ral
ssBioMed CentMalaria Journal
Open AcceResearch
Complement activation in Ghanaian children with severe 
Plasmodium falciparum malaria
Gideon K Helegbe1,4,6, Bamenla Q Goka2, Joergen AL Kurtzhals3, 
Michael M Addae4, Edwin Ollaga5, John KA Tetteh4, Daniel Dodoo4, 
Michael F Ofori4, George Obeng-Adjei2, Kenji Hirayama6, 
Gordon A Awandare7 and Bartholomew D Akanmori*4
Address: 1Department of Biochemistry and Molecular Medicine, SMHS, UDS, Tamale, Ghana, 2Department of Child Health, Korle-Bu Teaching 
Hospital, Accra, Ghana, 3Centre for Medical Parasitology, Department of Clinical Microbiology, Copenhagen University Hospital (Righospitalet), 
Copenhagen, Denmark, 4Immunology Dept., NMIMR, College of Health Sciences, University of Ghana, Legon, Ghana, 5Blood Bank, Korle-Bu 
Teaching Hospital, Accra, Ghana, 6Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Japan and 
7Biochemistry Department, University of Ghana, Legon, Ghana, and Division of Military Casualty Research, Department of Cellular Injury, Walter 
Reed Army Institute of Research, Silver Spring, MD, Ghana
Email: Gideon K Helegbe - kofigidi@yahoo.com; Bamenla Q Goka - bamenla@yahoo.co.uk; 
Joergen AL Kurtzhals - joergen.kurtzhals@rh.regionh.dk; Michael M Addae - maddae@excite.com; Edwin Ollaga - maddae@excite.com; 
John KA Tetteh - jtetteh@noguchi.mimcom.net; Daniel Dodoo - ddodoo@noguchi.mimcom.net; 
Michael F Ofori - mofori@noguchi.mimcom.net; George Obeng-Adjei - goadjei@yahoo.com; Kenji Hirayama - hiraken@nagasaki-u.ac.jp; 
Gordon A Awandare - gordon.awandare@us.army.mil; Bartholomew D Akanmori* - bakanmori@noguchi.mimcom.net
* Corresponding author    
Abstract
Background: Severe anaemia (SA), intravascular haemolysis (IVH) and respiratory distress (RD)
are severe forms of Plasmodium falciparum malaria, with RD reported to be of prognostic
importance in African children with malarial anaemia. Complement factors have been implicated in
the mechanism leading to excess anaemia in acute P. falciparum infection.
Methods: The direct Coombs test (DCT) and flow cytometry were used to investigate the mean
levels of RBC-bound complement fragments (C3d and C3bαβ) and the regulatory proteins
[complement receptor 1 (CD35) and decay accelerating factor (CD55)] in children with discrete
clinical forms of P. falciparum malaria. The relationship between the findings and clinical parameters
including coma, haemoglobin (Hb) levels and RD were investigated.
Results: Of the 484 samples tested, 131(27%) were positive in DCT, out of which 115/131 (87.8%)
were positive for C3d alone while 16/131 (12.2%) were positive for either IgG alone or both. 67.4%
of the study population were below 5 years of age and DCT positivity was more common in this
age group relative to children who were 5 years or older (Odds ratio, OR = 3.8; 95%CI, 2.2–6.7,
p < 0.001). DCT correlated significantly with RD (β = -304, p = 0.006), but multiple regression
analysis revealed that, Hb (β = -0.341, p = 0.012) and coma (β = -0.256, p = 0.034) were stronger
predictors of RD than DCT (β = 0.228, p = 0.061). DCT was also not associated with IVH, p =
0.19, while spleen size was inversely correlated with Hb (r = -402, p = 0.001). Flow cytometry
Published: 17 December 2007
Malaria Journal 2007, 6:165 doi:10.1186/1475-2875-6-165
Received: 11 July 2007
Accepted: 17 December 2007
This article is available from: http://www.malariajournal.com/content/6/1/165
© 2007 Helegbe 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.Page 1 of 9
(page number not for citation purposes)
showed similar mean fluorescent intensity (MFI) values of CD35, CD55 and C3bαβ levels on the
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
surfaces of RBC in patients and asymptomatic controls (AC). However, binding of C3bαβ
correlated significantly with CD35 or CD55 (p < 0.001).
