Association of severe malaria outcomes with platelet-mediated clumping and adhesion to a novel host receptor.
ABSTRACT Severe malaria has been attributed partly to the sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs. Identification of P. falciparum cytoadherence phenotypes that are associated with severe malaria may lead to the development of novel strategies against life-threatening malaria.
Forty-six P. falciparum isolates from Mozambican children under 5 years of age with severe malaria (cases) were examined and compared to 46 isolates from sex and age matched Mozambican children with uncomplicated malaria (controls). Cytoadherence properties such as platelet-mediated clumping, rosetting and adhesion to purified receptors (CD36, ICAM1 and gC1qR), were compared between these matched pairs by non-parametric tests. The most common clinical presentation associated with severe malaria was prostration. Compared to matched controls, prevalence of platelet-mediated clumping was higher in cases (P = .019), in children presenting with prostration (P = .049) and in children with severe anaemia (P = .025). Prevalence of rosetting and gC1qR adhesion were also higher in isolates from cases with severe anemia and multiple seizures, respectively (P = .045 in both cases), than in controls.
These data indicate a role for platelet-mediated clumping, rosetting and adhesion to gC1qR in the pathogenesis of severe malaria. Inhibition of these cytoadherence phenotypes may reduce the occurrence or improve the prognosis of severe malaria outcomes.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Interest in mapping the global distribution of malaria is motivated by a need to define populations at risk for appropriate resource allocation and to provide a robust framework for evaluating its global economic impact. Comparison of older and more recent malaria maps shows how the disease has been geographically restricted, but it remains entrenched in poor areas of the world with climates suitable for transmission. Here we provide an empirical approach to estimating the number of clinical events caused by Plasmodium falciparum worldwide, by using a combination of epidemiological, geographical and demographic data. We estimate that there were 515 (range 300-660) million episodes of clinical P. falciparum malaria in 2002. These global estimates are up to 50% higher than those reported by the World Health Organization (WHO) and 200% higher for areas outside Africa, reflecting the WHO's reliance upon passive national reporting for these countries. Without an informed understanding of the cartography of malaria risk, the global extent of clinical disease caused by P. falciparum will continue to be underestimated.Nature 04/2005; 434(7030):214-7. · 38.60 Impact Factor
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ABSTRACT: BACKGROUND: In the treatment of severe malaria, intravenous artesunate is more rapidly acting than intravenous quinine in terms of parasite clearance, is safer, and is simpler to administer, but whether it can reduce mortality is uncertain. METHODS: We did an open-label randomised controlled trial in patients admitted to hospital with severe falciparum malaria in Bangladesh, India, Indonesia, and Myanmar. We assigned individuals intravenous artesunate 2.4 mg/kg bodyweight given as a bolus (n=730) at 0, 12, and 24 h, and then daily, or intravenous quinine (20 mg salt per kg loading dose infused over 4 h then 10 mg/kg infused over 2-8 h three times a day; n=731). Oral medication was substituted when possible to complete treatment. Our primary endpoint was death from severe malaria, and analysis was by intention to treat. FINDINGS: We assessed all patients randomised for the primary endpoint. Mortality in artesunate recipients was 15% (107 of 730) compared with 22% (164 of 731) in quinine recipients; an absolute reduction of 34.7% (95% CI 18.5-47.6%; p=0.0002). Treatment with artesunate was well tolerated, whereas quinine was associated with hypoglycaemia (relative risk 3.2, 1.3-7.8; p=0.009). INTERPRETATION: Artesunate should become the treatment of choice for severe falciparum malaria in adults.The Lancet 366(9487):717-25. · 39.06 Impact Factor
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ABSTRACT: Despite the use of potent antimalarial drugs, the fatality rate associated with severe malaria remains high. Adjunctive therapies that target the immunopathological responses to infection may decrease mortality associated with severe malaria. We hypothesized that peroxisome proliferator-activated receptor gamma agonists (eg, rosiglitazone) would modulate the host's innate immune response to malaria and improve outcome. In a randomized, double-blind, placebo-controlled, phase I/II trial of treatment for malaria acquired in Thailand, we investigated the safety, tolerability, and efficacy of rosiglitazone use for parasite clearance and for reducing malaria-induced inflammation. Sequential patients with uncomplicated Plasmodium falciparum malaria were randomly assigned to 1 of 2 groups: 70 patients received rosiglitazone 4 mg twice daily for 4 days, and 70 patients received a placebo twice daily for 4 days. Both groups also received standard antimalarial therapy (ie, a fixed combination of 1000 mg of atovaquone per day for 3 days and 400 mg of proguanil per day for 3 days). Primary efficacy outcomes were 50% and 90% parasite clearance times (PCTs). Secondary outcomes