uniformly established [30].
The finding that parasitaemia prevalence varied only
minimally among AA, AC and AS genotypes in the full
cohort is consistent with previous studies conducted in
various parts of Africa [9,31-33]. Malaria premunition
Table 5 Incidence of malaria episodes (fever and parasitemia >2500 parasites/μl) by haemoglobin genotype and age
at enrollment
Age Haemoglobin
genotype
Number of
children
Number of
episodes
Incidence rate (95% CI)
(per person-year)
District-adjusted
rate ratio (95% CI)
P
value
0-1 yrs AA 52 115 2.7 (2.2, 3.2) ——
AC 15 31 2.5 (1.7, 3.5) 0.93 (0.62, 1.38) 0.72
AS 2 2 1.1 (0.1, 3.9) 0.41 (0.10, 1.65) 0.21
1-2 yrs AA 66 131 2.3 (2.0, 2.8) ——
AC 16 22 1.5 (1.0, 2.3) 0.66 (0.42, 1.04) 0.07
AS 9 19 2.5 (1.5, 3.9) 0.93 (0.56, 1.54) 0.77
2-3 yrs AA 70 145 2.5 (2.1, 2.9) ——
AC 10 8 0.9 (0.4, 1.7) 0.37 (0.18, 0.75) 0.01
AS 14 20 1.6 (1.0, 2.5) 0.63 (0.40, 1.01) 0.06
3-4 yrs AA 77 95 1.4 (1.1, 1.7) ——
AC 17 32 2.2 (1.5, 3.1) 1.61 (1.08, 2.41) 0.02
AS 6 9 1.7 (0.8, 3.2) 1.05 (0.53, 2.09) 0.89
4-5 yrs AA 66 56 0.9 (0.7, 1.2) ——
AC 10 11 1.2 (0.6, 2.2) 1.27 (0.66, 2.44) 0.48
AS 6 0 0.0 (0.0, 0.6) 0.00 1.00
Figure 2 Time to first malaria episode by haemoglobin abnormality among children aged 0–5 years, over one year follow-up (low
transmission season and high transmission season).
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has also been found comparable among haemoglobin
groups among patients greater than six years of age [30].
Age-specific associations of malariometric indices with
Hb gentoypes may be hypothesized to reflect the devel-
opment of anti-malarial immunity [30] as the protective
action of haemoglobin may be masked by maternally
transmitted antibodies among children less than six
months of age [34]. While age-specific patterns of asso-
ciation were not apparent in the current cross-sectional
measures, further inferences are limited due to the small
number of children within age and genotype strata.
Mean parasite density was markedly lower in children
AS relative to AA genotype normal haemoglobin, con-
sistent with previous reports [17,27]. Indeed, abnormal
haemoglobin may not allow for optimal development of
Plasmodium in deep organs where oxygen pressure is
reduced. Parasite density was however higher among AC
relative to AA genotype, suggesting potential mechanis-
tic variation among protection afforded by abnormal
genotypes in early childhood. Malaria-risk reduction
associated with HbAS genotype has been reported in
Mali[27,35] Burkina [17], Ghana [36] and Kenya[11]; a
similar protective advantage of HbAC has been less con-
sistently supported.
Abnormal Hb genotype in this cohort was further
associated with lower incidence of clinical malaria epi-
sodes and delayed first episode among children aged one
to three years. These effects were more subtle in infants
and older children. Several studies have identified a pro-
tective effect of abnormal Hb against clinical malaria
[17,35]. Although HbAS was found unassociated with
time to first malaria episode in Gabon [37], HbAS was
associated with reduced time to all-cause mortality in
Kenya, an effect attributed to reduction in malaria-
specific outcomes including severe malarial anaemia and
high-density parasitaemia[38]. Proposed mechanisms of
protection [39] include decreased red blood cell invasion
or poor growth under low-oxygen tension[40]; and accel-
erated acquisition of antibodies specific for P. falciparum
erythrocyte membrane protein-1 (PfEMP-1) and other
variant surface antigens[41].
Alternative findings among cohorts may in part reflect
variation in age distribution and associated development
of acquired immunity, malaria endemicity, and adminis-
tration of anti-malarial drugs. Estimates of malaria bur-
den in the current study are likely underestimates, due
to prompt treatment of malaria episodes identified by
active surveillance. This study is further limited by the
small number of children with abnormal genotypes and
restriction of follow-up to one year, preventing stronger
inferences related to changing risk associated with alter-
native genotypes throughout early childhood.
Conclusions
In this cohort of children under five years of age, AC or
AS Hb genotypes was associated with lower risk of
Figure 3 Time to first malaria episode by haemoglobin abnormality among children aged 1–3 years, over one year follow-up (low
transmission season and high transmission season).
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clinical malaria relative to normal genotype among chil-
dren aged one to three years. This age-specific associ-
ation may suggest influences of HbC and HbS genotypes
in the development of naturally acquired immunity in
early childhood. Evaluations of anti-malarial interventions
in endemic regions should consider Hb genotype as a po-
tentially important confounder, particularly among young
children.
Abbreviations
AA: Wild type; SS: Sickle cell; AS: Sickle trait; AC: Heterozygous for
haemoglobin C; CC: Homozygote for haemoglobin C; SC: Haemoglobins S
and C; Non-AA: Abnormal haemoglobin (AS, AS, SC, SS and CC);
PCR: Polymerase chain reaction.
Competing interest
The authors declare that they have no competing interests.
Authors’contributions
ECB designed the study, collected data and coordinated the study,
performed statistical analysis and wrote the first draft of the manuscript. SBS,
AT, IN, AO and ATK coordinated and participated in the design of the study,
participated in the statistical analysis and procedures and the drafting of the
manuscript. EBC, IS, SS and AD participated in the laboratory work and data
interpretation. JBY, OE and ECB carried out the study. NW, MS and TJTD
contributed to analysis and interpretation and to contribute to writing the
paper. All the authors read and approved the final version.
Acknowledgements
We thank the population of the study villages and local authorities of the
health district of Sapone for their cooperation, and the Ministry of Health,
Burkina Faso. We are grateful to the staff of the Centre National de
Recherche et de Formation sur le Paludisme (CNRFP) whose participation
has made this study possible and particularly to SERME Samuel for skilful
technical assistance. We are also grateful to Mr. Walter Jones and Dr. Steven
Rosenthal from NIAID/DMID for their helpful support.
This study was supported by the regular budget of the National Institutes of
Allergy and Infectious Diseases, National Institutes of Health, Department of
Infectious Disease, Bethesda, MD (NIH, NIAID,DMID (DMID Contract
HHSN266200400016C).
Author details
1
Centre National de Recherche et de Formation sur le Paludisme,
Ouagadougou, Burkina Faso.
2
Groupe de Recherche et Action en Santé,
Ouagadougou, Burkina Faso.
3
The EMMES Corporation Rockville, Maryland,
USA.
Received: 17 January 2012 Accepted: 19 April 2012
Published: 4 May 2012
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doi:10.1186/1475-2875-11-154
Cite this article as: Bougouma et al.:Haemoglobin variants and
Plasmodium falciparum malaria in children under five years of age living
in a high and seasonal malaria transmission area of Burkina Faso.
Malaria Journal 2012 11:154.
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