PARA 3897 No.

of Pages 8, Model 5G
26 September 2016

International Journal for Parasitology xxx (2016) xxx–xxx

1

Contents lists available at ScienceDirect

International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

2 Invited Review
5
4 Severe malaria: what’s new on the pathogenesis front?
6

7 Samuel Crocodile Wassmer a,b,⇑, Georges Emile Raymond Grau b

8 a
Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, London, United Kingdom
9 b
Vascular Immunology Unit, Department of Pathology, School of Medical Sciences & Marie Bashir Institute, The University of Sydney, Camperdown, Australia

11
10
12
a r t i c l e i n f o a b s t r a c t
1 8
2 4
15 Article history: Plasmodium falciparum causes the most severe and fatal form of malaria in humans with over half a mil- 29
16 Received 10 May 2016 lion deaths each year. Cerebral malaria, a complex neurological syndrome of severe falciparum malaria, is 30
17 Received in revised form 9 August 2016 often fatal and represents a major public health burden. Despite vigorous efforts, the pathophysiology of 31
18 Accepted 15 August 2016
cerebral malaria remains to be elucidated, thereby hindering the development of adjunctive therapies. In 32
19 Available online xxxx
recent years, multidisciplinary and collaborative approaches have led to groundbreaking progress both in 33
the laboratory and in the field. Here we review the latest breakthroughs in severe malaria pathogenesis, 34
20 Keywords:
with a specific focus on new pathogenetic mechanisms leading to cerebral malaria. The most recent find- 35
21 Plasmodium spp.
22 Pathophysiology
ings point towards specific parasite phenotypes targeting brain microvasculature, endothelial dysfunc- 36
23 Cerebral malaria tion and subsequent oedema-induced brain swelling. 37
24 Endothelial dysfunction Ó 2016 Published by Elsevier Ltd on behalf of Australian Society for Parasitology. 38
25 Sequestration 39
26 Malaria in pregnancy
27
40
41
42 1. Introduction target different receptors, the expression of which varies depend- 65
ing on the organ, could explain why some patients with malaria 66
43 Malaria is still a leading cause of morbidity and mortality in the develop organ-specific syndromes. Researchers have speculated 67
44 developing world. The virulence of Plasmodium falciparum is that a specific PfEMP1 variant could bind receptors that are prefer- 68
45 caused by several factors including parasite proteins on the surface entially expressed in cerebral microvasculature, and could account 69
46 of infected erythrocytes (IE). These allow the binding of these cells for the focal manifestations observed in CM, the most lethal com- 70
47 to the microvascular endothelium of various organs and tissues plication of P. falciparum infection. Two recent reports simultane- 71
48 during infection. Proteins of the P. falciparum erythrocyte mem- ously shed new light on the pathogenetic mechanisms leading to 72
49 brane protein 1 (PfEMP1) family mediate this adhesion through CM. First, endothelial protein C receptor (EPCR) was identified as 73
50 specific binding to multiple cell receptors. These include intercellu- a binding partner for PfEMP1. Second, normally low levels of EPCR 74
51 lar adhesion molecule-1 (ICAM-1), CD36, E-selectin, neural cell in brain microvessels were shown to be further down-regulated in 75
52 adhesion molecule (NCAM) and CD31 (PECAM-1) for endothelial CM, with a loss of EPCR and thrombomodulin at sites of IE seques- 76
53 beds, as well as chondroitin sulfate A (CSA) for placental syncy- tration. These studies provided new clues towards parasite and 77
54 tiotrophoblasts. Binding to endothelium results in widespread host cell interactions leading to CM, and connected for the first 78
55 sequestration of IE, which can lead to lead to endothelial activation time brain-specific sequestration of EPCR-binding parasites to the 79
56 as well as pro-inflammatory and pro-coagulant responses. loss of the protein C anti-coagulant function and endothelial cyto- 80
57 Severe falciparum malaria encompasses a broad range of dis- protective pathways (Aird et al., 2014). 81
58 eases, the development of which may be influenced by age, expo- While the relative frequency of severe malaria is low, its 82
59 sure and immune status (Wassmer et al., 2015). It includes reported case fatality rate has not substantially changed over dec- 83
60 complications that affect specific organs such as the brain in cere- ades, especially for CM (Manning et al., 2014). Due to the lack of 84
61 bral malaria (CM) or the placenta in malaria in pregnancy (MiP). specific neuro- and vasculoprotective therapies, treatments for 85
62 Histopathology and laboratory studies allowed investigators to CM are currently still precariously limited to antimalarial drugs 86
63 establish a causal link between placenta-specific sequestration of and emergency supportive care. The former are quickly dwindling, 87
64 P. falciparum and MiP. Indeed, the ability of PfEMP1 variants to as the resistance of P. falciparum malaria against artemisinin com- 88
bination treatments, the recommended first-line therapy for 89
infected patients, is on the rise in southeastern Asia. Multi-drug- 90
⇑ Corresponding author at: Department of Immunology and Infection, London resistant falciparum malaria is increasingly difficult to treat and 91
School of Hygiene and Tropical Medicine, London, United Kingdom.
new antimalarials are not expected to become available within 92
E-mail address: [email protected] (S.C. Wassmer).

http://dx.doi.org/10.1016/j.ijpara.2016.08.002
0020-7519/Ó 2016 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