were fever clearance time, levels of inflammatory mediators, blood glucose measurements, aminotransferase levels, admission to intensive care, and subjective tolerability of study drug. For the 70 patients who received rosiglitazone, parasite clearance from peripheral blood was significantly enhanced, compared with the 70 patients who received a placebo (mean 50% PCT, 19.0 h vs. 24.6 h [p = .029]; mean 90% PCT, 30.9 h vs. 40.4 h [p = .004]). Also, the patients who received rosiglitazone had reduced inflammatory responses to infection, compared with the patients who received a placebo (ie, interleukin-6 levels at 24 h [p < .005] and at 48 h [p = .013] and monocyte chemoattractant protein-1 level at 48 h [p = .05]). There were no significant differences between the 2 groups with regard to safety and tolerability of treatment, and there were no admissions the intensive care unit or deaths. The use of rosiglitazone is a well-tolerated adjunct to standard therapy for nonsevere P. falciparum malaria. Treatment with rosiglitazone increased parasite clearance and decreased inflammatory biomarkers associated with adverse malaria outcomes. ClinicalTrials.gov identifier NCT00149383 .Clinical Infectious Diseases 09/2009; 49(6):841-9. · 9.37 Impact Factor
Association of Severe Malaria Outcomes with Platelet-
Mediated Clumping and Adhesion to a Novel Host
Alfredo Mayor1,2,3., Abdul Hafiz4., Quique Bassat1,2,3, Eduard Rovira-Vallbona1,3, Sergi Sanz1, So ´nia
Machevo2, Ruth Aguilar1,2, Pau Cistero ´1, Betuel Sigau ´que2, Clara Mene ´ndez1,2,3, Pedro L. Alonso1,2,3,
Chetan E. Chitnis4*
1Barcelona Centre for International Health Research (CRESIB), Hospital Clı ´nic/Institut d’Investigacions Biome `diques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona,
Barcelona, Spain, 2Centro de Investigac ¸a ˜o em Sau ´de de Manhic ¸a (CISM), Maputo, Mozambique, 3Consorcio de Investigacio ´n Biome ´dica de Epidemiologı ´a y Salud Pu ´blica
(CIBERESP), Madrid, Spain, 4International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, I´ndia
Introduction: Severe malaria has been attributed partly to the sequestration of Plasmodium falciparum-infected
erythrocytes (IEs) in the microvasculature of vital host organs. Identification of P. falciparum cytoadherence phenotypes
that are associated with severe malaria may lead to the development of novel strategies against life-threatening malaria.
Methods and Findings: Forty-six P. falciparum isolates from Mozambican children under 5 years of age with severe malaria
(cases) were examined and compared to 46 isolates from sex and age matched Mozambican children with uncomplicated
malaria (controls). Cytoadherence properties such as platelet-mediated clumping, rosetting and adhesion to purified
receptors (CD36, ICAM1 and gC1qR), were compared between these matched pairs by non-parametric tests. The most
common clinical presentation associated with severe malaria was prostration. Compared to matched controls, prevalence of
platelet-mediated clumping was higher in cases (P=.019), in children presenting with prostration (P=.049) and in children
with severe anaemia (P=.025). Prevalence of rosetting and gC1qR adhesion were also higher in isolates from cases with
severe anemia and multiple seizures, respectively (P=.045 in both cases), than in controls.
Conclusions: These data indicate a role for platelet-mediated clumping, rosetting and adhesion to gC1qR in the
pathogenesis of severe malaria. Inhibition of these cytoadherence phenotypes may reduce the occurrence or improve the
prognosis of severe malaria outcomes.
Citation: Mayor A, Hafiz A, Bassat Q, Rovira-Vallbona E, Sanz S, et al. (2011) Association of Severe Malaria Outcomes with Platelet-Mediated Clumping and
Adhesion to a Novel Host Receptor. PLoS ONE 6(4): e19422. doi:10.1371/journal.pone.0019422
Editor: Anne Charlotte Gruner, Agency for Science, Technology and Research (A*STAR), Singapore
Received November 18, 2010; Accepted April 2, 2011; Published April 29, 2011
Copyright: ? 2011 Mayor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study received partial support from Instituto de Salud Carlos III (PS0901113) and Biomalpar grant of the European Commission (CEC). The Manhic ¸a
Health Research Centre receives core support from the Spanish Agency for International Cooperation. AH was supported by a Fellowship from the Council of
Scientific and Industrial Research (09/512 (70)/2004), AM, QB and ERV by the Instituto de Salud Carlos III (CP-04/00220, CM05/00134 and FI06/00019, respectively).
CEC is a recipient of a TATA Innovation Fellowship from Department of Biotechnology, Government of India. Work in the laboratory of CEC is partially supported
by EVIMalaR, PregVax, PreMalStruct and MalSig grants from the European Commission. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Plasmodium falciparum causes approximately 500 million clinical
episodes of malaria per year . A small fraction (1–2%) of these
malaria attacks develop into severe malaria, specially in children
less than 5 years of age . Case fatality rates for severe malaria
remain unacceptably high even after effective anti-malarial drugs
are administered . Several ancillary treatments have been tested
in combination with anti-malarial drugs [4–6] but, to date, there is
little evidence to recommend their use. A greater understanding of
the pathogenesis of severe malaria is essential for the rational
development of novel interventions.
