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Maeder et al. BMC Hematology (2016) 16:30 Page 2 of 9

Background Methods
During its 59th World Health Assembly held in 2006, Study design and population
WHO recognized sickle-cell disease (SCD), an inherited This is an observational cross-sectional study with pro-
disorder of haemoglobin as a priority of public health. A spective data collection of children aged between 2 to
resolution was adopted to develop and strengthen efforts 59 months presenting with fever (axillary temperature
for its prevention and management. Sickle-cell anaemia, =37.5 °C according to WHO criteria). The study was
one of the most common forms of SCD, is due to a conducted from May 2011 to November 2013 in the
point mutation within the sixth codon of the ß-globin rural village of Ampasimanjeva located in the south east
chain. The produced abnormal variant – haemoglobin cost of Madagascar. Ampasimanjeva is 320 km away
S(HbS) – is responsible for chronic haemolytic an- from the capital city Antananarivo with a total inhabit-
aemia and vaso-occlusion which are the underlying ant estimated to be 22,000 according to the last census.
causes of the clinical presentation of SCD. Individuals The east coast has hot and humid subequatorial climate
who express the homozygous form (HbSS) manifest the and an annual high cumulative rainfall of 4,000 mm per
disease, while those with the heterozygous form year. Transmission of malaria in this region is reported
(HbAS), also known as sickle-cell trait (SCT), are usu- to be strong and intense all over time. A standardized
ally asymptomatic carriers. questionnaire on socio-demographic and clinical data
Initially limited to the sub-Saharan Africa, the Middle- was fulfilled for each individual. The Ampasimanjeva
East and some parts of India [1], SCD has currently community hospital is a Hospital District Centre receiv-
spread to all continents with the migration of popula- ing each year around 950 children between 2 and 5 years
tions. The ßS-globin gene is found on five common hap- old. Acute Respiratory Infections were the main cause of
lotypes in Africa (Bantu, Benin, Cameroon and Senegal) morbidity accounting for 35% of consultation all ages
and Asia (Saudi Arab-Indian) [2] that can be used as a combined.
marker of genetic diversity and population origin. Ap- Ethical approval for the study was obtained from the
proximately 300,000 children with severe haemoglobin National Ethical Committee of the Ministry of Health of
disorders are born every year worldwide [3] and over Madagascar (authorization 019-CE/MINSAN as of 09/
75% of SCD occur in Africa where carrier frequency 04/2010). Individual written informed consent was pro-
ranged from 1% (Central African Republic, Senegal) to vided by the parents of all study participants.
38% (United Republic of Tanzania) [1]. In Madagascar,
several studies conducted in 1950s had estimated the Sample collection
general prevalence of SCD from 4% in the Central For each child, venous blood was collected with safety
highlands [4] to 11-13% in the South-eastern coast blood containers on dry and spray-dried EDTA. The sam-
[5]. Significant ethnical variation and high prevalence ples were transported in a refrigerated container at +4 °C
in asymptomatic carrier up to 28% were also reported to the Centre d’Infectiologie Charles Mérieux for analysis.
[6]. To our knowledge, the last published study was Transport of samples was organized twice a week.
done in the early 1980s [7]. These studies, however,
were based on the detection of sickle red blood cells Genotyping of haemoglobin
in hypoxic conditions, also known as “sickling test”, After DNA extraction (QIAamp DNA Mini Kit, Qiagen,
and the sickle solubility test [3] now considered as Germany) of the EDTA blood samples, the mutation re-
obsolete techniques. sponsible for the generation of HbS was identified by
In area where malaria is endemic, its relation with Restriction Fragment Length Polymorphism (RFLP)
SCT was often studied, in particular the high degree of assay of PCR-amplified DNA (Bio-Rad, USA) [11]. The
resistance to severe and complicated malaria in sickle RFLP pattern of the normal profile (HbAA) of healthy
cell trait [8, 9]. Malaria is endemic in Madagascar with subject is characterized by the presence of the restriction
strong and persistent transmission in the east coast [10]. endonuclease DdeI site and cleaved in 2 fragments (189
From May 2011 to November 2013, a cross-sectional and 93 bp). The homozygous profile (HbSS) responsible
study was conducted in the rural village of Ampasiman- of the disease has no restriction site and only one frag-
jeva in the south east cost of Madagascar aiming to ment of 282 bp is observed. The heterozygous profile
identify blood-borne protein biomarkers that can differ- (HbAS) also known as sickle-cell trait combines restric-
entiate the causes of unexplained acute febrile illness in tion and no restriction site and reveals 3 fragments (93,
children. The study site is known for its high endemicity 189, and 282 bp).
to malaria, acute respiratory infections and SCD. The
main objective of this study was to estimate the preva- Malaria assays
lence of SCD; the secondary objective was to evaluate its Rapid Diagnostic Test (RDT) of malaria was performed
association with malaria and respiratory infections. on site using CareStart™ Malaria (Access Bio, Inc., New