2 S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx

93 the next few years. This underlines the necessity for molecular tions for pathogenic mechanisms in severe malaria. Full‐length 156
94 markers for surveillance of partner drug resistance, in conjunction sequences of PfEMP1 encoding transcripts were characterised in 157
95 with the implementation of new biomarkers for early diagnosis clinical isolates from children with severe malaria admitted to hos- 158
96 and outcome prediction, as well as effective adjunct therapies. pital in Tanzania, and EPCR-binding CIDRa1 domains dominated 159
97 Here we review some recent data with a focus on newly devel- PfEMP1 transcript profiles of children suffering from CM and/or 160
98 oped research approaches aimed at a better understanding of the severe malarial anaemia, further strengthening the evidence for a 161
99 pathogenetic mechanisms of severe malaria in general and CM in crucial pathogenic role of the PfEMP1–EPCR interaction in severe 162
100 particular. malaria (Jespersen et al., 2016). 163
EPCR is not the only receptor suspected to play an important 164
role in the development of severe malaria. Studies of parasite iso- 165
101 2. Parasite-brain microvasculature specificity in CM: a virulence lates have demonstrated high rates of in vitro ICAM-1 binding 166
102 factor? among wild strains but reported correlations between ICAM-1 167
binding and disease severity have been inconsistent. Recent results 168
103 The severity of P. falciparum is linked to sequestration of IEs suggest that ICAM-1 is a co-receptor for a subset of EPCR-binding 169
104 within the microvasculature of various organs including the brain. parasites (Avril et al., 2016). Indeed, Avril et al. (2016) showed that 170
105 This sequestration is driven both by the expressed var gene in the parasites expressing DC13 have dual binding specificity for EPCR 171
106 parasite, leading to the expression of a specific variant of PfEMP-1, and ICAM-1, suggesting for the first time that ICAM-1-binding vari- 172
107 and the presence of its associated receptors on microvascular walls ants can be grouped into CD36 and EPCR co-receptor-binding 173
108 (Hviid and Jensen, 2015). Since there are considerable variations in traits. This leads to different cytoadherence abilities on TNF- 174
109 both adhesion molecule expression and functional properties of stimulated endothelial cells, which has important implications 175
110 endothelial cells depending on their position within the vascular for understanding parasite organ-specific microvascular bed trop- 176
111 bed of a particular tissue, it has been hypothesised that CM may ism in pro-inflammatory conditions. 177
112 result from a brain endothelial-specific adhesive type of parasite
113 (Moxon et al., 2014). Indeed, IE expressing the domain cassettes
114 (DC) 8 and 13 of the cytoadherent ligand PfEMP-1 adhere to EPCR 3. Microvascular endothelial dysfunction: new causes and 178
115 (Turner et al., 2013). By interfering with EPCR anti-coagulant and repercussions 179
116 pro-endothelial barrier functions, IE adhesion could promote coag-
117 ulation and vascular permeability that contribute to the pathogen- In the recent years, convincing evidence has been presented to 180
118 esis of CM (Moxon et al., 2014). To better understand parasite support the role of both endothelial cell activation and platelets in 181
119 factors that contribute to disease severity, Gillrie et al. (2015) modulating the pathogenesis of severe P. falciparum malaria. 182
120 developed in vitro binding models for different microvascular beds Thrombin, a common factor in both processes, is now thought to 183
121 to examine the adhesion of DC8- and DC13-expressing parasite be a driver of pathology in CM. The relative contribution of 184
122 lines to endothelial cells from different microvasculature, and the EPCR-binding parasites versus loss of EPCR from the endothelial 185
123 consequences of EPCR engagement on endothelial cell function. surface in mediating CM is not known, although both are associ- 186
124 They reported that IE from IT4var19 (DC8) and IT4var07 (DC13) ated with disease in clinical studies (Turner et al., 2013; Moxon 187
125 parasite lines adhere to human brain, lung and dermal endothelial et al., 2014). The key mediator of inflammation in both cases 188
126 cells under shear stress. However, the relative contribution of EPCR appears to be thrombin, which is a potent mediator of both pro- 189
127 to parasite cytoadherence on different types of endothelial cell and anti-inflammatory pathways, depending on the context of 190
128 varied. endothelial signalling. Thrombin engages thrombomodulin on the 191
129 Divergent functional outcomes for DC8 cysteine-rich interdo- plasma membrane of intact endothelium, where it promotes acti- 192
130 main region (CIDR) a1.1 and DC13 CIDRa1.4 domains were also vation of protein C. The latter is accelerated by the presence of 193
131 observed. IT4var07 CIDRa1.4 inhibited generation of activated pro- EPCR, which binds to protein C and presents it for optimal activa- 194
132 tein C (APC) on lung and dermal endothelial cells and blocked the tion by the thrombin-thrombomodulin complex. Further to its 195
133 APC-EPCR binding interaction on brain endothelial cells. IT4var19 anticoagulant activity, generated APC can also trigger numerous 196
134 CIDRa1.1 inhibited thrombin-induced endothelial barrier dysfunc- cell-signaling pathways initiating protective cellular responses 197
135 tion in lung endothelial cells, whereas IT4var07 CIDRa1.4 inhibited upon exposure to pro-inflammatory, pro-apoptotic, or toxic insult. 198
136 the protective effect of APC on thrombin-induced permeability. IEs expressing PfEMP1 compete with protein C and APC for EPCR, 199
137 Overall, these findings reveal a much greater complexity of how thereby down-regulating protein C activation by the thrombin- 200
138 CIDRa1-expressing parasites may modulate malaria pathogenesis thrombomodulin complex. The resulting loss of EPCR-APC– 201
139 through EPCR adhesion (Gillrie et al., 2015). DC8 PfEMP1 encode induced cytoprotective signalling is consistent with a decrease in 202
140 multiple endothelial binding domains, including binding activity blood–brain barrier (BBB) properties, potentially leading to vaso- 203
141 for EPCR. These results show that PfEMP1 domains compete with genic oedema in CM. 204
142 protein C for EPCR binding but the extent of competition differs Direct protein C pathway alteration by binding of EPCR-specific 205
143 between domains. IEs is not the only cause of endothelial dysfunction in CM. Indeed, 206
144 Bernabeu et al. (2016) recently investigated these parasite viru- P. falciparum infection also initiates early endothelial and platelet 207
145 lence factors in adult patients in India and demonstrated that activation, leading to coagulation dysregulation and microvascular 208
146 specific EPCR-binding parasites lead to severe malaria in that pop- lesions locally (Wassmer et al., 2011a). Furthermore, IEs elicit a 209
147 ulation. In addition, parasite phenotype and biomass are associated myriad of signalling pathways leading to aberrant pro-coagulant 210
148 with patient hospitalisation and disease severity. The authors show effects, ultimately resulting in enhanced endothelial activation, 211
149 a broad range of EPCR binding activity from severe malaria isolates damage and apoptosis (O’Sullivan et al., 2016). Lastly, severe falci- 212
150 and even parasite domains that partially obstructed the interaction parum malaria has been associated with a down-regulation of nor- 213
151 between EPCR and APC were sufficient to interfere with the cyto- mal endogenous anticoagulant pathways. EC surface expression of 214
152 protective functions of APC (Bernabeu et al., 2016). Taken together, thrombomodulin and EPCR are both reduced, likely through 215
153 their findings suggest that parasites may be under selection for cytokine-enhanced shedding, leading to an increase of their soluble 216
154 phenotypic variation in a key host pathway that regulates coagula- levels in plasma (Moxon et al., 2013). Together, these effects 217
155 tion and endothelial barrier properties, and has important implica- combine and lead to a significant reduction in generation of 218

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx 3

219 anti-inflammatory and cytoprotective APC on the endothelial sur- molecules, thereby increasing their combined avidity for carbohy- 281
220 face. These findings suggest new avenues for acute therapeutic drate receptors on surrounding erythrocytes and promoting the 282
221 intervention and match well with post-mortem observations and formation of rosettes. These results suggest that P. falciparum sol- 283
222 magnetic resonance imaging (MRI) findings linked to mortality in uble host proteins for its own benefit, and avoids immune destruc- 284
223 CM. tion by manipulating those to facilitate adhesion of IEs to low- 285
224 Such an endothelial dysfunction is not only observed in severe affinity carbohydrate receptors. In addition, another report 286
225 falciparum malaria, but was also reported in severe Plasmodium recently showed that PfEMP1 is not the only parasite ligand used 287
226 vivax (Barber et al., 2015) and Plasmodium knowlesi (Yeo et al., in rosetting, potentially opening new rosette-disrupting 288
227 2007) cases, which raises the question of possible common patho- approaches. Goel et al. (2015) showed in a very elegant study that 289
228 genic pathways in these various types of infection. All plasmodia the repetitive interspersed family of proteins (RIFINs) mediates 290
229 species can cause severe and fatal malaria. In falciparum malaria, binding of IEs with a preference for blood group A, leading to the 291
230 the most common cause of severe malaria, impaired tissue perfu- formation of large rosettes of 10 or more IEs. This was not observed 292
231 sion has long been recognised to arise from microvascular obstruc- with IEs from group O. Indeed, blood group O is common in malaria 293
232 tion by IE adherent to endothelial cells. More recently recognised is endemic areas and IEs of that blood group were shown to form 294
233 the contribution of concurrent impairment of nitric oxide (NO) small, weak rosettes (Rowe et al., 2007). The role of RIFINs was 295
234 bioavailability, endothelial activation and microvascular dysfunc- confirmed by the disruption of rosettes in the presence of anti- 296
235 tion to impaired tissue perfusion and severe disease (Yeo et al., RIFIN antibodies. These results suggest that RIFINs not only play 297
236 2014). Angiopoietin-2, released from endothelial cell Weibel- a fundamental role in the development of severe malaria but also 298
237 Palade bodies and an NO-inhibited autocrine mediator of endothe- contribute to the virulence of P. falciparum. 299
238 lial activation, is markedly elevated in severe falciparum malaria Puzzlingly, formed rosettes have never been described in the 300
239 and consistently associated with impaired tissue perfusion and peripheral bloodstream, suggesting that they sequester in the 301
240 fatal outcome in both adult and paediatric severe malaria. This is microvasculature and aggravate microvascular obstructions during 302
241 independent of both total and sequestered parasite biomass, sug- severe malaria in general, and CM in particular. Understanding the 303
242 gesting that microvascular obstruction and microvascular dysfunc- mechanisms by which rosettes sequester (i.e., by direct binding of 304
243 tion make separate contributions to pathogenesis. Microvascular rosetting IE to endothelial cells, to platelets on endothelial cells or 305
244 function, the capacity to increase flow and oxygen delivery in to non-rosetting cytoadherent IE is fundamental, as it may open 306
245 response to ischaemia, is decreased in severe falciparum malaria new adjunct therapies to reduce the biomass of sequestered IE, a 307
246 and associated with an increased risk of death. Endothelial activa- parameter associated with disease severity (Hendriksen et al., 308
247 tion, decreased endothelial NO bioavailability and microvascular 2012). IE of the IT/R29 strain expressing a rosette-mediating 309
248 dysfunction are also associated with impaired tissue perfusion in PfEMP1 variant (IT4var09) were recently shown to cytoadhere to 310
249 vivax and knowlesi malaria, a factor likely to contribute to organ human brain microvascular endothelial cells using heparan sulfate 311
250 dysfunction and severe malaria caused by infection with these spe- proteoglycans as ligands (Adams et al., 2014). This process is dis- 312
251 cies. Initial clinical trials of agents aimed at restoring NO bioavail- tinct from rosetting, which is primarily mediated by interactions 313
252 ability in severe falciparum malaria have recently been completed with between the NTS-DBL1a domain of PfEMP1 and complement 314
253 (Hawkes et al., 2011; Serghides et al., 2011), and microvascular receptor 1. This study shows for the first time that IT4var09- 315
254 function can be improved by L-arginine infusion in severe falci- expressing parasites are capable of dual interactions with both 316
255 parum malaria (Yeo et al., 2013). These results suggest that new endothelial cells and uninfected erythrocytes via distinct 317
256 compounds aimed at increasing microvascular NO and microvas- receptor-ligand interactions, and therefore could aggravate 318
257 cular functions have potential clinical benefits as adjunctive treat- microvascular obstruction in severe malaria by facilitating the 319
258 ments in severe malaria from all species. sequestration of platelet-mediated clumps. 320
One of the most recently described P. falciparum cytoadherence 321
phenotypes is the ability of IEs to bind to platelets in suspension 322
259 4. Rosetting and clumping: consequences for sequestration and
in vitro to form platelet-mediated clumps. Similar to rosetting, the 323
260 microvascular obstruction
formation of clumps is a common phenotype and has been reported 324
in a variety of endemic settings (Rowe et al., 2009). This is mediated 325
261 Parasite adhesion interactions in severe falciparum malaria are
by the binding of IEs to platelet receptors which include CD36 (Pain 326
262 not restricted to the endothelium. Indeed, IEs can bind uninfected
et al., 2001), P-selectin (Wassmer et al., 2008) and globular C1q 327
263 erythrocytes to form rosettes (Handunnetti et al., 1989), or plate-
receptor (gC1qR) (Biswas et al., 2007). In all cases, the parasite 328
264 lets to form clumps (Pain et al., 2001). Both processes have been
ligands are unknown, although PfEMP1 is a likely candidate mole- 329
265 associated with severe malaria (Rowe et al., 1995; Pain et al.,
cule. A recent study performed using clinical isolates from Mozam- 330
266 2001) and CM (Carlson et al., 1990; Wassmer et al., 2008).
bican patients to evaluate cytoadherence properties such as 331
267 In this context, recent advances have been made in elucidating
platelet-mediated clumping, rosetting and adhesion to purified 332
268 the molecular mechanisms underlying rosetting, with a view to
receptors (CD36, ICAM1 and gC1qR) revealed that, compared with 333
269 development of rosette-reversing therapies. Rosetting generally
matched controls, prevalence of both rosetting and platelet- 334
270 requires the presence of soluble serum factors such as IgM, and
mediated clumping and adhesion to gC1qR was higher in severe 335
271 the ability of IE to form rosettes and to bind non-immune IgM is
cases (Mayor et al., 2011). Inhibition of these cytoadherence pheno- 336
272 correlated (Rowe et al., 2002). While the role of IgM binding in
types may therefore reduce the occurrence or improve the prognosis 337
273 rosetting remains unclear, it appears to strengthen the bond of
of severe malaria outcomes. Similar to rosettes, platelet-mediated 338
274 the central IE with the surrounding erythrocytes. Stevenson et al.
clumps have not been observed in the bloodstream and it is likely 339
275 (2015) recently reported that the serum protein a2-
that they sequester in the microvasculature using IE or platelet 340
276 macroglobulin (a2M) is able to induce rosetting in vitro and
receptors (or both) to cytoadhere on endothelial cells (Wassmer 341
277 ex vivo, using several parasite isolates. In contrast to IgM, a2M elic-
et al., 2011a). Since this may potentially aggravate microvascular 342
278 its rosetting alone, while the presence of IgM significantly lowers
obstruction in CM, further studies aimed at understanding the 343
279 the concentration of a2M required. The authors of the study postu-
molecular process involved in their sequestration are warranted. 344
280 late that a2M allows the crosslinking of several individual PfEMP1