The following two theories are proposed to explain the patho-
physiological mechanisms leading to severe malaria. The cytokine-
based hypothesis suggests that inflammatory mediators such as
cytokines and nitric oxide released by host cells following exposure
to malaria ‘toxins’ have a major role in the onset of the pathology of
severe malaria . In contrast, the mechanical theory suggests that
severe malaria is attributable to excessive sequestration of P.
falciparum-infected erythrocytes (IEs) in the microvasculature of vital
host organs leading to mechanical obstruction of blood flow,
hypoxia, tissue damage and, ultimately, organ failure . Seques-
tration of parasites is suggested to be mediated through the
adherence of mature forms of P. falciparum IEs to host receptors
expressed on the endothelium lining host capillaries, on uninfected
erythrocytes to form ‘rosettes’  and on platelets to form platelet-
mediated clumps . The following observations support cy-
toadherence as the main process leading to severe malaria. First,
PLoS ONE | www.plosone.org1 April 2011 | Volume 6 | Issue 4 | e19422
among all human malaria parasites, only P. falciparum, which is
responsible for most of the malaria related deaths, is known to
sequester . Second, autopsy studies have suggested that parasite
sequestration plays an important role in cerebral malaria (CM)
[11,12]. Third, estimationof the sequestered parasite biomass based
on plasma histidine-rich protein-2 has shown that disease severity
increases with increasing parasite sequestration . Finally, organ-
specific accumulation of distinct P. falciparum variant types suggests
that IEs can exhibit preferential adhesion in the body .
In vitro analysis of the cytoadherence phenotypes of P. falciparum
isolates derived from infected individuals has shown a wide range
of binding affinities to numerous host receptors such as in-
tercellular adhesion molecule ICAM-1 , CD36 , chon-
droitin sulphate A (CSA) , gC1qR  and CR1 .
However, the patho-physiological significance of these cytoadher-
ence phenotypes remains unclear as associations with severe
malaria or specific signs of severity have been found in some
studies [10,20–26] but not in others [24,27–29]. Moreover, some
cytoadherence phenotypes, such as adhesion to CD36, have been
associated with uncomplicated malaria [22,24,29], suggesting a
protective role in the context of severe disease [30,31].
Here we have examined the relationship between specific ad-
hesion phenotypes of P. falciparum isolates, namely, adhesion to
ICAM1, CD36 and gC1qR, rosetting and platelet-mediated
clumping, and disease severity in children less than 5 years of
age residing in a malaria-endemic area of Mozambique.
Among the 142 parasite isolates included in the study, 111 (78%)
matured to trophozoite stages. The proportion of isolates that did
not recover from cryopreservation was similar for parasite isolates
collected from children with severe (n=19, 27%) or uncomplicated
malaria (n=12, 17%, p=0.223). Parasitemia at thawing was higher
for isolates from children with severe malaria (7.3%, interquartile
range [IQR] [2.2–12.4]) than in children with uncomplicated
malaria (3.5%, IQR [1.7–5.9], p=0.011). Of the 111 isolates that
matured to trophozoite stage, 46 matched case-control pairs of
parasites could be used for analysis. The characteristics of the 92
patients that provided the parasite isolates used are summarized in
Table 1. Paired analysis showed that children with severe and
uncomplicated malaria were not significantly different in the
prevalence of previous malaria treatment, days of fever, history of
cough or vomiting, platelet count and multiplicity of infection
(MOI) at recruitment, although parasitemia was higher in severe
malaria cases than controls. Among the 46 cases of severe malaria,
19 (41%) had a single criteria of malaria severity, 15 (33%) had two,
and 12 (26%) three or more. Of the 19 cases with a single criterion
ofmalariaseverity,13had prostration,5hadmultipleseizuresand 1
had severe anemia. Prostration was the most prevalent of the severe
malaria signs (Table 1), followed by acute respiratory distress
(ARD), severe anemia and multiple seizures. Only 3 (6%) patients
had CM and only 2 (4%) had hypoglycaemia. Two severe malaria
patients died, yielding a case fatality rate of 2.2%. Final outcome
could not be ascertained for two severe patients, as one absconded
in a very serious condition and the other was transferred to
Maputo’s Central Hospital.
Isolates were considered positive for rosetting if frequency of
rosettes was higher than 2%  and for platelet-mediated
clumping if frequency of clumps was higher in presence of platelets
than in buffer-control. Adhesion to host receptors was determined
by measuring binding to purified receptors coated onto Petri
dishes. Non-specific background adhesion was determined by
measuring binding to Duffy antigen coated on Petri dishes. Based
on the mean number of IEs bound to Duffy antigen (1.91per mm2;
standard deviation 8.78), we established the cut-off for positive
binding to endothelial host receptors as 19.5 per mm2(mean+ 2
I was doing it and I will do my best to finish Sixty out of 92 P.
falciparum isolates tested (65%) formed clumps in presence of
platelets. Among those isolates showing platelet-mediated clumping,
patient’s platelet count decreased with increasing frequency
of clumps (rho=20.258, p=0.051). No correlation was fo-
und between frequency of clumps and parasitemia at thawing
(rho=0.09, P=0.457). Results from platelet-mediated clumping
assays performed without adjusting parasitemia correlated well with
results from the same assay performed with parasitemias adjusted to
1% (n=72, rho=0.754, p,0.001). Both assays gave similar results
in terms of prevalence of clumping (unadjusted: 44/72 [61%];
adjusted: 47/72 [65%]; p=0.405), although medians for frequency
of clumping were slightly higher in assays with unadjusted
parasitemias (1.9%, IQR [0.0–6.4]) compared to assays performed
at 1% parasitemia (1.6%, IQR [0.0–13.7]; p=0.006).