Conclusion: These results suggest that complement activation contributed to anaemia in acute
childhood P. falciparum malaria, possibly through induction of erythrophagocytosis and haemolysis.
In contrast to other studies, this study did not find association between levels of the complement
regulatory proteins, CD35 and CD55 and malarial anaemia. These findings suggest that
complement activation could also be involved in the pathogenesis of RD but larger studies are
needed to confirm this finding.
Background
The mortality associated with malaria largely occurs in
children as a result of complications, such as severe anae-
mia (SA), intravascular haemolysis (IVH), cerebral
malaria (CM) and metabolic acidosis, clinically mani-
fested as respiratory distress (RD) [1-5]. Sub-Saharan
Africa accounts for 90% of the world's 300–500 million
malaria cases and 1.5–2.7 million deaths annually [6]. A
recent study has shown that in Ghana, the most common
manifestations of severe malaria (SM) are SA (36.5%), fol-
lowed by RD (24.4%) and CM (5.4%) [7]. There are no
reports of the relative contribution of IVH to SM cases in
Ghana.
IVH due to P. falciparum is a condition with high case-
fatality if diagnosis and treatment are not optimal [8]. It is
usually considered a rare complication of malaria in
endemic areas, but recent studies have highlighted its
importance [5,9]. Although it appears that the direct RBC
destruction due to IVH is a minor contributor to malarial
anaemia it is nonetheless strongly associated with eryth-
rophagocytosis and with a poor prognosis [5,9]. Most
studies of IVH in malaria have focussed on the influence
of glucose 6-phosphate dehydrogenase (G6PD) [10-12],
and the role played by antimalarial drugs such as chloro-
quine [13,14]. Mechanical trauma from a damaged
endothelium, complement fixation and activation on the
RBC surface, and infectious agents may cause direct mem-
brane degradation and cell destruction [4].
It has been observed that the degree of red blood cell
(RBC) breakdown during acute malaria cannot be
explained solely by the direct destruction of RBC by
malaria parasite schizogony [15]. Thus depletion of RBC
is thought to be partly immune-mediated [15]. Infected
erythrocytes bind to endothelial cells, and P. falciparum
antigens known as erythrocyte membrane protein 1
(PfEMP1), inserted into the infected erythrocyte surface,
mediate this interaction. It has been argued that these
antigens are recognized by IgG subclasses that activate the
classical complement pathway [16]. This pathway may
also be triggered by binding of immune complexes or
surfaces, are activated to phagocytose the infected RBC.
Thus, complement does not seem to kill parasites directly,
but could play a role as an opsonin for neutrophils and
macrophages [18]. In line with this, previous studies have
shown that binding of complement factor C3d to RBC is
common in childhood malaria, whereas IgG binding is
rare [19].
A role for complement activation in RBC breakdown dur-
ing malaria is supported by reports of positive DCT in
patients with anaemia [20-23]. In addition, the balance
between the beneficial immune activation functions of
the complement cascade and its detrimental role in dis-
ease pathogenesis is maintained by a large number of reg-
ulatory proteins. Some of these include, complement
receptor 1 (CD35), which binds C3b, and decay accelerat-
ing factor (CD55) and membrane attack complex inhibi-
tor factor (CD59), which play a role in regulating
haemolysis due to deposition of immune complexes on
the surface of RBC [24]. Studies have shown that, deficien-
cies of these membrane-bound complement-regulatory
proteins on infected and uninfected erythrocytes are asso-
ciated with SM [25-28]. However, these studies did not
use strictly defined patient categories and, in some cases
malaria diagnosis was not confirmed in the control
groups. In the present study, the role of complement acti-
vation in the pathogenesis of SM was investigated in a
group of Ghanaian children presenting at hospital with
strictly defined manifestations of malaria, including
uncomplicated malaria (UM), SA, CM, IVH and RD. In
addition, the binding of complement factors and the
expression of complement regulatory proteins on RBC
was investigated by flow cytometry. Given the high rate of
mortality associated with RD in Ghanaian children [29],
and the association between SA and RD [7], the relation-
ship between complement activation and RD was also
examined.
