healthmedicinet

Study population

Study participants were enrolled at the Korogwe District Hospital and from two villages,
Kwamasimba and Mkokola, in northeastern Tanzania. From Korogwe District Hospital,
children under the age of 5 years were enrolled, if presenting with any symptoms of
malaria, after obtaining informed consent from a parent or legal guardian. For diagnostics
and research, blood samples were collected and treatment was initiated if the patients
were positive by a malaria rapid diagnostic test (mRDT), following national guidelines.
Children were considered as having severe malaria if haemoglobin levels were 5 g/dl
(severe anaemia), assessed with a Blantyre coma score below 3 (cerebral malaria),
showing clinical signs of respiratory distress, or reaching a parasitaemia level above
200.000 asexual stages/µl (hyperparasitaemia). Children not fulfilling the above criteria
were defined as having uncomplicated malaria, as previously described 29]. Furthermore, convalescent blood samples from a sub-set of malaria patients were
collected after recovery. These samples were randomly selected from a database of
the original study of which all the hospitalized patients of this study were part
30], 31].

As part of an ongoing, annual, malariometric, cross-sectional study in the study communities,
blood samples were collected from volunteers residing in the villages of Mkokola and
Kwamasimba, Korogwe district, northeastern Tanzania in 2007 who tested negative for
P. falciparum by blood smear microscopy. The study area has previously been described by others
32], 33].

Determination of the EPCR haplotypes H1, H2, H3, and H4 of the PROCR gene

After collection of whole blood, the plasma and buffy coat were each separated from
the erythrocytes and stored at ?80 and ?20 °C, respectively. DNA was extracted from
the buffy coat with the NucleoSpin Blood Kit (Machery–Nagel) according to manufacturer’s
instructions.

EPCR is encoded by the PROCR gene on chromosome 20 and consists of four exons and three introns 34], 35]. Primers for the PCRs were designed to amplify two regions of the PROCR gene which define the H1, H3 and H4 haplotypes: the T(4414)C and C(4868)T SNPs in
intron 1, and the A(6936)GSNP in exon 4. H1, H3 and H4 were defined based on earlier
publications 18], 19], whereas H2 was defined as being none of the above (Table 1) 18], 19]. Size and specificity were tested by agarose gel electrophoreses. For each PCR reaction,
1 µl DNA was amplified in a 10 µl reaction consisting of 1.5 µl H
₂
O, 2.5 µl 3 µM primermix, and 5.0 µl TEMPase Hot Start Master (Ampliqon). The reaction
was performed in a 96-well PCR plate (Starlab GmbH, Hamburg, Germany) in a VWRi Duo
Cycler (VWR/Bie Berntsen, Radnor, PA, USA).

Table 1. Haplotypes of the PROCR gene encoding endothelial protein C receptor (EPCR)

Following the PCR reactions, the products were mixed, and 2 µl used in the succeeding
analysis; a ligase detection reaction-fluorescent microsphere assay (LDR-FMA). The
LDR-FMA was developed similar to a previously described method 36] to analyse the samples for the EPCR haplotypes with minor modifications; 2 µl PCR
product was used in the following ligase detection reaction (LDR), added to a 13 µl
mastermix consisting of 11.3 µl H
₂
O, 0.15 µl 2 µM LDR primermix, 1.5 µl ligase reaction buffer, and 0.05 µl (2 units)
Taq DNA ligase (both from New England Biolabs, Beverly, MA, USA). The reaction was
performed in a 96-well PCR plate (Starlab GmbH, Hamburg, Germany) in a VWRi Duo Cycler
(VWR/Bie Berntsen). The conditions of the following microsphere assay were modified
on a few parameters; 12 µl TMAC (3 M tetramethyl-ammonium chloride, 50 mM Tris–HCl
(pH 8.0), 3 mM EDTA (pH 8.0), 0.1 % SDS)/1.2 µl Streptavidin-R-phycoerythrin conjugate
(Sigma Aldrich, St Louis, MO, USA) (10 µg/ml) solution was added to each well in the
second hybridization; 75 of each microsphere were analysed. All primers were designed
based on the GenBank accession number AF106202 and are shown in Table 2 along with reaction conditions and tags complementary to Luminex MagPlex-TAG™ microspheres
(Luminex Corp, Austin, TX, USA). Control samples were kindly provided by Dr Shirley
Uitte de Willige from the Department of Haematology, Erasmus University Medical Centre
Rotterdam, The Netherlands.

Table 2. Primers and conditions for the PROCR polymerase chain reaction (PCR) and ligase detection reactions (LDR)

Determination of sEPCR in patient plasma samples

The levels of sEPCR in the patient plasma samples were determined by enzyme-linked
immunosorbent assay (ELISA) (Asserachrom, Stago, France) according to manufacturer’s
instructions.

Statistical analysis

Differences in mean age and distribution of gender between the groups were analysed
with Mann–Whitney and Chi square test, respectively. The genotype distributions for
each SNP were analysed for deviations from the Hardy–Weinberg Equilibrium with The
Court Lab Calculator 37]. Chi square test was used to analyse differences in allele or haplotype frequencies.
Differences in sEPCR levels were analysed by Mann–Whitney tests. All analyses were
done using SigmaPlot 12.3 (SPSS Inc.). P values  0.05 were considered statistically
significant.

Ethical clearance

Ethical clearance for the study was granted by the Tanzania Medical Research Coordinating
Committee and Ministry of Health and Social Welfare, Tanzania. Informed consent was
obtained from parents or legal guardians.