Adhesion to CD36 was the most frequent cytoadherence phe-
notype among P. falciparum isolates (80/92, 87%), followed by
platelet-mediated clumping (60/92, 65%), adhesion to ICAM1
(41/92, 45%), to gC1qR (39/92, 42%) and rosetting (31/86,
36%). Among the isolates showing cytoadherence, median ad-
hesion (parasites per mm2) was highest for CD36 (179, IQR [95–
332]), followed by gC1qR (60, IQR [45–155]) and ICAM1 (55,
IQR [35–105]). Median frequency of rosetting and platelet-
mediated clumping was 3.0% (IQR [1.9–5.3]) and 7.2% (IQR
Cytoadherence phenotypes were compared between isolates
from children presenting with severe malaria or specific severe
malaria syndromes and their matched controls. Tables 2 and 3
show the prevalence and median levels of cytoadherence
phenotypes respectively. Although not statistically significant,
intensities of adhesion to CD36 were always lower in isolates
from children with severe malaria than in their matched controls
(Table 3). Prevalence and level of platelet-mediated clumping was
significantly higher in isolates from severe cases compared to
matched controls, both when the assays were conducted with
unadjusted parasitemias (Table 2 and 3) and when parasitemias
were adjusted to 1% (Table S1).
For the rest of the cytoadhesion phenotypes, a uniform trend
towards higher levels of cytoadherence among parasites isolated
from children with severe malaria was observed, although these
differences were not statistically significant. Platelet-mediated
clumping assessed at unadjusted parasitemias was also higher for
those parasites isolated from children with prostration and severe
anemia, as compared to their matched controls (Table 2 and 3).
Similar results were found when the assay was conducted at 1% of
parasitemia, although differences reached statistical significance
for ARD but not for prostration (Table S1). These differences
probably reflect different sample sizes (46 pairs for unadjusted and
36 for adjusted parasitemias). Adhesion to gC1qR was found to be
significantly higher in isolates from children with multiple seizures
compared to matched controls (Table 2). Rosetting was also higher
among isolates from children with severe anemia (Table 2 and 3).
Adhesion phenotypes of isolates from CM and hypoglycemia cases
could not be statistically compared to matched controls due to low
numbers (n=3 and 2, respectively).
The results of this case-control study suggest that platelet-
mediated clumping is associated with severe malaria. Moreover, to
Cytoadherence in Severe falciparum Malaria
PLoS ONE | www.plosone.org2 April 2011 | Volume 6 | Issue 4 | e19422
our knowledge, this study reports for the first time that platelet-
mediated clumping is associated with prostration, and that
adhesion to the recently identified cytoadherence receptor gC1qR
 is associated with multiple seizures, one of the criteria that
defines severe malaria.
The 46 patients with severe falciparum malaria participating
in this study are representative of this condition in Manhic ¸a
(Mozambique) , which is characterized by a high prevalence of
prostration, severe anemia and ARD, and a lower prevalence of
CM. Malaria-associated case fatality rates were slightly lower than
previously reported , possibly due to the small number of
patients and intensive follow-up. The adequate likelihood of
malaria being the principal cause of disease was guaranteed by
using a minimum parasitaemia threshold (500 parasites/ml) with a
proven high sensitivity/specificity balance  to enroll the
children in the study, as well as the exclusion from the study of
those cases with concomitant bacterial infections. Furthermore, no
difference between severe malaria cases and non-severe controls
was found in the prevalence of previous malaria treatment or
the number of days with symptoms of malaria before arrival
to hospital, suggesting that severity was not due to a longer
progression of the disease among cases.
Table 1. Clinical and biological characteristics of the study participants at the time of admission.
Severe malaria Non-severe malaria
Age (months) 32.5 (17–43)32 (17–42).871
Males, n (%)31 (67.4) 31 (67.4)1.000
Anti-pyretic, n (%)3 (6) 0 (0).083
Antibiotic, n (%)2 (4%)1 (2).564
Antimalarial, n (%)1 (2)0 (0) .317
Temperature (uC)38.6 (37.9–39.2) 38.2 (36.8–39.6) .880
Weight (Kg) 11.4 (9.4–13.2)11.9 (10.0–12.9)1.000
Splenomegaly, n (%)23 (50.0) 10 (21.7).067
Hepatomegaly, n (%)8 (17.4)2 (43.5) .033
Previous days of fever1 (1–2) 1 (1–2).860
Previous days of cough1 (0.5–1)1 (0–1) .250
Previous days of vomits0 (0–0) 1 (1–1).250
Microscopic parasitaemia (103/mL) 53.5 (28.1–129.4) 40.5 (18.2–73.7).007
Multiplicity of infection3 (3–4)3 (2–4).405
Packed cell volume (%)28 (16–32) 32 (28–34).019
Platelets (109/L) 116 (76–184) 143 (92–181).164
Glucose (mM)6.0 (5.2–7.0) 5.9 (5.4–6.8).532
White blood cells (109/L) 10.1 (7.4–12.7) 5.9 (5.4–6.8).059
Lymphocytes (%)31.4 (21.3–43.1)37.4 (23.6–50.6).014
Neutrophiles (%) 63.9 (46.2–71.1)56.5 (38.3–65.9).065
Creatinin (m/L) 35 (30–38) 34 (30–38).542
Bilirubin (mM) 24 (17–38)15 (10–24).009
ALT (m/L)33 (21–39) 25 (18–35).371
Lactate (mM) 3.7 (2.3–5.0)2.4 (1.9–3.7).009
Severe malaria syndromes
Prostration, n (%)37 (80)-
ARD, n (%) 19 (41)-
Severe anemia, n (%)14 (30)-
Multiple seizures, n (%)13 (28)-
Cerebral malaria, n (%) 3 (6)-
Hypoglycemia, n (%)2 (4)-
All continuous data is presented as the Median (Interquartile range).