Materials and methods
Study design and patient population
Children between one and twelve years of age with severe
forms of malaria as described previously [30], were con-Page 2 of 9
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dead merozoites to the RBC surface [17]. As a result,
monocytes, which have C3b and C3bαβ receptors on their
secutively recruited into an unmatched case control study
in July-August, 2000 at the Emergency Unit of the Depart-
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
ment of Child Health (DCH), Korle-Bu teaching Hospital,
Accra, Ghana. Apparently healthy children, randomly
selected from a database of children in the same age range
from a nearby community, Dodowa were enrolled as con-
trol subjects (AC) [29,30]. Malaria transmission in the
study area, a coastal savannah, is perennial with consider-
able seasonal variation, peaking during and immediately
after the rains (May-October). Residents are estimated to
receive about 20 infective bites per year and P. falciparum
constitutes 98% of all infections [31]. The Scientific and
Technical Committee of the Noguchi Memorial Institute
for Medical Research and the Ethics and Protocol Review
Committee of the University of Ghana Medical School,
Korle Bu, Accra approved the study. All patients and con-
trol subjects were enrolled in the study only after signed,
informed, parental consent was obtained.
Clinical investigation and inclusion criteria
Patients with axillary temperature > 37.5°C of no other
obvious cause than malaria were screened for inclusion by
a project physician. Clinical parameters were documented
on standard written forms. Spleen enlargement was
assessed by palpation and quantified as cm below the left
costal margin along the mid-clavicular line. Patients with
P. falciparum parasitaemia ≥ 10,000 parasites/µL were
enrolled into the study if they fell into one of the follow-
ing patient categories [29]: SA: haemoglobin (Hb) < 5 g/
dL, fully conscious with no episodes of severe bleeding,
reported or observed convulsions; CM: Blantyre coma
score ≤ 3 and duration of coma > 60 minutes, any Hb
value and no record of recent severe head trauma and
other cause of coma or neurological diseases; IVH: evi-
dence of haemoglobinuria detected by the urine dipstick
test (Roche Diagnostics Ltd, Great Britain) followed by
microscopy as described elsewhere [21]; RD: rapid breath-
ing plus one or more of the following: alar flare, chest
recessions, use of accessory muscles for respiration, or
abnormally deep breathing; UM: Hb > 8 g/dL, fully con-
scious, no other features of SM. Patients with Hb between
5 and 8 g/dL were only included if they fell into one or
more of the patient categories: IVH, CM and RD.
Exclusion criteria
Patients who were sickling positive were excluded. Other
haemoglobinopathies were not taken into consideration.
Patients were also excluded from the study based on evi-
dence of other infectious disease such as typhoid or upper
respiratory tract infections or any other identified cause of
anaemia than malaria. Patients with a history of antima-
larial treatment within 2 weeks prior to admission were
excluded [13,14].
Management of patients
at a total dose of 25 mg/kg body weight, given over three
days. In the event of treatment failure, treatment with
amodiaquine (10 mg/kg body weight per day, as single
daily doses for three days) was instituted. All patients with
SM were treated with either amodiaquine syrup via
nasogastric tube at the same dosage as described, or intra-
muscular quinine dihydrochloride (10 mg/kg body
weight, 8-hourly). Parenteral quinine was changed to
syrup at the same dosage when patients regained full con-
sciousness or after 72 hours (which ever was earlier), to
complete a 7-day course. Patients with SA or RD were
given humidified oxygen and children with Hb < 5 g/dL
received blood transfusions [29].
Blood sampling and laboratory analysis
Immediately after admission, 5 ml of venous blood was
collected into EDTA tubes from all patients screened for
inclusion. Haematological profile was determined using
an 18-parameter, automatic haematology analyzer (Sys-
mex KX-21, Japan). Sickling test was done using the
sodium metabisulphite test. Giemsa-stained thick and
thin blood films were used for microscopic detection and
identification of Plasmodium parasites. Parasites were
counted against 300 WBC, and the value was converted
into parasites per µL of peripheral blood, based on indi-
vidual WBC count. Quality control (QC) was ensured by
checking the autoanalyser daily and testing of 10% slides
by second look. Errors were below 10% variation or disa-
greement and without systematic deviations.