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

4 S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx

345 5. Clinical consequences of BBB opening in CM: vasogenic Lastly, it is entirely plausible that both mechanisms are not 409
346 oedema and brain swelling mutually exclusive. A study in live mice using high-field MRI with 410
whole-brain coverage showed that vasogenic oedema occurs first 411
347 Following the report by Seydel et al. (2015), a study was initiated in infected animals, and starts in the olfactory bulb before spread- 412
348 to investigate the different mechanisms potentially responsible for ing deeper into the brain along a specific path called the rostral 413
349 brain swelling in both pediatric and adult CM patients in India migratory stream, eventually reaching the brain stem. Microvascu- 414
350 (Wassmer et al., 2015). Brain swelling was identified by MRI in over lar pathology and ischemic brain injury develop only secondarily, 415
351 50% of patients enrolled in the ongoing study, irrespective of their after vasogenic oedema formation (Hoffmann et al., 2016). Addi- 416
352 age group. The frequent occurrence of brain swelling in CM has tional MRI studies are currently underway in CM patients from 417
353 been previously reported in separate studies on Indian adults using Asia and Africa, and are aiming to elucidate these pathogenetic 418
354 computed tomography (CT) (Mohanty et al., 2011), as well as in processes (Wassmer et al., 2015). 419
355 Bangladeshi adults (Maude et al., 2014) and Malawian children
356 using MRI (Potchen et al., 2012). The cause of the swelling, which 6. New adjunct therapies and critical care approaches in severe 420
357 can lead to brain herniation in fatal cases (Seydel et al., 2015), malaria 421
358 remains to be identified and may be due to cytotoxic or vasogenic
359 causes. Recent results show that patients who develop CM exhibit Even under optimal conditions, the case–fatality rate in severe 422
360 an over-reaction of their endothelial cells to systemic inflammation, malaria treated with either artemisinin derivatives or quinine 423
361 which is not observed in uncomplicated malaria (Wassmer et al., remains high. In addition, multi-drug-resistant falciparum malaria 424
362 2011b). Such vulnerability, coupled to a loss of cytoprotective EPCR is increasingly difficult to treat and new effective antimalarial 425
363 in the brain, could lead to a disruption of the BBB and the leakage of agents are not expected to become available within the next few 426
364 fluids and proteins from the vascular system into the extracellular years. In an effort to reduce malaria-related mortality, numerous 427
365 space. Also called vasogenic oedema, this process is consistent with adjunctive therapies that may alter severe malaria-induced physi- 428
366 the description of ring haemorrhages, a characteristic sign of BBB ological abnormalities are being evaluated, some of which have 429
367 disruption, in pediatric CM (Taylor et al., 2004; Dorovini-Zis et al., been described in other sections of this review. Such therapies 430
368 2011). In addition, these ring hemorrhages correlate positively are, in nature, highly specific to distinct syndromes of severe 431
369 between retinal and cerebral tissues, owing to their common malaria, as their aim is to target precise pathophysiological pro- 432
370 embryological origin from the neuroectoderm (Barrera et al., cesses. For example, protein C system defects inflicted by the 433
371 2015; Greiner et al., 2015). Malaria retinopathies seen in pediatric malaria parasite protein PfEMP1 in CM can be overcome by a sol- 434
372 CM are therefore a direct reflection of the neurovascular disease uble EPCR variant (Petersen et al., 2015), and could therefore rep- 435
373 process, and the high frequency of ring haemorrhages both in the resent a revolutionary approach to dampen the pathogenetic 436
374 brain and in the retina in fatal disease point towards the occurrence mechanisms and decrease mortality in affected patients. Promising 437
375 of vasogenic oedema. However, evidence for a generalised increase advances in adjunct therapies for other severe malaria syndromes 438
376 in BBB permeability leading to vasogenic oedema is still debated. A such as acute respiratory distress, acute kidney injury, severe anae- 439
377 study performed in Thailand showed no evidence of BBB impair- mia and metabolic acidosis are still lacking. 440
378 ment, as radioactively-labelled albumin given intravenously was The potential clinical benefit of fluid resuscitation was recently 441
379 not found in the cerebrospinal fluid (CSF) of CM patients during investigated and liberal fluid loading in adults with severe malaria 442
380 coma (Warrell et al., 1986). Additionally, post-mortem analyses of showed no improvement in the acidosis and acute kidney injury, 443
381 adult Vietnamese patients who died of CM showed that its patho- but increased the incidence of acute pulmonary oedema (Hanson 444
382 physiology only involved subtle functional changes in BBB integrity et al., 2014). However, a more conservative fluid strategy, using a 445
383 (Brown et al., 2000), and evidence from a different cohort demon- simple weight-based algorithm, led to a low incidence of acute res- 446
384 strated that localised loss of vascular integrity did not correlate piratory distress without significant deterioration in acid-base sta- 447
385 with the occurrence of pre-mortem coma (Medana et al., 2011). tus, renal function, electrolyte profile or systemic haemodynamics, 448
386 Brain swelling may also occur as a result of cytotoxic oedema, and was associated with increased survival (Aung et al., 2015). 449
387 which can result from cells in the cerebral tissue being unable to In addition to P. falciparum, severe cases of P. vivax and P. know- 450
388 maintain membrane potential after the failure of Na+/K+ ATP- lesi have also been reported, although sequestration is not a speci- 451
389 dependent pumps, due to hypoxia or nutrient deprivation. This fic feature of either infection and the relative contribution of co- 452
390 may cause a redistribution of water from the extra-cellular to the morbidities to clinical manifestations, particularly in vivax malaria, 453
391 intracellular compartments, ultimately leading to cell swelling, cell remains to be investigated (Wassmer et al., 2015). Both parasitic 454
392 death and tissue damage. Ischaemic or hypoxic insults due to infections can cause acute pulmonary oedema with a clinical phe- 455
393 mechanical effects of microvascular obstruction by IEs, rosettes notype similar to that seen in severe falciparum malaria. No stud- 456
394 and platelet-mediated clumps, as well as nutrient ‘‘steal” by local ies of fluid resuscitation have been performed in these patients, but 457
395 metabolically active parasites could account for cytotoxic oedema those are warranted. 458
396 in CM. This mechanism is consistent with abundant sequestration
397 in the cerebral microvasculature during the neurologic syndrome 7. Novel parasite factors involved in malarial pathogenesis and 459
398 (White et al., 2013), and the first in vivo magnetic resonance study possible therapeutic targets 460
399 of experimental CM revealed a preponderant role for cytotoxic
400 oedema in fatal outcome (Penet et al., 2005). In parallel to the development of adjunct therapies, a growing 461
401 An increase in intravascular fluid volume within the brain due effort in the search for new and effective pharmacotherapies has 462
402 to sludging of blood flow with sequestration of IEs, rosettes and been triggered by the emergence of multi-drug resistance in P. fal- 463
403 platelet-mediated clumps could also cause brain swelling and ciparum. While still in their infancy, these approaches are promis- 464
404 would explain the diffuse mild brain swelling reported in severe ing and provide a wide range of new therapeutic targets. Several 465
405 malaria patients from Bangladesh (Maude et al., 2014), without new parasite factors were recently identified and have emerged 466
406 evidence of either cytotoxic or vasogenic oedema. However, as potential drug target candidates. Among those, G-quadruplex 467
407 cytoadherence itself may not be directly or solely responsible for (G4) DNA motifs and RecQ helicases are newly described players 468
408 this clinical syndrome (Storm and Craig, 2014). in virulence gene control in P. falciparum. G4s are four-stranded 469