ALT: Alanine aminotransferase; ARD: Acidosis or respiratory distress; IQR: Interquartile Range.
a, McNemar’s chi-squared (categorical) and Singtest (continuous).
Cytoadherence in Severe falciparum Malaria
PLoS ONE | www.plosone.org3 April 2011 | Volume 6 | Issue 4 | e19422
In accordance with previous studies from other sites [10,18,21–
23,27–29], P. falciparum isolates infecting Mozambican children
were characterized by their predominant adhesion to CD36
(87%), followed by platelet-mediated clumping (65%), adhesion to
ICAM1 (45%), to gC1qR (42%) and rosetting (36%). It could be
argued that cryopreservation of parasites may reduce the adhesive
ability of IEs, although it has been reported that it does not alter
the amount of parasite proteins on the surface of IEs as assessed by
flow cytometry . Platelet-mediated clumping was the most
prominent cytoadherence phenotype associated with severe
malaria in this study. This finding was observed when the
clumping data was analyzed in terms of prevalence and frequency
of clumps, and also when the assays were conducted at both
adjusted and unadjusted parasitemias. These results show that
Table 2. Number (%) of isolates showing cytoadherence in children with severe and non-severe malaria.
Clinical malariaRosetting PM-Clumping CD36ICAM1 gC1qR Duffy
Uncomplicated (n=46) 15a(34.9) 24 (52.2)39 (84.8)18 (39.1) 17 (36.9) 2 (4.3)
Severe (n=46)16a(37.2)36 (78.3) 41 (89.1)23 (50.0)22 (47.8)1 (2.2)
P .808 .019 .527.297 .297 .564
Uncomplicated (n=37) 13a(38.2) 20 (54.0)31 (83.8)14 (37.8)13 (35.1) 1 (2.7)
Prostration (n=37)13a(38.2) 29 (78.5) 33 (89.2)18 (48.7)17 (45.9) 1 (2.7)
P 1.000 .049.479 .355 .3551.000
Uncomplicated (n=19)5b(27.8)11 (57.9) 17 (89.5)9 (47.4) 9 (47.4) 1 (5.3)
ARD (n=19)7b(38.9) 15 (78.9)17 (89.5) 8 (42.1)5 (26.3)0 (0.0)
P .317 .1571.000 .781 .205.317
Uncomplicated (n=14)2b(15.4) 8 (57.1) 13 (92.8)6 (42.9) 5 (35.7) 2 (14.3)
Severe anaemia (n=14)6b(46.2)13 (92.8) 12 (85.7)6 (42.8)6 (42.9)0 (0.0)
P .045.025 .564 1.000.739.157
Uncomplicated (n=13)4 (30.8)5 (38.5) 8 (61.5)5 (38.5)2 (15.4) 0 (0.0)
Multiple seizures (n=13)4 (30.8) 9 (84.6)11 (84.6)9 (69.2) 6 (46.2) 1 (7.7)
P 1.000.206.179 .157 .045 0.317
a: 3 missing;
b: 1 missing.
PM-Clumping: Platelet-mediated clumping (unadjusted); ARD: Acidosis and/or respiratory distress.
Table 3. Median (interquartile range) intensities of cytoadherence phenotypes by clinical malaria manifestation.
Clinical malariaRosetting PM-ClumpingCD36ICAM1 gC1qR
UM (n=46)0.6a(0.0; 2.6)0.2 (0.0; 8.7) 178.1 (50.0; 405.0)2.5 (0.0; 40.0)0.0 (0.0; 45.0)
SM (n=46)1.0a(0.2; 4.6) 5.1 (0.4; 20.2) 123.4 (50.0; 245.0)15 (0.0; 55.0) 8.5 (0.0; 70.0)
P .627 .0321.000 .499.229
UM (n=37) 0.6a(0.0; 3.0) 0.2 (0.0; 8.7) 171.6 (50.0; 405.0)0.0 (0.0; 45.0)0.0 (0.0; 48.6)
P (n=37)1.0a(0.2; 4.0) 4.7 (0.4; 20.4)127.1 (50.0; 245.0)10.0 (0.0; 55.0) 0.0 (0.0; 75.0)
P.855 .041 1.000.701 .327
UM (n=19)0.2b(0.0; 2.2)2.6 (0.0; 16.9) 275.0 (125.0; 533.4) 15.0 (0.0; 54.2)0.0 (0.0; 55.0)
ARD (n=19)1.1b(0.0; 5.3) 6.3 (0.4; 20.2) 110.0 (44.9; 175.0) 0.0 (0.0; 35.0)0.0 (0.0; 75.0)
P .791 .323.167 .803.267
UM (n=14)0.9b(0.0; 2.7) 0.3 (0.0; 11.6)178.1 (52.5; 423.5) 0.0 (0.0; 37.9)0.0 (0.0; 45.0)
SM (n=14)2.0b(0.2; 7.7) 6.6 (2.0; 15.5)75.0 (45.0; 175.0) 0.0 (0.0; 47.7)2.5 (0.0; 55.0)
P .065 .092 .424 .5491.000
UM (n=13) 0.2 (0.0; 2.2) 2.6 (0.0; 16.9)275.0 (125.0; 533.4) 15.0 (0.0; 54.2)0.0 (0.0; 55.0)
MS (n=13) 1.0 (0.2; 2.2) 2.5 (0.0; 7.0) 120.3 (45.0; 255.0)22.4 (0.0; 51.6) 12.0 (0.0; 50.0)
P 1.000 .774.774 .387.125
a: 3 missing;
b: 1 missing.