Direct Coombs' test
DCT was done using the method described by Goka et al.
[21]. RBC suspensions were washed 4 times in compara-
tively large volumes (4 ml per wash) of 0.9% saline by
centrifugation and reconstituted with saline to 5% PCV.
One drop of this suspension was mixed with two drops of
poly-antiglobulin sera (DIAGAST laboratories, Cedex,
France), followed by centrifugation at 1,200 rpm for 1
minute. The samples were immediately inspected macro-
scopically for agglutination and negative or doubtful pos-
itive/weak results were re-examined by light microscopy.
The positive samples were re-tested using mono-specific
antiserum against IgG and C3d. Test results were graded as
+4 (complete agglutination), +3 (several large aggluti-
nates, few cells), +2 (large agglutinates in a sea of smaller
clumps and free cells), +1 (many small agglutinates) or 0
(no agglutination). Positive and negative controls were
run in parallel.
Flow cytometry
Packed RBC were washed twice by centrifugation and
resuspended in PBS (pH 7.2) to 2.0 × 107 cells/ml. The
RBC suspension was stained with ethidium bromide (50Page 3 of 9
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Based on institutional practice at the time, all patients
with UM were treated with a standard chloroquine regime
µg/ml final concentration), followed by surface staining
with FITC-conjugated antibodies in the dark [32]. The fol-
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
lowing antibodies were used: Mouse IgG1 FITC (Pharmi-
gen International, 33814X, San Diego, CA, USA); CD35
(Pharmigen, 30961A); IgG (Becton Dickinson, San Jose,
CA, USA, 345140{5140}); Mouse IgG2a FITC (Pharmi-
gen, 33034X); CD55 (Pharmigen, 33571A); Mouse IgG1
pure (DAKO, Glostrup, Denmark, X 0931); C3bαβ (Cym-
bus Biotechnology Ltd., Hampshire, UK, CBL 189 for α;
CBL 190 for β); Rabbit IgG pure (Zymed Laboratories,
INC., San Franscisco, California, USA, 81245172); C3d
pure (DAKO, A 0063); C3d FITC (DAKO, F 0323) and
controls (Goat anti-mouse FITC (DAKO, F0479); Swine
anti-rabbit FITC (DAKO, F0054). After incubation, cells
were washed twice with cellwash (Becton Dickinson) by
centrifugation and samples were stored at 4°C in the dark
till acquisition on a FACScan flow cytometer (Becton
Dickinson, Japan). A minimum of 10,000 RBC were
acquired and analysis was done by the CellQuest pro-
gramme. The cut-off point was 101 on both axes.
Statistical analysis
Statistical analysis was performed using SigmaStat soft-
ware package (Jandel Scientific). Continuous variables
were compared between groups using the student t-test or
one way analysis of variance (ANOVA). For data that were
not normally distributed and could not be normalised by
logarithmic transformation a one-way ANOVA on ranks
was used. Correlation between parameters was deter-
mined by Spearman rank order test. Proportions were
compared using the Chi-square test, or Fisher's exact test.
Variables that showed significant or near-significant dif-
ferences between groups by univariate analysis or that
were otherwise considered relevant for the study were
entered in a conditional logistic regression analysis and in
a multiple regression analysis. P-values < 0.05 were con-
sidered significant.
Results
Characteristics of the patient categories and controls
A total of 484 parasitaemic patients were screened for
enrolment of which 87 patients with distinct clinical pres-
entations of SM were included, 36 SA, 18 IVH, 27 CM and
6 RD. Their clinical characteristics together with those of
UM patients and AC are summarised in Table 1. SA
patients were younger (mean age = 2.5 yrs.) than the other
categories of patients and the AC group (mean age 5.7
yrs.) (p < 0.001). SA had the lowest mean Hb (4.1 g/dL),
followed by RD (5.5 g/dL) and IVH (6.5 g/dL). The high-
est mean Hb was found in AC (10.9 g/dL). All the patient
categories had high parasitaemia whilst AC had the lowest
parasite density (geometric mean, 1.4 × 103 parasites/µL).