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx 5

470 structures formed by the stacking of quartets of guanines, and gametocytogenesis, also represents a promising target to disrupt 536
471 recent work has shown that they can form in vivo as well as malaria transmission (Coleman et al., 2014). Extensive characteri- 537
472 in vitro, affecting DNA replication, transcription, translation and sations of the parasite epigenetic factors, as well as its post- 538
473 telomere maintenance. Harris and Merrick (2015) first demon- transcriptional and translational control processes, are likely to 539
474 strated that DNA helicases, as well as histone-modifying proteins, open new avenues for drug development against P. falciparum. 540
475 can influence var gene dynamics in P. falciparum. Understanding
476 the G4-mediated regulation of the parasite virulence may open
477 the door to novel therapeutic interventions. 8. Pathologies other than CM: MiP 541
478 Plasmodium falciparum exports parasite-encoded proteins
479 involved in structural and functional remodelling of the host cell. Plasmodium falciparum infection during pregnancy can result in 542
480 This process is essential for the development of the parasite and a pathology caused by the accumulation of IEs in the placental 543
481 is also associated with its virulence (Maier et al., 2009). Molecular intervillous space and the infiltration of maternal monocytes/- 544
482 chaperones of the heat shock protein (Hsp) family are prominent macrophages (Rogerson et al., 2003), with detrimental outcomes 545
483 members of the exportome, including various Hsp40s and one for both the mother and the foetus. Expression of 546
484 Hsp70. The first biochemical evidence for a specific functional co- PfEMP1-var2csa at the surface of IEs mediates their adhesion to 547
485 chaperone interaction between the exported malarial PFA0660w the placenta. Adaptive immunity is progressively acquired during 548
486 and PfHsp70-x was recently reported (Daniyan et al., 2016). sequential malaria infections in pregnancy and is mediated by 549
487 PFA0660w can stimulate the ATPase activity of PfHsp70-x and the production of anti-VAR2CSA antibodies, which promote IE 550
488 work additively with it in a co-chaperone/chaperone interaction, adhesion blocking and opsonisation (Desai et al., 2007). This natu- 551
489 resulting in protein aggregation suppression. The authors also rally acquired immunity is the key basis for development of a vac- 552
490 showed that PFA0660w could potentially act independently as a cine to protect women during pregnancy, and VAR2CSA is 553
491 chaperone. These findings support the proposed role of PfHsp70- currently the leading candidate (Fried and Duffy, 2015). However, 554
492 x and PFA0660w in parasite protein trafficking and folding in IEs. recent findings indicate that broadly neutralising antibodies of 555
493 Further studies are underway to determine the molecular basis multigravidae are not depleted on VAR2CSA recombinant antigens. 556
494 for the specificity of this interaction, and to identify inhibitors cap- Using a new approach to assess VAR2CSA domains for functional 557
495 able of disrupting it. All of the modifications to the IEs are induced epitopes recognised by naturally acquired antibodies, 558
496 by parasite proteins, most of which initially traffic from the para- Doritchamou et al. (2016) recently demonstrated that different 559
497 site via the secretory pathway to the parasitophorous vacuole. Duffy binding-like (DBL) domain-specific IgG could react to both 560
498 There, proteins interact with a translocon complex called PTEX homologous as well as heterologous antigens and parasites, sug- 561
499 (Plasmodium translocon of exported proteins) that transports them gesting that conserved epitopes are shared between allelic vari- 562
500 across the vacuole membrane into the IE. Blocking protein export ants. In addition, IE binding was blocked by ID1-DBL2-ID2a, DBL4 563
501 through blocking PTEX function leads to the arrest of parasite and DBL5-specific IgG, while partial cross-inhibition activity was 564
502 growth and loss of virulence (Kalanon et al., 2016), making PTEX observed with purified IgG specific to ID1-DBL2-ID2a and DBL4 565
503 an excellent drug target (Gilson et al., 2016). antigens. Interestingly, plasma from patients still showed broadly 566
504 Post-translational modification of histones is one of the key adherence-blocking activity after complete depletion of these 567
505 gene regulation mechanisms during the intra-erythrocyte develop- VAR2CSA specificities. These results suggest that VAR2CSA vacci- 568
506 ment cycle of P. falciparum. Studies of proteins, which recognise nes based on a single construct and variant might induce antibod- 569
507 and interact with histone post-translational modifications, are piv- ies with limited broad blocking activity, and confirm that a 570
508 otal for understanding P. falciparum pathogenesis. Bromodomain multivalent vaccine comprised of up to five different alleles or with 571
509 proteins bind to acetylated lysines, often on histones, and fre- the addition of multiple placental malaria vaccine candidates may 572
510 quently play a role in regulation of gene expression. Plasmodium be needed to elicit the broad blocking activity observed in African 573
511 falciparum-specific bromodomain protein 1 (PfBDP1) binds to chro- multigravidae (Avril et al., 2010; Hommel et al., 2010). 574
512 matin at transcriptional start sites of invasion-related genes and The generation of protective vaccines is becoming a priority, 575
513 directly controls their expression. Conditional PfBDP1 knockdown especially in areas where the prevalence of malaria has decreased 576
514 causes a significant defect in parasite invasion and growth due to control and elimination campaigns. Indeed, a recent study 577
515 (Josling et al., 2015). In parallel to these studies, several small conducted in Mozambique showed a close relationship between 578
516 molecule inhibitors have recently been reported to have a high antibody levels and the intensity of malaria transmission. Mayor 579
517 affinity and specificity to bromodomains, and could represent a et al. (2015) showed convincing evidence that a decline in the 580
518 new therapeutic avenue in P. falciparum infection (Padmanabhan prevalence of malaria documented in the study area was accompa- 581
519 et al., 2016). nied by reductions in levels of IgG antibodies not only against 582
520 The unique plasticity of the epigenetic regulation in P. falci- VAR2CSA, but also against non-pregnancy-specific malaria anti- 583
521 parum has also emerged as a pivotal virulence and pathogenicity gens. In pregnant women with MiP, this was associated with an 584
522 factor in recent years. Karmodiya et al. (2015) performed increase in parasite densities and a higher adverse effect of P. falci- 585
523 genome-wide mapping of multiple histone modifications of P. fal- parum infection on maternal haemoglobin levels and newborn 586
524 ciparum and reported H3K36me2 as a global repressive mark, with weights (Mayor et al., 2015). Although they also suggest that 587
525 gene regulation being fine-tuned by the ratio of activation marks to immunity may be regained as exposure increases, the findings of 588
526 H3K36me2 (Karmodiya et al., 2015). Moreover, var genes are this study indicate that malaria control and elimination pro- 589
527 mostly poised and marked by a unique set of activation (H4ac) grammes could precede a resurgence of pregnancy-associated 590
528 and repression (H3K9me3) marks, which are mutually exclusive malaria pathologies. 591
529 to other P. falciparum housekeeping genes. A better characterisa- Additionally, MiP has also recently been linked to placental 592
530 tion of epigenetic regulation in P. falciparum will lead to the iden- pathology in a low malaria transmission area in Brazil, where P. 593
531 tification of potential therapeutic targets (Ay et al., 2015). Indeed, vivax is predominant (Souza et al., 2013). While MiP was not asso- 594
532 disrupting the function of proteins responsible for maintaining ciated with severe outcome in a second study performed by the 595
533 heterochromatin, such as HP1 (Brancucci et al., 2014), could be same team in this region, an increased ratio of peripheral receptor 596
534 an effective strategy to block parasite replication during the tyrosine kinase Tie-2 to angiopoietin (Ang-1) was associated with 597
535 asexual cycle. PfAP2-G, a transcription factor shown to drive the occurrence of MiP. Both Ang-1 and Ang-2 had similar magni- 598