PM-Clumping: Platelet-mediated clumping; UM: Uncomplicated malaria; SM: severe malaria; P: Prostration; ARD: Acidosis and/or respiratory distress; SA: Severe anemia;
MS: Multiple seizures.
P-values for cytoadherence to Duffy receptors are as follows: 0.453 for SM; 1.000 for P, D and MS; and 0.625 for SA.
Cytoadherence in Severe falciparum Malaria
PLoS ONE | www.plosone.org4 April 2011 | Volume 6 | Issue 4 | e19422
both assays correlated fairly well (rho=0.754), probably due to the
fact that children with low density parasitemias (,500 parasites/ml
of blood) were not recruited into the study. The association
between severe malaria and platelet-mediated clumping was
similar when the analyses were based on data from assays
conducted with or without adjusted parasitemias (1%). This study
thus provides clear evidence for association of platelet-mediated
clumping with severe malaria.
Since pathogenesis may be quite different among the syndromes
used to define severe malaria, we have also analyzed cytoadher-
ence separately for each syndrome. Prostration, the most prevalent
severe malaria presentation in this study population, was found to
be associated with platelet-mediated clumping. Multiple seizures
were observed to be associated with adhesion to gC1qR. Seizures
are frequent among severe malaria cases. They may occur as part
of CM or independently , and have been associated with
increased neuro-cognitive deficits and mortality [37,38], suggest-
ing neurological involvement . It is possible that IEs pre-
ferentially sequester in the brain using gC1qR, which is expressed
on brain endothelial cells . Local obstruction of blood flow
may lead to the manifestation of multiple seizures. However,
adhesion to ICAM1, which is also expressed on brain endothe-
lial cells , was not associated with seizures in this study.
Alternatively, engagement of gC1qR by sequestered parasite
ligands may reduce the local availability of gC1qR for normal
physiological functions, such as inhibition of the complement
cascade . Reduced availability of gC1qR may result in over-
activation of complement cascade, which may evoke seizures .
The role of adhesion to ICAM1, whichhas been previously
implicated in the pathogenesis of CM , and of gC1qR on CM
could not be evaluated as there were only 3 cases of CM among
Severe anemia was associated with higher platelet-mediated
clumping and rosetting, suggesting a role for these cytoadherence
phenotypes in the etiology of malaria-related anemia. Destruction of
uninfected erythrocytes attached to IEs in rosettes or down
regulation of erythrocyte production by abnormal erythrocyte
aggregates might also contribute to severe anemia. The role of
platelet-mediated clumping in severe anemia is unclear and requires
further investigation. If platelet-mediated clumping is found to have
a causative role in severe anemia, novel anti-adhesion interventions
that prevent platelet-mediated clumping might have an impact on
reducing severe anemia in malaria endemic areas. Such interven-
tions might be of public health relevance, as severe anemia is an
important contributor to malaria-related morbidity and mortality in
children residing insub-Saharan Africa and the most important
risk factor for death in Mozambican children younger than 8
months with malaria .
Role of adhesion to CD36 in the physiopathology of severe
malaria is still a subject of debate, as some studies have found higher
binding in isolates infecting children with severe malaria [43,44]
and others in parasites infecting children with non-severe malaria
[22,24,29]. Although not statistically significant, these results show a
consistent trend towards higher levels of adhesion to CD36 in
parasites isolated from children with uncomplicated malaria than
from children with severe malaria, suggesting that adherence to
CD36 might be an indicator of a less pathogenic infection.
Observations from this study suggest that anti-adhesion therapies
should be considered with caution, as blocking adhesion to CD36
may potentially lead to selection of parasites with affinities for other
receptors which might be detrimental to the host.
In summary, the results of this case-control study support the
hypothesis that cytoadherence properties of P. falciparum may be an
important virulence factor. The potential for inhibiting or
reversing platelet-mediated clumping and adhesion to gC1qR
with antibodies or receptor analogs could improve severe malaria
outcomes and should be actively explored.
Materials and Methods
The study protocol was approved by the National Ethics
Review Committee of Mozambique and the Ethics Review
Committee of the Hospital Clinic of Barcelona.
The study took place in Manhic ¸a District, southern Mozam-
bique. The study area, which is characterized by perennial malaria
transmission with some seasonality, has been extensively described
elsewhere . Malaria in Manhic ¸a is primarily caused by P.
falciparum infections with an average entomological inoculation
rate of 38 infective bites per person per year in 2002 .