The geometric mean parasite densities of SA and UM cases
(63.0 and 57.3 × 103parasites/µL respectively) were signif-
icantly lower than those of the IVH, CM and RD cases
3 3 3 
complement activation on RD, the data were re-analysed
for all patients with SM, including those with overlapping
clinical manifestations. The characteristics of these 104
children is summarised in Figure 1. Forty-six children had
Hb < 5 g/dL, 34 had coma score ≤ 3, 22 had haemoglob-
inuria and 20 had RD.
Association of variables in children with severe malaria
Of all the parameters tested, only age and spleen size
showed a significant correlation with Hb (Table 2). Age
was positively correlated with Hb (r = 0.379, p = 0.002),
and spleen size was inversely correlated to Hb (r = -0.402,
p = 0.001).
Clinical characteristics of DCT positive and negative 
patients and controls
Of all the patients screened (484), 27.0% (131) were DCT
positive. Most of the sensitisation was due to C3d alone
(87.8%), with a small proportion being ascribed to IgG
alone (2.3%) or to both (9.9%). Children below 5 years
formed 84.7% (111/131) of the DCT positives relative to
those of 5 years and older, OR = 3.8 (95% CI, 2.2–6.7, p
< 0.001). There was a high prevalence of DCT positive
cases in the RD (50.0%), IVH (38.8%) and SA (27.8%)
patients whereas UM and CM had 9.5% and 11.8% prev-
alence respectively. Surprisingly, 26.3% of the AC patients
had positive DCT (Table 1). When overlapping cases were
included in the analysis, RD patients still had the highest
DCT positive rate with (50.0%, (10/20)), followed by IVH
(45.5%, (10/22)); SA (34.8%, (16/46)) and CM (20.6%,
(7/34)), Figure 1. Of all the children with severe forms of
malaria (SM, n = 104), 33 (31.6%) were DCT positive
(Table 3). There were no significant differences in age and
parasitaemia between the DCT positive and the DCT neg-
ative patients of the SM (p > 0.05, Table 3), whereas the
mean Hb was significantly lower in the DCT positive
patients (p < 0.001). Although there was a trend toward
higher prevalence of a positive DCT in RD, IVH, and SA,
compared with CM, these associations were not statisti-
cally significant (Table 3).
Relationship between DCT result and RD
Out of 20 RD cases, 50.0% (10/20) were DCT positive
compared with 27.4% (23/84) non RD cases, p = 0.092.
Furthermore, the strength of the DCT result (0–4) corre-
lated significantly with RD (standard coefficient β = -
0.304, p = 0.006). Since children with RD appeared to be
more likely to have DCT positive results compared to
those without RD, the relationship between DCT result
and RD was further investigated using multiple linear
regression where the strength of the DCT result, Hb, coma
score, and age were tested for their contribution to RD. In
this model, Hb (β = -0.341, p = 0.012) and coma score (βPage 4 of 9
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(98.7 × 10 , 114.9 × 10 and 80.9 × 10 parasites/µL,
respectively, p < 0.001). In order to study the effect of
= -0.256, p = 0.031, Table 4) emerged as the strongest pre-
dictors of RD compared with DCT grade (β = 0.228, p =
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
Page 5 of 9
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Table 1: Demographic, clinical and laboratory characteristics of patients and healthy controls
Characteristics SA IVH CM RD UM AC p value
n 36 18 27 6 21 19 -
Female sex, % 43.7 21.1 40.7 60.0 42.8 47.8 -
Mean Age, years (95% CI) 2.5** (1.8–3.1) 4.8 (3.1–6.6) 4.3 (3.3–5.3) 2.7** (2.0–5.5) 5.2 (3.6–6.8) 5.7 (4.4–7.1) <0.001b
DCT Positive, n (%) 10* (27.8%) 7* (38.8%) 4 (11.8%) 3* 50.0% 2 (9.5%) 5.0* (26.3%) <0.05a
Mean Hb, g/dL (95% CI) 4.1 (3.8–4.3) 6.5 (5.6–7.4) 7.2 (6.4–8.0) 5.5 (4.6–6.2) 9.2 (8.5–9.8) 10.9 (10.6–11.4) -
Parasite density, geometric mean 
parasites × 103/µL (95% CI) 63.0*** (34.3–94.1) 98.7 (47.6–284.5) 114.9 (67.4–162.6) 80.9 (12.1–256.9) 57.3*** (41.0–107.3) 1.4 (0–2.9) <0.001
b
Spleen size, cm (95% CI) 2.3 (1.5–3.0) 0.6(0–1.6) 0.7 (0.1–1.4) ND 1.9 (0.6–3.2) ND -
Non-overlapping patient groups (see text). SA, severe anaemia (Hb < 5 g/dL). IVH, intravascular haemolysis with evidence of haemoglobinuria. CM, 
cerebral malaria (coma score ≤ 3). RD, respiratory distress. UM, uncomplicated malaria with Hb > 8 g/dL. AC, healthy, asymptomatic controls. 