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

6 S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx

599 tudes but inverse associations with placental barrier thickness. MiP modal imaging to quantify molecular markers of disturbed energy 662
600 is an effect modifier of the association between Ang-1 and placen- metabolism and peroxidative stress thus provides new insights 663
601 tal barrier thickness (Ataide et al., 2015). These findings provide a into understanding CM pathogenesis (Hackett et al., 2015). 664
602 possible pathway through which placental pathological changes
603 occur during MiP.
10. Conclusions and future perspectives 665
604 Lastly, further to imbalances in cytokine cascades, IE cytoadhe-
605 sion and angiogenic dysregulation, excessive or dysregulated com-
Despite the recent leap in our understanding of pathogenetic 666
606 plement activation as part of the host innate immune response to
mechanisms leading to severe malaria, the translational outputs 667
607 malaria infection can also exacerbate the severity of MiP, leading to
to improve the clinical outcome of patients remain meager. Collab- 668
608 poor pregnancy outcomes (McDonald et al., 2015b). Using an
orative and multidisciplinary approaches using clinical samples 669
609 experimental model of MiP in conjunction with micro-CT and HPLC
from field sites in endemic areas, in vitro and ex vivo models, as 670
610 analysis of neurotransmitter levels, McDonald et al. (2015a)
well as animal models of the disease, are crucial to allow global 671
611 showed that complement activation, in particular C5a, contributes
advances in the fight not only against severe falciparum malaria, 672
612 to foetal neuropathologic outcomes during MiP. The offspring of
but also emerging public health issues such as severe malaria 673
613 infected animals showed persistent neurocognitive deficits in
caused by P. vivax and P. knowlesi. 674
614 memory and affective-like behaviour compared with unexposed
615 controls (McDonald et al., 2015a). These impairments were linked
616 with decreased tissue levels of neurotransmitters in regions of the Acknowledgements 675

617 brain associated with the deficits. The inhibition of maternal C5a
618 complement receptor signaling restored the levels of neurotrans- Research reported in this publication was supported by the 676

619 mitters and reversed the associated phenotype, suggesting new National Institute of Allergy and Infectious Diseases of the National 677

620 targets for intervention in MiP aimed at decreasing foetal neu- Institutes of Health (USA) under Award Number U19AI089676, as 678

621 ropathologic outcomes. well as by the National Health and Medical Research Council (Aus- 679
tralia), the Rebecca L. Cooper Foundation (Australia) and the Aus- 680
tralian Research Council (Australia). 681

622 9. New investigative tools and experimental models

References 682
623 A vast array of new tools and models has recently become avail-
Adams, Y., Kuhnrae, P., Higgins, M.K., Ghumra, A., Rowe, J.A., 2014. Rosetting 683
624 able to facilitate the investigation of severe malaria pathogenesis, 684
Plasmodium falciparum-infected erythrocytes bind to human brain
625 with a particular focus on CM. These are detailed elsewhere microvascular endothelial cells in vitro, demonstrating a dual adhesion 685
626 (Sahu et al., 2015). Further to this, the rise of the ‘omic’ era during phenotype mediated by distinct P. falciparum erythrocyte membrane protein 686
1 domains. Infect. Immun. 82, 949–959. 687
627 the past decade has provided the malaria research community 688
Aird, W.C., Mosnier, L.O., Fairhurst, R.M., 2014. Plasmodium falciparum picks (on)
628 with unprecedented approaches and technologies to better under- EPCR. Blood 123, 163–167. 689
629 stand the biology, evolution and pathogenesis of different Plasmod- Anderson, D.C., Lapp, S.A., Akinyi, S., Meyer, E.V., Barnwell, J.W., Korir-Morrison, C., 690
Galinski, M.R., 2015. Plasmodium vivax trophozoite-stage proteomes. J. 691
630 ium spp. The Malaria Host-Pathogen Interaction Centre (MaHPIC) is
Proteomics 115, 157–176. 692
631 a large international systems biology consortium developed in Ataide, R., Murillo, O., Dombrowski, J.G., Souza, R.M., Lima, F.A., Lima, G.F., Hristov, 693
632 2012 and based in the USA (http://www.systemsbiology.emory. A.D., Valle, S.C., Di Santi, S.M., Epiphanio, S., Marinho, C.R., 2015. Malaria in 694
633 edu/index.html), which allowed the development of a variety of pregnancy interacts with and alters the angiogenic profiles of the placenta. PLoS 695
Negl. Trop. Dis. 9, e0003824. 696
634 distinctive hypothesis-generating and hypothesis-driven collabo- Aung, N.M., Kaung, M., Kyi, T.T., Kyaw, M.P., Min, M., Htet, Z.W., Anstey, N.M., Kyi, M. 697
635 rations with scientific teams in malaria-endemic regions. These M., Hanson, J., 2015. The safety of a conservative fluid replacement strategy in 698
636 collaborations involve investigators from several countries in adults hospitalised with malaria. PLoS One 10, e0143062. 699
Avril, M., Cartwright, M.M., Hathaway, M.J., Hommel, M., Elliott, S.R., Williamson, K., 700
637 South America, southeastern Asia and sub-Saharan Africa, with a Narum, D.L., Duffy, P.E., Fried, M., Beeson, J.G., Smith, J.D., 2010. Immunization 701
638 focus on infections caused by the predominant, less predominant, with VAR2CSA-DBL5 recombinant protein elicits broadly cross-reactive 702
639 or mixed species of Plasmodium (Anderson et al., 2015; Lapp antibodies to placental Plasmodium falciparum-infected erythrocytes. Infect. 703
Immun. 78, 2248–2256. 704
640 et al., 2015), to study disease states, pathogenesis and physiologi- 705
Avril, M., Bernabeu, M., Benjamin, M., Brazier, A.J., Smith, J.D., 2016. Interaction
641 cal or immunobiological questions through the use of untargeted between endothelial protein C receptor and intercellular adhesion molecule 1 706
642 high-resolution metabolomics, as well as clinical and demographic to mediate binding of plasmodium falciparum-infected erythrocytes to 707
endothelial cells. mBio 7, e00615. 708
643 metadata (Salinas et al., 2014). In addition, the MaHPIC team is 709
Ay, F., Bunnik, E.M., Varoquaux, N., Vert, J.P., Noble, W.S., Le Roch, K.G., 2015.
644 intensively studying malaria using non-human primate model sys- Multiple dimensions of epigenetic gene regulation in the malaria parasite 710
645 tems (macaque and New World monkey species, (Joyner et al., Plasmodium falciparum: gene regulation via histone modifications, nucleosome 711
positioning and nuclear architecture in P. falciparum. Bioessays 37, 182–194. 712
646 2015)). An overarching goal, beyond the team’s specific research
Barber, B.E., William, T., Grigg, M.J., Parameswaran, U., Piera, K.A., Price, R.N., Yeo, T. 713
647 quests, is to develop and make available unique large ‘omic’ data- W., Anstey, N.M., 2015. Parasite biomass-related inflammation, endothelial 714
648 sets (e.g., transcripts, proteins, lipids, metabolites and immune activation, microvascular dysfunction and disease severity in vivax malaria. 715
649 responses), integrated models and tools for the use and benefit of PLoS Pathog. 11, e1004558. 716
Barrera, V., Hiscott, P.S., Craig, A.G., White, V.A., Milner, D.A., Beare, N.A., 717
650 the research community at large. This unique and integrated com- MacCormick, I.J., Kamiza, S., Taylor, T.E., Molyneux, M.E., Harding, S.P., 2015. 718
651 bination of ‘omic’ approaches represents a revolutionary platform Severity of retinopathy parallels the degree of parasite sequestration in the eyes 719
652 to identify, develop and evaluate new diagnostic tools, antimalarial and brains of malawian children with fatal cerebral malaria. J. Infect. Dis. 211, 720
1977–1986. 721
653 drugs and vaccines for different types of malaria parasites. Bernabeu, M., Danziger, S.A., Avril, M., Vaz, M., Babar, P.H., Brazier, A.J., Herricks, T., 722
654 In parallel, live imaging, including multi-photon approaches, Maki, J.N., Pereira, L., Mascarenhas, A., Gomes, E., Chery, L., Aitchison, J.D., 723
655 has contributed to the understanding of CM pathogenesis, notably Rathod, P.K., Smith, J.D., 2016. Severe adult malaria is associated with specific 724
PfEMP1 adhesion types and high parasite biomass. Proc. Natl. Acad. Sci. U.S.A. 725
656 its haemodynamics (Nacer et al., 2014) and its immune cell mobil- 726
113, E3270–E3279.
657 isation (Pai et al., 2014) components. Recently, in experimental CM, Biswas, A.K., Hafiz, A., Banerjee, B., Kim, K.S., Datta, K., Chitnis, C.E., 2007. 727
658 vibrational spectroscopies provided evidence of peroxidative stress Plasmodium falciparum uses gC1qR/HABP1/p32 as a receptor to bind to 728
vascular endothelium and for platelet-mediated clumping. PLoS Pathog. 3, 729
659 and protein oxidation within cerebellar gray matter, which were
1271–1280. 730
660 co-localised with elevated non-haeme iron at the site of micro- Brancucci, N.M., Bertschi, N.L., Zhu, L., Niederwieser, I., Chin, W.H., Wampfler, R., 731
661 haemorrhages. A novel combination of chemical probe-free, multi- Freymond, C., Rottmann, M., Felger, I., Bozdech, Z., Voss, T.S., 2014. 732