Respiratory distress and anaemia, together with the clinical sign of
prostration, are the most prevalent presentations of severe malaria,
with the majority of cases occurring in younger children, as
opposed to coma, which is infrequent and slightly shifted to older
Between April and November 2006, a sex and age (+/23
months) matched case-control study was conducted, the cases
being children under five years of age with severe malaria (n=71)
and controls being outpatient children with non-severe malaria
(n=71). Children attending the Manhic ¸a District Hospital with a
primary clinical diagnosis of P. falciparum malaria were recruited
into the study after written informed consent was given by their
parents or guardians. Blood smears were stained with Giemsa and
examined by microscopy according to quality-controlled proce-
dures . Clinical malaria was defined as the presence of fever
(axillary temperature$37.5uC) with an asexual parasitemia of P.
falciparum$500/mLon thin blood film examination. This definition
has both a sensitivity and specificity of greater than 90% in
children from Manhic ¸a . Children with severe malaria were
those presenting with at least one of the following clinical
definitions : CM (Blantyre Coma Score#2), severe anaemia
(packed cell volume ,15% or hemoglobin,5 g/dL), acidosis or
respiratory distress (ARD; lactate.5 mM and/or chest in-drawing
or deep breathing), prostration (inability to sit or breastfeed in
children old enough to do so based on their age), hypoglycemia
(blood glucose,2.2 mM) and multiple seizures ($2 convulsions in
the preceding 24 h). Children with non-severe malaria were those
with clinical malaria not showing any of the mentioned signs of
severity and able to take oral medication. All severe cases were
tested for bacteremia by blood cultures, and children with positive
bacteremia were excluded from the study. All severe and
uncomplicated cases were reviewed by the study pediatrician to
confirm that malaria was the sole or principal cause of the disease.
Children with severe disease were admitted at the Manhic ¸a
District Hospital and treated with parenteral quinine, and non-
severe malaria controls were treated with a combination of oral
amodiaquine and sulfadoxine-pyrimethamine (FansidarH) follow-
ing national guidelines of Mozambique at the time.
Collection of parasites
Before treatment, 10 mL of peripheral blood was collected by
venipuncture into a tube containing lithium heparin. All blood
samples were collected by trained persons in presence of a
paediatrician. The amount of blood collected was less than the
Cytoadherence in Severe falciparum Malaria
PLoS ONE | www.plosone.org5 April 2011 | Volume 6 | Issue 4 | e19422
limit of 3 ml per kg of bodyweight as recommended by ethical
guidelines . Blood samples were used for biochemical analysis
and blood culture at the study site for clinical management.Bio-
chemical determinations (alanine aminotransferase, bilirubin,
lactate and creatinine) and a full blood count were performed
for each patient using Vitros DT60 and Sysmex Kx21 analyzers,
respectively. Blood samples were centrifuged, IEs were buffy coat
depleted, washed three times in phosphate-buffered saline (PBS)
and cryopreserved in multiple aliquots in glycerolyte solution at
280uC. Frozen samples were transported to the International
Centre for Genetic Engineering and Biotechnology (ICGEB) in
New Delhi, and kept in liquid nitrogen until thawed for
cytoadherence assays. Ten ml of blood was required for bio-
chemical analysis and blood culture and to provide sufficient
parasite aliquots for testing in cytoadherence assays. One aliquot
was needed to conduct each of the following assays: 1.)
cytoadherence to purified receptors, 2.) cytoadherence to cells
expressing receptors CD36, ICAM1, gC1qR, 3.) rosette forma-
tion, 4.) platelet-mediated clump formation. Each assay was
repeated at least once. Two drops were also spotted onto filter
paper (Schleicher & Schuell number 903TM; Dassel, Germany)
and used for extraction of DNA for use in PCR analysis.
Multiplicity of infection
DNA was extracted from blood onto filter papers using the
QIAGEN QIAamp DNA Mini Kit. The number of concurrent
infections (multiplicity of infection, MOI) was estimated as the
highest number of msp-1 ormsp-2 alleles detected in the sample by
Thawing and culture of isolates
Cryopreserved P. falciparum isolates were thawed by serial
dilutions in a gradient of salt solutions following standard methods.
Erythrocytes were washed in RPMI 1640 medium containing
2 mM L-glutamine, 25 mM HEPES, 25 mM sodium bicarbonate
and 10 mg/mL gentamicin (incomplete RPMI) and cultured at
37uC in complete RPMI (incomplete RPMI supplemented with
0.5% Albumax I and 50 mM hypoxanthine) as previously
described . Cultures were monitored by Giemsa-stained thin
smears for 18–36 hours and only those that matured to the
pigmented trophozoite stage were included in the study.
Researchers were blinded to the case-control identification of the
Measurement of parasite adhesion to purified receptors was
done as previously described  with slight modifications. Briefly,
25 mL of CD36, ICAM1 (R & D Systems, 20 mg/mL) and gC1qR
(50 mg/mL) were spotted in duplicate onto 35-mm Petri dishes
(BD Labware). Duffy antigen fused to Fc of human IgG (Duffy-Fc
; 50 mg/mL) and bovine serum albumin (BSA) (50 mg/mL)
were used as negative controls. After overnight incubation at 4uC
in moist environment, dishes were blocked with PBS-1% BSA.
Parasites were suspended in 1.5 mL adhesion media (RPMI-1640
with 0.5% BSA, pH6.8) at 1% hematocrit and 1% parasitaemia,
and incubated for 1 h with the receptors coated on Petri dishes.
Samples with less than 1% parasitaemia were enriched using 0.7%
Gelatin (Sigma Aldrich) at the pigmented trophozoite stage .