DCT, direct Coombs' test. *Significantly higher than UM and CM. ** Significantly lower than CM, UM and AC. *** Significantly lower than IVH and 
CM. ND, not done.
a = Calculated using X2
b = Calculated using ANOVA
Clinical categories of children with severe malariaFigure 1
Clinical categories of children with severe malaria. Children admitted to the paediatric ward of the Korle-Bu teaching 
hospital, Accra, with severe malaria (SM). Symptoms of SM included coma (score ≤ 3 on the Blantyre coma scale), severe anae-
mia (SA) (haemoglobin [Hb] < 5.0 g/dL), intravascular haemoloysis (IVH, evidence of haemoglobinuria), and respiratory distress 
(RD). The number of children showing the various symptoms, as well as the mean (SEM) Hb (in g/dL) and parasitaemia (Para, in 
parasites per µL) plus prevalence of a direct Coombs' test (DCT) is shown. NP, no patient.
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
0.061) and age (β = 0.208 and p = 0.820). These results
suggest that the association between RD and DCT may be
related to the role of DCT in precipitating anaemia.
Relationship between opsonins (C3d and C3bαβ) and 
complement regulatory proteins (CD35 and CD55) in 
different patient categories
The association between the DCT method and flow
cytometry was confirmed by a positive correlation
between DCT grade and MFI after surface staining of RBC
with anti-C3d (r = 0.53, p < 0.001, Figure 2). In order to
study the influence of complement regulatory proteins on
clinical outcome, binding of C3d and C3bαβ and expres-
sion of CD35 and CD55 on the RBC surface were meas-
ured by flow cytometry in a subset of the patients (Table
5). As shown in Table 5, there were no differences in any
of the tested parameters between the main clinical groups
(p > 0.7). The binding of C3bαβ correlated significantly
with CD35 and CD55 (p < 0.001) in children with SM,
Figure 3. There was no correlation between C3d and either
CD35 or CD55 (p > 0.05, data not shown).
Discussion
Results from the present study show that infection with P.
falciparum parasites stimulates complement activation,
consistent with previous studies [21-23,33]. The comple-
ment activation was associated with reduced Hb-levels
[21,22,24]. A central molecule in this association appears
to be C3d that can opsonise the RBC for erythrophagocy-
tosis. To a large extent, the erythrophagocytosis is medi-
ated by macrophages, as indicated by increased levels of
neopterin [29,30,34], which is a marker of macrophage
activation [35]. The combination of activated macro-
phages and RBC opsonised by complement after activa-
tion will lead to increased erythrophagocytosis and
decreased Hb as demonstrated in other studies [21,36].
This was reflected in an inverse correlation between
spleen size and Hb and a trend toward larger spleen size
in DCT positive than in DCT negative patients. Thus,
phagocytosis of iRBC caused by macrophages in the
spleen, enhanced by the opsonisation of RBC by C3d,
may be one of the mechanisms by which Hb drops due to
P. falciparum infection. However, a prospective study com-
paring DCT positive and negative patients in relation to
drop in Hb after initiation of treatment would be relevant
to confirm a causal relationship.