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx 7

733 Heterochromatin protein 1 secures survival and transmission of malaria Joyner, C., Barnwell, J.W., Galinski, M.R., 2015. No more monkeying around: primate 818
734 parasites. Cell Host Microbe 16, 165–176. malaria model systems are key to understanding Plasmodium vivax liver-stage 819
735 Brown, H.C., Chau, T.T., Mai, N.T., Day, N.P., Sinh, D.X., White, N.J., Hien, T.T., Farrar, biology, hypnozoites, and relapses. Front. Microbiol. 6, 145. 820
736 J., Turner, G.D., 2000. Blood-brain barrier function in cerebral malaria and CNS Kalanon, M., Bargieri, D., Sturm, A., Matthews, K., Ghosh, S., Goodman, C.D., 821
737 infections in Vietnam. Neurology 55, 104–111. Thiberge, S., Mollard, V., McFadden, G.I., Menard, R., de Koning-Ward, T.F., 2016. 822
738 Carlson, J., Helmby, H., Hill, A.V., Brewster, D., Greenwood, B.M., Wahlgren, M., 1990. The Plasmodium translocon of exported proteins component EXP2 is critical for 823
739 Human cerebral malaria: association with erythrocyte rosetting and lack of establishing a patent malaria infection in mice. Cell. Microbiol. 18, 399–412. 824
740 anti-rosetting antibodies. Lancet 336, 1457–1460. Karmodiya, K., Pradhan, S.J., Joshi, B., Jangid, R., Reddy, P.C., Galande, S., 2015. A 825
741 Coleman, B.I., Skillman, K.M., Jiang, R.H., Childs, L.M., Altenhofen, L.M., Ganter, M., comprehensive epigenome map of Plasmodium falciparum reveals unique 826
742 Leung, Y., Goldowitz, I., Kafsack, B.F., Marti, M., Llinas, M., Buckee, C.O., mechanisms of transcriptional regulation and identifies H3K36me2 as a 827
743 Duraisingh, M.T., 2014. A Plasmodium falciparum histone deacetylase regulates global mark of gene suppression. Epigenet. Chromatin 8, 32. 828
744 antigenic variation and gametocyte conversion. Cell Host Microbe 16, 177–186. Lapp, S.A., Mok, S., Zhu, L., Wu, H., Preiser, P.R., Bozdech, Z., Galinski, M.R., 2015. 829
745 Daniyan, M.O., Boshoff, A., Prinsloo, E., Pesce, E.R., Blatch, G.L., 2016. The malarial Plasmodium knowlesi gene expression differs in ex vivo compared to in vitro 830
746 exported PFA0660w is an Hsp40 co-chaperone of PfHsp70-x. PLoS One 11, blood-stage cultures. Malar. J. 14, 110. 831
747 e0148517. Maier, A.G., Cooke, B.M., Cowman, A.F., Tilley, L., 2009. Malaria parasite proteins 832
748 Desai, M., ter Kuile, F.O., Nosten, F., McGready, R., Asamoa, K., Brabin, B., Newman, R. that remodel the host erythrocyte. Nat. Rev. Microbiol. 7, 341–354. 833
749 D., 2007. Epidemiology and burden of malaria in pregnancy. Lancet Infect. Dis. Manning, L., Laman, M., Davis, W.A., Davis, T.M., 2014. Clinical features and outcome 834
750 7, 93–104. in children with severe Plasmodium falciparum malaria: a meta-analysis. PLoS 835
751 Doritchamou, J.Y., Herrera, R., Aebig, J.A., Morrison, R., Nguyen, V., Reiter, K., Shimp, One 9, e86737. 836
752 R., MacDonald, N.J., Narum, D.L., Fried, M., Duffy, P.E., 2016. VAR2CSA domain- Maude, R.J., Barkhof, F., Hassan, M.U., Ghose, A., Hossain, A., Abul Faiz, M., 837
753 specific analysis of naturally acquired functional antibodies to P. falciparum Choudhury, E., Rashid, R., Abu Sayeed, A., Charunwatthana, P., Plewes, K., 838
754 placental malaria. J. Infect. Dis. 214, 577–586. Kingston, H., Maude, R.R., Silamut, K., Day, N.P., White, N.J., Dondorp, A.M., 2014. 839
755 Dorovini-Zis, K., Schmidt, K., Huynh, H., Fu, W., Whitten, R.O., Milner, D., Kamiza, S., Magnetic resonance imaging of the brain in adults with severe falciparum 840
756 Molyneux, M., Taylor, T.E., 2011. The neuropathology of fatal cerebral malaria in malaria. Malar. J. 13, 177. 841
757 malawian children. Am. J. Pathol. 178, 2146–2158. Mayor, A., Hafiz, A., Bassat, Q., Rovira-Vallbona, E., Sanz, S., Machevo, S., Aguilar, R., 842
758 Fried, M., Duffy, P.E., 2015. Designing a VAR2CSA-based vaccine to prevent placental Cistero, P., Sigauque, B., Menendez, C., Alonso, P.L., Chitnis, C.E., 2011. 843
759 malaria. Vaccine 33, 7483–7488. Association of severe malaria outcomes with platelet-mediated clumping and 844
760 Gillrie, M.R., Avril, M., Brazier, A.J., Davis, S.P., Stins, M.F., Smith, J.D., Ho, M., 2015. adhesion to a novel host receptor. PLoS One 6, e19422. 845
761 Diverse functional outcomes of Plasmodium falciparum ligation of EPCR: Mayor, A., Bardaji, A., Macete, E., Nhampossa, T., Fonseca, A.M., Gonzalez, R., 846
762 potential implications for malarial pathogenesis. Cell. Microbiol. 17, 1883– Maculuve, S., Cistero, P., Ruperez, M., Campo, J., Vala, A., Sigauque, B., Jimenez, 847
763 1899. A., Machevo, S., de la Fuente, L., Nhama, A., Luis, L., Aponte, J.J., Acacio, S., 848
764 Gilson, P.R., Chisholm, S.A., Crabb, B.S., de Koning-Ward, T.F., 2016. Host cell Nhacolo, A., Chitnis, C., Dobano, C., Sevene, E., Alonso, P.L., Menendez, C., 2015. 849
765 remodelling in malaria parasites: a new pool of potential drug targets. Int. J. Changing trends in P. falciparum burden, immunity, and disease in pregnancy. 850
766 Parasitol. [This issue] N. Engl. J. Med. 373, 1607–1617. 851
767 Goel, S., Palmkvist, M., Moll, K., Joannin, N., Lara, P., Akhouri, R.R., Moradi, N., McDonald, C.R., Darling, A.M., Conroy, A.L., Tran, V., Cabrera, A., Liles, W.C., Wang, 852
768 Ojemalm, K., Westman, M., Angeletti, D., Kjellin, H., Lehtio, J., Blixt, O., Idestrom, M., Aboud, S., Urassa, W., Fawzi, W.W., Kain, K.C., 2015a. Inflammatory and 853
769 L., Gahmberg, C.G., Storry, J.R., Hult, A.K., Olsson, M.L., von Heijne, G., Nilsson, I., angiogenic factors at mid-pregnancy are associated with spontaneous preterm 854
770 Wahlgren, M., 2015. RIFINs are adhesins implicated in severe Plasmodium birth in a cohort of Tanzanian women. PLoS One 10, e0134619. 855
771 falciparum malaria. Nat. Med. 21, 314–317. McDonald, C.R., Tran, V., Kain, K.C., 2015b. Complement activation in placental 856
772 Greiner, J., Dorovini-Zis, K., Taylor, T.E., Molyneux, M.E., Beare, N.A., Kamiza, S., malaria. Front. Microbiol. 6, 1460. 857
773 White, V.A., 2015. Correlation of hemorrhage, axonal damage, and blood-tissue Medana, I.M., Day, N.P., Sachanonta, N., Mai, N.T., Dondorp, A.M., Pongponratn, E., 858
774 barrier disruption in brain and retina of Malawian children with fatal cerebral Hien, T.T., White, N.J., Turner, G.D., 2011. Coma in fatal adult human malaria is 859