After washing with adhesion media till no unbound erythrocytes
were seen in the Petri dish under inverted microscope observation,
the adherent cells were fixed with 2% glutaraldehyde and stained
with Giemsa. The number of adherent IEs per mm2was counted
by observation of 10 fields (using 1006 objective) under light
microscopy. Adhesion to BSA was subtracted to yield specific
binding to each receptor.
An aliquot of culture suspension at pigmented trophozoite stage
was stained with 20 mg/mL of ethidium bromide in rosetting
media (incomplete RPMI supplemented with 10% AB+ human
sera). Twenty mL of the suspension at 2% hematocrit was exa-
mined by fluorescence and direct light microscopy, and the
proportion of IEs in rosettes was measured for each sample in
triplicate. Two hundred IEs were scored in each experiment, with
the adhesion of two or more uninfected erythrocytes to an IE
constituting a rosette. The level of rosetting is reported as the
percentage of IEs forming rosettes.
Preparation of non-activated platelets
Blood was collected from donors by venipuncture in citrate-
phosphate dextrose solution (Sigma Aldrich), and centrifuged at
250 g for 5 min. Platelet rich plasma was transferred to a separate
tube, equal volume of fresh CCDAT buffer (920 mM tri-sodium
citrate, 80 mM citric acid, 150 mM glucose, 5 mM adenosine and
3 mM theophylline) was added drop wise and incubated for
15 min. Platelets were collected by centrifugation at 1000 g for
20 min and resuspended in CCDAT buffer. Homogenous platelet
suspension was transferred to new tubes and centrifuged at 1000 g
for 20 min. Platelets were finally resuspended in Hanks buffer at
108platelets per mL, stored in sterile condition at room
temperature and used within 4 days of collection. All steps were
performed at room temperature. P-selectin (CD62) was not
detected on the surface of platelets by flow cytometry ,
indicating that platelets prepared by this way were inactivated.
Platelet-mediated clumping assays
Parasites were thawed and used for clumping assays as described
below. The assays were performed at thawing parasitemias and 2%
hematocrit, as well as at 1% adjusted parasitemias (with gelatin
enrichment for those samples with less than 1% parasitemia )
and 5% hematocrit to discard the confounding effect of different
parasitemias between cases and controls . Culture suspensions
were stained with ethidium bromide, washed and resuspended in
Hanks buffer. Five mL of platelet suspension or Hanks buffer (buffer-
control) were added to 45 mL of the IE suspension and the mixture
was rotated for 30 min. Five hundred IEs were counted in triplicate
for each sampleby fluorescence and direct light microscopy, with the
adhesion of three or more IEs constituting a clump. The level of
clumping was expressed as percentage of IEs in clumps. Platelet-
mediated clumping frequency was defined as the difference between
clumping frequency in presence of platelets and in buffer-control.
To account for day-to-day variations, adhesion values were
normalized using data obtained from parasite lines with known
cytoadherence phenotypes: ITG-ICAM1, which binds CD36 and
ICAM1, IGHCR14, which binds gC1qR and forms clumps, and
R29R+, which form rosettes. These parasite lines were expanded
by standard culture and frozen at ring-stage in multiple aliquots
previous confirmation of their adhesion profile. Six field isolates
were tested per experiment along with control parasite lines used
Definitions and statistical methods
All data collected was entered into the Excel software (Micro-
soft Co.) and was analysed using the Stata version 9.0 (Stata
Cytoadherence in Severe falciparum Malaria
PLoS ONE | www.plosone.org6 April 2011 | Volume 6 | Issue 4 | e19422
Corporation). A P-value less than .05 was considered statistically
significant. Data from cytoadherence experiments were expressed
as the mean of duplicate/triplicate measurements. Cytoadherence
to purified receptors was considered to be positive if the number of
IEs bound per mm2to purified receptors coated on Petri dishes was
higher than the mean binding plus 2 standard deviations to Duffy-
Fc coated Petri dishes. Isolates were considered positive for
rosetting if frequency of rosettes was higher than 2%  and for
platelet-mediated clumping if frequency of clumps was higher in
presence of platelets than in buffer-control. Categorical and
continuous data were compared between matched case/control
pairs by McNemar’s chi-squared test and Signtest, respectively.
Correlations between variables were assessed by Spearman’s rank
coefficient. Primary analysis consisted in the comparison of
cytoadherence data between parasites isolated from children with
severe and uncomplicated malaria. For the secondary analysis,
severe malaria cases were grouped according to their criteria for
malaria severity, and compared separately to matched controls.
at 1% parasitemia and 5% hematocrit.
Platelet-mediated clumping results in assays conducted
We are grateful to the children who participated in the study; the staff of
the Manhic ¸a District Hospital; the clinical officers, field supervisors and
data managers; G. Cabrera, L. Mussacate, N. Ernesto Jose ´ and A.
Nhabomba for their contribution to the collection of parasites; L. Puyol for
her laboratory management; Professor Kasturi Datta, School of Environ-
mental Sciences, Jawaharlal Nehru University (JNU) New Delhi for
providing gC1qR construct and Khalid Wasim for help with expression
and purification of gC1qR.
Conceived and designed the experiments: CEC AM AH. Performed the
experiments: AM AH ER-V PC. Analyzed the data: AM AH SS.
Contributed reagents/materials/analysis tools: AM AH CEC PLA CM
SM BS QB RA. Wrote the paper: CEC AM AH.
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