Complement activation could additionally contribute to
malarial anaemia by direct lysis of infected RBC [5]. How-
ever, both this and a previous study [21] only showed a
weak and insignificant association between DCT results
and haemoglobinuria. Other factors apart from comple-
ment that may trigger IVH in malaria are drugs such as
chloroquine [13,14], genetic factors [10,37] and autoim-
Table 3: Clinical characteristics of all children with severe malaria grouped by DCT results
Characteristics DCT positive DCT negative P value
n (%) 33 (31.6%) 71 (68.2%) -
Age, years 3.3 (2.5–4.0) 4.0 (3.4–4.6) 0.11b
Hb, g/dL 5.0 (4.3–5.6) 6.6 (6.1–7.1) <0.001b
Parasite density, × 103/µL 125.6 (81.5–169.7) 139.7 (107.6–171.8) 0.33b
Mean spleen size, cm 2.1 (1.1–3.1) 1.2 (0.6–1.8) 0.09b
SA, n (%) 16 (48.5%) 30 (42.3%) 0.70a
CM, n (%) 7 (21.2%) 27 (38.0%) 0.15a
Haemoglobinuria, n (%) 10 (30.3%) 12 (16.9%) 0.19a
Mortality, n (%) 4 (12.1%) 15 (21.1%) 0.40a
Data are mean (95% confidence interval) unless otherwise indicated. Patients include all patients in Figure 1. Thus, the same patient can appear in 
several categories.
Table 2: Correlation between variables in children with severe or uncomplicated malaria
Hb Spleen Coma Log para
Variable r p r p r p r p
Age 0.379 0.002 -0.066 0.610 -0.142 0.265 -0.0618 0.630
Hb -0.402 0.001 -0.201 0.114 -0.053 0.678
Spleen 0.270 0.032 -0.170 0.183
Coma -0.208 0.102
Hb, haemoglobin. Spleen size, cm below left costal margin. Coma, score ≤ 3 on the Blantyre coma scale. Para, parasite (per µL). Patients analysed 
here are all the SM and UM cases (n = 125). Asymptomatic controls (AC) are not included.Page 6 of 9
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a = Calculated using X2 or Fischer's exact test
b = Calculated using, student t-test
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
munity [38]. Apart from excluding sickle cell genes, other
factors were not controlled for in this study. However, the
lack of association between DCT and IVH suggests that the
main contribution of complement activation to the
pathogenesis of malarial anaemia is opsonisation for
erythrophagocytosis. The surprisingly high prevalence of
DCT positives in the AC group indicates that a number of
apparently healthy children may have an activated com-
plement system. In a related study [39], it was observed
that asymptomatic P. falciparum infection resulted in
immune activation and anaemia in semi-immune chil-
dren. The present study suggests that complement activa-
tion could contribute to the low Hb in the semi-immune
children.
In the present study the DCT [40] was used alongside flow
cytometry to determine the relationship between the lev-
els of complement fragments (C3bαβ) and regulatory
proteins (CD35 and CD 55). Other investigators have
shown that activation and levels of regulatory proteins on
surfaces of RBC are related to RBC loss in malaria [25,28].
In contrast, the present study did not find any relationship
between anaemia and CD35 and CD55 levels. Unlike the
current study, the previous studies did not use strictly
defined patient categories and, in some cases malaria
diagnosis was not confirmed. However, the discrepancy
could also be due to differences in responses to P. falci-
parum of children from distinct geographical locations or
exposed to varying levels of malaria transmission. There is
a need to perform additional investigations of the role of
complement regulatory proteins in the pathogenesis of
malaria anaemia. The correlation between C3 fragment
deposition and CD35 and CD55 levels reported here con-
firms results from previous studies [41], and suggests an
adequate role of CD55 in regulating the extent of haemo-
lysis in the studies patients.