775 malaria. Front. Cell. Infect. Microbiol. 5, 18. not caused by cerebral oedema. Malar. J. 10, 267. 860
776 Hackett, M.J., Aitken, J.B., El-Assaad, F., McQuillan, J.A., Carter, E.A., Ball, H.J., Tobin, Mohanty, S., Mishra, S.K., Patnaik, R., Dutt, A.K., Pradhan, S., Das, B., Patnaik, J., 861
777 M.J., Paterson, D., de Jonge, M.D., Siegele, R., Cohen, D.D., Vogt, S., Grau, G.E., Mohanty, A.K., Lee, S.J., Dondorp, A.M., 2011. Brain swelling and mannitol 862
778 Hunt, N.H., Lay, P.A., 2015. Mechanisms of murine cerebral malaria: multimodal therapy in adult cerebral malaria: a randomized trial. Clin. Infect. Dis. 53, 349– 863
779 imaging of altered cerebral metabolism and protein oxidation at hemorrhage 355. 864
780 sites. Sci. Adv. 1, e1500911. Moxon, C.A., Wassmer, S.C., Milner Jr., D.A., Chisala, N.V., Taylor, T.E., Seydel, K.B., 865
781 Handunnetti, S.M., David, P.H., Perera, K.L., Mendis, K.N., 1989. Uninfected Molyneux, M.E., Faragher, B., Esmon, C.T., Downey, C., Toh, C.H., Craig, A.G., 866
782 erythrocytes form ‘‘rosettes” around Plasmodium falciparum infected Heyderman, R.S., 2013. Loss of endothelial protein C receptors links coagulation 867
783 erythrocytes. Am. J. Trop. Med. Hyg. 40, 115–118. and inflammation to parasite sequestration in cerebral malaria in African 868
784 Hanson, J., Anstey, N.M., Bihari, D., White, N.J., Day, N.P., Dondorp, A.M., 2014. The children. Blood 122, 842–851. 869
785 fluid management of adults with severe malaria. Crit. Care 18, 642. Moxon, C.A., Chisala, N.V., Wassmer, S.C., Taylor, T.E., Seydel, K.B., Molyneux, M.E., 870
786 Harris, L.M., Merrick, C.J., 2015. G-quadruplexes in pathogens: a common route to Faragher, B., Kennedy, N., Toh, C.H., Craig, A.G., Heyderman, R.S., 2014. 871
787 virulence control? PLoS Pathog. 11, e1004562. Persistent endothelial activation and inflammation after Plasmodium 872
788 Hawkes, M., Opoka, R.O., Namasopo, S., Miller, C., Conroy, A.L., Serghides, L., Kim, H., falciparum Infection in Malawian children. J. Infect. Dis. 209, 610–615. 873
789 Thampi, N., Liles, W.C., John, C.C., Kain, K.C., 2011. Nitric oxide for the adjunctive Nacer, A., Movila, A., Sohet, F., Girgis, N.M., Gundra, U.M., Loke, P., Daneman, R., 874
790 treatment of severe malaria: hypothesis and rationale. Med. Hypotheses 77, Frevert, U., 2014. Experimental cerebral malaria pathogenesis–hemodynamics 875
791 437–444. at the blood brain barrier. PLoS Pathog. 10, e1004528. 876
792 Hendriksen, I.C., Mwanga-Amumpaire, J., von Seidlein, L., Mtove, G., White, L.J., O’Sullivan, J.M., Preston, R.J., O’Regan, N., O’Donnell, J.S., 2016. Emerging roles for 877
793 Olaosebikan, R., Lee, S.J., Tshefu, A.K., Woodrow, C., Amos, B., Karema, C., hemostatic dysfunction in malaria pathogenesis. Blood 127, 2281–2288. 878
794 Saiwaew, S., Maitland, K., Gomes, E., Pan-Ngum, W., Gesase, S., Silamut, K., Padmanabhan, B., Mathur, S., Manjula, R., Tripathi, S., 2016. Bromodomain and 879
795 Reyburn, H., Joseph, S., Chotivanich, K., Fanello, C.I., Day, N.P., White, N.J., extra-terminal (BET) family proteins: new therapeutic targets in major diseases. 880
796 Dondorp, A.M., 2012. Diagnosing severe falciparum malaria in parasitaemic J. Biosci. 41, 295–311. 881
797 African children: a prospective evaluation of plasma PfHRP2 measurement. Pai, S., Qin, J., Cavanagh, L., Mitchell, A., El-Assaad, F., Jain, R., Combes, V., Hunt, N.H., 882
798 PLoS Med. 9, e1001297. Grau, G.E., Weninger, W., 2014. Real-time imaging reveals the dynamics of 883
799 Hoffmann, A., Pfeil, J., Alfonso, J., Kurz, F.T., Sahm, F., Heiland, S., Monyer, H., leukocyte behaviour during experimental cerebral malaria pathogenesis. PLoS 884
800 Bendszus, M., Mueller, A.K., Helluy, X., Pham, M., 2016. Experimental cerebral Pathog. 10, e1004236. 885
801 malaria spreads along the rostral migratory stream. PLoS Pathog. 12, e1005470. Pain, A., Ferguson, D.J., Kai, O., Urban, B.C., Lowe, B., Marsh, K., Roberts, D.J., 2001. 886
802 Hommel, M., Elliott, S.R., Soma, V., Kelly, G., Fowkes, F.J., Chesson, J.M., Duffy, M.F., Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a 887
803 Bockhorst, J., Avril, M., Mueller, I., Raiko, A., Stanisic, D.I., Rogerson, S.J., Smith, J. common adhesive phenotype and is associated with severe malaria. Proc. Natl. 888
804 D., Beeson, J.G., 2010. Evaluation of the antigenic diversity of placenta-binding Acad. Sci. U.S.A. 98, 1805–1810. 889
805 Plasmodium falciparum variants and the antibody repertoire among pregnant Penet, M.F., Viola, A., Confort-Gouny, S., Le Fur, Y., Duhamel, G., Kober, F., Ibarrola, 890
806 women. Infect. Immun. 78, 1963–1978. D., Izquierdo, M., Coltel, N., Gharib, B., Grau, G.E., Cozzone, P.J., 2005. Imaging 891
807 Hviid, L., Jensen, A.T., 2015. PfEMP1 – A parasite protein family of key importance in experimental cerebral malaria in vivo: significant role of ischemic brain edema. 892
808 Plasmodium falciparum malaria immunity and pathogenesis. Adv. Parasitol. 88, J. Neurosci. 25, 7352–7358. 893
809 51–84. Petersen, J.E., Bouwens, E.A., Tamayo, I., Turner, L., Wang, C.W., Stins, M., Theander, 894
810 Jespersen, J.S., Wang, C.W., Mkumbaye, S.I., Minja, D.T., Petersen, B., Turner, L., T.G., Hermida, J., Mosnier, L.O., Lavstsen, T., 2015. Protein C system defects 895
811 Petersen, J.E., Lusingu, J.P., Theander, T.G., Lavstsen, T., 2016. Plasmodium inflicted by the malaria parasite protein PfEMP1 can be overcome by a soluble 896
812 falciparum var genes expressed in children with severe malaria encode EPCR variant. Thromb. Haemost. 114, 1038–1048. 897
813 CIDRalpha1 domains. EMBO Mol. Med. 8, 839–850. Potchen, M.J., Kampondeni, S.D., Seydel, K.B., Birbeck, G.L., Hammond, C.A., Bradley, 898
814 Josling, G.A., Petter, M., Oehring, S.C., Gupta, A.P., Dietz, O., Wilson, D.W., Schubert, W.G., DeMarco, J.K., Glover, S.J., Ugorji, J.O., Latourette, M.T., Siebert, J.E., 899
815 T., Langst, G., Gilson, P.R., Crabb, B.S., Moes, S., Jenoe, P., Lim, S.W., Brown, G.V., Molyneux, M.E., Taylor, T.E., 2012. Acute brain MRI findings in 120 Malawian 900
816 Bozdech, Z., Voss, T.S., Duffy, M.F., 2015. A Plasmodium falciparum bromodomain children with cerebral malaria: new insights into an ancient disease. AJNR Am. 901
817 protein regulates invasion gene expression. Cell Host Microbe 17, 741–751. J. Neuroradiol. 33, 1740–1746. 902