Increased levels of inflammatory mediators have been
shown to play a role in RD [29]. High levels of comple-
ment factors being reported here may implicate a synergis-
tic role of these complement factors and inflammatory
Association of CD55 and CD35 with C3αβ in children with severe malariaFigure 3
Association of CD55 and CD35 with C3αβ in children 
with severe malaria. Correlation between C3bαβ and 
complement regulatory proteins (CD35 (complement recep-
tor type 1) and CD55 (decay accelerating factor)) measured 
by flow cytometry on surfaces of RBC from 30 children with 
SM. Solid line: C3bαβ vs CD55, r = 0.777, p < 0.001; dotted 
regression line: C3bαβ vs CD35, r = 0.624, p < 0.001. MFI, 
mean fluorescence intensity.
Table 4: Multiple regression analysis of variables predicting 
respiratory distress
Characteristics Standard coefficient (β) P-value
Age (years) 0.208 0.820
Coma score -0.256 0.031
Hb (g/dL) -0.341 0.012
DCT grade (0–4) 0.228 0.061
NB: Patients analysed here are all the SM and UM cases (n = 125). AC 
were not included.
DCT results compared with flow cytometry using anti-C3dFigure 2
DCT results compared with flow cytometry using 
anti-C3d. RBC suspensions from 30 DCT positive and nega-
tive SM patients were stained with ethidium bromide and 
monoclonal antibodies to detect surface-bound C3d. Associ-
ation between DCT results and flow cytometry results for 
C3d are shown by scatter plots with regression line. MFI, 
mean fluorescence intensity. DCT was graded as accordingly 
as +4 (complete agglutination), +3 (several large agglutinates, 
few cells), +2 (large agglutinates in a sea of smaller clumps 
and free cells), +1 (many small agglutinates), 0 (no sign of Page 7 of 9
(page number not for citation purposes)
mediators in causing the RD due to P. falciparum infec-
tion. Such a synergistic interaction has been shown to take
agglutination).
Malaria Journal 2007, 6:165 http://www.malariajournal.com/content/6/1/165
place during cardiac surgery with cardiopulmonary
bypass (CBP) [42]. Similarly, in addition to complement
activation during severe malaria infection, there is an
imbalance between pro- and anti-inflammatory
cytokines. Thus in children with SA complicated by RD,
institution of effective treatment during the early phase of
infection appears to be critical to survival [3,43]. The
application of simple clinical and laboratory guidelines
identifying children likely to develop SM and thus most in
need for rapid interventions may thus improve survival
and reduce unnecessary use of blood transfusion [3]. This
study demonstrates that DCT can be used as a simple and
reliable method of measuring complement activation in
children with acute malaria and may be used to predict
progression of UM to SA-RD and SA+RD. Prospective
studies are needed to validate this diagnostic approach.
Conclusion
The data presented here, supports a role for complement-
mediated removal of RBC through erythrophagocytosis in
the pathogenesis of malarial anaemia. In contrast to pre-
vious studies there was no relationship between the sever-
ity of anaemia and levels of complement receptor 1 or
decay accelerating factor. Complement activation could
also be involved in the pathogenesis of RD but larger stud-
ies are needed to confirm this finding. In addition the
study demonstrates that DCT might be used as a simple
method of predicting development of complications but
this need to be studied prospectively in subsequent inves-
tigations.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
GKH designed the study, carried out laboratory work, data
analyses and interpretation and drafted the manuscript.
BQG and GOA assisted with design of study, selection,
clinical examination and management of patients, as well
was involved with data analysis and extensive revision of
manuscript for intellectual content. JKAT, DD and MFO
designed the flow cytometry with GKH, acquired and ana-
lyzed the data. GAA was involved in data analyses, and
preparation and revision of manuscript. JALK and BDA
were involved in the design of experiment, data analysis
and revision of manuscript for intellectual content.
Acknowledgements
Children and their guardian/parents are thanked for participating in this 
study. Messrs John Arko-Mensah and William Vanderpuije are thanked for 
technical assistance. This study received financial support from United 
Nations Development Programme/World Bank/World Health Organiza-
tion Special Programme for Research and Training in Tropical Diseases 
(TDR) (Multilateral Initiative on Malaria/TDR grant 980037); Programme 
for Enhancement of Research Capacity in Developing Countries, Danish 
International Development Assistance (DANIDA).
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