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002
 PARA 3897 No. of Pages 8, Model 5G
26 September 2016

8 S.C. Wassmer, G.E.R. Grau / International Journal for Parasitology xxx (2016) xxx–xxx

903 Rogerson, S.J., Pollina, E., Getachew, A., Tadesse, E., Lema, V.M., Molyneux, M.E., Taylor, T.E., Fu, W.J., Carr, R.A., Whitten, R.O., Mueller, J.S., Fosiko, N.G., Lewallen, S., 944
904 2003. Placental monocyte infiltrates in response to Plasmodium falciparum Liomba, N.G., Molyneux, M.E., 2004. Differentiating the pathologies of cerebral 945
905 malaria infection and their association with adverse pregnancy outcomes. Am. J. malaria by postmortem parasite counts. Nat. Med. 10, 143–145. 946
906 Trop. Med. Hyg. 68, 115–119. Turner, L., Lavstsen, T., Berger, S.S., Wang, C.W., Petersen, J.E., Avril, M., Brazier, A.J., 947
907 Rowe, A., Obeiro, J., Newbold, C.I., Marsh, K., 1995. Plasmodium falciparum rosetting Freeth, J., Jespersen, J.S., Nielsen, M.A., Magistrado, P., Lusingu, J., Smith, J.D., 948
908 is associated with malaria severity in Kenya. Infect. Immun. 63, 2323–2326. Higgins, M.K., Theander, T.G., 2013. Severe malaria is associated with parasite 949
909 Rowe, J.A., Shafi, J., Kai, O.K., Marsh, K., Raza, A., 2002. Nonimmune IgM, but not IgG binding to endothelial protein C receptor. Nature 498, 502–505. 950
910 binds to the surface of Plasmodium falciparum-infected erythrocytes and Warrell, D.A., Looareesuwan, S., Phillips, R.E., White, N.J., Warrell, M.J., Chapel, H.M., 951
911 correlates with rosetting and severe malaria. Am. J. Trop. Med. Hyg. 66, 692– Areekul, S., Tharavanij, S., 1986. Function of the blood-cerebrospinal fluid 952
912 699. barrier in human cerebral malaria: rejection of the permeability hypothesis. 953
913 Rowe, J.A., Handel, I.G., Thera, M.A., Deans, A.M., Lyke, K.E., Kone, A., Diallo, D.A., Am. J. Trop. Med. Hyg. 35, 882–889. 954
914 Raza, A., Kai, O., Marsh, K., Plowe, C.V., Doumbo, O.K., Moulds, J.M., 2007. Blood Wassmer, S.C., Taylor, T., Maclennan, C.A., Kanjala, M., Mukaka, M., Molyneux, M.E., 955
915 group O protects against severe Plasmodium falciparum malaria through the Grau, G.E., 2008. Platelet-induced clumping of Plasmodium falciparum-infected 956
916 mechanism of reduced rosetting. Proc. Natl. Acad. Sci. U.S.A. 104, 17471–17476. erythrocytes from Malawian patients with cerebral malaria-possible 957
917 Rowe, J.A., Claessens, A., Corrigan, R.A., Arman, M., 2009. Adhesion of Plasmodium modulation in vivo by thrombocytopenia. J. Infect. Dis. 197, 72–78. 958
918 falciparum-infected erythrocytes to human cells: molecular mechanisms and Wassmer, S.C., Combes, V., Grau, G.E., 2011a. Platelets and microparticles in cerebral 959
919 therapeutic implications. Expert Rev. Mol. Med. 11, e16. malaria: the unusual suspects. Drug. Discov. Today Dis. Mech. 8, e15–e23. 960
920 Sahu, P.K., Satpathi, S., Behera, P.K., Mishra, S.K., Mohanty, S., Wassmer, S.C., 2015. Wassmer, S.C., Moxon, C.A., Taylor, T., Grau, G.E., Molyneux, M.E., Craig, A.G., 2011b. 961
921 Pathogenesis of cerebral malaria: new diagnostic tools, biomarkers, and Vascular endothelial cells cultured from patients with cerebral or 962
922 therapeutic approaches. Front. Cell. Infect. Microbiol. 5, 75. uncomplicated malaria exhibit differential reactivity to TNF. Cell. Microbiol. 963
923 Salinas, J.L., Kissinger, J.C., Jones, D.P., Galinski, M.R., 2014. Metabolomics in the fight 13, 198–209. 964
924 against malaria. Mem. Inst. Oswaldo Cruz 109, 589–597. Wassmer, S.C., Taylor, T.E., Rathod, P.K., Mishra, S.K., Mohanty, S., Arevalo-Herrera, 965
925 Serghides, L., Kim, H., Lu, Z., Kain, D.C., Miller, C., Francis, R.C., Liles, W.C., Zapol, W. M., Duraisingh, M.T., Smith, J.D., 2015. Investigating the pathogenesis of severe 966
926 M., Kain, K.C., 2011. Inhaled nitric oxide reduces endothelial activation and malaria: a multidisciplinary and cross-geographical approach. Am. J. Trop. Med. 967
927 parasite accumulation in the brain, and enhances survival in experimental Hyg. 93, 42–56. 968
928 cerebral malaria. PLoS One 6, e27714. White, N.J., Turner, G.D., Day, N.P., Dondorp, A.M., 2013. Lethal malaria: Marchiafava 969
929 Seydel, K.B., Kampondeni, S.D., Valim, C., Potchen, M.J., Milner, D.A., Muwalo, F.W., and Bignami were right. J. Infect. Dis. 208, 192–198. 970
930 Birbeck, G.L., Bradley, W.G., Fox, L.L., Glover, S.J., Hammond, C.A., Heyderman, R. Yeo, T.W., Lampah, D.A., Gitawati, R., Tjitra, E., Kenangalem, E., McNeil, Y.R., Darcy, C. 971
931 S., Chilingulo, C.A., Molyneux, M.E., Taylor, T.E., 2015. Brain swelling and death J., Granger, D.L., Weinberg, J.B., Lopansri, B.K., Price, R.N., Duffull, S.B., 972
932 in children with cerebral malaria. N. Engl. J. Med. 372, 1126–1137. Celermajer, D.S., Anstey, N.M., 2007. Impaired nitric oxide bioavailability and 973
933 Souza, R.M., Ataide, R., Dombrowski, J.G., Ippolito, V., Aitken, E.H., Valle, S.N., L-arginine reversible endothelial dysfunction in adults with falciparum malaria. 974
934 Alvarez, J.M., Epiphanio, S., Marinho, C.R., 2013. Placental histopathological J. Exp. Med. 204, 2693–2704. 975
935 changes associated with Plasmodium vivax infection during pregnancy. PLoS Yeo, T.W., Lampah, D.A., Rooslamiati, I., Gitawati, R., Tjitra, E., Kenangalem, E., Price, 976
936 Negl. Trop. Dis. 7, e2071. R.N., Duffull, S.B., Anstey, N.M., 2013. A randomized pilot study of L-arginine 977
937 Stevenson, L., Laursen, E., Cowan, G.J., Bandoh, B., Barfod, L., Cavanagh, D.R., infusion in severe falciparum malaria: preliminary safety, efficacy and 978
938 Andersen, G.R., Hviid, L., 2015. Alpha2-macroglobulin can crosslink multiple pharmacokinetics. PLoS One 8, e69587. 979
939 Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) molecules Yeo, T.W., Lampah, D.A., Kenangalem, E., Tjitra, E., Weinberg, J.B., Granger, D.L., 980
940 and may facilitate adhesion of parasitized erythrocytes. PLoS Pathog. 11, Price, R.N., Anstey, N.M., 2014. Decreased endothelial nitric oxide 981
941 e1005022. bioavailability, impaired microvascular function, and increased tissue oxygen 982
942 Storm, J., Craig, A.G., 2014. Pathogenesis of cerebral malaria-inflammation and consumption in children with falciparum malaria. J. Infect. Dis. 210, 1627–1632. 983
943 cytoadherence. Front. Cell. Infect. Microbiol. 4, 100. 984

Please cite this article in press as: Wassmer, S.C., Grau, G.E.R. Severe malaria: what’s new on the pathogenesis front?. Int. J. Parasitol. (2016), http://dx.doi.
org/10.1016/j.ijpara.2016.08.002